WO2007139956A2

WO2007139956A2 - Methods and systems for the quantitative analysis of biomarkers

Info

Publication number: WO2007139956A2
Application number: PCT/US2007/012525
Authority: WO
Inventors: Russell Philip Grant; Andrew Dennis Wagner; Mary Katherine Morr; Milan Rameshkumar Patel
Original assignee: Laboratory Corporation Of America Holdings
Priority date: 2006-05-26
Filing date: 2007-05-25
Publication date: 2007-12-06
Also published as: WO2007139956A3; US20080128606A1; US7935921B2; CA2654319C; CA2820957A1; US20110240842A1; CA2654319A1

Abstract

Disclosed are methods and systems using liquid chromatography/tandem mass spectrometry (LC-MS/MS and 2D-LC-MS/MS) for the analysis of endogenous biomarkers, including steroid hormones, such as estrone and estradiol, thyroid hormones, such as free thyroxine, and metabolites, such as 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, in biological samples.

Description

METHODS AND SYSTEMS FOR THE QUANTITATIVE ANALYSIS OF

BIOMAKKERS

PRIORITY CLAIM TO RELATED APPLICATIONS This application claims priority to U.S. provisional patent application Serial No.

60/808,812, filed May 26, 2006. The disclosure of U.S. provisional patent application Serial No. 60/808,812 is incorporated by reference in its entirety herein.

FIELD OF INVENTION

The presently disclosed subject matter relates to methods and systems for the analysis of biomarkers. In certain embodiments, the biomarkers are endogenous to human subjects such that the measurement may be used for clinical diagnosis.

BACKGROUND

Biomarkers, such as hormones, vitamins, metabolites, can be used for the clinical diagnosis of multiple disorders and as endogenous biomarkers in endocrinology. For example, the measurement of estrogen compounds, such as estrone and estradiol can be used to evaluate ovarian function and to evaluate excess or diminished estrogen levels in a patient. Also, measurement of thyroxine can be used to quantify thyroid function.

Requirements for the clinical diagnostic testing of endogenous biomarkers in endocrinology may include highly sensitive and specific assays, the ability to analyze small sample volumes (e.g., pediatric sample volumes can be limited to less than about 200 μL), and the ability to screen for multiple analytes to accurately diagnose a disease state, e.g., an endocrine disorder. Historically, radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA) methods have been used in such clinical diagnostic testing. Immunoassay methods (IA), such as RIA and EIA, however, may suffer from low throughput, antibody cross- reactivity, which can require extra preparation for specificity, and poor scalability. Also, the analysis of endogenous biomarkers by RIA may require multiple serial dilutions for the analysis of each individual marker, which can lead to the need to make multiple adjustments to normalize sample volumes and/or the need for multiple separate tests. Also, immunoassay tesing is not particularly conducive to the analysis of multiple biomarkers in each sample. The analysis for multiple analytes in a single assay can allow for using samples of reduced size which results in assays of increased sensitivity and efficiency per sample.

An important class of hormones are the steroid hormones, such as testosterone and estrogens. Testosterone develops and maintains the male secondary sex characteristics, and promotes growth and development of sperm. Estrogen is the term used for a group of hormones of which there are three principle forms, estrone, estradiol, and estriol.

For example, relatively small variations in estrogen levels may be clinically significant. Generally, the level of estrogen in post-menopausal women, adult males, and prepubescent children is ≤ 10 pg/mL. Elevated estrogen levels in children may lead to precocious puberty (and short stature). In post-menopausal women, low estrogen levels may require replacement, where as levels greater than 5 pg/mL may be prognostic for certain cancers. In adult males, elevated estrogen levels may be indicative of certain disease states (testicular cancer). In adult females, reduced or elevated levels may also be indicative of certain cancers (e.g., ovarian cancer). A level of serum estrogen of 15 pg/mL is clinically different from 10 pg/mL and thus, measurement of estrogen compounds (e.g., estradiol and estrone) requires an LLOQ of 1-5 pg/mL irrespective of sample type, patient age, gender and diet.

Another important class of hormones are the thyroid hormones. Thyroxine (T4) and triiodothyronine (T3) are examples of thyroid hormones. T4 and T3 enter cells and bind to intracellular receptors. T4 and T3 are important in regulation of a number of factors including growth and development, carbohydrate metabolism, oxygen consumption, and protein synthesis. T4 acts as a prohormone, as the bulk of T3 present in blood is produced by monodeiodination of T4 by intracellular enzymes. Thyroid hormone concentrations in blood are essential tests for the assessment of thyroid function.

Thus, there is a need to develop analytical techniques that can be used for the measurement of endogenous biomarkers, and for methods that provide more sensitivity and higher throughput than RIA. Until recently, however, only GC-MS or LC-MS/MS with derivatization has been successful for small sample volumes. Thus, there is a need in the art for LC-MS/MS techniques for the analysis of endogenous biomarkers for clinical diagnosis in endocrinology capable of providing detection limits at acceptable levels, without the need for the cumbersome derivatization processes.

SUMMARY In some embodiments, the presently disclosed subject matter provides methods and systems for the quantitative analysis of endocrine biomarkers in a test sample. The quantification of such markers may, in certain embodiments, be used for clinical diagnosis in endocrinology. For example, in some embodiments, the methods and systems of the present invention may be used for the quantitative analysis of total levels of certain hormones, including steroid hormones, such as estrone and estradiol, and their metabolites, such as estrone sulfate. In other embodiments, the methods and systems of the present invention provide for the quantitative analysis of biomarkers that can be difficult to detect in their active state. For example, the systems and methods of the present invention may be used to quantify free (i.e., not bound to protein) serum hormones, such as free thyroxine (T4) in biological samples. Or, in other embodiments, the systems and methods of the present invention may be used to quantify free triiodothyronine (T3) or testosterone. In an embodiment, the methods and systems of the present invention allow for measuresment of such hormones without the need for derivatation processes. In some embodiments, the biomarkers of interest are estradiol and/or estrone. Thus, in one embodiment, the present invention comprises a method for determining the presence or amount of estradiol in a sample by tandem mass spectrometry, comprising: (a) generating a dehydrated precursor ion of the estradiol; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the presence or amount of one or more of the ions generated in step (a) or (b) or both, and relating the detected ions to the presence or amount of the estradiol in the sample. In an embodiment, the sample comprises a mixture of estradiol and estrone.

In other embodiments, the biomarker comprises free thyroxine (T4) or triiodothyronine (T3). In certain embodiments, the present invention provides a high- throughput assay for free thyroxine (T4). Thus, in one embodiment, the present invention comprises a method for determining the presence or amount of free thyroxine in a plurality of samples by tandem mass spectrometry, comprising: (a) dialyzing the plurality of samples to separate the free thyroxine from the protein-bound thyroxine in the samples; (b) generating a precursor ion of the thyroxine; (b) generating one or more fragment ions of the thyroxine; and (c) detecting the presence or amount of one or more of the ions generated in step (b) or (c) or both, and relating the detected ions to the presence or amount of the free thyroxine in the plurality of samples.

In some embodiments, the methods and systems of the present invention comprise liquid chromatography (LC) methods in combination with other analytical techniques as a means to measure such biomarkers with high sensitivity and high throughput. In certain embodiments, the present invention comprises quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of endocrine biomarkers in a test sample. In some embodiments, two-dimensional or tandem LC is used. The method may include, in alternate emboiments, liquid-liquid extractions, dialysis, sample dilution, and/or sample dehydration steps prior to analysis by tandem mass spectrometry.

Accordingly, embodiments of the present invention may provide methods for the quantitative LC-MS/MS and 2D-LC-MS/MS analysis of hormones, including steroid hormones, such as estrone and estradiol. Additionally or alternatively, embodiments of the present invention may provide methods for the quantitative determination of a free (i.e., nonprotein bound) hormone or metabolite using dialysis in combination with LC-MS/MS analysis for hormones that in biological samples, may be predominantly protein-bound. Such hormones may include free thyroxine (T4), free triiodothyronine (3), or free testosterone. Certain objects of the present invention, having been stated hereinabove, will become further evident as the description proceeds when taken in connection with the accompanying figures and examples as described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale.

FIG. 1 shows a flow chart of a method for quantitative analysis of a biomarker of interest in accordance with one embodiment of the present invention.

FIG. 2 shows dehydration of estradiol and the effect on mass spectrometry (MS) analysis in accordance with an embodiment of the present invention. FIG. 3 shows potential isobaric interferences for measurement of estrone and estradiol due to dehydration of dehydroepiandrosterone (DHEA) in accordance with one embodiment of the present invention.

FIG. 4 shows an example of heart-cutting from a primary separation gradient to remove compounds that comprise isobaric interference in the analysis of estrone and estradiol in accordance with one embodiment of the present invention.

FIG. 5 shows a method for the quantification of estrone and estradiol in accordance with an embodiment of the present invention.

FIG. 6 shows a method for the quantification of free thyroxine (T4) in accordance with an embodiment of the present invention. FIG. 7 shows a system for quantitative analysis of a metabolite in accordance with one embodiment of the present invention (Panel A), and a system for multiplex analysis (Panel B) in accordance with alternate embodiments of the present invention. FIG. 8 shows a LC-MS/MS chrornatogram of estrone sulfate at a limit of quantification of 100 pg/mL in accordance with one embodiment of the present invention.

FIG. 9 shows a LC-MS/MS chromatogram of free thyroxine at a limit of quantification of 2 pg/mL in accordance with one embodiment of the present invention. FIG. 10 shows a 2D-LC-MS/MS chromatogram of 25-hydroxyvitamin D2 at a limit of quantification of 1 ng/mL in accordance with one embodiment of the present invention.

FIG. 11 shows a 2D-LC-MS/MS chromatogram of 25-hydroxyvitamin D3 at a limit of quantification of 1 ng/mL in accordance with one embodiment of the present invention.

FIG. 12 shows a 2D-LC-MS/MS chromatogram of estrone at a limit of quantification of 2.5 pg/mL in accordance with one embodiment of the present invention.

FIG. 13 shows a 2D-LC-MS/MS chromatogram of estradiol at a limit of quantification of 1 pg/mL in accordance with one embodiment of the present invention.

FIG. 14 shows a LC-MS/MS chromatogram of estrone sulfate at an upper limit of quantification of 50 ng/mL in accordance with one embodiment of the present invention. FIG. 15 shows a LC-MS/MS chromatogram of free thyroxine at an upper limit of quantification of 100 pg/dL in accordance with one embodiment of the present invention.

FIG. 16 shows a 2D-LC-MS/MS chromatogram of 25-hydroxyvitamin D2 at an upper limit of quantification of 250 ng/mL in accordance with one embodiment of the present invention. FIG. 17 shows a 2D-LC-MS/MS chromatogram of 25-hydroxyvitamin D3 at an upper limit of quantification of 250 ng/mL in accordance with one embodiment of the present invention.

FIG. 18 shows a 2D-LC-MS/MS chromatogram of estrone at an upper limit of quantification of 500 pg/mL in accordance with one embodiment of the present invention. FIG. 19 shows a 2D-LC-MS/MS chromatogram of estradiol at an upper limit of quantification of 500 pg/mL in accordance with one embodiment of the present invention.

FIG. 20 shows a calibration curve obtained by LC-MS/MS for estrone sulfate in accordance with one embodiment of the present invention.

FIG. 21 shows a calibration curve obtained by LC-MS/MS for free thyroxine in accordance with one embodiment of the present invention.

FIG. 22 shows a calibration curve obtained by 2D-LC-MS/MS for 25-hydroxyvitamin D2 in accordance with one embodiment of the present invention. FIG. 23 shows a calibration curve obtained by 2D-LC-MS/MS for 25-hydroxyvitamin D3 in accordance with one embodiment of the present invention.

FIG. 24 shows a calibration curve obtained by 2D-LC-MS/MS for estrone in accordance with one embodiment of the present invention. FIG. 25 shows a calibration curve obtained by 2D-LC-MS/MS for estradiol in accordance with one embodiment of the present invention.

FIG. 26 shows cross-validation data for LC-MS/MS as compared to radioimmunoassay (RIA) for estrone sulfate in accordance with one embodiment of the present invention.

FIG. 27 shows cross-validation data for LC-MS/MS as compared to immunoassay (IA) for free thyroxine in accordance with one embodiment of the present invention.

FIG. 28 shows cross-validation data for 2D-LC-MS/MS as compared to a competitive binding protein assay (CBP) (Panel A) or immunoassay (IA) (Panel B) for total 25- hydroxyvitamin D (25-hydroxyvitamin D2+D3) in accordance with alternate embodiments of the present invention. FIG. 29 shows cross-validation data for 2D-LC-MS/MS as compared to RIA for

Estrone in accordance with one embodiment of the present invention.

FIG. 30 shows cross-validation data for 2D-LC-MS/MS as compared to RIA for Estradiol in accordance with one embodiment of the present invention.

FIG. 31 shows a comparison of Estradiol (E2) cross-validation of LC-MS/MS with derivatization to 2D-LC-MS/MS without derivatization in accordance with an embodiment of the present invention.

FIG. 32 shows the measured concentration (pg/mL) of free thyroxine vs. dialysis time (hours). The squares (■) show dialysis losses and the diamonds (♦) show effective dialysis for free thyroxine using 96-well equilibrium dialysis plates in accordance with one embodiment of the present invention.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying description and drawings, in which some, but not all embodiments of the presently disclosed subject matter are shown. The presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. Many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. The disclosure utilizes the abbreviations shown below.

ABBREVIATIONS

APCI atmospheric pressure chemical ionization

CBP competitive binding protein

El Estrone

E2 17B-Estradiol or Estradiol

FT4 Free Thryoxine

HTLC high turbulence (throughput) liquid chromatography

HPLC high performance liquid chromatography

LLE liquid-liquid extraction

LOQ limits of quantification

LLOQ lower limit of quantification

IA immunoassay

ELISA enzyme linked immunoassay

RIA radioimmunoassay

SST system suitability test

ULOQ upper limit of quantification

2D-LC-MS/MS two-dimensional liquid chromatography hyphenated to tandem mass spectrometry

(LC)-LC-MS/MS two-dimensional liquid chromatography tandem hyphenated to mass spectrometry (LC)-MS/MS liquid chromatography hyphenated to tandem mass spectrometry

Definitions

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter. Other definitions are found throughout the specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g. 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. Additionally, any reference referred to as being "incorporated herein" is to be understood as being incorporated in its entirety.

The terms "a", "an", and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to "a cell" includes a plurality of such cells, unless the context clearly is to the contrary (e.g., a plurality of cells), and so forth. As used herein, the term "biomarker" is any biomolecule that may provide biological information about the physiological state of an organism. In certain embodiments, the presence or absence of the biomarker may be informative. In other embodiments, the level of the biomarker may be informative. A biomarker may be a hormone, such as an estrogen (e.g., estradiol, estrone), testosterone, thyroxine (T4), triiodothyronine (T3), or a metabolite of a hormone (estrogen sulfate). A biomarker may also be a vitamin or a metabolite of a vitamin. For example, in one embodiment, the measured biomarker may comprise a vitamin D compound such as 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.

As used herein, the terms "purify" or "separate" or derivations thereof do not necessarily refer to the removal of all materials other than the analyte(s) of interest from a sample matrix. Instead, in some embodiments, the terms "purify" or "separate" refer to a procedure that enriches the amount of one or more analytes of interest relative to one or more other components present in the sample matrix. In some embodiments, a "purification" or "separation" procedure can be used to remove one or more components of a sample that could interfere with the detection of the analyte, for example, one or more components that could interfere with detection of an analyte by mass spectrometry.

As used herein, "derivatizing" means reacting two molecules to form a new molecule. Derivatizing agents may include isothiocyanate groups, dansyl groups, dinitro-fluorophenyl groups, nitrophenoxycarbonyl groups, and/or phthalaldehyde groups. As used herein, "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

As used herein, "liquid chromatography" (LC) means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). "Liquid chromatography" includes reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and high turbulence liquid chromatography (HTLC).

As used herein, the term "HPLC" or "high performance liquid chromatography" refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column. The chromatographic column typically includes a medium (i.e., a packing material) to facilitate separation of chemical moieties (i.e., fractionation). The medium may include minute particles. The particles include a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties such as the biomarker analytes quantified in the experiments herein. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded surface. Alkyl bonded surfaces may include C-4, C-

8, or C-18 bonded alkyl groups, preferably C- 18 bonded groups. The chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample. In the method, the sample (or pre-purifϊed sample) may be applied to the column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port. Different solvent modes may be selected for eluting different analytes of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode. In one embodiment, HPLC may performed on a multiplexed analytical HPLC system with a Cl 8 solid phase using isocratic separation with wateπmethanol as the mobile phase. As used herein, the term "analytical column" refers to a chromatography column having sufficient chromatographic plates to effect a separation of the components of a test sample matrix. Preferably, the components eluted from the analytical column are separated in such a way to allow the presence or amount of an analyte(s) of interest to be determined. In some embodiments, the analytical column comprises particles having an average diameter of about 5 μm. In some embodiments, the analytical column is a functionalized silica or polymer-silica hybrid, or a polymeric particle or monolithic silica stationary phase, such as a phenyl-hexyl functionalized analytical column. Analytical columns can be distinguished from "extraction columns," which typically

• are used to separate or extract retained materials from non-retained materials to obtained a "purified" sample for further purification or analysis. In some embodiments, the extraction column is a functionalized silica or polymer-silica hybrid or polymeric particle or monlithic silica stationary phase, such as a Poroshell SBC-18 column. The term "heart-cutting" refers to the selection of a region of interest in a chromatogram and subjecting the analytes eluting within that region of interest to a second separation, e.g., a separation in a second dimension.

The term "electron ionization" as used herein refers to methods in which an analyte of interest in a gaseous or vapor phase interacts with a flow of electrons. Impact of the electrons with the analyte produces analyte ions, which may then be subjected to a mass spectrometry technique.

The term "chemical ionization" as used herein refers to methods in which a reagent gas (e.g. ammonia) is subjected to electron impact, and analyte ions are formed by the interaction of reagent gas ions and analyte molecules. The term "field desorption" as used herein refers to methods in which a non-volatile test sample is placed on an ionization surface, and an intense electric field is used to generate analyte ions.

The term "matrix-assisted laser desorption ionization," or "MALDI" as used herein refers to methods in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo-ionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates desorption of analyte molecules.

The term "surface enhanced laser desorption ionization," or "SELDI" as used herein refers to another method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo- ionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is typically bound to a surface that preferentially retains one or more analytes of interest. As in MALDI, this process may also employ an energy-absorbing material to facilitate ionization. The term "electrospray ionization," or "ESI," as used herein refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Upon reaching the end of the tube, the solution may be vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplet can flow through an evaporation chamber which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

The term "Atmospheric Pressure Chemical Ionization," or "APCI," as used herein refers to mass spectroscopy methods that are similar to ESI, however, APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electric discharge between the spray capillary and a counter electrode. Then, ions are typically extracted into a mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated N₂ gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than

ESI for analyzing less-polar species.

The term "Atmospheric Pressure Photoionization" ("APPI") as used herein refers to the form of mass spectroscopy where the mechanism for the photoionization of molecule M is photon absorption and electron ejection to form the molecular M+. Because the photon energy typically is just above the ionization potential, the molecular ion is less susceptible to dissociation. In many cases it may be possible to analyze samples without the need for chromatography, thus saving significant time and expense. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+. This tends to occur if M has a high proton affinity. This does not affect quantitation accuracy because the sum of M+ and MH+ is constant. Drug compounds in protic solvents are usually observed as MH+, whereas nonpolar compounds such as naphthalene or testosterone usually form M+ (see e.g., Robb et al., 2000, Anal. Chem. 72(15): 3653-3659).

The term "inductively coupled plasma" as used herein refers to methods in which a sample is interacted with a partially ionized gas at a sufficiently high temperature to atomize and ionize most elements.

The term "ionization" and "ionizing" as used herein refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those ions having a net negative charge of one or more electron units, while positive ions are those ions having a net positive charge of one or more electron units.

The term "desorption" as used herein refers to the removal of an analyte from a surface and/or the entry of an analyte into a gaseous phase. As used herein, the term "immunoassay" (IA) refers to a method for measuring the amount of an analyte of interest by quantifying the binding, or the inhibition of binding, of a substance to an antibody. Where an enzyme is used to detect the amount of binding of the substance (e.g. antigen) to an antibody, the assay is an enzyme-linked immunoassay (ELISA). As used herein, the term "radioimmunoassay" (RIA) refers to a method for measuring the amount of an analyte of interest by quantifying the binding, or the inhibition, of binding, of a radiolabled substance to an antibody.

As used herein, the term "hemolysed" refers to the rupturing of the red blood cell membrane, which results in the release of hemoglobin and other cellular contents into the plasma or serum and the term "lipemic" refers to an excess of fats or lipids in blood. Analysis of Biomarkers bv LC-MS/MS

Thus, embodiments of the present invention relate to methods and systems for the quantitative analysis of endogenous biomarkers for clinical diagnosis. The present invention may be embodied in a variety of ways.

In one embodiment, the present invention comprises a method for determining the presence or amount of at least one biomarker of interest in a biological sample, the method comprising: providing a biological sample believed to contain at least one biomarker of interest; chromatographically separating the at least one biomarker of interest from other components in the sample; and analyzing the chromatographically separated at least one biomarker of interest by mass spectrometry to determine the presence or amount of the at least one biomarker of interest in the sample.

In an embodiment, the at least one biomarker comprises a steroid hormone or a thyroid hormone. For example, in one embodiment, the at least one biomarker comprises estradiol and estrone. Or, the at least one biomarker may comprise free thyroxine (T4) or triiodothyronine (T3). In cetain embodiments, the chromatography may comprise high performance liquid chromatography (HPLC). In an embodiment, the chromatography may comprises extraction and/or analytical liquid chromatography. In an embodiment, the method may comprise purifying the biomarker of interest prior to chromatography. For example, the sample may be partially purfied by at least one of liquid-liquid extraction. Also, the method may comprise the step of diluting the sample into a solvent or solvents used for LS and/or MS. In some embodiments, the method may comprise the use of two liquid chromatography steps. For example, in certain embodiments, the method for determining the presence or amount of one or more biomarkers in a test sample may comprise the steps of: (a) providing a sample suspected of containing one or more biomarkers of interest; (b) partially purifying the one or more biomarkers of interest from other components in the sample by at least one of liquid-liquid extraction or by diluting the sample; (c) transferring the extracted one or more hormones or metabolites onto an extraction column (i.e., on-line or off-line); (d) transferring the one or more biomarkers of interest from the extraction column onto an analytical column and chromatographically separating the one or more biomarkers of interest from other components in the sample; and (e) analyzing the chromatographically separated biomarkers of interest by mass spectrometry to determine the presence or amount of the one or more biomarkers in the test sample.

In certain embodiments, the present invention comprises methods for measuring at least one of estradiol and/or estrone in a sample. In certain embodiments, the estradiol is dehydrated to reduce the complexity of the MS/MS spectrum, such that the sensitivity of estradiol detection is increased. For example, in one embodiment, the present invention comprises a method for determining the presence or amount of estradiol in a sample by tandem mass spectrometry, comprising: (a) generating a dehydrated precursor ion of the estradiol; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the presence or amount of one or more of the ions generated in step (a) or (b) or both, and relating the detected ions to the presence or amount of the estradiol in the sample.

In an embodiment, the sample may be subjected to a purification step prior to ionization.

For example, in certain embodiments, the purification step may comprises chromatography. As discussed herein, in certain embodiments, the chromatography comprises high performance liquid chromatography (HPLC). The LC step may comprise one LC separation, or multiple LC separations. In one embodiment, the chromatographic separation comprises extraction and analytical liquid chromatography. Additionally or alternatively, high turbulence liquid chromatography (HTLC) (also known as high throughput liquid chromatography) may be used.

The purification may comprise steps in addition to HPLC or other types of chromatographic separation techniques. In alternate embodiments, the method may comprise at least one of liquid-liquid extraction or dilution. In one embodiment, the sample is diluted into a solvent or solvent mixture that may be used for LC and/or MS (e.g., LC-MS/MS or 2D- LC-MS/MS).

In an embodiment, the treatment of estradiol to form a dehydrated form of the compound reduces the molecular weight of the estradiol by about 18 mass units. Thus, in an embodiment, the precursor ion has a mass/charge ratio (m/z) of about 255.2. Also, in an embodiment, treatment of estradiol to form a dehydrated form of the compound reduces the complexity of the mass spectrum. Thus, in a embodiment the fragment ions comprise ions having a mass/charge ratio (m/z) of about 159.0 and 133.0. By reducing the complexity of the spectrum, the sensitivity of the procedure may be increased. The method may comprise detection of estradiol over a range of from a LOQ of about 1 pg/ml to an ULOQ of about 500 pg/mL as a single assay (i.e., as a linear assay without multiple dilution of the samples). Also, the method may comprise detection of estrone over a range of from a LOQ of about 2.5 pg/mL to and ULOQ of about 500 pg/mL as a single assay (i.e., as a linear assay without multiple dilution of the samples). Also, since the spectrum of the estradiol is simplified, the analysis may further comprise a determination of the amount of other estrogens, such as estrone, in the sample.

The sample may only require heating for a relatively brief period of time to form the dehydrated estradiol. For example, the sample may be heated within the range of 300⁰C to 1000⁰C. In an embodiment, the sample is heated in the interface where the sample is transferred to the mass spectrometer. In alternate embodiments, the heating step is done for less than 1 second, or less than 100 milliseconds (msec), or less than 10 msec, or less than 1 msec, or less than 0.1 msec, or less than 0.01 msec, or less than 0.001 msec.

In other embodiments, the present invention comprises methods for determining the presence or amount of a free thyroxine in a sample or a plurality of samples. In an embodiment, the present invention may comprise a method for determining the presence or amount of free thyroxine in a plurality of samples by tandem mass spectrometry, comprising: (a) dialyzing the plurality of samples to separate the free thyroxine from the protein-bound thyroxine in the samples; (b) generating a precursor ion of the thyroxine in each sample; (b) generating one or more fragment ions of the thyroxine in each sample; and (c) detecting the presence or amount of one or more of the ions generated in step (b) or (c) or both in each sample, and relating the detected ions to the presence or amount of the free thyroxine in the plurality of samples. In an embodiment, the method may comprise detection of thyroxine over a range of from a LLOQ of about 2.0 pg/mL to an ULOQ of about 100 pg/mL as a single assay (i.e., without dilution of the samples). In an embodiment, and as described in more detail herein, the dialysing step may comprise the use of a buffer, and wherein the buffer comprises and sufficient salts such that the buffer is isotonic. In an embodiment, the sample may be subjected to a purification step prior to ionization.

For example, in certain embodiments, the purification step may comprises chromatography. As discussed herein, in certain embodiments, the chromatography comprises high performance liquid chromatography (HPLC). The LC step may comprise one LC separation, or multiple LC separations. In one embodiment, the chromatographic separation comprises extraction and analytical liquid chromatography. Additionally or alternatively, high turbulence liquid chromatography (HTLC) may be used.

The purification may comprise steps in addition to HPLC or other types of chromatographic separation techniques. In alternate embodiment, the purification may comprise at least one of liquid-liquid extraction or dilution. In alternate embodiment, the sample may diluted in a solvent or solvents used for LC or MS, rather than undergoing LLE. In other embodiments, the present invention comprises a system for determining the presence or amount of one or more biomarkers in a sample. In an embodiment, the system for determining the presence or amount of one or more biomarkers in a sample may comprise a station for chromatographically separating the one or more biomarkers from other components in the sample. For example, in some embodiments, the present invention may comprise system for determining the presence or amount of at least one biomarker of interest in a sample, the system comprising: a station for providing a sample believed to contain at least one biomarker of interest; a station for chromatographically separating the at least one biomarker of interest from other components in the sample; and a station for analyzing the chromatographically separated at least one biomarker of interest by mass spectrometry to determine the presence or amount of the one or more biomarkers in the sample. In an embodiment, the system may comprise a station for partially purifying the at least one biomarker of interest from other components in the sample. In an embodiment, the mass spectrometry is operated in an atmospheric pressure chemical ionization (APCI) mode. In an embodiment, the system may further comprise a station for dialyzing a plurality of samples as a means to separate the at least one biomarker of interest that is bound to proteins in the sample from the portion of the biomarker of interest that is free in solution (i.e., "free"). Also in certain embodiments, at least one of the stations is automated and/or controlled by a computer. For example, as described herein, in certain embodiments, at least some of the steps are automated such that little to no manual intervention is required.

In one embodiment, the station for chromatographic separation comprises at least one apparatus to perform liquid chromatography (LC). In one embodiment, the station for liquid chromatography comprises a column for extraction chromatography. Additionally or alternatively, the station for liquid chromatography comprises a column for analytical chromatography. In certain embodiments, the column for extraction chromatography and analytical chromatography comprise a single station or single column. For example, in one embodiment, liquid chromatography is used to purify the biomarker of interest from other components in the sample that co-purify with the biomarker of interest after extraction or dilution of the sample.

The system may also include a station for analyzing the chromatographically separated one or more biomarkers of interest by mass spectrometry to determine the presence or amount of the one or more biomarkers in the test sample. In certain embodiments, tandem mass spectrometry is used (MS/MS). For example, in certain embodiments, the station for tandem mass spectrometry comprises an Applied Biosystems API4000 or API5000 or thermo quantum oτ Agilent 7000 triple quadrupole mass spectrometer.

The system may also comprise a station for extracting the one or more hormones or metabolites from the test sample and/or diluting the sample. In an embodiment, the station for extraction comprises a station for liquid-liquid extraction. The station for liquid-liquid extraction may comprise equipment and reagents for addition of solvents to the sample and removal of waste fractions. In some cases a isotopically-labeled internal standard is used to standardize losses of the biomarker that may occur during the procedures. Thus, the station for liquid-liquid extraction may comprise a hood or other safety features required for working with solvents.

Additionally, the system may comprise a station for dialyzing sample as a means to separate the free hormone or metabolite from a sample that comprises free and protein-bound hormone or metabolite for measurement. The station for dialysis may comprise equipment for aliquoting samples into dialysis chambers. Also, the station for dialysis may comprise a mixing chamber to effect dialysis of the free analyte (e.g., free homone) from the sample.

In embodiments of the methods and systems of the present invention, the biomarker is a hormone or a metabolite. The methods and systems of the present invention may be used to measure the amount of either total and/or free biomarkers of intersest in serum. In an embodiment, the hormone may comprise a steroid hormone. Or, the hormone may comprise a thyroid hormone. Or, the hormone may comprise a protein or peptide hormone. For example, in alternate embodiments, the steroid hormone may comprise an estrogen, androgen, mineralcorticoid, or glucocorticoid hormone. In certain embodiments, the hormone may comprise at least one of estrone or estradiol. In other embodiments, the hormone may comprise an estrogen metabolite. For example, the hormone may comprise estrone sulfate and/or glucoronidated and sulphated metabolites of estradiol, estrone or estriol. Or, other steroid hormones or steroid hormone metabolites may be measured. For example, the hormone may comprise testosterone. Or, non-steroid hormones may be measured. For example, in certain embodiments, the methods and systems may be used to measure a thyroid hormone, such as free thyroxine (T4) or triiodothyronine (T3). Or, pre- hormones (such as 25 hdroxyvitamin D) may be measured. For examples, the methods and systems of the present invention may be used to measure vitamins or other metabolites. In some embodiments, the metabolite may comprise a vitamin D compound such as 25- hydroxyvitamin D2 or 25-hydroxyvitamin D3, 1,25 dihydroxyvitamin D2 and 1,25 dihydroxyvitamin D3. In yet other embodiments, the methods and systems of the present invention may be used to measure a non-hormone compound.

In certain embodiments, the test samples suitable for analysis by the methods and systems of the present invention can include any liquid sample that can contain one or more target analytes of interest. In an embodiment, the biomarker is endogenous to a subject. For example, in some embodiments, the test sample comprises a biological sample. As used herein, the term "biological sample" refers to a sample obtained from a biological source, including, but not limited to, an animal, a cell culture, an organ culture, and the like. Suitable samples include blood, plasma, serum, urine, saliva, tear, cerebrospinal fluid, organ, hair, muscle, or other tissue sample.

As used herein, a subject may comprise an animal. Thus, in some embodiments, the biological sample is obtained from a mammalian animal, including, but not limited to a dog, a cat, a horse, a rat, a monkey, and the like. In some embodiments, the biological sample is obtained from a human subject. In some embodiments, the subject is a patient, that is, a living person presenting themselves in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition. In some embodiments, the test sample is not a biological sample, but comprises a non-biologicial sample, e.g., obtained during the manufacture or laboratory analysis of a synthetic steroid, which can be analyzed to determine the composition and/or yield of the manufacturing and/or analysis process.

A variety of methods may be used to extract the biomarker of interest from the sample. In certain embodiments, extracting the one or more hormones or metabolites from the test sample comprises a liquid-liquid extraction procedure. For example, for the analysis of estrone and estradiol in serum, a hexane:ethyl acetate is used for extraction. For example, in one embodiment, a 9:1 hexane:ethyl acetate solution may be used.

In certain embodiments, purifying the at least one biomarker of interest from the test sample may also comprise the use of a liquid chromatography extraction column. In one embodiment, the column is on-line. In an embodiment, purification of the biomarker of interest using a extraction column may comprises the steps of: (i) transferring the test sample on an extraction column; and (ii) eluting the biomarker of interest from the extraction column.

In certain embodiments, the methods and systems of the present invention may comprise multiple liquid chromatography steps. Thus, in certain embodiments, a two- dimensional liquid chromatography (LC) procedure is used. For example, in one embodiment, the method and systems of the present invention may comprise transferring the biomarker of interest from the LC extraction column to an analytical column. In one embodiment, the transferring of the at least one biomarker of interest from the extraction column to an analytical column is done by a heart-cutting technique. In another embodiment, the biomarker of interest is transferred from the extraction column to an analytical column by a chromatofocusing technique. Alternatively, the biomarker of interest is transferred from the extraction column to an analytical column by a column switching technique. These transfer steps may be done manually, or may be part of an on-line system. Various columns comprising stationary phases and mobile phases that may be used for extraction or analytical liquid chromatography are described herein. The column used for extraction liquid chromatography may be varied depending on the biomarker of interest. In some embodiments, the extraction column is a fiinctionalized silica or polymer-silica hybrid or polymeric particle or monlithic silica stationary phase, such as a Poroshell SBC-18 column. The column used for analytical liquid chromatography may be varied depending on the biomarker of interest and/or the column that was used for the extraction liquid chromatography step. For example, in certain embodiments, the analytical column comprises particles having an average diameter of about 5 μm. In some embodiments, the analytical column is a functionalized silica or polymer-silica hybrid, or a polymeric particle or monolithic silica stationary phase, such as a phenyl-hexyl functionalized analytical column.

A variety of methods may be used to quantify the at least one biomarker of interest once the biomarker of interest has been substantially purified (i.e., substantially separated away from other components that may have been present in the sample). In some embodiments, mass spectrometry is used to quantify the at least one biomarker of interest. In certain embodiments, the mass spectrometer may comprise a tandem mass spectrometer (MS/MS). For example, in one embodiment of the methods and systems of the present invention, the tandem MS/MS spectrometry comprises a triple quadrupole tandem mass spectrometer.

The tandem MS/MS may be operated in a variety of modes. In one embodiment, the tandem MS/MS spectrometer is operated in an atmospheric pressure chemical ionization (APCI) mode. In some embodiments, the quantification of the analytes and internal standards is performed in the selected reaction monitoring mode (SRM). Thus, embodiments of the present invention comprise methods and systems for applying liquid chromatography and mass spectrometry as a means to separate a biomarker analyte of interest from other components that may be present in a biological sample. In certain embodiments, two liquid chromatography (LC) steps are used in tandem. Also, the method may comprise an off-line liquid-liquid extraction and/or sample dilution step as a means to partially purify the sample prior to liquid chromatography. In some embodiments, tandem mass spectrometry is used to quantify the analyte of interest. The methods and systems may be used for clinical diagnosis.

The systems and methods of the present invention may, in certain embodiments, provide for a multiplexed assay. For example, certain embodiments of the present invention may comprise a multiplexed liquid chromatography tandem mass spectrometry (LC-MS/MS) or two-dimensional or tandem liquid chromatography-tandem mass spectrometry (LC)-LC- MS/MS) methods for the quantitative analysis of one or more analytes, including steroid hormones, such as estrone and estradiol and/or thyroid hormones, such as free thyroxine (T4) or triiodothyroine (T3) in biological samples.

An example of a method (2) of the present invention is shown iri FIG. 1. Thus, in an embodiment, the method may include a step of providing a biological sample, for example, a serum sample believed to contain one or more analytes of interest (4). In some embodiments, an appropriate internal standard is added to the sample (6). For example, in some embodiments of the presently disclosed method for analyzing estrone and estradiol in serum samples, deuterated D₄-estrone and Ds-estradiol are added as internal standards. Or, C ₁3- estrone and Ci₃-estradiol stable labeled isotopes may be used. Or, for thyroxine, a deuterated or Ci ₃ derviative may be used. For example, in one embodiment, Thyroxine Ring- ¹³Ce may be used. In yet other embodiments, structural analogue of the biomarker of interest may be used. For example, such structural analogues may comprise compounds wherein a first chemical group is replaced with a second chemical group. In general, the groups are of similar chemical reactivity, but different mass, as for example, the replacement of a methyl (— CH₃) group with an ethyl (-CH₂CH₃) group.

In some embodiments, the analytes of interests are partially purified by liquid-liquid extraction of the sample (8). Or, the sample may be diluted (9) in a solvent that can be used for LC or MS in subsequent purification steps.

In an embodiment, the liquid-liquid extraction is used to concentrate and partially purify the analyte. For example, for estradiol/estrone analysis, the liquid extraction may be used to remove conjugated estrogens, such as sulfated and glucoronidated estrogens. Also, the liquid extraction may remove lipids and/or fibrinogen from the samples. In some embodiments, estrone and estradiol can be extracted from a serum sample with an organic solvent that can separate estrone and estradiol from conjugated estrogens. For example, in an embodiment, an alkane mixed with a more polar solvent is used. For example, in certain embodiments, hexane is mixed with a more polar solvent. In an embodiment, the polar solvent comprises ethyl acetate or a similar solvent. In an embodiment, 9:1 hexanerethyl acetate is used.

Or, other solvents may be used. As is known in the art, the solvents employed may be optimized to separate the analyte of interest from the sample. For example, the solvents used to extract estrone and estradiol from serum may not be the same solvent or solvent mix as used to extract estrone and estradiol from urine. Or, the solvents used to extract estrone and estradiol from serum may not be the same solvent or solvent mix as used to extract thyroxine (T4), triiodothyronine (T3), or vitamin-D compounds from serum. For example, in certain embodiments, acetonitrile is used for liquid extraction of vitamin-D compounds, and ethyl acetate:hexane:methanol is used for extraction of T4.

Certain biomolecules may have a propensity to nonspecifically bind to proteins or other biomolecules. For example, thyroid hormones can non-specifically bind to proteins such as serum albumin, sex hormone binding globulin, and the like. For determination of tree thyroxine (T4), the sample may be treated to separate the free thyroxine from thyroxine that is bound to proteins in the biological sample (e.g., serum).

In one embodiment, the sample may initially be dialyzed to separate the free hormone or metabolite from a mixture of free and protein-bound hormone or metabolite (5). In certain embodiments, multiple samples may be processed concerrently. For example, the dialysis may be performed using a multiwell dialysis plate which allows for the dialysis of multiple samples at one time. In certain embodiments 96 well plates are used. In this way, multiple samples are processed to comprise a high throughput assay. For example, samples of serum that may contain free thyroxine and protein-bound thyroxine may be introduced into the individual sample chambers which are on one side of the membrane and a buffer solution introduced into the diluent chambers on the other side of the membrane from the sample. The 96 well plate is then positioned vertically and rotated to facilitate transfer of the free thyroxine across the membrane. The dialysis buffer may, in certain embodiments, be isotonic and contain gelatin. The gelatin may be used over a range of concentrations depending upon the nature of the membranes and hardware used for dialysis. In alternate embodients, the gelatin may be in a range of from about 0.1 to 10 mg/mL. In an embodiment, the gelatin is at about 1 mg/mL. In certain embodiments, the buffer used for dialysis comprises multiple endogenous salts to provide a buffer that is isotonic with the serum sample to thereby negate any potential dilution effects and/or disruptions to the ratio of bound thyroxine to free thyroxine in the sample. Also, gelatin may be include to prevent adsorptive losses of free thyroixine onto the dialysis membrane or the sample chamber. Gelatin may act as a carrier on the dialysate side of the 96-well plate to ensure free thyroxine remains in the dialysate solution. Gelatin does not bind free thyroxine and thus, does not affect the ratio of bound throxine to free thyroxine in the sample on the sample side of the membrane.

For the analysis of free thyroxine, a liquid extraction step may be performed after the dialysis. The liquid extraction may be designed to remove residual salts and/or other additives which are used in the dialysis solution and/or remain from the sample, but that may interfere with the MS analysis. Thus, in one embodiment, the dialysate comprising free thyroxine is extracted with 71.25:23.75:5 ethyl acetate:hexane:methanol. In another embodiment, the dialysate may be diluted with a solution of methanol containing a stable labeled internal standard and directly injected onto the LC-MS/MS system for analysis.

Where the sample is extracted, the internal standard addition may include a protein to prevent the free thyroxin from sticking to the walls of the sample container. Addition of protein (e.g., bovine serum albumin) can minimize loss in extraction and recovery for liquid- liquid extraction. Where extraction is not performed, the internal standard may be added in methanol or a similar solvent used for LC.

As is known in the art, in some embodiments, the organic extract may be transferred to a fresh tube and then back-washed. For example, in an embodiment where the analyte of interest is estradiol and/or estrone, the solvent may be back-washed with aqueous sodium hydroxide (pH of about 12) to further purify the sample. Or, for extraction of other biomarkers, back-extraction may employ other solvents. The back- wash may, in certain embodiments, remove additional lipids or interfering analytes from the sample.

The extract supernatant may then be evaporated and the sample reconstituted. For example, for analysis of estradiol and/or estrone, the sample may be reconstituted in 70:30 wateπmethanol. Or, for analysis of thyroxine, the solvent used for liquid-liquid extraction may be evaporated and the sample reconstituted in 50:50 wateπmethanol.

Still referring to FIG. 1, the method may further include liquid chromatography as a means to separate the analyte of interest from other components in the sample. In an embodiment, two liquid chromatography steps are used. For example, the method may comprise a first extraction column liquid chromatography (10), transfer of the biomarker of interest to a second analytical column (12), and an analytical column liquid chromatography

(16). In other embodiments, only one liquid chromatography step is used.

The first extraction liquid chromatography column may, in certain embodiments, comprise a step whereby the analytes of interest are separated from a majority of contaminants. Thus, in certain embodiments, the first column provides the majority of selectivity for the procedure. The second analytical liquid chromatography column may, in certain embodiments, comprise a step whereby the analytes of interest are concentrated, to thereby increase sensitivity for analysis by mass spectrometry (MS). For example, the reconstituted extract may be applied onto a high performance liquid chromatography (HPLC) system, wherein the analytes are eluted using an isocratic separation through an extraction column. In certain embodiments, the mobile phase that is used comprises a gradient. For example, in an embodiment for the separation of estradiol and estrone from other components in serum, the stationary phase comprises a Poroshell

300SBC-18 column. Thus, the inventors have found that surprisingly, a stationary phase designed for large molecules such as proteins may be used to separate smaller molecules such as estrone and estradiol. The mobile phase may comprise methanol and water.

Depending upon the biomarker of interest, a variety of analytical columns known in the art may be used as needed to provide good purification. In cetain embodiments, the analytical column may comprise particles having an average diameter of about 5 μm. In some embodiments, the analytical column is a functionalized silica or polymer-silica hybrid, or a polymeric particle or monolithic silica stationary phase, such as a phenyl-hexyl functionalized analytical column. For example, in one embodiment, estrone and estradiol are separated from isobaric substances by separation using a Poroshell 300SBC10 column (7.5 mm by 2.1mm) with 5 micron particle size using a gradient separation using methanol and water for elution at 1 mL per minute flow rate. Estrone and estradiol are transferred from the extraction column after 2.5 minutes and chromatofocused onto a phenyl-hexyl column (50mm by 2.1 mm) with 5 micron particles using water for 45 seconds. The transferred and purified analytes are chromatographed using an accelerated gradient employing methanol and water to improve sensitivity prior to introduction into the mass spectrometer interface and subsequent detection.

For liquid chromatography of thyroxin, a single liquid chromatography step may be used. Thus, for liquid chromatography of thyroxin, a phenyl hexyl column (50mm by 2.1 mm) with 5 micron particle size may be used. Thus, following either: (a) liquid-liquid extraction, evaporation and reconstitution; or (b) post-dialysis sample dilution with internal standard solution; samples are injected onto the liquid chromatography column. The transferred analyte and internal standard are chromatographed using a methanol: water gradient separation at 1 mL per minute. To enable further sensitivity gains, a post-separation additional flow of 90:10 methanol: water containing ammonium carbonate (1 mM) is introduced at 200 microliters per minute prior to introduction into the mass spectrometer (MS) electrospray interface. If two liquid chromatography steps are employed, the eluted analytes may be transferred to the analytical column in a manner such that the sample is concentrated upon application to the analytical column. In some embodiments, the eluted analytes are transferred to the analytical column via a heart-cutting technique. In some embodiments, a chromato-focusing procedure is used to transfer and focus the analytes on the analytical column. Also in some embodiments, a column-switching procedure is used to transfer the analytes to the analytical column. The analytes may then be separated on the analytical column (16) and the fraction containing the analyte of interest is eluted. In an embodiment, the second column in run in a manner to maximize throughput, and to provide the sample in a reduced volume.

The separated analytes are then introduced into a mass spectrometer (MS) system (20). In some embodiments, a tandem MS/MS system is used. As is known by those of skill in the art, in tandem MS spectrometry, the precursor ion is selected following ionization, and that precursor ion is subjected to fragmentation to generate product (i.e., fragment) ions, whereby one or more product ions are selected for detection.. A sample may therefore be analyzed for both estradiol and estrone since the compounds have different precursor and product ions in tandem mass spectrometric methodologies (i.e., different transitions).

The analyte of interest may then be quantified based upon the amount of the characteristic transitions measured by tandem MS. In some embodiments, the tandem mass spectrometer comprises a triple quadrupole mass spectrometer. In some embodiments, the tandem mass spectrometer is operated in a positive ion Atmospheric Pressure Chemical Ionization (APCI) mode. In some embodiments, the quantification of the analytes and internal standards is performed in the selected reaction monitoring mode (SRM). Or, other methods of ionization such as the use of inductively coupled plasma, or MALDI, or SELDI, ESI, or APPI may be used for ionization.

In some embodiments, the back-calculated amount of each analyte in each sample may determined by comparison of unknown sample response or response ratio when employing internal standardization to calibration curves generated by spiking a known amount of purified analyte material into a standard test sample, e.g., charcoal stripped human serum. In one embodiment, calibrators are prepared at known concentrations and analyzed as per the biomarker methodology to generate a response or response ratio when employing internal standardization versus concentration calibration curve. In one embodiment, the sample may be treated so as to chemically modify the analyte of interest to allow for improved detection in the MS system. For example, in one embodiment, a sample being analyzed for estrone and/or estradiol may be heated to the extent that the estradiol loses a molecule of water thereby converting the estradiol to a dehydrated form of the compound (FIG. 2, Panels A and B, respectively). This conversion can reduce the number of major product ion peaks seen for estradiol from about 60 to 3 (FIG. 2, panels C and D). For MS analysis, the sensitivity of the analysis is generally inversely proportional to the number of product ion peaks. Thus, with fewer peaks, the sensitivity of detection using tandem mass spectrometry is increased. For example, as illustrated in FIG. 2, estradiol may be quanfied by measuring the transition from the precursor ion at a mass to charge (m/z) 255.3 ±

0.5 mass units to the two product (fragment) ions at a mass to charge (m/z) of 159.0 ± 0.5 mass units and 133.0 ± 0.5.

In alternate embodiments, the sensitivity obtained for measurement of estradiol is increased more than 10 fold, or more than 20 fold, or more than 50 fold, or more than 100 fold, or more than 150 fold, or more than 200 fold, or more than 500 fold, or more than 1,000 fold. For example, in alternate embodiments, the sensitivity is increased by about 5-1,000 fold, or a by about 20-500 fold, or by about 50-150 fold, or by about 100 fold.

The temperature for heating the sample may, in alternate embodiments range from 300⁰C to about 1000⁰C and includes all ranges therein.. In an embodiment, the dehydration step is performed within the interface of the mass spectrometer employed in APCI or electrospray mode at 500 degrees C + 100 degrees. In an embodiment, the sample is heated for several microseconds at the interface for dehydration to occur. In alternate embodiments, the heating step is done for less than 1 second, or less than 100 milliseconds (msec), or less than 10 msec, or less than 1 msec, or less than 0.1 msec, or less than 0.01 msec, or less than 0.001 msec.

In an embodiment, the tandem liquid chromatograhy (LC) steps help reduce isobaric interferences. For example, in one embodiment, there are 24 potential isobaric interferences in estradiol (transition m/z 255 -> 159, 133), and 16 potential interferences for estrone (transition m/z 273 -> 159, 133). For example, dehydroepiandrosterone (DHEA) undergoes thermal dehydration forming MH-H20J+ and MH-2H₂O]+ (FIG. 3). There may be DHEA concentrations that are about 300-1,500 times the levels of estrone and estradiol in healthy patients. Thus, the M+2 isotopic overlap of dehydrated DHEA may become an isobaric interference. Heart cutting from the primary separation using isocratic or gradient separation resolves most isobaric interferences (FIG. 4). Thus, as shown in FIG. 4, heart cutting combined with chromatofocusing may be used to separate estradiol (E2) and estrone (El) from all but one potential isobaric contaminant which is separated within the analytical (second) liquid chromatography separation dimension. An example of a method for measuring estradiol and estrone is provided in FIG. 5. For example, in an embodiment, a method (40) of measuring estrone and estradiol comprise providing a sample believed to contain at least one of estrone and estradiol (44). The method may also comprise adding an internal standard of D₄-estrone and Ds-estradiol to the sample (46). Also, the method may optionally comprise partial purification of the estrone and estradiol by liquid-liquid extraction of the estrone and/or estradiol from the serum with 9:1 hexane-ethyl acetate (48). Or, the sample may be diluted (50) as a means to improve sensitivity in subsequent purification and/or analysis steps (e.g. LC and/or MS).

After initial purification by liquid-liquid extraction or dilution, the sample may be further purified by liquid chromatography. Thus, in one embodiment, the solvent is evaporated and the extracted estrone/estradiol is reconstituted in 30:70 methanol water for application to a liquid chromatography extraction column (52). The estradiol/estrone may be eluted from the extraction column. For example, in alternate embodiments, the estradiol/estrone may be eluted by heart cutting, chromatofocusing or column switching. Next, the fraction containing the estrone/estradiol may, in certain embodiments, be applied to an analytical LC column (54). The fraction containing the estrone/estradiol may then be transferred to the LC-MS/MS interface to undergo ionization and dehydration of the estradiol (60) prior to MS/MS detection in SRM mode (62).- In an embodiment, heating the estradiol removes a molecule of water, and changes the resultant MS/MS profile such that it comprises only three major product ions. Thus, the methods provide the ability to quantify estrone and/or estradiol at physiologically relevant levels. As discussed herein, the difference between a serum level of 10 pg/mL and 15 pg/mL may be clinically relevant. In one embodiment, the method is able to measure estrone and/or estradiol at levels of about 2.5pg/mL and 1 pg/mL respectively.

An example of a method for measuring free thyroxine (T4) (70) is provided in FIG. 6. In an embodiment, the method may comprise providing a sample that includes thyroxine (both free and protein-bound) (74). The method may also comprise dialyzing the sample (76) to separate the free thyroxine from the protein bound thyroxine. Also, the method may comprise adding an internal standard such as ⁶Ci₃-thyroxine (78) to allow for the measured amount of thyroxine to be correlated to the actual amount present in the sample (i.e., to quantify the amount lost during the extraction and measurement procedures).

The method may also comprise an optional step whereby the free thyroxine present in the dialysate is extracted by liquid-liquid extraction (80), Alternatively, the sample may be diluted into the solvent used for LC-MS/MS as a means to reduce interference from non-T4 or non-T3 analytes (81). At this point, the solvent used for extraction may be evaporated, and the extracted thyroxine reconstituted in 50:50 methanol: water for application to an LC column (82). The free thyroxine may then be eluted from the column (84) and then quantified by MS/MS (86). Thus, the methods provide the ability to quantify free thyroxine at physiologically relevant levels. The difference between a serum level of 8 pg/mL and 12 pg/mL T4 may be clinically relevant. The method is able to measure free thyroxine (T4) at levels of 2 pg/mL . Systems for Quantification of Endogenous Biomarkers FIG. 7 A shows an embodiment of a system of the present invention. As shown in FIG. 7, the system may comprise a station for aliquoting a sample (104) that may comprise a biomarker of interest into sampling containers. In one embodiment, the sample is aliquoted into a container or containers to facilitate liquid-liquid extraction or sample dilution. The station for aliquoting may comprise receptacles to discard the portion of the biological sample that is not used in the analysis. Alternatively or additionally, the sample may be aliquoted into a container for dialysis. As described above, the container for dialysis may comprise a multi-well plate. Thus, in addition to the station for aliquoting, the system may comprise a station for dialysis (106). The station for dialysis may comprise a rotator oven, multi-chamber pipettes for sample transfer, as well as receptacles to discard the portion of the biological sample that is not used in the analysis.

The system may further comprise a station for adding an internal standard to the sample (108). In an embodiment, the internal standard comprises the biomarker of interest labeled with a non-natural isotope. Thus, the station for adding an internal standard may comprise safety features to facilitate adding an isotopically labeled internal standard solutions to the sample. The system may also, in some embodiments, comprise a station (110) for liquid-liquid extraction and/or dilution of the sample.

The system may also comprise a station for liquid chromatography (LC) of the sample. As described herein, in an embodiment, the station for liquid chromatography may comprise an extraction liquid chromatography column (112). The station for liquid chromatography may comprise a column comprising the stationary phase, as well as containers or receptacles comprising solvents that are used as the mobile phase, In an embodiment, the mobile phase comprises a gradient of methanol and water, acetonitrile and water, or other miscible solvents with aqueous volatile buffer solutions. Thus, in one embodiment, the station may comprise the appropriate lines and valves to adjust the amounts of individual solvents being applied to the column or columns. Also, the station may comprise a means to remove and discard those fractions from the LC that do not comprise the biomarker of interest. In an embodiment, the fractions that do not contain the biomarker of interest are continuously removed from the column and sent to a waste receptacle for decontamination and to be discarded.

A variety of extraction LC systems may be used. For example, in the embodiment where the system is being used to measure estrone or estradiol, a Poroshell 300SBC18 extraction column with a phenyl hexyl analytical column, with mobile phases comprising a gradient of methanol and water are used. Or, for measurement of thyroxine, a phenyl hexyl column, with a mobile phase of methanol:water is used with post-column addition of a methanol:water solution containing ammonium carbonate. Or, for vitamin D metabolites, a Fluophase WP extraction column, with a mobile phase of methanol: water is used and an Extent Cl 8 analytical column is used with a mobile phase of methanol:water is used. The system may also comprise an analytical LC column (114). The analytical column may facilitate further purification and concentration of the biomarker of interest as may be required for further characterization and quantfication.

Also, the system may comprise a station for characterization and quantification of the biomarker of interest. In one embodiment, the system may comprise a station for mass spectrometry (MS) of the biomarker. In an embodiment, the station for mass spectrometry comprises a station for tandem mass spectrometry (MS/MS). Also, the station for characterization and quantification may comprise a computer and software for analysis of the MS/MS results. In an embodiment, the analysis comprises both identification and quantification of the biomarker of interest. In some embodiments, one or more of the purification or separation steps can be preformed "on-line." As used herein, the term "on-line" refers to purification or separation steps that are performed in such a way that the test sample is disposed, e.g., injected, into a system in which the various components of the system are operationally connected and, in some embodiments, in fluid communication with one another. The on-line system may comprise an autosampler foτ removing aliquots of the sample from one container and transferring such aliquots into another container. For example, an autosampler may be used to transfer the sample after extraction onto an LC extraction column. Additionally or alternatively, the on-line system may comprise one or more injection ports for injecting the fractions isolated from the LC extraction columns onto the LC analytical column. Additionally or alternatively, the on-line system may comprise one or more injection ports for injecting the LC purified sample into the MS system. Thus, the on-line system may comprise one or more columns, including but not limited to, an extraction column, including an HTLC extraction column, and in some embodiments, an analytical column. Additionally or alternatively, the system may comprise a detection system, e.g., a mass spectrometer system. The on-line system may also comprise one or more pumps; one or more valves; and necessary plumbing. In such "on-line" systems, the test sample and/or analytes of interest can be passed from one component of the system to another without exiting the system, e.g., without having to be collected and then disposed into another component of the system.

In some embodiments^ the on-line purification or separation method can be automated. In such embodiments, the steps can be performed without the need for operator intervention once the process is set-up and initiated. For example, in one embodiment, the system, or portions of the system may be controlled by a computer or computers (102). Thus, in certain embodiments, the present invention may comprise software for controlling the various components of the system, including pumps, valves, autosamplers, and the like. Such software can be used to optimize the extraction process through the precise timing of sample and solute additions and flow rate.

Although some or all of the steps in the method and the stations comprising the system may be on-line, in certain embodiments, some or all of the steps may be performed

"off-line." In contrast to the term "on-line", the term "off-line" refers to a purification, separation, or extraction procedure that is performed separately from previous and/or subsequent purification or separation steps and/or analysis steps. In such off-line procedures, the analytes of interests typically are separated, for example, on an extraction column or by liquid/liquid extraction, from the other components in the sample matrix and then collected for subsequent introduction into another chromatographic or detector system. Off-line procedures typically require manual intervention on the part of the operator. Liquid chromatography may, in certain embodiments, comprise high turbulence liquid chromatography or high throughput liquid chromatography (HTLC). See, e.g., Zimmer et al., J. Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos. 5,968,367; 5,919,368; 5,795,469; and 5,772,874. Traditional HPLC analysis relies on column packings in which laminar flow of the sample through the column is the basis for separation of the analyte of interest from the sample. In such columns, separation is a diffusional process. Turbulent flow, such as that provided by HTLC columns and methods, may enhance the rate of mass transfer, improving the separation characteristics provided. In some embodiments, high turbulence liquid chromatography (HTLC), alone or in combination with one or more purification methods, may be used to purify the biomarker of interest prior to mass spectrometry. In such embodiments, samples may be extracted using an HTLC extraction cartridge which captures the analyte, then eluted and chromatographed on a second HTLC column or onto an analytical HPLC column prior to ionization. Because the steps involved in these chromatography procedures can be linked in an automated fashion, the requirement for operator involvement during the purification of the analyte can be minimized. Also, in some embodiments, the use of a high turbulence liquid chromatography sample preparation method can eliminate the need for other sample preparation methods including liquid-liquid extraction. Thus, in some embodiments, the test sample, e.g., a biological fluid, can be disposed, e.g., injected, directly onto a high turbulence liquid chromatography system. For example, in a typical high turbulence or turbulent liquid chromatography system, the sample may be injected directly onto a narrow (e.g., 0.5mm to 2mm internal diameter by 20 to 50mm long) column packed with large (e.g., > 25 micron) particles. When a flow rate (e.g., 3-500 niL per minute) is applied to the column, the relatively narrow width of the column causes an increase in the velocity of the mobile phase. The large particles present in the column can prevent the increased velocity from causing back pressure and promote the formation of vacillating eddies between the particles, thereby creating turbulence within the column.

In high turbulence liquid chromatography, the analyte molecules may bind quickly to the particles and typically do not spread out, or diffuse, along the length of the column. This lessened longitudinal diffusion typically provides better, and more rapid, separation of the analytes of interest from the sample matrix. Further, the turbulence within the column reduces the friction on molecules that typically occurs as they travel past the particles. For example, in traditional HPLC, the molecules traveling closest to the particle move along the column more slowly than those flowing through the center of the path between the particles. This difference in flow rate causes the analyte molecules to spread out along the length of the column. When turbulence is introduced into a column, the friction on the molecules from the particle is negligible, reducing longitudinal diffusion. The methods and systems of the present invention may use mass spectrometry to detect and quantify the biomarker of interest. The terms "mass spectrometry" or "MS" as used herein generally refer to methods of filtering, detecting, and measuring ions based on their mass-to- charge ratio, or "m/z." In MS techniques, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrometer where, due to a combination of electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge

("z").

In certain embodiments, the mass spectrometer uses a "quadrupole" system. In a "quadrupole" or "quadrupole ion trap" mass spectrometer, ions in an oscillating radio frequency (RF) field experience a force proportional to the direct current (DC) potential applied between electrodes, the amplitude of the RF signal, and m/z. The voltage and amplitude can be selected so that only ions having a particular m/z travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments can act as both a "mass filter" and as a "mass detector" for the ions injected into the instrument.

In certain embodiments, tandem mass spectrometry is used. See, e.g., U.S. Pat. No. 6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry," which is hereby incorporated by reference in its entirety. Further, the selectivity of the MS technique can be enhanced by using "tandem mass spectrometry," or "MS/MS." Tandem mass spectrometry (MS/MS) is the name given to a group of mass spectrometric methods wherein "parent or precursor" ions generated from a sample are fragmented to yield one or more "fragment or product" ions, which are subsequently mass analyzed by a second MS procedure. MS/MS methods are useful for the analysis of complex mixtures, especially biological samples, in part because the selectivity of MS/MS can minimize the need for extensive sample clean-up prior to analysis. In an example of an MS/MS method, precursor ions are generated from a sample and passed through a first mass filter to select those ions having a particular mass-to-charge ratio. These ions are then fragmented, typically by collisions with neutral gas molecules in a suitable ion containment device, to yield product (fragment) ions, the mass spectrum of which is recorded by a electon multiplier detector. The product ion spectra so produced are indicative of the structure of the precursor ion, and the two stages of mass filtering can eliminate ions from interfering species present in the conventional mass spectrum of a complex mixture.

In an embodiment, the methods and systems of the present invention use a triple quadrupole MS/MS (see e.g., Yost, Enke in Ch. 8 of Tandem Mass Spectrometry, Ed. McLafferty, pub. John Wiley and Sons, 1983). Triple quadrupole MS/MS instruments typically consist of two quadrupole mass filters separated by a fragmentation means. In one embodiment, the instrument may comprise a quadrupole mass filter operated in the RP only mode as an ion containment or transmission device. In an embodiment, the quadropole may further comprise a collision gas at a pressure of between 1 and 10 millitorr. Many other types of "hybrid" tandem mass spectrometers are also known, and can be used in the methods and systems of the present invention including various combinations of magnetic sector analyzers and quadrupole filters. These hybrid instruments often comprise high resolution magnetic sector analyzers (i.e., analyzers comprising both magnetic and electrostatic sectors arranged in a double-focusing combination) as either or both of the mass filters. Use of high resolution mass filters may be highly effective in reducing chemical noise to very low levels.

For the methods and systems of the present invention, ions can be produced using a variety of methods including, but not limited to, electron ionization, chemical ionization, fast atom bombardment, field desorption, and matrix-assisted laser desorption ionization ("MALDI"), surface enhanced laser desorption ionization ("SELDI"), photon ionization, electrospray ionization, and inductively coupled plasma.

In those embodiments, such as MS/MS, where precursor ions are isolated for further fragmentation, collision-induced dissociation ("CID") may be used to generate the fragment ions for further detection. In CID, precursor ions gain energy through collisions with an inert gas, and subsequently fragment by a process referred to as "unimolecular decomposition."

Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy.

In some embodiments, to attain the required analytical selectivity and sensitivity, the presently disclosed 2D-LC-MS/MS methods include multiplexed sample preparation procedures. For example, in certain embodiments dialysis of the sample is performed using a 96 well plate having a dialysis membrane in each well or multiple sample tubes (FIG. 7B). Additionally or alternatively, the multiplex system may comprise staggered multiplexed LC and MS sample inlet systems. Also, the methods and systems of the present invention may comprise multiple column switching protocols, and/or heart-cutting (LC-LC or 2D-LC) techniques, and/or LC separations prior to MS detection. In some embodiments, the methods and systems of the present invention may include a multiplexed two-dimensional liquid chromatographic system coupled with a tandem mass spectrometer (MS/MS) system, for example a triple quadrupole MS/MS system. Such embodiments provide for staggered, parallel sample input into the MS system.

Thus, as shown in FIG. 7B, four samples (132 A-D) may each be applied to individual extraction columns (134 A-D). Once the samples have each run through the extraction column, they may each be transferred directly (e.g., by column switching) to a second set of analytical columns (136 A-D). As each sample elutes from the analytical column, it may be transferred (138) to the mass spectrometer (140) for identification and quantification.

A plurality of analytes can be analyzed simultaneously or sequentially by the presently disclosed LC-MS/MS and 2D-LC-MS/MS methods. Exemplary analytes amenable to analysis by the presently disclosed methods include, but are not limited to, steroid hormones, such as estradiol, estrone, and metabolites, such as estrone sulfate. In other embodiments, thyroid hormones, such as free thyroxine (T4) and triiodothyronine (T3) can be measured. In the other embodiments, metabolites, such as 25-Hydroxyvitamin D2₅ 25 -Hydroxy vitamin D3, may be measured. One of ordinary skill in the art would recognize after a review of the presently disclosed subject matter that other similar analytes could be analyzed by the methods and systems disclosed herein. Thus, in alternate embodiments, the methods and systems may be used to quantify steroid hormones, protein and peptide hormones, peptide and protein biomarkers, drugs of abuse and therapeutic drugs. For example, optimization of key parameters for each analyte can be performed using a modular method development strategy to provide highly tuned bioanalytical assays. Thus, certain steps may be varied depending upon the analyte being measured as disclosed herein.

Also, embodiments of the methods and systems of the present invention may provide greater sensitivity than the sensitivities previously attainable for many of the analytes being measured. For example, through using this optimization procedure, an LOQ of about 1 picogram per milliliter (pg/mL), or less than 5 pg/mL, or less that 10 pg/mL, or less than 25 pg/mL is attained for the analysis of at least one of estradiol, estrone or free thyroxine without the cumbersome derivatization processes historically required for LC-MS/MS analyses of steroids. Importantly, the low levels of detection allow for the analysis of small sample volumes, for example 100 μL, 200 μL, 500 μL, or less than 1 rαL, which can be necessary to analyze pediatric sample volumes. Thus, the presently disclosed LC-MS/MS and (LC)-LC-MS/MS methods can be used to measure levels of steroid hormones, such as estrone and estradiol, or other hormones or metabolites (e.g., free thyroxine, vitamin D metabolites and the like) in serum or plasma samples from children, women, and men. Embodiments of the present invention may provide certain advantages. In certain embodiments, the methods and systems of the present invention may provide greater sensitivity than the sensitivities previously attainable for many of the analytes being measured.

Also, embodiments of the methods and systems of the present invention may provide for rapid throughput that has previously not been attainable for many of the analytes being measured. For example, using the methods and systems of the present invention, multiple samples may be analysed for free thyroxine using 96 well plates and a multiplex system of four LC-MS/MS systems, significantly increasing the throughput.

As another advantage, the specificity and sensitivity provided by the methods and systems of the present invention may allow for the analysis of analytes from a variety of biological materials. For example, the 2D-LC-MS/MS methods of the present invention can be applied to the quantification of analytes of interest in complex sample biological matrices, including, but not limited to, blood, serum, plasma, urine, saliva, and the like. Thus, the methods and systems of the present invention are suitable for clinical applications and/or clinical trials.

As additional potential advantages, in certain embodiments, the systems and methods of the present invention provide approaches for addressing isobaric interferences, varied sample content, including hemolysed and lipemic samples, while attaining low pg/mL limits of quantification (LOQ) of the target analytes. Accordingly, embodiments of the methods and systems of the present invention may provide for the quantitative, sensitive, and specific detection of clinical biomarkers used in the clinical diagnosis of endocrine disorders. Validation of LC-MS/MS and 2D-LC-MS/MS Assays for Endogenous Biomarkers

A general strategy for the validation of the presently disclosed LC-MS/MS and 2D-LC- MS/MS methods for endogenous biomarkers is provided in Scheme 1. Thus, Scheme 1 shows the different tests that were used to validate the procedures. Matrix specificity testing was performed by analyzing 6 different lots of charcoal stripped matrix in quadruplicate for the presence of residual analyte, absence of analyte enables the charcoal stripped matrix to be spiked with known concentrations of target analytes to generate calibration curves. Internal standard specificity was performed by spiking the stable labeled internal standard into analyte- free charcoal stripped matrix and measuring for the presence of analyte in quadruplicate. Absence of unlabeled analyte confirms the purity of internal standard materials. Endogenous (hormones) and exogenous (drugs) are spiked into analyte free matrix to confirm the selectivity of the method.

Accuracy and precision was determined using 6 replicates per level in spiked charcoal stripped serum at the LLOQ, 2 levels within the analytical range and the ULOQ in 3 different batches. Precision was determined using 6 replicates in 3 separate runs of pooled matrix samples at concentrations of approximately 3 to 10 times the LLOQ, the mid point of the analytical range and approximately 80% of the ULOQ. Accuracy was determined in pooled matrix samples using spike and recovery (standard addition) at 3 different concentrations throughout the analytical range using 4 replicates per level.

Linearity was confirmed using multi-level calibrators over 5 separate runs. Sample mixing experiments were also undertaken mixing pooled matrix samples with fortified stripped matrix samples to ensure the assays were free of matrix interferences in quadruplicate.

Recovery was undertaken using both spiked stripped matrix and pooled matrix samples in quadruplicate as .confirmation of linearity and also further proof that the assay was free of matrix effects. The effect of matrix content on measurement was also tested following post- column infusion, addition of lipemia and hemolysis content, alternate sample types (e.g. serum and or plasma) and sample draw-tubes in quadruplicate. Sample stability was undertaken using both spiked stripped matrix samples and pooled matrix samples at storage conditions expected from sample collection to final analysis. Each condition was compared against baseline samples drawn and frozen at— 70⁰C for comparison and analyzed in quadruplicate at each concentration. Inter-assay comparison was performed using at least 50 samples representing physiological range during comparison of LC-MS/MS and LC-MS/MS assays with alternate techniques. Reference range generation and/or transference was undertaken using guidance from the National Committee on Clinical Laboratory Standards (NCCLS).

Scheme 1. Bioanalytical Validation Strategy

Specificity Testing: Matrix, Internal Standard, Endogenous/Exogenous Analytes

Accuracy and Precision: Stripped Matrix: LLOQ, Mid levels (x2) ULOQ; Pooled Matrix: 3x LLOQ, mid and 80% of ULOQ; Spike and Recovery at 3 levels

Representative LC-MS/MS and 2D-LC-MS/MS chromatograms of selected analytes at the limit of quantification (LOQ) obtained by using the LC-MS/MS and 2D-LC-MS/MS methods of the present invention are shown in FIGS. 8-13. In these figures the X axis is time, and the Y axis corresponds to the amount of material (i.e., the response). Thus, it can be seen that estrone sulfate was detected at 100 pg/mL (FIG. 8); free thyroxine was detected at 2 pg/mL (FIG. 9); 25-hydroxy vitamin D2 was detected at 1 ng/mL (FIG. 10); 25- hydroxy vitamin D3 was detected at 1 ng/mL (FIG. 11); estrone was detected at 2.5 pg/mL (FIG. 12); and estradiol was detected at 1 pg/mL (FIG. 13). Similarly, representative LC-MS/MS and 2D-LC-MS/MS chromatograms of selected analytes at the upper limit of quantification (ULOQ) (the level of analyte above which the assay is outside of linear range) obtained by using the LC-MS/MS and 2D-LC-MS/MS methods and systems of the present invention are shown in FIGS. 14-19. Thus, it can be seen that estrone sulfate has an ULOQ of 50 ng/mL (FIG. 14); free thyroxine has an ULOQ of 100 pg/mL (FIG. 15); 25 -hydroxy vitamin D2 has an ULOQ of 250 ng/mL (FIG. 16); 25- hydroxyvitamin D3 has an ULOQ of 250 ng/mL (FIG. 17); estrone has an ULOQ of 500 pg/mL (FIG. 18); and estradiol has an ULOQ of 500 pg/mL (FIG. 19).

Representative LC-MS/MS and 2D-LC-MS/MS calibration curves for selected analytes are shown in Figures 20-25. FIG. 20 shows a calibration curve for estrone sulfate where it is seen that the assay is linear over a 1000-fold range. Calibration curves for thyroxine (FIG. 21), 25-hydroxyvitamin D2 (FIG. 22), 25-hydroxyvitamin D3 (FIG. 23), estrone (FIG. 24) and estradiol (FIG. 25) also show linearity over at 100 to 250 fold range. In these figures, the X axis is the concentration, and the Y axis is the ratio of the analyte to an internal standard response. Representative inter-assay comparison results for the presently disclosed LC-MS/MS and 2D-LC-MS/MS methods versus RIA, CBP, or IA methods are shown in Figures 26-30. Thus, it can be seen that using LC-MS/MS provides excellent correlation with RIA for measurement of estrone sulfate (FIG. 26); good correlation but a bias (slope offset) with IA for measurement of free thyroxine (FIG. 27), excellent correlation but a bias (slope offset) for measurement of total 25-hydroxy vitamin D by CBP and average correlation with IA (FIG. 28, panels A and B, respectively); excellent correlation with RIA for measurement of estrone (FIG. 29); and excellent correlation with RIA for measurement of estradiol (FIG. 30). Also, FIG. 31 shows good correlation between the 2D-LC-MS/MS assays described herein for estradiol with an alternate LC-MS/MS strategy involving derivatization. Excellent correlation is observed for samples within the higher analytical range of the derivatization assay (lOpg/mL LLOQ).

FIG. 32 shows results of dialysis losses for thyroxine. The squares (■) show dialysis losses and the diamonds (♦) show effective dialysis for free thyroxine using 96-well equilibrium dialysis plates in accordance with one embodiment of the present invention. This indicates that during the dialysis experiments, free T4 does not degrade or bind to the 96-well plate apparatus (i.e. losses). Further, FIG.31 indicates that dialysis is complete after approximately 16 hours.

Data showing the validation bias due to ionization effect and recovery for selected analytes are provided in Tables 1 through 7, below. Data showing the accuracy and precision of the presently disclosed LC-MS/MS and 2D-LC-MS/MS methods are provided in Tables 8 through 13. As known by those in the art, acceptable values based on the FDA and CLIA regulations are < 20% bias or imprecision (% CV) at the LLOQ and < 15% over the remainder of the assay. See e.g., FDA Guidance: 1.1 Guidance for Industry, Bioanalytical Method Validation, FDA, May 2001, BP, and CLIA Regulation: 42 CFR 493.1253 Standard:

Establishment and verification of performance specifications. Subart K, Quality System for Non-waived Testing. Thus, as used herein, "acceptable" or "good" indicates that the assay or aspect of the method being measured meets the NCCLS, FDA and CLIA critieria.

Each of the presently disclosed LC-MS/MS and 2D-LC-MS/MS methods was evaluated for specificity against multiple steroids and/or other potential interferences for a total of up to 60 different analytes (see Examples 1 and 2) at excessive concentrations, for example, 100 μg/dL, to ensure accurate measurement in each assay independent of clinical levels of endogenous and therapeutic agents, such as steroids, which either cross-react in RIA assays or are not discriminated using the specificity provided by MS/MS detection. Additionally, analyte stability was evaluated for all conditions expected from original patient sampling to final result. Proven stability includes sample shipment (-20⁰C), sample processing (2O⁰C; > 3 freeze/thaw cycles) and post processing (20⁰C and autosampler at 10⁰C) to ensure accurate and precise determination of analytes, such as hormone steroids, derived from patient samρles(see Examples 1 and 2).

Table 1 Estrone Sulfate

Validation Bias (%)

Serum -3.8 - 2.6

Lipemia -0.2

Hemolysis 0.7

Recovery 85.0 - 97.1

Table 2 Free Thyroxine

Validation Bias (%)

Serum -6.9 - 4.8

Lipemia 1.0

Hemolysis 12.8

Recovery 90.5 - 95.8

Table 3 25-Hydroxyvitamin D2

Validation Bias (%)

Serum -3.8 - 2.2

Lipemia -5.0

Hemolysis -5.4

Recovery 93.2 - 100.5

Table 4 25-Hydroxyvitamin D3

Validation Bias (%)

Serum -8.8 - 1 1 .9

Lipemia -0.3

Hemolysis -1.6

Recovery 88.8 - 94.1 Table 5 Estrone

Table 6 Estradiol

Table 7

Table 8

Table 9

Table 11

Estrone

Accuracy (% ) Precision (%)

Cone. (pg/mL) 2.5 250 500 2.5 250 500

Intra - 1 12.4 1.1 -1.4 2.9 5.5 3.3

Intra - 2 -0.3 9.2 4.0 7.3 2.8 2.4

Intra - 3 3.6 6.3 5.1 6.3 3.8 1.6

Inter 4.8 5.5 2.6 7.4 5.1 3.7

EXAMPLES

Additional data from the analytical validation and standard operating procedures for the presently disclosed method for the quantification of estrone and estradiol by liquid-liquid extraction and 2D-LC-MS/MS, or free thyroxine by dialysis, an optional liquid-liquid extraction, and LC -MS/MS are set forth in the following Examples. The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. Example 1: 2D-LC-MS/MS Analysis for Estrone, Estradiol, and Estrone Sulfate

Estrone and estradiol were validated to 2.5 pg/mL and 1 pg/mL, respectively, from a 1-mL serum sample. Sensitivity and selectivity were generated using heat assisted and guided fragmentation of estradiol to optimize sensitivity. Analytical specificity was generated via 2D LC using gradients in both LC dimensions, heart-cutting and chromato-focusing prior to

MS/MS detection. Optimum selectivity was generated following titration of buffer pH and chromatography to enable separation from co-extracted isobaric endogenous interferences, while retaining necessary sensitivity (S/N >20) for accurate quantification quantification in estrone sulfate analysis using LC-MS/MS. More particularly, estrone (El) and estradiol (E2) were measured by two-dimensional

(2D) liquid chromatography with tandem mass spectrometry detection (2D-LC-MS/MS) after liquid-liquid extraction (LLE). Deuterated D₄-Estrone and Ds-Estradiol were added as internal standards to serum aliquots. Estrone and Estradiol were extracted from 1 mL serum samples with 8 mL of 9:1 Hexane:Ethyl Acetate. The organic extract was transferred to a fresh tube and back washed with 1 mL aqueous sodium hydroxide solution (pH of about 12), then evaporated and reconstituted in 70:30 Water.Methanol.

Duplicate sets of stripped serum calibrators were analyzed in each batch. All injections were made in singlicate. All samples are injected onto the ARIA TX4 or Transcend TX4 HTLC system (Thermo Fisher, Franklin, Massachusetts, United States of America), where the analytes were first eluted using a separation gradient through an extraction column. Analytes of interest were then transferred onto the analytical column, where chromatographic separation was continued via a gradient. An API5000 triple quadrupole mass spectrometer (MDS-SCIEX, Concord, Ontario, Canada), operating in positive ion Atmospheric Pressure Chemical Ionization (APCI) mode was used for detection. Quantification of analyte and internal standard was performed in selected reaction monitoring mode (SRM). The back-calculated amount of each analyte in each sample was determined from duplicate calibration curves generated by spiking known amounts of purified estrone and estradiol into charcoal stripped human serum.

Measurement of estrone and estradiol was used to evaluate ovarian function and to evaluate excess or diminished estrogen levels. Analysis of estrone and estradiol by 2D-LC-

MS/MS detection was developed to measure levels in serum or plasma samples from children, women and men. The lower limit of detection using the default sample aliquot of ImL was 1.0 pg/mL for Estradiol and 2.5 pg/mL for Estrone. Definitions

APCI - Atmospheric Pressure Chemical Ionizationl LLE - Liquid-Liquid Extraction; SST - System Suitability Test; 2D -LC-MS/MS - Two-dimensional liquid chromatography with tandem mass spectrometry detection; El - Estrone; E2 - 17β-Estradiol or Estradiol; HTLC - High Throughput Liquid Chromatography Specimens

A recommended sample is 1.5 inL serum or plasma. Separate within one hour. Store and ship frozen in plastic vial.

Draw into red top vacutainer tube. Allow clotting to occur for 20 minutes at room temperature (or until clot has retracted). Spin and remove serum to labeled plastic vial. Freeze immediately. Storage is for short term storage (2 weeks): Frozen (< -20⁰C); for long term storage (6 months): Frozen (< -20°C). Shipping of specimens is:frozen (< -2O⁰C) - on dry ice. Generally, samples drawn into SST tubes are unacceptable for this procedure. Equipment & Materials

The following materials were used: Standard manual pipetting devices; 1.2ml MBlock Polypropylene 96 Well Collection Plate (SPE Ware, Inc. Product No. SPE0210); Heat Sealing Foil (SPE Ware, Inc. Product No. AB-0589); Mechanical Shaker, Eberbach Inc.; Rotary

Evaporator (rotovap), Speed Vac SC 200, Savant (or equivalent); Class A Volumetric Glass Containers, various sizes; API 5000 Tandem Mass Spectrometer, Sciex, (Toronto, Canada); Turbo V™ Ion Source with APCI probe, Sciex, (Toronto, Canada); Aria TX4 HTLC System, Cohesive Technologies, (MA, USA) consisting of 4 each: 1100 Series Quaternary Pump, 1100 Series Binary Pump, 1100 Series Vacuum Degasser; HTS Twin PAL System Autosampler,

CTC Analytics AG (Switzerland); Luna Phenyl-Hexyl Analytical Column, 50 X 2.0 mm, 5μm particle size, Phenomenex, (USA) Product No. 00B-4257-B0; Poroshell 300SB-C18 Column, 2.1 x 75mm, 5μm particle size, Agilent Inc., (USA) Product No. 660750-902; 16mm Flange Caps, Stockwell Scientific, (AZ, USA) Product No. 8558; Vortex Mixer, VWR or equivalent; Combi-thermo Heat Sealer, Abgene Inc., Product No. AB-0559; 96-Well Centrifuge 5804R Eppendorf or equivalent; 16x100 mm borosilicate glass tubes; Thermo Hot pocket column oven, Thermo Electron Corp. (USA) Product No. 92016-150; Analyst Version 1.4 or greater. Applied Biosystems, (CA, USA); Aria OS Version 1.4 or greater, Cohesive Technologies (MA, USA). Reagents The following reagents were used. Water — Type II, Millipore MiIIiQ or equivalent;

Charcoal Stripped Human Serum (Bioreclamation, Inc); Ethyl alcohol USP (AAPER Alcohol, USA); Acetonitrile HPLC grade (EM Science AXO 142-1); Methanol (Reagent A.C.S., Fisher Scientific); Isopropanol HPLC Grade (Fisher Scientific, Catalog #A451-4); 17β-Estradiol, Sigma-Aldrich, (USA) Product # E8875 or USP, (USA) Product #1250008; Estrone, Sigma-Aldrich, (USA) Product # E1274 or USP, (USA) Product #1255001 ; 17β-

Estradiol-2,4,16,16,17-D₅, CDN Isotopes, (USA) Product # D-5403; Estrone-2,4,16,16-D₄, CDN Isotopes, (USA) Product # D-3650; Acetone HPLC Grade (Burdick & Jackson, Catalog #AH010-4); Total Estrogen Stock Solution^"(10μg/mL in ethanol) prepared by serial dilution.

The following solvents were used as the mobile phases for liquid chromatography. Eluting and Loading Pump A Mobile Phase (90% Water and 10% Methanol); Loading Pump B Mobile Phase (90% Methanol and 10% Water); and Loading Pump C Mobile Phase (60:30:10 Acetonitrile: Isopropanol: Acetone).

Internal Standard Solution (1 ng/mL Ds-Estradiol and 2 ng/mL D₄-Estrone) was prepared in 99:1 wateπacetonitrile solution. The reconstitution solution (for reconstituting the sample after liquid-liquid extraction) was 70:30 Millipore Water: Methanol. Two needle wash solutions were used: Needle Wash Solution 1 (Aqueous 1% Formic Acid); and Needle Wash Solution 2 (70:30 Acetonitrile: IN Ammonium Hydroxide).

Total Estrogen Stock Solution (lOμg/mL) - 5μg/mL estrone and 5μg/mL estradiol was used to prepare intermediate stock solutions for preparation of calibrators. To prepare standards, 0.5mL of Total Estrogen Stock (lOμg/mL) was diluted to 10OmL with stripped human serum to yield 25 ng/mL El E2 solution, and 2mL of the 25ng/mL solution was diluted to 10OmL in charcoal stripped human serum to yield a 0.5 ng/mL E1E2 solution. The diluted stocks (0.5 ng/mL and 25 ng/mL) were stored at -20⁰C. Calibration Standards were then prepared as shown in the following table. All standards were prepared in charcoal stripped human serum. The standard curve back-fit data should be 80-120% at LLOQ, 85 —

1 15% at other concentrations of expected curve values. Standard Preparation Procedure

Quality Control

Control pools are prepared in human serum and introduced into use according to the analytically robust procedures. Each run contained duplicates of four control pools, each with a nominal target value.

Quality Control Concentrations

Control Target concentration (pg/mL)

Name Estrone Estradiol

Pool l 10 10

Pool 2 25 25

Pool 3 115 115

Pool 4 300 300

Assay Procedure

The assay was performed as follows. Pipetted 1.0 ml standard, control or patient into the tube with an Eppendorf pipette (or equivalent). Using an Eppendorf Plus repeating pipette with 5 ml tip (or equivalent), added 50 μl Internal Standard Solution to each tube except double blanks. Mix all samples on multi-vortexer for 30 seconds. Added 8 mL 9:1 Hexane: Ethyl Acetate extraction solvent to all tubes and shook on mechanical shaker for 10 minutes. Removed tubes from shaker and spun down all samples in centrifuge up to 2000 rpm. Labeled a separate set of 16x100 mm test tubes, and add 1 mL redistilled water and 1 drop of IN NaOH to each. Froze aqueous layer of extracted samples and poured into the tubes containing the water and NaOH. Capped and shook on mechanical shaker for 10 minutes. Removed from shaker and spin down all samples in centrifuge up to 2000rpm. Froze aqueous layer of extracted samples and poured into new tubes. Placed all tubes into a Rotovap (or equivalent) and allow solvent to evaporate for 45 to 60 minutes. Once there was no trace of solvent left in the tubes, remove from Rotovap and reconstitute each with 120μL

E1E2 Reconstitution solution. Covered tubes with parafilm and mixed on multivortexer, 4 x 30 seconds.

Using an Eppendorf pipette, or equivalent, transfered the reconstituted tubes to a 96- well plate. Placed a heat-sealing foil over the plate and seadl the plate with the heated plate sealer. Centrifuged plate at 3700 rpm at 10⁰C for 10 minutes and placed 96-well plate in LC- MS/MS Autosampler. Liquid chromatography procedures

After liquid-liquid extraction and reconstitution in 100 μL, 80 μL of extracted sample was injected into the HTLC system using methanol .water in the mobile phase. The HTLC system is logically divided into two functions: (1) first dimension extraction/separation using a highly selective LC column using a binary gradient; and (2) second dimension separation using an sharper binary gradient of methanol and water and a 5 μm reverse phase analytical column. In this example, a Poroshell 300SBC18 75 x 2.1, 5 μm column was used for extraction. In the extraction mode of the HTLC system, the sample was first pumped through the extraction column at a 1 ml/min flow rate using the HTLC loading pump. This separation ensures optimized separation of isobaric interferences and the passage of unwanted coextracted analytes to waste.

After the first dimension separation step, the flow rate was reduced to 0.5mL/min and combined with 0.5mL/min of water during transfer and chromatofocussing onto the second dimension phenyl hexyl column. Such HPLC columns are commercially available (e.g., Thermo Hypersil Phenyl hexyl, Phenomenex Luna Phenyl Hexyl)). In the analytical mode of the HTLC, after the sample was chromatofocussed onto the analytical column. A binary gradient from 0% to 90% methanol at 1 mL/min was used, resulting in the separation and increase in peak concentration of estrone and estradiol from other analytes contained in the sample. The separated sample was then transferred to the MS/MS for quantitation. The 2D- LC method is summarized below. Estrone, estradiol, D₄-estrone and Ds-estradiol elute from the second column at approximately 5.2 minutes (+ or - 1 minute). Estrone and Estradiol 2D-LC Method

Step 3: Transfer of eluent from the extraction column at 0.5mL/minute and chromatofocus with 0.5mL/Mminute of milliporc water provided by the eluting pump.

"""Loading and Eluting Buffers: Loading Pump Buffer A 90:10 Millipore Water: Methanol; Loading Pump Buffer B 10:90 Millipore Water: Methanol; Eluting Pump Buffer A Millipore Water; Eluting Pump Buffer B 10:90 Millipore Water: Methanol

Mass Spectrometry

The flow of liquid solvent from the HTLC system entered the heated nebulizer (APCI) interface of the MS/MS analyzer. The solvent/analyte mixture was first converted to vapor in the heated tubing of the interface. The analytes, contained in the nebulized solvent, were ionized and a positive charge added by the corona discharge needle of the interface, which applies a large voltage to the nebulized solvent/analyte mixture. During heating and ionization of estradiol (E2), the selected interface heater settings of 500⁰C enable dehydration of estradiol to the dehydrated moiety at m/z 255.3 that enables the sensitivity gains observed within the underivatized assay described here. The ions passed through the orifice of the instrument and entered the first quadrupole. Quadrupoles 1 and 3 (Ql and Q3) were the mass filters, allowing selection of ions based on their mass to charge ratio (m/z). Quadrupole 2 (Q2) was the collision cell, where ions were fragmented.

The first quadrupole of the MS/MS (Ql) selected for molecules with the mass to charge ratio of estrone/estradiol (271.3/255.3 ± 0.5 m/z or mass units). Ions with these m/z passed to the collision chamber (Q2), while ions with any other m/z collided with the sides of the quadrupole and were fragmented. Ions entering Q2 collided with neutral gas molecules and fragment. This process is called Collisionally Activated Dissociation (CAD). The CAD gas used in this example was nitrogen resulting in the generation of fragment (products). The fragment ions generated were passed into quadrupole 3 (Q3), where the two fragment ions of estradiol to be measured (tn/z 159.0 ±.0.5 m/z and 133.0 ± 0.5 m/z or mass units) and 2 ions for estrone (m/z 159.0 ± 0.5/n/z and 133.0 ±+-0.5 tn/z or mass units) were selected for, while other ions were screened out. The selected fragment ions were collected by the detector. The same process was carried out for an internal standards, which were 5-deuterated estradiol and

4-deuterated estrone molecules. Thus, the selected MS/MS transitions (nominal masses) measured weTβ as follows: Estradiol m/z 255 to 159 and 133; Estrone m/z 21\ to 159 and 133; D₅- Estradiol m/z 260 to 161; and D₄-Estrone m/z 275 to 161.

Selected MS/MS parameters were as follows: Dwell time: 100 msec for each estradiol and estrone transition, and 50 msec for each internal standard transition; Unit mass resolution in both resolving g quadrupoles (Ql and Q3); Curtain Gas: 10; CADGas: 6; NC: μA; Temp: 500⁰C; GSl: 20; GS2: 0; CE: 27 for estradiol and internal standards and 35 for estrone and internal standards.

As ions collide with the detector, they produce a pulse of electrons. The pulse was converted to a digital signal, which was counted to provide an ion count. The acquired data was relayed to the computer, which plotted counts of the ions collected vs. time. Areas of the chromatohgraphic peaks generated were computer-measured, response factors (ratio of analyte to internal standard responses) were generated from calibration materials spiked into stripped matrix calibrators at known concentrations, and estradiol and estrone concentrations in unknown samples were thereby quantitated by back-calculating the area response ratios of analyte to internal standards against the constructed calibration curves.

The HTLC system can be operated with 1 to 4 channels in parallel, with each channel incorporating 1 or more columns. Given that a single assay requires about 3-6 minutes minutes to traverse the column or columns, by staggering the start time on each column, a 4- fold multiplexed system can inject four times as many test samples into the MS/MS instrument than with a single column. Thus, a set of 1000 samples may be assayed for estrone and estradiol in 1 day using HTLC 4 channel muliplexing, as opposed to 4 days when using a single channel system. Furthermore, following transfer of samples to the autosampler, no further operator handling of samples is required, as the HTLC may be computer-controlled to perform the subsequent purification and analysis steps in a fully on-line configuration. Calculations

Calibration curves are constructed using the software system (Analyst) that controls the mass spectrometer. Calibration curves are generated by assigning the known concentrations to calibrators to generate a response ratio of analyte to internal standard versus concentration of analyte added [FIG. 20 — 26]. The concentrations of unknown samples are automatically calculated by comparing the response ratio of analyte to internal standard observed in measuring unknown samples to the calibration curve generated above. Samples that have an initial value greater than 500 pg/mL are diluted and re-extracted/analyzed to provide a result within the linear range of the assay. Assay Performance Characteristics

A quantitative bioanalytical method for the determination of estrone and estradiol in human serum using LLE and 2D LC-MS/MS detection was developed and validated. The assay accuracy and precision was shown to be specific for the analysis of estrone and estradiol.

Spiked standard samples at nine (estrone) and ten (estradiol) concentrations were used to generate a weighted (1/x) linear regression calibration curve, which covered the range from 2.5 to 500 pg/mL for estrone and 1 to 500 pg/mL for estradiol. Average inaccuracies and imprecision < 20% at the LLOQ and <15% throughout the remainder of the range were observed. The correlation coefficients of the curves were greater than 0.98. Sample dilution was evaluated for both estrone and estradiol. Acceptable bias was observed for all samples within the analytical ranges of both assays. Selectivity Blank Matrix Interference

Quadruplicate injections of stripped serum were injected to determine the degree of blank matrix interference for each analyte. Matrix responses were <20% of the mean LLOQ for estrone in all 6 lots tested and <20% of the mean LLOQ for estradiol in 5 of 6 lots tested.

Anticoagulant Effect on Matrix. The effect of EDTA and heparin as anticoagulants was tested by drawing four healthy volunteers using red top serum collection tubes and vacutainers containing Sodium Heparin and EDTA anticoagulants. The results of the heparin and EDTA tubes were compared to the results from the serum collection tubes. Variation of sample type and anticoagulant exhibited a bias < ±15% for measurement of estrone and estradiol. Thus, plasma samples collected with Heparin and EDTA are acceptable specimen types. Anticoagulant Effect on Estrone and Estradiol Measurement

Concentration (pg/mL)

Sample Serum Heparin EDTA

32.200 27.953 28.590 19.387 20.425

Estrone 20.213 162.481 151.423 156.020 17.925 17.841 16.521

Mean 58.205 54.151 55.389 Mean Matrix Effect (%) NA -6.96 -4.84 n 4 4 4

22.475 21.226 20.300 40.975 42.657 42.183

Estradiol 181.435 184.180 193.202 13.833 13.635 13.092

Mean 64.680 65.425 67.194 Mean Matrix Effect (%) NA 1.15 3.89 n 4 4 4

Effect of Lipemia and Hemolysis in the Matrix.

The effect of lipemia and hemolysis on the quantitative result was determined by spiking pooled patient serum with 5% by volume of lipid solution or lysed red blood cells. The samples were run in quadruplicate and the results compared to the results of the pool before contamination. Bias was < ±15% following addition of lipemic or hemolyzed material was observed for both estrone and estradiol. Thus, lipemic and hemolyzed samples may be processed using this assay. Internal Standard Interference.

Internal standards (D4-estrone and Ds-estradiol) working solution was spiked into charcoal stripped serum and tested in quadruplicate to evaluate the presence of unlabeled analyte. Internal standards (D4-estrone and Ds-estradiol) interference responses were less than 20% of the mean LLOQ response for both estrone and estradiol. Matrix Effect.

The matrix effect was calculated at low, mid and high level concentrations. The matrix effect for the internal standard was measured at a single concentration. A minimum of 4 samples per QC level were analyzed for both analytes to determine matrix effect on quantitative result. Matrix effects were less than 15% for all analytes (estrone, estradiol and D4-estrone and Ds-estradiol internal standards) tested when comparing water and pooled samples. Effect of Lipemia and Hemolysis on Estrone and Estradiol Concentration (pg/mL)

Sample Serum Lipemic Hemolyzed

118.286 118.877 113.430

108.600 120.709 114.765

Estradiol

108.772 120.159 114.246

113.406 126.448 119.172

Mean 1 12.266 121.548 115.403

Mean Matrix Effect (%) NA 8.27 2.79 n 4 4 4

108.159 97.741 114.344

108.131 103.348 96.851

Estrone

105.072 98.358 104.242

102.476 99.496 96.149

Mean 105.960 99.736 102.897

Mean Matrix Effect (%) NA -5.87 -2.89 n 4 4 4

Estrone Matrix Effect

Sample Matrix Millipore Water Pooled Human Serum

Post-Column Analyte Internal Standard Analyte Internal Standard

Ratio Ratio Infusion Level Response (cps) Response (cps) Response (cps) Response (cps)

13704.285 13282.465 1.032 12977.458 14357.420 0.904

1 1983.207 12280.880 0.976 12726.749 15154.084 0.840

13447.370 14129.109 0.952 13866.153 1401 1.588 0.990

12912.166 13543.690 0.953 12798.316 14614.364 0.876

MARR per Cone. NA NA 0.978 NA NA 0.902 % Change NA NA NA NA NA -7.76

34680.714 12444.598 2.787 37110.060 13962.366 2.658

34353.842 11496.604 2.988 35363.380 13567.318 2.607

38284.618 13119.045 2.918 37102.053 14459.360 2.566

36175.359 12448.411 2.906 37599.353 15799.305 2.380

MARR per Cone. NA NA 2.900 NA NA 2.553 % Change NA NA NA NA NA -11.98

123737.835 1 1465.898 10.792 132777.319 14635.791 9.072

109476.758 11070.122 9.889 123422.614 14005.516 8.812

High Infusion

130085.379 12808.997 10.156 142380.844 14729.975 9.666

124071.128 1 1506.322 10.783 133162.192 13750.007 9.685

MARR per Cone. NA NA 10.405 NA NA 9.309 % Change NA NA NA NA NA -10.54 Estradiol Matrix Effect

Sample Matrix Millipore Water Low Pooled Human Serum

Post-Column Analyte Internal Standard Analyte Internal Standard

Infusion Level Response (cps) Response (cps) Response (cps) Response (cps)

9289.430 15134.202 0.614 10756.094 16273.794 0.661

9142.882 14970.197 0.61 1 9623.939 15742.986 0.61 1

9178.822 14245.804 0.644 10323.741 15672.658 0.659

91 15.187 14769.048 0.617 10657.31 1 16270.173 0.655

MARR per Cone. NA NA 0.622 NA NA 0.646

% Change NA NA NA NA NA 4.02

32753.289 14367.449 2.280 34773.500 15118.142 2.300

33992.565 14615.840 2.326 33326.1 10 14131.532 2.358

34038.400 14387.971 2.366 34970.558 14653.828 2.386

33191.028 13699.972 2.423 33447.218 14509.296 2.305

MARR per Cone. NA NA 2.348 NA NA 2.338

% Change NA NA NA NA NA -0.47

123906.209 13814.999 8.969 147602.652 15918.071 9.273

129674.464 13636.345 9.509 140981.287 16008.385 8.807

High Infusion

126576.356 13173.822 9.608 136601.192 15250.626 8.957

128043.106 13263.078 9.654 149499.158 16614.989 8.998

MARR per Cone. NA NA 9.435 NA NA 9.009

% Change NA NA NA NA NA -4.52

For both the estrone and estradiol matrix effect tables, matrix effect = [(Mean Analyte to Internal standard ratio in pooled serum)/ Mean Analyte to Internal standard ratio in water)]-l, expressed as a percentage

A. Intra-assay Precision

The intra-assay precision of the analytical method was calculated for three assays using patient pools (Pool 1 at 10 pg/mL, Pool 2 at 25 pg/mL, Pool 3 at 115 pg/mL, and Pool 4 at 300 pg/mL, all concentrations are approximate). The following tables show the data for these pools, as well as, the data from quality controls made from spiked charcoal stripped human serum at four concentrations (1.0 pg/mL, 2.5 pg/mL, 250 pg/mL, and 500 pg/mL).

Estrone Intra-assay Precision

] [ntra-Assay %CV

Assay No. Pool l Pool 2 Pool 3 Pool 4 1.0 2.5 250 500

1 4.50 5.03 4.42 2.69 15.35 2.91 5.52 3.30

2 5.21 4.71 3.64 4.30 19.56 7.16 2.76 2.39

3 3.47 2.91 1.87 2.95 29.91 6.30 3.81 1.63

Estradiol Intra-assay Precision

] ntra-Assay %CV

Assay No. Pool l Pool 2 Pool 3 Pool 4 1.0 2.5 250 500

1 2.27 3.43 1.80 3.23 4.72 4.77 2.81 1.15

2 4.31 3.04 1.34 2.56 4.44 6.26 1.60 4.33

3 5.59 1.35 3.64 4.86 4.70 3.92 2.61 3.46

Reproducibility

The inter-assay precision was calculated from the overall data from the precision assays for each of the QC samples. As shown in the inter-assay tables, the method has good inter-assay precision.

Estrone Inter-Assay Precision

Pool l Pool 2 Pool 3 Pool 4 1.0 2.5 250 500

Average 10.34 26.16 116.91 309.26 1.22 2.62 263.79 512.80

%CV 4.88 4.59 4.65 5.37 29.23 7.39 5.08 3.67

N 18 18 18 18 18 18 18 18

Estradiol Inter-Assay Precision

Pool l Pool 2 Pool 3 Pool 4 1.0 2.5 250 500

Average 10.83 23.94 114.18 293.45 0.97 2.57 254.82 513.87

%CV 4.36 3.53 . 3.33 4.86 7.39 5.22 3.94 4.10

N 18 18 18 18 18 18 18 18

Accuracy

The inter-assay accuracy was determined by calculating the percent bias for samples of known concentrations. Stripped human serum was spiked to 1.0 pg/mL, 2.5 pg/mL, 250 pg/mL, and 500 pg/mL and then assayed 6 times in three different runs. As shown in the intra-assay tables, the method has good inter-assay accuracy. Estrone Inter-assay Accuracy Data

System Precision l.O pg/mL 2.5 pg/mL 250 pg/mL 500 pg/mL

Expected Result 1.0 2.5 250 500

Average Result 1.22 2.62 263.79 512.80

% Bias 22.13 4.79 5.51 2.56

Estradiol Inter-assay Accuracy Data

System Precision l.O pg/mL 2.5 pg/mL 250 pg/mL 500 pg/mL

Expected Result 1.0 2.5 250 500 Average Result 0.97 2.57 254.82 513.87 % Bias -3.28 2.70 1.93 2.77

Spike and Recovery

A patient pool and stripped human serum calibrator were spiked with Estrone and Estradiol. Recovery was performed by comparing the measured results of samples spiked with 50, 200 and 400 pg/mL of standard material against expected values. Samples were analyzed in quadruplicate. Both estrone and estradiol exhibited recoveries >85% and <115%. Specificity

Specificity was tested by adding 1 μg of the steroids listed below to 1 mL (equivalent to lOOμg /dL) of water before extraction and injection. Where specificity is being further evaluated, lower concentrations of analytes are used (i.e. 100ng/mL or IOng/mL). Acceptability Criteria: Response less than the LLOQ at the appropriate retention time in excess of physiologically significant amounts of potential interfering analytes.

It was found that estrone analysis is not affected by the presence of circulating hormones or drugs at physiological concentrations in the testing. The liquid-liquid extraction step employed is known to exclude extraction of estrone sulfate during sample processing, thus, it is apparent that the estrone sulfate material tested contains estrone as an impurity at approximately 0.03% and thus, the assay is considered specific for the measurement of estrone. Also, estradiol analysis was not affected by the presence of the circulating hormones and drugs tested at relevant physiological levels. Estrone and Estradiol Spike and Recovery

Concentration Added (pg/mL)

0.000 50.000 200.000 400.000

Concentration Measured (pg/mL)

10.716 61.395 199.547 394.156

Estrone Stripped 11.578 58.841 198.228 423.264

Serum 12.133 65.149 216.259 400.486

10.381 59.695 206.891 431.507

Mean 11.202 61.270 205.231 412.353

Expected Cone. NA 61.202 211.202 411.202

Recovery (%) NA 100.1 97.2 100.3 n 4 4 4 4

24.513 83.123 234.620 434.374

Estrone Pooled 27.357 82.876 228.706 431.772

Serum 27.574 81.036 230.123 434.208

25.478 78.861 231.405 433.010

Mean 26.231 81.474 231.214 433.341

Expected Cone. NA 76.231 226.231 426.231

Recovery (%) NA 106.9 102.2 101.7 n 4 4 4 4

10.029 54.400 197.858 362.120

Estradiol Stripped 9.950 56.014 197.599 373.907

Serum 10.542 59.706 200.453 373.998

9.757 56.920 204.747 366.630

Mean 10.070 56.760 200.164 369.164

Expected Cone. NA 60.070 210.070 410.070

Recovery (%) NA 94.5 95.3 90.0 n 4 4 4 4

21.627 67.816 207.658 375.399

Estradiol Pooled 23.183 70.755 202.194 390.105

Serum 24.317 70.378 206.603 41 1.400

23.876 71.583 221.472 400.773

Mean 23.251 70.133 209.482 394.419

Expected Cone. NA 73.251 223.251 423.251

Recovery (%) NA 95.7 93.8 93.2 n 4 4 4 4

Estrone Hormone Specificity

Androstenediol 1000000 0.000 0.00000

5-androsten-3, 11,17-trione 1000000 0.000 0.00000

Androstenedione 1000000 0.000 0.00000

17a-Methyltestosterone 1000000 0.000 0.00000

Cortisone 1000000 0.000 0.00000

Epitestosterone 1000000 0.000 0.00000

Dehyd roepi androstenedi on e 1000000 0.000 0.00000

Dexamβthasone 1000000 0.000 0.00000

5a-androstan-3b, 17b-diol 1000000 0.000 0.00000

5b-androstan-3a, 17b-diol 1000000 0.000 0.00000

Epiandrosterone 1000000 0.000 0.00000

17a-Hydroxyprogesterone 1000000 0.000 0.00000

1 1-DesoxycortisoI 1000000 0.000 0.00000

Prednisone 1000000 0.000 0.00000

Estriol 1000000 7.741 0.00077

Corticosterone 1000000 0.385 0.00004

Androsterone 1000000 0.000 0.00000

Prednisolone 1000000 0.000 0.00000

17-Hydroxypregπenoloπe 1000000 0.000 0.00000

Progesterone 1000000 0.000 0.00000

20a-Hydroxy-progesterone 1000000 0.000^' 0.00000

20b-Hydroxy-progesterone 1000000 0.000 0.00000

Beclomethasone 1000000 0.000 0.00000

Triamcinolone Acetonide 1000000 0.000 0.00000

Fluticasone Propionate 1000000 0.000 0.00000

Pregnanetriol 1000000 2.385 0.00024

Tctrahydrocortisol 1000000 0.000 0.00000

Tetrahydrocortisone 1000000 0.000 0.00000

Pregnenolone sulphate 1000000 0.000 0.00000

Ethinyl Estradiol 100000 0.845 0.00085

Budesonide 1000000 0.000 0.00000

Pregnanediol 1000000 1.828 0.00018

Desoxycorticosterone 1000000 0.951 0.00010

Cortisol 1000000 0.000 0.00000

21 - Desoxycorti sol 1000000 0.000 0.00000

Pregnenolone 1000000 0.000 0.00000

Andrenosterone 1000000 0.000 0.00000

Aldosterone 1000000 0.000 0.00000

Dihydroandrosterone 1000000 0.000 0.00000

11 a Hydroxy-Progesterone 1000000 0.000 0.00000

Testosterone 1000000 0.000 0.00000

Estrone-3-Sulfate 1000000 339.301 0.03393 Estradiol Hormone Specificity (%) =

Androstenediol 100000 2.763 0.00276

5-androsten-3, 11,17-trione 1000000 0.000 0.00000

Androstenedione 1000000 0.000 0.00000

17a-Mcthyltcstostcronc 1000000 0.000 0.00000

Cortisone 1000000 0.000 0.00000

Epitestosterone 1000000 0.000 0.00000

Dehydroepiandrostenedione 1000000 0.000 0.00000

Dexamethasone 1000000 0.000 0.00000

5a-androstan-3b, I7b-diol 1000000 0.000 0.00000

5b-androstan-3a, 17b-diol 1000000 0.000 0.00000

Epiandrosterone 1000000 0.000 0.00000

17a-Hydroxyprogesterone 1000000 0.000 0.00000

11-Desoxycortisol 1000000 1.739 0.00017

Prednisone 1000000 0.000 0.00000

Bstriol 10000 2.937 0.02937

Corticosterone I 000000 0.000 0.00000

Androsterone IOOOOOO 0.000 0.00000

Prednisolone 1000000 0.000 0.00000

17-Hydroxypregnenolone 1000000 4.560 0.00046

Progesterone 1000000 0.000 0.00000

20a-Hydroxy-progesterone 1000000 0.000 0.00000

20b-Hydroxy-progesterone 1000000 0.000 0.00000

Beclomethasone 1000000 0.000 0.00000

Triamcinolone Acetonide 1000000 0.61 1 0.00006

Fluticasone Propionate 1000000 0.000 0.00000

Pregnanetriol 1000000 1.434 0.00014

Tetrahydrocortisol 1000000 9.51 1 0.00095

Tetrahydrocortisone 1000000 0.000 0.00000

Pregnenolone sulphate 1000000 0.000 0.00000

Ethinyl Estradiol 100000 0.937 0.00094

Budesonide 1000000 0.417 0.00004

Pregnanediol 1000000 0.000 0.00000

Desoxycorticosterone 1000000 0.000 O.00000

Cortisol 1000000 0.000 0.00000

21-Desoxycortisol 1000000 0.000 0.00000

Pregnenolone 1000000 0.000 0.00000

Andrenosterone 1000000 0.000 0.00000

Aldosterone t 000000 0.000 0.00000

Dihydroandrosterone ^' 1000000 0.000 0.00000

1 1 a Hydroxy- Progesterone 1000000 0.000 0.00000

Testosterone 1000000 0.000 0.00000

Estrone-3-Sulfate I 00000 0.000 0.00000 Stability

Stability in human serum was demonstrated at the following conditions for the times shown below.

Sensitivity

The lower limit of quantitation was determined to be 1.Opg/mL for estradiol and 2.5pg/mL for estrone using a sample size of ImL with 80μL being injected into the 2D-LC- MS/MS system. Inter-assay comparison A. Radioimmunoassay Compared to 2D LC-MS/MS.

A minimum of 50 routine samples representing the physiological range were analyzed by 2D LC-MS/MS and RIA following extraction/off-line chromatographic separation for estrone and estradiol and LC-MS/MS with derivatization for estradiol.

The inter-assay comparison of estrone RIA to LC-LC-MS/MS yielded an average bias of 5.46% for samples within the analytical range of both assays. Comparison of data throughout the range generated a slope of 0.9005 with a correlation coefficient of 0.8962; thus, estrone assay-to-assay cross-validation was successful (FIG. 29). Inter-assay comparison of estradiol RIA to LC-LC-MS/MS yielded an average bias of 2.22% for samples within the analytical range of both assays. Comparison of data throughout the range generated a slope of 1.1392 with a correlation coefficient of 0.9776; thus, estradiol inter- assay comparison was successful (FIG. 30). Inter-assay comparison of LC-MS/MS with derivatization compared to LC-LC-MS/MS yielded an average bias of -0.51% (combined data for both Tables below) for samples within the analytical range of both assays. Comparison of data throughout the range generated a slope of 0.9844 with a correlation coefficient of 0.9926 (FIG. 31). Estrone Inter-assay Comparison of Radioimmunoassay to 2D LC -MS/MS

Concentration (pg/mL) Concentration (pg/mL)

LC-LC- LC-LC-

Sample # RJA Bias (%) Sample # RIA Bias (%)

MS/MS MS/MS

Sample 12 53 58.387 10.16 Sample 59 68 62.183 -8.55

Sample 14 45 40.046 -11.01 Sample 60 36 49.805 ^■ 38.35

Sample 15 60 49.222 -17.96 Sample 61 130 151.848 16.81

Sample 16 12 1 1.171 -6.91 Sample 62 76 79.513 4.62

Sample 17 82 67.171 -18.08 Sample 63 73 84.343 15.54

Sample 19 BLQ 8.561 NA Sample 64 102 106.993 4.90

Sample 20 8 12.399 54.99 Sample 66 33 23.136 -29.89

Sample 25 59 52.737 -10.62 Sample 67 100 80.956 -19.04

Sample 31 8 13.281 66.01 Sample 68 17 17.714 4.20

Sample 33 44 43.813 -0.42 Sample 69 62 46.039 -25.74

Sample 38 45 45.408 0.91 Sample 70 41 48.489 18.27

Sample 39 6 13.919 131.98 Sample 71 29 36.534 25.98

Sample 42 52 54.994 5.76 Sample 72 57 47.071 -17.42

Sample 44 49 51.138 4.36 Sample 73 28 39.740 41.93

Sample 46 22 25.341 15.19 Sample 74 25 30.986 23.94

Sample 47 31 35.076 13.15 Sample 75 51 47.044 -7.76

Sample 48 BLQ 15.587 NA Sample 76 42 43.604 3.82

Sample 51 90 76.809 -14.66 Sample 77 9 1 1.251 25.01

Sample 52 57 57.380 0.67 Sample 78 37 33.795 -8.66

Sample 53 27 28.932 7.16 Sample 79 42 44.081 4.95

Sample 54 43 30.371 -29.37 Sample 80 19 21.918 15.36

Sample 55 21 21.693 3.30 Sample 81 88 64.153 -27.10

Sample 56 42 30.132 -28.26 Sample 82 78 75.737 -2.90

Sample 57 27 28.123 4.16 Sample 83 107 90.226 -15.68

Sample 58 48 53.190 10.81 Sample 84 57 51.286 -10.02

Average Bias = 5.46

Bias (%) = (Total LC-LC-MS/MS result - RIA result)/ RIA result, expressed as a percentage Samples were selected in sequential order following cross-validation of Estradiol LC-MS/MS to LC-LC- MS/MS to coincide with anticipated measurable levels in the Estrone RIA assay. BLQ = below limit of quantification.

Estradiol Inter-assay Comparison of Radioimmunoassay to 2D LC-MS/MS

Concentration (pg/mL) Concentration (pg/mL)

LC- LC- LC-LC-

Sample # RIA Bias (%) Sample # RlA Bias (%) MS/MS MS/MS

Sample 12 14 13.512 -3.49 Sample 59 44 44.229 0.52

Sample 14 19 1 1.612 -38.88 Sample 60 26 27.235 4.75

Sample IS 18 9.950 -44.72 Sample 61 223 248.650 1 1.50

Sample 16 18 17.069 -5.17 Sample 62 134 139.889 4.39

Sample 17 18S 203.458 8.22 Sample 63 138 144.874 4.98

Sample 19 25 21.031 -15.88 Sample 64 216 235.982 9.25

Sample 20 10 10.688 6.88 Sample 66 12 7.893 -34.23

Sample 25 92 149.479 62.48 Sample 67 46 41.866 -8.99

Sample 31 12 8.816 -26.53 Sample 68 21 18.206 -13.30

Sample 33 28 24.187 -13.62 Sample 69 61 70.482 15.54

Sample 38 66 79.085 19.83 Sample 70 57 65.890 15.60

Sample 39 10 8.092 -19.08 Sample 71 27 36.189 34.03

Sample 42 78 82.430 5.68 Sample 72 43 50.018 16.32

Sample 44 74 86.914 17.45 Sample 73 37 40.562 9.63

Sample 46 23 22.228 -3.36 Sample 74 72 61.767 -14.21

Sample 47 28 25.616 -8.51 Sample 75 81 104.333 28.81

Sample 48 17 16.038 -5.66 Sample 76 28 28.832 2.97

Sample 51 152 181.717 19.55 Sample 77 42 47.134 12.22

Sample 52 84 89.387 6.41 Sample 78 33 31.675 -4.02

Sample 53 42 45.092 7.36 Sample 79 48 55.149 14.89

Sample 54 48 50.658 5.54 Sample 80 15 15.629 4.19

Sample 55 17 12.738 -25.07 Sample 81 65 75.748 16.54

Sample 56 32 30.153 -5.77 Sample 82 79 89.239 12.96

Sample 57 34 33.255 -2.19 Sample 83 35 35.169 0.48

Sample 58 27 25.110 -7.00 Sample 84 21 27.692 31.87

Average Bias = 2.22

Bias (%) = (Total LC-LC-MS/MS result - RIA result)/ RJA result, expressed as a percentage Samples were selected in sequential order following cross-validation of Estradiol LC-MS/MS to LC-LC- MS/MS to coincide with measurable levels in the Estradiol RIA assay.

Estradiol Inter-assay Comparison of LC-MS/MS to 2D LC -MS/MS

Concentration (pg/mL) Concentration (pg/mL)

LC- LC-LC- LC- LC-LC-

Sample # Bias (%) Sample # Bias (%) MS/MS MS/MS MS/MS MS/MS

Sample 1 BLQ 5.166 NA Sample 26 BLQ BLQ NA

Sample 2 BLQ 1.754 NA Sample 27 BLQ BLQ NA

Sample 3 BLQ 6.888 NA Sample 28 BLQ BLQ NA

Sample 4 BLQ 1.697 NA Sample 29 BLQ BLQ NA

Sample 5 BLQ 2.488 NA Sample 30 BLQ 6.283 NA

Sample 6 ALQ ALQ NA Sample 31 10.22 8.816 -13.75

Sample 7 BLQ 1.91 1 NA Sample 32 BLQ 1.1 10 NA

Sample 8 BLQ 8.004 NA Sample 33 23.94 24.187 1.02

Sample 9 BLQ 3.811 NA Sample 34 BLQ 6.641 NA

Sample 10 BLQ 1.115 NA Sample 35 BLQ 8.473 NA

Sample 11 BLQ 3.387 NA Sample 36 BLQ BLQ NA

Sample 12 14.56 13.512 -7.22 Sample 37 BLQ BLQ NA

Sample 13 ALQ ALQ NA Sample 38 79.90 79.085 -1.02

Sample 14 12.06 11.612 -3.73 Sample 39 11.06 8.092 -26.86

Sample 15 10.04 9.950 -0.86 Sample 40 BLQ BLQ NA

Sample 16 18.56 17.069 -8.01 Sample 41 BLQ 4.344 NA

Sample 17 210.69 203.458 -3.43 Sample 42 78.66 82.430 4.79

Sample 18 BLQ 3.202 NA Sample 43 12.37 1 1.371 -8.10

Sample 19 21.13 21.031 -0.46 Sample 44 87.28 86.914 -0.42

Sample 20 11.05 10.688 -3.23 Sample 45 BLQ 1.043 NA

Sample 21 BLQ 1.906 NA Sample 46 21.21 22.228 4.80

Sample 22 BLQ BLQ NA Sample 47 25.18 25.616 1.74

Sample 23 BLQ 2.716 NA Sample 48 15.81 16.038 1.43

Sample 24 BLQ BLQ NA Sample 49 BLQ 4.677 NA

Sample 25 143.77 149.479 3.97 Sample 50 ALQ ALQ NA

Average Bias (samples 1-50)= -3.30

Bias (%) = (Total LC-LC-MS/MS result - LC-MS/MS result)/ LC-MS/MS result, expressed as a percentage BLQ = Below limit of quantification (lOpg/mL for LC-MS/MS, lpg/mL for LC-LC-MS/MS) ALQ = Above limit of quantification (lOOOpg/mLfor LC-MS/MS, 500pg/mL for LC-LC-MS/MS)

Estradiol Cross-Validation of LC-MS/MS to 2D LC-MSWIS Continued

Concentration (pg/mL) Concentration (pg/mL)

LC- LC-LC- LC- LC- LC-

Sample # Bias (%) Sample # Bias (%) MS/MS MS/MS MS/MS MS/MS

Sample 51 187.11 181.717 -2.88 Sample 76 28.66 28.832 0.59

Sample 52 91.41 89.387 -2.21 Sample 77 50.36 47.134 -6.41

Sample 53 47.76 45.092 -5.59 Sample 78 34.31 31.675 -7.68

Sample 54 53.73 50.658 -5.71 Sample 79 54.11 55.149 1.91

Sample 55 14.32 12.738 -11.07 Sample 80 15.43 15.629 1.28

Sample 56 26.19 30.153 15.14 Sample 81 75.17 75.748 0.78

Sample 57 34.38 33.255 -3.28 Sample 82 92.92 89.239 -3.96

Sample 58 27.69 25.1 10 -9.30 Sample 83 34.64 35.169 1.52

Sample 59 41.44 44.229 6.73 Sample 84 28.92 27.692 -4.23

Sample 60 27.36 27.235 -0.46 Sample 85 107.78 105.278 -2.32

Sample 61 260.1 1 248.650 -4.41 Sample 86 77.28 80.854 4.62

Sample 62 142.53 139.889 -1.85 Sample 87 29.52 36.282 22.92

Sample 63 142.95 144.874 1.34 Sample 88 61.28 60.413 -1.42

Sample 64 236.23 235.982 -0.1 1 Sample 89 56.01 58.909 5.18

Sample 65 ALQ ALQ NA Sample 90 49.78 49.144 -1.27

Sample 66 12.14 7.893 -34.99 Sample 91 56.81 65.677 15.62

Sample 67 44.69 41.866 -6.31 Sample 92 102.40 104.305 1.86

Sample 68 21.81 18.206 -16.51 Sample 93 39.18 34.476 -12.00

Sample 69 69.20 70.482 1.85 Sample 94 58.75 63.487 8.06

Sample 70 60.36 65.890 9.16 Sample 95 66.04 79.660 20.63

Sample 71 50.51 36.189 -28.35 Sample 96 20.45 20.995 2.65

Sample 72 47.80 50.018 4.65 Sample 97 49.59 47.858 -3.49

Sample 73 41.42 40.562 -2.08 Sample 98 12.34 14.914 20.88

Sample 74 58.87 61.767 4.92 Sample 99 40.30 49.841 23.69

Sample 75 100.37 104.333 3.95 Sample 100 93.42 108.173 15.79

Average Bias (samples 51 -100) = 0.36

Bias (%) = (Total LC-LC-MS/MS result - LC-MS/MS result)/ LC-MS/MS result, expressed as a percentage BLQ = Below limit of quantification (10pg/mL for LC-MS/MS, lpg/mL for LC-LC-MS/MS) ALQ = Above limit of quantification (lOOOpg/mLfor LC-MS/MS, 500pg/mL for LC-LC-MS/MS)

Reference Interval

A. Reference Range Sample Groups and Results

Reference range transfer for estrone, estradiol and total estrogens was evaluated using NCCLS guidance (see references). Transfer of the reference range was established using the samples listed below.

Children samples will include 25 boys <10 years old and 25 girls <9 years old.

B. Reference Interval of Patient Test Results

Estrone Reference Ranges

Estradiol Reference Ranges

C. Reference Range Transfer

Guidance provided by NCCLS allows reference range transfer where no more than 2 out of 20 (10%) of samples fall outside the original reference range. A total of 22 out of 23 normal adult female samples were within range for reference range transfer of estrone, estradiol and total estrogens. Adult female reference ranges are transferable. All normal adult male samples were within range for reference range transfer of estrone and estradiol. A total of 22 out of 23 normal adult male samples were within range for reference range transfer of total estrogens. Adult male reference ranges are transferable. All 50 pre-pubertal reference samples were within range for reference range transfer for estrone, estradiol and total estrogens. Pre-pubertal reference ranges are transferable. A total of 24 out of 25 normal postmenopausal female samples were within range for reference range transfer of estrone and total estrogens. A total of 23 out of 25 normal post-menopausal female samples were within range for reference range transfer of estradiol. Post-menopausal female reference ranges are transferable.

Estrone and Estradiol Adult Reference Range Verification

Concentration (pg/mL) Concentration (pg/τnL)

Adults Estrone Estradiol Total Adults Estrone Estradiol Total

Female 1 70.883 95.809 166.692 Male 1 30.575 21.764 52.339

Female 2 76.600 127.096 203.696 Male 2 28.002 20.025 48.027

Female 3* 13306.414 14156.104 27462.518 Male 3 32.190 22.324 54.514

Female 4 66.318 60.373 126.691 Male 4 28.509 31.160 59.669

Female 5 65.789 132.145 197.934 Male 5 29.666 19.525 49.191

Female 6 95.624 109.358 204.982 Male 6 40.293 18.999 59.292

Female 7 81.402 114.658 196.060 Male 7 20.721 16.842 37.563

Female 8 45.846 33.105 78.951 MaIe S 46.467 19.506 65.973

Female 9 37.735 39.985 77.720 Male 9* 77.912 48.095 126.007

Female 10 30.071 38.714 68.785 Male 10 24.086 20.251 44.337

Female 11 58.483 65.622 124.105 Male 11 30.402 14.473 44.875

Female 12 119.945 345.081 465.026 Male 12 40.782 23.593 64.375

Female 13 112.298 246.219 358.517 Male 13 48.644 34.843 83.487

Female 14 103.689 51.824 155.513 Male 14 29.029 19.883 48.912

Female 15 86.195 109.157 195.352 Male 15 38.322 34.511 72.833

Female 16 43.546 30.348 73.894 Male 16 26.460 34.060 60.520

Female 17 83.344 96.485 179.829 Male 17 38.597 28.584 67.181

Female 18 31.321 31.050 62.371 Male 18* 71.676 55.531 127.207

Female 19 64.664 158.151 222.815 Male 19 49.944 26.688 76.632

Female 20 48.442 30.094 78.536 Male 20 37.529 24.837 62.366

Female 21 45.145 30.001 75.146 Male 21 33.808 19.570 53.378

Female 22* 46.019 1 1.005 57.024 Male 22 33.282 25.675 58.957

Female 23 26.042 35.425 61.467 Male 23 18.813 14.025 32.838

Female 24 52.566 64.966 1 17.532 Male 24 26.792 17.124 43.916

Female 25 44.51 1 142.452 186.963 Male 25 30.602 33.692 64.294

^♦ = Abnormal results using alternate assay, excluded from reference range calculations. Estrone Female reference range = 30 - 100 pg/mL (luteal), 90-160 pg/mL (follicular) Estrone Male reference range = 10 — 50 pg/mL. Estradiol Female reference range = 30 - 100 pg/mL (luteal), 70-300 pg/mL (follicular)

Estrone Male reference range = 8 — 35 pg/mL

Total Estrogens Female reference range = 60- 200 pg/mL (luteal), 160-400 pg/mL (follicular) Total Estrogens Male reference range = 20 - 80 pg/mL Estrone and Estradiol Prepubertal Reference Range Verification

Concentration (pg/mL) Concentration (pg/mL)

Children Estrone Estradiol Total Children Estrone Estradiol Total

Female 1 BLQ BLQ BLQ Male 1 BLQ BLQ BLQ

Female 2 BLQ BLQ BLQ Male 2 BLQ BLQ BLQ

Female 3 BLQ BLQ BLQ Male 3 BLQ BLQ BLQ

Female 4 BLQ BLQ BLQ Male 4 BLQ BLQ BLQ

Female 5 BLQ 1.052 1.052 Male 5 BLQ BLQ BLQ

Female 6 BLQ BLQ BLQ Male 6 BLQ BLQ BLQ

Female 7 BLQ BLQ BLQ Male 7 BLQ BLQ BLQ

Female 8 BLQ BLQ BLQ MaIe S BLQ BLQ BLQ

Female 9 BLQ BLQ BLQ Male 9 BLQ BLQ BLQ

Female 10 BLQ BLQ BLQ Male 10 BLQ BLQ BLQ

Female 1 1 BLQ BLQ BLQ Male 11 BLQ BLQ BLQ

Female 12 BLQ BLQ BLQ Male 12 BLQ BLQ BLQ

Female 13 BLQ BLQ BLQ Male 13 BLQ BLQ BLQ

Female 14 BLQ BLQ BLQ Male 14 BLQ BLQ BLQ

Female 15 BLQ 1.718 1.718 Male 15 BLQ BLQ BLQ

Female 16 BLQ BLQ BLQ Male 16 BLQ BLQ BLQ

Female 17 BLQ BLQ BLQ Male 17 2.577 BLQ 2.577

Female 18 BLQ BLQ BLQ Male 18 BLQ BLQ BLQ

Female 19 BLQ BLQ BLQ Male 19 BLQ BLQ BLQ

Female 20 BLQ BLQ BLQ Male 20 BLQ BLQ BLQ

Female 21 BLQ BLQ BLQ Male 21 BLQ BLQ BLQ

Female 22 BLQ BLQ BLQ Male 22 BLQ BLQ ^' BLQ

Female 23 BLQ BLQ BLQ Male 23 BLQ BLQ BLQ

Female 24 BLQ BLQ BLQ Male 24 BLQ BLQ BLQ

Female 25 BLQ BLQ BLQ Male 25 BLQ BLQ BLQ

Estrone Pre-pubertal reference range = < 15 pg/mL Estradiol Pre-pubertal reference range = < 15 pg/mL Total Estrogens Pre-pubertal reference range = < 25 pg/mL

Estrone and Estradiol Post-menopausal Reference Range Verification

Concentration (pg/mL)

Post Menopausal Estrone Estradiol Total Estrogens

Post Menopausal Female 1 15.512 6.174 21.686

Post Menopausal Female 2 26.081 14.535 40.616

Post Menopausal Female 3 17.392 5.680 23.072

Post Menopausal Female 4 1 1.037 4.297 15.334

Post Menopausal Female 5 26.321 12.717 39.038

Post Menopausal Female 6 22.282 4.653 26.935

Post Menopausal Female 7 10.769 5.123 15.892

Post Menopausal Female 8 34.193 14.398 48.591

Post Menopausal Female 9 8.257 4.016 12.273

Post Menopausal Female 10 25.306 1 1.681 36.987

Post Menopausal Female 1 1 22.873 10.268 33.141

Post Menopausal Female 12 7.838 14.060 21.898

Post Menopausal Female 13 12.848 3.788 16.636

Post Menopausal Female 14 8.348 21.916 30.264

Post Menopausal Female 15 8.502 3.173 11.675

Post Menopausal Female 16 17.502 1 1.371 28.873

Post Menopausal Female 17 9.837 5.903 15.740

Post Menopausal Female 18 34.756 14.946 49.702

Post Menopausal Female 19 24.933 6.502 31.435

Post Menopausal Female 20 10.280 6.123 16.403

Post Menopausal Female 21 30.275 19.200 49.475

Post Menopausal Female 22 15.434 4.680 20.114

Post Menopausal Female 23 8.889 9.670 18.559

Post Menopausal Female 24 7.437 5.139 12.576

Post Menopausal Female 25 44.117 14.607 58.724

Estrone Post-menopausal female reference range = < 40 pg/mL Estradiol Post-menopausal female reference range = < 15 pg/mL Total Estrogens Post-menopausal female reference range = < 50 pg/mL

Standard Curve Fitting and Reproducibility

The reproducibility of the standard curve was evaluated by comparing the back- calculated concentrations to the theoretical concentration of the standard in five analytical runs using the concentrations listed below. Calibrator Concentrations for estrone and estradiol (pg/mL) were as follows: 1; 2.5; 5; 10; 25; 50; 100; 200; 350; and 500.

The reproducibility of the standard curve was evaluated by comparing the back- calculated concentrations to the actual concentration of the standard in five analytical runs. The curve was fit with a straight line with weighted 1/x fit, as established during method development. Acceptability Criteria: Imprecision of < 20% at the LLOQ and less than <_15% at other concentrations. Correlation coefficient (r) greater than 0.98. It was found that estrone calibration curves exhibited mean imprecision <15% for all concentrations between 2.5pg/mL and 500pg/mL. Correlation coefficients were greater than 0.98. Estradiol calibration curves exhibited mean imprecision <15% for all concentrations between 1.0pg/mL and 500pg/mL. Correlation coefficients were greater than 0.98. Analytical Reportable Range A. LLOQ (Lower Limit of Quantification)

The lower limit of quantification for estrone using this assay was 2.5 pg/mL as determined during evaluation of inaccuracy, imprecision and calibration curve reproducibility. The lower limit of quantification for estradiol using this assay was 1.0 pg/mL as determined during evaluation of inaccuracy, imprecision and calibration curve reproducibility.

B. ULOQ (Upper Limit of Quantification)

The upper limit of quantification using this assay was 500 pg/mL for both estrone and estradiol, as determined during evaluation of inaccuracy, imprecision and calibration curve reproducibility.

Estrone Standard Curve Fitting and Reproducibility

Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Std 8 Std 9 Std 1.0

Actual Concentration (pg/mL)

Batch 1.000 2.500 5.000 10.000 25.000 50.000 100.000 200.000 350.000 500.000

1.925 2.768 5.648 9.71 1 23.467 46.373 94.259 185.715 349.442 469.565

1 1.390 2.754 4.873 1 1.121 23.507 47.062 92.564 191.601 366.589 557.980

0.461 2.173 4.906 9.681 24.933 52.284 97.879 208.321 373.433 466.693

2 1.511 2.321 5.407 10.333 24.749 52.497 103.488 200.953 355.922 489.025

0.886 2.613 4.448 10.294 25.019 50.470 97.538 203.637 352.701 495.023

3 1.897 2.896 4.602 9.475 24.639 50.202 99.235 209.807 340.983 501.419

0.110 2.514 5.357 9.913 24.999 50.807 100.802 202.390 354.388 473.363

4 1.146 2.371 5.183 ^■ 9.199 24.605 48.966 101.284 204.313 354.972 509.573

IE 2.728 5.174 9.417 23.121 46.892 98.169 188.782 344.288 500.983

5 1.204 2.372 5.230 10.185 24.417 54.467 102.299 203.899 363.129 499.448

Mean 1.170 2.551 5.083 9.933 24.346 50.002 98.752 199.942 355.585 496.307

Accuracy (%RE) 17.00 2.04 1.66 -0.67 -2.62 0.00 -1.25 -0.03 1.60 -0.74

Precision (%RSD) 52.09 9.28 7.33 5.71 2.91 5.37 3.49 4.15 2.79 5.29 n 9 10 10 10 10 10 10 10 10 10

IE = Injection error

Estrone Standard Curve Fitting and Reproducibility Continued

Batch K₀ (Y-intercept) Ki (slope) Correlation Coefficient (R)

1 0.0071 0.0148 0.9973 2 0.0227 0.0126 0.9989 3 0.0168 0.0134 0.9998 4 0.0210 0.0135 0.9996 5 0.0153 0.0136 0.9995

Mean 0.0166 0.0136 0.9990

Precision (%RSD) NA 5.81 0.10 n 5 5 5

Estradiol Standard Curve Fitting and Reproducibility

Std l Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Std 8 Std 9 Std 10

Actual Concentration (pg/mL)

Batch 1.000 2.500 5.000 10.000 25.000 50.000 100.000 200.000 350.000 500.000

0.957 2.550 5.264 10.062 24.338 48.037 96.655 208.854 349.140 495.339

1 0.939 2.590 4.946 10.258 26.356 48.204 100.102 202.014 362.357 488.038

0.948 2.281 5.005 10.065 26.660 51.180 100.076 203.386 361.785 493.088

2 0.838 2.168 5.524 10.089 25.499 51.815 1 10.920 228.732 329.844 467.097

0.998 2.550 5.306 10.203 23.766 51.117 100.742 206.641 341.105 516.785

3 0.951 2.500 5.171 9.725 24.066 50.488 98.928 195.879 349.439 490.639

1.083 2.494 5.032 9.294 22.005 44.878 95.496 190.745 348.234 477.884

4 1.073 2.463 5.346 9.807 25.939 51.655 105.187 208.906 365.338 514.143

IE 2.685 4.853 9.247 25.130 47.971 97.174 196.876 349.441 500.232

5 0.995 2.517 5.178 9.999 25.929 49.433 102.279 203.003 355.375 497.684

Mean 0.976 2.480 5.163 9.875 24.969 49.478 100.756 204.504 351.206 494.093

Accuracy (%RE) -2.42 -0.81 3.25 -1.25 -0.12 -1.04 0.76 2.25 0.34 -1.18

Precision (%RSD) 7.59 6.06 3.98 3.61 5.73 4.47 4.52 5.05 3.05 3.02 n 9 10 10 10 10 10 10 10 10 10

IE = Injection error

Estradiol Standard Curve Fitting and Reproducibility Continued

Batch Ko (Y- intercept) Ki (slope) Correlation Coefficient (R)

1 0.0069 0.0234 0.9997

2 0.0167 0.0217 0.9980

3 0.0058 0.0229 0.9997

4 0.0035 0.0223 0.9991

5 0.0014 0.0226 0.9999

Mean 0.0088 0.0226 0.9990

Precision (%RSD) NA 2.83 0.08 n 5 5 5

Example 2: 96-Well Equilibrium Dialysis Followed by Isotope Dilution LC-MS/MS for Free Thyroxine

Development and validation of free thyroxine (FT4) was developed using a high throughput 96-well based equilibrium dialysis techniques to provide a generational improvement in assay performance over historical radioimmunoassays. Equilibrium dialysis of free thyroxine was chosen over ultrafiltration during method development. Significant variance between ultrafiltration and equilibrium dialysis with RIA detection was observed at 37°C. Further, filtrate yield (and analytical sensitivity) was limited for sample types containing significant lipemic content, resulting in blockage of the filtration membrane. Optimization of dialysis parameters (rotator speed, dialysate buffer composition and dialysis time) was undertaken using pooled normal calibrators, controls of known free thyroxine concentration and spiked dialysis buffer (50% yield of original result when completely dialysed). Dialysis losses were evaluated using spiked dialysis buffer. Referring now to FIG. 31, the measured concentration of free thyroxine versus dialysis time is shown. Free thyroxine (FT4) was measured by liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) after 96-well plate based equilibrium dialysis and either sample dilution or liquid-liquid extraction (ED-LLE). Thyroxine Ring- C₁₃ is added as internal standard to post dialysis aliquots. For liquid liquid extraction protocols, free thyroxine was extracted from dialysate and calibrator samples with 71.25: 23.75: 5 Ethyl Acetate :Hexane: Methanol. The organic extract was transferred to a fresh tube and then evaporated and reconstituted in 50:50 Water: Methanol. For the sample dilution preparative protocols, dialysate duffer without gelatin was prepared and only internal standard solution in methanol was added, prior to LC-MS/MS analysis.

Duplicate sets of FT4 calibrators were analyzed in each batch. All injections were made in singlicate. All samples were injected onto the ARIA TX4 or Transcend TX4 system where the analyte of interest was chromatographed through an analytical column via a gradient separation. An MDS-SCIEX API5000 triple quadrupole mass spectrometer, operating in negative ion electrospray ionization (ESI) mode (Turboionspray) was used for detection. Quantification of analyte and internal standard was performed in selected reaction monitoring mode (SRM). The back-calculated amount of each analyte in each sample was determined from duplicate calibration curves generated by spiking known amounts of purified thyroxine into dialysis buffer or methanol: water solutions. Measurement of free thyroxine is used to evaluate hyperthyroidism and hypothyroidism, to differentiate and evaluate disorders involving variance in levels of circulating proteins, such as, familial dysalbuminemic hyperthyroximenia, euthyroid hypothyroxemias, transthyretin excess and variant thyroxine binding globulin. Analysis of free thyroxine by ED-LLE-LC-MS/MS or ED-LC-MS/MS detection was developed to measure levels in serum samples from children, women and men. The lower limit of detection using the default sample aliquot of 200μL is 0.2ng/dL (2pg/mL) for free thyroxine. Definitions

T4- Thryroxine, ED - Equilibrium Dialysis, LC-MS/MS — Liquid Chromatography tandem mass spectrometry detection, ESI- Electrospray Ionization (Turboionspray), L-

ThyToxine, Sigma-Aldrich, (USA) Product # T2376 or USP, (USA) Product #044K1436; Stable Labeled Thyroxine, Tyrosine Ring-¹³C₆, CDN Isotopes, (USA) Product # CLM-6725- O- Specimen Requirements Recommended: 0.5mL serum or plasma. Separate within one hour. Store and ship frozen in a plastic vial.

Blood was drawn into red top vacutainer tube and clotting allowed to occur for 20 minutes at room temperature (or until clot had retracted). The sample was then centrifuged and the serum transferred to a labeled plastic vial, and immediately frozen. Storage is short term storage (2 weeks); frozen (< -20⁰C). Shipping is Frozen (< -20⁰C) - on dry ice. Equipment & Materials

The following materials were used: Standard manual pipetting devices; Big Shot II Hybridization Oven (Rotator, Boekel Scientific Model No. 230401); 96-Well Equilibrium 1OkD Dialyzer Plates (Harvard Apparatus, Product No. 74-2331); 1.2ml MBlock

Polypropylene 96 Well Collection Plate (SPE Ware, Inc. Product No. SPE0210); Heat Sealing Foil (SPE Ware, Inc. Product No. AB-0589); Mechanical Shaker, Eberbach Inc.; Rotary Evaporator (rotovap), Speed Vac SC 200, Savant; Volumetric Glass Bottles for mobile phases, various sizes; API 5000 Tandem Mass Spectrometer, Sciex, (Toronto, Canada); Turbo V™ Ion source with ESI probe, Sciex, (Toronto, Canada); Aria TX4 HTLC System, Cohesive Technologies, (MA, USA) consisting of 4 each: 1100 Series Quaternary Pump, 1100 Series Binary Pump, 1100 Series Vacuum Degasser; HTS Twin PAL System Autosampler, CTC Analytics AG (Switzerland); BETASIL Phenyl-Hexyl Analytical Column, 50 X 2.1 mm, 5μm particle size, Thermo Electron Corporation, (USA) Product No.

73005-052130; Vortex Mixer, VWR or equivalent; Combi-thermo Heat Sealer, Abgene Inc., Product No. AB-0559; 96-Well Centrifuge 5804R Eppendorf or equivalent; Solvent washed 12x75mm borosilicate glass tubes with polypropylene snap caps; Analyst Version 1.4 or greater. Applied Biosystems, (CA, USA); Aria OS Version 1.4 or greater, Cohesive Technologies (MA, USA). Reagents

Water — Type II, Millipore MiIIiQ or equivalent; Water - Glass Distilled; Acetonitrile HPLC Grade (EM Science, Catalog#AX0142-l); Methanol HPLC Grade (Fisher Scientific, Catalog#A452-4); Hexane HPLC Grade (Fisher Scientific, Catalog #H302-4); Ethyl Acetate OPTIMA Grade (Fisher Scientific, Catalog #E196-4); Ammonium Hydroxide 28.0-30.0%

(JT Baker, Catalog#9721-04); Formic Acid 88% (Fisher Scientific, Catalog#Al 18-500); lmg/mL stock L-Thyroxine (Sigma, Catalog#T2376) (Dissolve appropriate amount of L- Thyroxine powder into the appropriate volume of 25% Ammonium Hydroxide in Methanol); Blue Dextran Dye Marker (18/mg/mL) in millipore water. Ammonium Carbonate (100 mM) in millipore water. Extraction solvent for liquid-liquid extraction (71.25% Ethyl Acetate, 23.75% Hexane, and 5% Methanol).

1 mg/mL stock Tyrosine Ring-¹³Cβ (Cambridge Isotope Labs, Catalog#CLM-6725-O) Dissolve appropriate amount of Tyrosine RiHg-¹³C₆ powder into the appropriate volume of 25% Ammonium Hydroxide in Methanol). The following protocol was used to prepare FT4 Dialysis Buffer (with and without gelatin). Mix 800 mL of Millipore Water or equivalent, add 1 mL Sodium DL-Lactate 60% (w/w) (Sigma-Aldrich, Catalog#L1375), 5.265g Sodium Chloride (EMD Science, Catalog#SX0420-l), 0.224g Potassium Chloride (EMD Science, Catalog#PXl 450-1), 0.180g Potassium Phosphate (EMD Science, Catalog#PXl 565-1), 0.246g Magnesium Sulfate 7H₂O (Sigma-Aldrich, Catalog#M1880), 0.300g Urea (Sigma-Aldrich, Catalog#U0631). Dissolved

0.275g of Calcium Chloride (EMD Science, Catalog#SX0130-1) into 5mL of Millipore Water in a separate glass vial and added to the above solution. Next (Step A) is performed: The buffer solution was heated to 50⁰C and while mixing, slowly added LOg Laboratory Grade Gelatin Type A (Fisher, Catalog#G8-500). The buffer solution was then allowed to. Step B: added 5.891g HEPES Sodium Salt (Sigma-Aldrich, Catalog#H7006). In a separate glass bottle dissolved 7.19Og of HEPES Acid (Sigma-Aldrich, Catalog#H3375) into 20OmL of Millipore Water. Added this to above solution slowly until the pH reached 7.4 and QS to 1000 mL with Millipore Water. The buffer is then aliquoted into glass vials and stored frozen at -20⁰C for up to 3 months. To prepare FT4 Dialysis Buffer without gelatin, the buffer is prepared as above except excluding step A.

To prepare internal standard diluent 1 , two Liters Millipore Water or equivalent are mixed with 0.20Og Calcium Chloride (EMD Science, Catalog#SX0130-1), I6.37g Sodium Chloride (EMD Science, Catalog#SX0420-l), 2.Og Sodium Azide (Sigma-Aldrich.

Catalog#S8032), 4.3Og Sodium Phosphate Dibasic (EMD Science, Catalog#SX0715). 0.380g Sodium Phosphate Monobasic (J.T. Baker, Catalog#3818-0), 26mL 10% BSA (Sigma- Aldrich, Catalog# A3803). The lot is tested to ensure minimal analyte response from buffer and stored refrigerated at 4°C for up to 6 months. The internal standard diluent 2 is methanol. To prepare an internal standard solution for liquid-liquid extraction (100pg/mL ¹³C_O-

Thyroxine) added 0.ImL of 100ng/ml ^l3C₆-Thyroxine to 100 mL internal standard diluent 1, and mixed well. The standard was then aliquoted into 2OmL glass scintillation vials. To prepare an internal standard solution for diluting and inject (lOOpg/mL ¹³C6-Thyroxine), added 0. ImL of 100ng/ml ^l3C₆-Thyroxine to 100 mL methanol. The FT4 Reconstitution Solution was 50:50 Millipore Water: Methanol. The following mobile phases were used: Eluting Pump A Mobile Phase (90% Water and 10% Methanol); Eluting Pump B Mobile Phase (90% Methanol and 10% Water); Loading Pump A Mobile Phase for post-column addition (90% Methanol and 10% Water with ImM Ammonium Carbonate). Two needle wash solutions were used: Needle Wash Solution 1 (Aqueous 1% Formic

Acid), Needle Wash Solution 2 (70:30 Acetonitrile: IN Ammonium Hydroxide). The needle wash solutions were stored at room temperature for up to 6 months. Calibration Procedures With Liquid-Liquid Extraction

Duplicate standard curves, as described in this procedure, are included with each analytical batch. An L-Thyroxine Stock Solution (lOOng/mL) - 100ng/ml L-Thyroxine stock, made by serially diluting the lmg/mL L-Thyroxine stock in methanol, is used to prepare intermediate stock solutions for preparation of calibrators. Dilute 1.OmL of L- Throxine Stock (lOOng/mL) to 10OmL with FT4 Dialysis Buffer to yield lng/mL thyroxine solution. This stock is stable when stored at -70⁰C. Next, dilute 1OmL of the lng/mL solution to 10OmL in FT4 Dialysis Buffer to yield a 100pg/mL free thyroxine solution. Stable when stored at -70⁰C. Using these stock solutions, calibration standards of 0.2, 0.5, 1.0, 2.5, 5.0, and 10 ng/dL were made. All standards were prepared in FT4 Dialysis Buffer with or without gelatin if performing liquid-liquid extraction. The calibration standards were then transferred into appropriately labeled glass vials in ImL aliquots, capped and stored frozen at -20⁰C. Calibration Procedures With Sample Dilution

Duplicate standard curves, as described in this procedure, are included with each analytical batch. An L-Thyroxine Stock Solution (lOOng/mL) - lOOng/ml L-Thyroxine stock, made by serially diluting the lmg/mL L-Thyroxine stock in methanol, was used to prepare intermediate stock solutions for preparation of calibrators. To make the intermediate stock solutions, diluted LOmL of L-Throxine Stock (lOOng/mL) to 10OmL with methanol to yield lng/mL thyroxine solution. This stock is stable when stored at -70⁰C. Next, diluted 1OmL of the lng/mL solution to 10OmL in methanol to yield a 100pg/mL thyroxine solution.

^• This stock is also stable when stored at -70⁰C. Using these stock solutions, calibration standards of 0.2, 0.5, 1.0, 2.5, 5.0, and 10 ng/dL were made. All standards were prepared in 1 :1 methanol:water. The calibration standards were transferred into appropriately labeled glass vials in ImL aliquots, capped and stored frozen at -20⁰C. Quality Control

Control pools are prepared in human serum as shown below and introduced into use according to analytically robust procedures

Quality Control Concentrations

The control data was recorded for each run on Levy- Jennings charts. Points were plotted and connected. The control chart is reviewed for shifts or trends. A warning situation may exists if one control falls within the ±2 SD and ± 3 SD interval; all test results may be released.

Test Procedure

A. Assay Procedure Dialysis of samples: Dialysis was performed as follows. Warmed the Big Shot II hybridization oven to reach 37⁰C. Capped sample side (blue side) of the dialysis plate. Turned the plate over and added 300 μL of FT4 Dialysis Buffer either with or without gelatin using an Eppendorf Plus repeating pipette, or equivalent, with a 1OmL tip to the buffer side (clear side) of the dialysis plate. Added the buffer gently to stop bubble formation. Capped the buffer side. Turned the plate over and uncapped the sample side of the dialysis plate.

Pipetted 10 μl of 18mg/mL blue dextran dye marker solution into each well. Pipette controls and patient samples (0.2 mL) into the wells with an Eppendorf pipette (or equivalent). Added the patient and QC samples gently to stop bubble formation. Recapped the sample side of the dialysis plate, and place the dialysis plate into the oven and turn on the rotator with the speed set at 15RPM. Let rotate overnight for 16 hours ± 1 hour.

After the dialysis procedure was complete, thawed and mix the internal standard solution to add to the samples for liquid-liquid extraction. Labeled a solvent washed 12x75 glass tube for each dialysis sample, duplicate tubes for each standard point, and four tubes for the double blanks. After dialysis was complete checked for any trace of blue dextran in the buffer side (clear side) of the plate. Note dany membrane leakage and did not transfer the dialysate from the wells with faulty membranes. Pipetted 200 μL of dialysate from the buffer side (clear side) of the dialysis plate for each sample into the corresponding 12x75 glass tube. Pipetted 200 μL of standard into the appropriate labeled tubes. Pipetted 200 μL of FT4 Dialysis Buffer into the double blanks. Liquid-liquid Extraction Procedure: Liquid-liquid extraction was done as follows. 50 μL of 100pg/mL FT4 Internal Standard 1 was added to all tubes, except the double blanks to which 50 μL of water was added. Tubes were mixed 10 times up and down (e.g., on a multi- tube vortexer) and let stand for 10 minutes.

For extraction, 2mL of extraction solvent was added to all tubes. Tubes were capped and mixed on a multi-tube vortexer, 4 times for 1 minute intervals. The tubes were removed from the shaker and spun down in centrifuge at 3000rpm for 10 minutes. A separate set of 12x75 solvent (methanol) washed glass tubes was labeled, and after freezing the aqueous layer, the extract was poured into the labeled tubes. The tubes were placed in a Rotovap to allow solvent to evaporate for at least 45 minutes.

Once there was no trace of solvent left in the tubes, the samples were reconstituted with 100 μL reconstitution solution. Tubes were then covered with parafilm and mixed on multivortexer, 4 times for 30 second intervals. Using an Eppendorf pipette, a robotic liquid handler, or equivalent, the reconstituted sample was transferred from the 12x75 tubes into a 96-well plate. A heat-sealing foil was placed over the plate and the plate sealed with a heated plate sealer. The sealed plate was then centrifuged at 3700 rpm (approximately 200Og) at 10⁰C for 10 minutes. 80 μL was used for injection. Dilute and Inject Procedure: For injection, 50 μL of lOOpg/mL FT4 Internal Standard solution 2 (methanol) was added to all tubes, except the double blanks to which 50 μL of water was added. Tubes were mixed (e.g. 10 times up and down on a multi-tube vortexer) and allowed to stand for 10 minutes. Place a heat-sealing foil over the plate and seal the plate with the heated plate sealer. Centrifuge plate at 3700 rpm (approximately 200Og) at 10⁰C for 10 minutes. Inject 100 μL.

LC-MS/MS Procedures: For LC-MS/MS, the 96-well plate is placed in LC-MS/MS Autosampler and the system filled with LC system reagents. After liquid-liquid extraction or sample dilution, 80 to 100 μL of processed sample was injected into the HTLC system using methanol .waterin the mobile phase. The HTLC system comprises two HPLC pumps per channel that can be employed in two functions: (1) Post HPLC -column addition of solvents to improve sensitivity; and (2) HPLC chromatography using a binary gradient and a 5 μm reverse phase analytical column. In this example a phenyl hexyl column was used for chromatography, which had a 5-μm particle size. Such HPLC columns are commercially available (e.g., Thermo Hypersil Phenyl Hexyl, Luna Phenyl Hexyl). In the analytical mode of the HTLC, the sample was first loaded onto the analytical column. A binary gradient of from 40% to 90% methanol at ImL per minute over 2 minutes was used, resulting in the separation of thyroxine and internal standard from matrix interferences and other analytes contained in the sample. Ionization efficiency and thus detection limits were enhanced by post-column addition of a 90: 10 Methanol:water solution containing ImM ammonium carbonate at 200 microliters per minute. The separated sample was then transferred to the

MS/MS for quantitation. The LC method is summarized in the table below. Thyroxine and internal standard elute at approximately 2.5 minutes from the start of the method (+^• or - 0.5 minutes) 2525

Free Thyroxine LC Method

^♦Loading Pump Buffer 10:90 Millipore Water: Methanol with 1 mM Ammonium Carbonate Eluting Pump Buffer A Millipore Water Eluting Pump Buffer B 10:90 Millipore Water: Methanol

Mass Spectrometry: The flow of combined liquid solvents from the HTLC entered the rurboionspray (ESI) interface of the MS/MS analyzer. The solvent/charged analyte mixture was first electrosprayed and converted to fine droplets after exiting the electrospray capillary. The residual solvent is removed from the charged analytes through a combination of heating and nitrogen gas flow to eventually yield gas phase analyte ions. The ions passed through the orifice of the instrument and entered the first quadrupole. Quadrupoles 1 and 3 (Ql and Q3) were the mass filters, allowing selection of ions based on their mass to charge ratio (m/z). Quadrupole 2 (Q2) was the collision cell, where ions were fragmented. The first quadrupole of the MS/MS (Ql) selected for molecules with the mass to charge ratio of thyroxine (775.5 ± 1.0 m/z or mass units). Ions with these m/z passed to the collision chamber (Q2), while ions with any other m/z collided with the sides of the quadrupole and were fragmented. Ions entering Q2 collided with neutral gas molecules and fragmented. This process is called Collisionally Activated Dissociation (CAD). The CAD gas used in this example was nitrogen resulting in the generation of fragments (product). The fragment ions generated were passed into quadrupole 3 (Q3), where the two fragment ions of thyroxine to be measured {m/z 574.6+-.1.0 m/z & 126.8+-1.0 m/z or mass units) were selected for, while other ions were screened out. The selected fragment ions were measured by the detector. The same process was carried out for the internal standard, which was ¹³Cβ- Thyroxine . Thus, the selected MS/MS transitions (nominal masses) measured were as follows: Thyroxine m/z 775 to 574 and 127, ¹³C₆-Thyroxine m/z 781 to 580 and 127. Selected MS/MS parameters were as follows: Dwell time: 100 msec for each transition, Unit mass resolution in both resolving quadrupoles (Ql and Q3) Curtain Gas: 20 CADgas: 6, Temp.: 45O.degree. C. Gasl: 80, GS2: 40 CE: -80 for m/z 127 product ions, -45 for m/z 574 and 580 product ions. Electrospray voltage = -4500V.

As ions collide with the detector, they produce a pulse of electrons. The pulse was converted to a digital signal, which was counted to provide an ion count. The acquired data was relayed to the computer, which plotted counts of the ions collected vs. time. Calculations

Calibration curves are constructed using the software system (Analyst) that controls the mass spectrometer. Calibration curves are generated by assigning the known concentrations to calibrators to generate a response ratio of analyte to internal standard versus concentration of analyte added [FIG. 20 - 26]. The concentrations of unknown samples are automatically calculated by comparing the response ratio of analyte to internal standard observed in measuring unknown samples to the calibration curve generated above.

Only results within the measurable range (2-100pg/ml, 0.2-10ng/dL) are valid. Assay Performance Characteristics

A quantitative bioanalytical method for the determination of free thyroxine (FT4) in human serum using ED, LLE or sample dilution, and LC-MS/MS detection was developed and validated.

The assay was shown to be specific for the analysis of free thyroxine. Quantitative interference was not observed in the FT4 Dialysis Buffer. The matrix effect for free thyroxine and its internal standard was within acceptable limits for all post-dialysis sample types including pooled serum, plasma containing heparin and EDTA anticoagulants, hemolyzed and lipemic samples. Assay specificity was shown for ¹³C_f,-Thyroxine internal standard .

The method was assayed and validated using four analytical batches. Replicates of spiked quality control (QC) samples at approximately 1.0, 1.5, 2.5 and 8.0ng/dL were prepared in pooled serum to determine imprecision for free thyroxine. Replicates of calibrator samples at approximately 0.1, 0.2, 5.0 and 10.0ng/dL were prepared in FT4 Dialysis Buffer to determine inaccuracy and imprecision for free thyroxine. The intra- and inter-assay inaccuracy (%bias) and imprecision (%CV) for the calibrators and QC (imprecision only) samples were < 20% at the LLOQ and < 15% at all other concentrations. The analytical range of the assay was validated between 0.2ng/dL (LLOQ) and 10.0ng/dL (ULOQ) for free thyroxine. ^■ ₎

Spike and recovery experiments were performed using a post-dialysis serum pool and a FT4 Dialysis Buffer Calibrator containing low levels of thyroxine. The dialysate from the pool and the calibrator were spiked with pure thyroxine standard material. Mean recoveries of between 85% and 115% were observed for l.Ong/dL, 2.0ng/dL and 8.0ng/dL spikes respectively.

Hormone specificity interference less than the LLOQ response was observed during evaluation of free thyroxine for all analytes tested at relevant circulating levels. Free thyroxine whole blood stability has been shown for up to 48 hours at room temperature. Free thyroxine calibrator stability has been shown for up to 16 hours at room temperature. Free thyroxine QC sample stability has been shown for up to 24 hours at room temperature. Free thyroxine calibrator and QC stability has been shown for up to 14 days when stored at -20⁰C. Free thyroxine calibrator and QC stability has been shown for up to three freeze/thaw cycles. Free thyroxine stock solution stability has been shown for up to 10 days frozen at -70⁰C. Free thyroxine working stock solution stability has been shown for up to 6 days when stored refrigerated at 4°C. ¹³C₆-Thyroxine internal standard solution stability has been shown for up to 7 days refrigerated at 4^UC. Free thyroxine autosampler stability has been shown for 24 hours at 10⁰C. Cross-validation of samples analyzed by Centaur (Free thyroxine) against ED-LLE followed by LC-MS/MS indicates acceptable bias. Comparison using scatter plots (FIG. 27) produces acceptable comparison for Centaur and ED-LLE followed by LC-MS/MS for Free thyroxine. Reference range transfer was successful for free thyroxine in adults and prepubertal children ages 2-8 years. Standard curve fitting and reproducibility was evaluated using spiked standard samples at six concentrations. The standards were used to generate a weighted (1/x) linear regression calibration curve, which covered the range from 0.2 to lO.Ong/dL for free thyroxine. Average inaccuracies and imprecision < 20% at the LLOQ and < 15% throughout the remainder of the range were observed. The correlation coefficients of the curves were greater than 0.98 (Tables 29-30).

Selectivity Blank Matrix Interference.

Quadruplicate injections of FT4 Dialysis Buffer and 1 : 1 methanol: water were injected to determine the degree of blank matrix interference for thyroxine. For each assay, matrix analyte responses were less than the mean LLOQ responses. Anticoagulant Effect on Measurement.

The effect of EDTA and heparin as anticoagulants was tested by drawing healthy volunteers using red top serum collection tubes and vacutainers containing sodium heparin and potassium EDTA anticoagulants. The results of the heparin and EDTA tubes were compared to the results from the serum collection tubes. It was found that variation of sample type and anticoagulant exhibited a bias < ±15% for measurement of free thyroxine. Thus, plasma samples collected with heparin and EDTA are acceptable specimen types. Anticoagulant Effect on Free Thyroxine Measurement

Concentration (ng/dL)

Sample Serum Heparin EDTA

273.094* 1.536 1.570

1.746 1.754 1.669

1.745 1.413 1.553

1.651 1.521 1.527

Mean 1.714 1.556 1.580

Mean Matrix Effect (%) NA -9.22 -7.83 n 4 4 4

* Dialysis error, excluded from calculations; NA = Not Applicable.

Effect of Lipemia and Hemolysis in the Matrix.

The effect of lipemia and hemolysis on the quantitative result was determined by spiking pooled patient serum with either a lipid solution or lysed red blood cells at a concentration of 5% by volume. The samples were run in quadruplicate and the results were compared to the results of the pool before contamination. A bias < ±15% following addition of lipemic or hernolyzed material was observed for free thyroxine. Thus, lipemic and hemolyzed samples may be processed using this assay.

Effect of Lipemia and Hemolysis on Free Thyroxine Measurement

Concentration (ng/dL)

Sample Serum Lipemic Hemolyzed

1.417 1.463 2.157*

PTiI 1.403 1.385 1.684

1.696 3.021* 1.621

1.285 1.547 1.602

Mean 1.450 1.465 1.636

Mean Matrix Effect (%) NA 1.02 12.79 n 4 4 4

* = Data point outside 3SD from mean, excluded from calculations. NA = Not Applicable. Internal Standard Interference.

Internal standard (¹³C₆-Thyroxine) working solution was spiked into FT4 Dialysis Buffer and tested in quadruplicate to evaluate the presence of unlabeled analyte. Internal standard (¹³C₆-Thyroxine) interference response < LLOQ response was observed. Thus, ¹³C₆-Thyroxine interference was considered acceptable. Matrix Effect.

Matrix effect was calculated at low, mid, and high level concentrations. Matrix effect for internal standard was measured at a single concentration. A minimum of 4 samples per QC level were analyzed for FT4 to determine matrix effect on quantitative result. Matrix effects were less than 15% for free thyroxine and ¹³C6-Thyroxine Internal Standard when comparing water and dialysate from pooled samples.

Free Thyroxine Matrix Effect

Sample Matrix Millipore Water Post-Dialysis Pooled Serum

Post-Column Analyte Peak IS Peak Analyte Peak IS Baseline

Ratio Ratio Infusion Level Height (cps) Height (cps) Height (cps) Height (cps)

24299.421 16100.000 1.509 26033.955 17600.000 1.479 24027.876 15400.000 1.560 25844.532 16800.000 1.538

Low Infusion 25823.905 15900.000 1.624 25012.124 17100.000 1.463 24917.386 15900.000 1.567 24618.647 15900.000 1.548

MARR per Cone. NA NA 1.565 NA NA 1.507

% Change NA NA NA NA NA -3.71

173144.290 24400.000 7.096 164420.482 21200.000 7.756

181732.088 23100.000 7.867 164337.907 20800.000 7.901

175921.645 23900.000 7.361 155907.875 20200.000 7.718

178237.273 23400.000 7.617 156578.143 20000.000 7.829

MARR per Cone. NA NA 7.485 NA NA 7.801

% Change NA NA NA NA NA 4.22

303787.094 23000.000 13.208 264941.287 19500.000 13.587

312136.060 21600.000 14.451 266588.958 19000.000 14.031

High Infusion

329631.304 24400.000 13.509 272643.856 19100.000 14.275

311677.872 22300.000 13.977 269085.967 19300.000 13.942

MARR per Cone. NA NA 13.786 NA NA 13.959

% Change NA NA NA NA NA 1.25

Matrix effect = [(Mean Analyte to Internal standard ratio in pooled serum)/ Mean Analyte to Internal standard ratio in water)]- 1 , expressed as a percentage

Inaccuracy and Imprecision

A. Intra-assay and Inter-assay Imprecision.

Intra-assay imprecision was calculated with replicate samples of spiked FT4 Buffer solutions (data from 4 runs) and replicate samples at different concentrations in post-dialysis pooled human serum (data from 4 runs). Inter-assay imprecision was calculated using data from each of the assay runs (n > 18). Free thyroxine exhibited intra and inter-assay imprecision < 20% at the LLOQ, which was established to be 0.2ng/dL, and < 15% throughout the remainder of the linear range (0.2 to 10.0 ng/dL) in both post-dialysis pooled serum and FT4 Dialysis Buffer.

B. Intra-assay and Inter-assay Inaccuracy.

Intra-assay inaccuracy was calculated in 4 assay runs with replicates at 4 different concentrations in FT4 dialysis buffer spiked with known amounts of analyte. Inter-assay inaccuracy was calculated using data from each of 4 assay runs (n > 18). Free thyroxine exhibited intra and inter-assay inaccuracy < ±20% at the LLOQ, which was established to be 0.2 ng/dL, and < +15% throughout the remainder of the linear range (0.2 to lO.Ong/dL) in FT4 Dialysis Buffer.

A. Intra-assay Precision

The intra-assay precision of the analytical method was calculated for four assays using patient pools (QC 1, QC 2, QC 3, and QC 4). The following tables show the data for these pools, as well as, the data from quality controls made from spiked FT4 Dialysis Buffer at four concentrations (O.lng/dL, 0.2ng/dL, 5.0ng/dL, and lO.Ong/dL).

Free Thyroxine Intra-assay Precision

Intra-day %CV

Batch Number QC1 QC2 QC3 QC4 0.1 0.2 5 10

1 9.61 4.75 3.89 3.79 153.4 4.98 6.20 5.53

2 12.16 9.55 5.53 4.41 39.65 5.12 5.24 2.19

3 4.96 2.31 5.44 6.42 22.20 4.37 4.05 5.47

4 3.92 3.51 4.29 5.89 117.7 2.48 0.53 3.90

B. Reproducibility

The inter-assay precision was calculated from the overall data from the precision assays for each of the QC samples. As shown in the table below, the method has acceptable inter-assay precision.

Free Thyroxine Inter-Assay Precision

QC 1 QC 2 QC 3 QC 4 0.1 0.2 5.0 10.0

Average 1.12 1.52 2.51 7.67 0.19 0.20 4.88 10.18 %CV 9.86 8.41 6.79 6.34 144.44 4.61 5.66 4.67 N 23 22 21 24 20 19 20 20

0.1 ng/dL standard failed inter-assay precision requirements, therefore 0.2ng/dL standard determined to be the LLOQ. Accuracy

The inter-assay accuracy was determined by calculating the percent bias for samples of known concentrations. FT4 Dialysis Buffer was spiked to O.lng/dL, 0.2ng/dL, 5.0ng/dL, and lO.Ong/dL and then assayed a total of at least 18 times in 4 different runs.

Free Thyroxine Accuracy Data

O.lng/dL standard failed Inter-Assay precision, therefore 0.2ng/dL standard determined to be the LLOQ.

Spike and Recovery

Spike and Recovery Preparation

A post dialysis low level QC sample and a FT4 dialysis buffer calibrator were spiked with thyroxine. Recovery was performed by comparing the measured results of samples spiked with 1.0, 2.0, and 8.0ng/dL of standard material against expected values. Samples were analyzed in quadruplicate. Free thyroxine assay exhibited recoveries >85% and <115%.

Thyroxine Spike and Recovery

Concentration Added (ng/dL)

0.000 1.000 2.000 8.000

Concentration Measured (ng/dL)

0.949 1.885 2.760 8.691

1.034 1.887 2.746 7.761

FT4 Calibrator

0.972 1.873 2.900 8.867

1.030 1.855 2.844 8.666

Mean 0.996 1.875 2.813 8.496

Expected Cone. NA 1.996 2.996 8.996

Recovery (%) NA 93.9 93.9 94.4 n 4 4 4 4

1.072 2.043 2.657 8.600

1.084 1.963 2.905 8.285

Post-Dialysis Pooled Serum 1.088 1.991 2.898 8.515

1.097 1.992 2.761 8.124

Mean 1.085 1.997 2.805 8.381

Expected Cone. NA 2.085 3.085 9.085

Recovery (%) NA 95.8 90.9 92.2 n 4 4 4 4

NA = Not Applicable Selectivity

Selectivity was tested by spiking Ing of the analytes listed below into ImL

(equivalent to 100ng/dL) of FT4 dialysis buffer before extraction and injection. Acceptability Criteria: Response less than the LLOQ at the appropriate retention time in excess of physiologically significant amounts of potential interfering substance.

The circulating concentrations of the cross reactants above are all at physiological concentrations less than 3ng/dL. This concentration would give a response equal to 0.03ng/dL of FT4 at most in the case of RT3. This is well below the level of detection for free thyroxine. Free thyroxine analysis is not affected by the presence of circulating hormones or drugs at physiological concentrations in the testing performed. Interfering substances are removed through sample purification, chromatography and selected reaction monitoring.

Free Thyroxine Hormone Specificity

Measured

Amount added Mean Relative

Steroid Concentration

(ng/dL) Response (%)

(ng/dL)

Cross Reactant T3 100 0.377

Cross Reactant T3 100 0.336 n Δft

Cross Reactant T3 100 0.519

Cross Reactant T3 100 0.365

Cross Reactant RT3 100 1.123

Cross Reactant RT3 100 1.032 1 f!7

Cross Reactant RT3 100 1.082

Cross Reactant RT3 100 1.025

Cross Reactant_3,5-DIT 100 0.162

Cross Reactant_3,5-DIT 100 0.043 o no

Cross Reactant_3,5-DIT 100 0.062

Cross Reactant_3,5-DIT 100 0.108

Cross Reactant 3-IT 100 0.104

Cross Reactant 3-IT 100 0.183

Cross Reactant 3-IT 100 0.176

Cross Reactant 3-IT 100 0.208 T3 = 3, 5, 3'-Triiodo-L-Thyronine; RT3 = 3, 3', 5'-Triiodo-L-Thyroninc; 3,5-DIT = 3, 5-Diiodo-L-Thyroninc;

3-IT = 3-Iodo-L-Thyroπiπe. Stability

Stability was demonstrated at the following conditions for the listed times.

Inter-assay Comparison

A. Bayer Centaur Immunoassay Compared to ED-LLE-LC-MS/MS.

A minimum of 25 routine samples representing the physiological range, were analyzed by ED-LLE-LC-MS/MS and Bayer Centaur for assay-to-assay comparison (see FIG. 27).

Free Thyroxine Cross-Validation of Centaur to ED-LLE-LC-MS/MS

Concentration (ng/dL)

Sample # Centaur ED LC-MS/MS Bias (%)

Sample I 1.16 2.231 92.33

Sample 2 1.29 1.781 38.06

Sample 3 1.31 2.072 ^' 58.17

Sample 4 0.99 1.265 27.78

Sample 5 1.37 2.128 55.33

Sample 6 1.58 2.274 43.92

Sample 7 0.95 1.325 39.47

Sample 8 1.12 1.272 13.57

Sample 9 0.90 1.191 32.33

Sample 10 1.03 1.305 26.70

Sample 11 0.95 1.212 27.58

Sample 12 1.63 2.399 47.18

Sample 13 1.03 1.417 37.57

Sample 14 1.1 1 1.545 39.19

Sample 15 0.81 1.035 27.78

Sample 16 1.15 1.647 43.22

Sample 17 0.98 1.288 31.43

Sample 18 1.36 2.145 57.72

Sample 19 1.38 1.817 31.67

Sample 20 1.43 2.591 81.19

Sample 21 1.14 1.927 69.04

Sample 22 1.00 1.159 15.90

Sample 23 1.98 3.066 54.85

Sample 24 0.38 0.297 -21.84

Sample 25 0.88 1.052 19.55

Average bias (%) 39.59

Bias (%) = (ED-LLE-LC-MS/MS result - Centaur result) / Centaur result, expressed as a percentage.

Inter-assay Comparison of Centaur to LC-MS/MS:

Cross-validation of free thyroxine Centaur analysis to ED-LLE-LC-MS/MS yielded an average bias of 39.59% for samples within the analytical range. Comparison of data throughout the range generated a slope of 1.8145 with a correlation coefficient of 0.8923 (FIG. 27).

Reference Interval A. Reference Range Sample Groups

Reference range transfer for free thyroxine was evaluated using NCCLS guidance (see references). Transfer of the reference range was established using the samples listed below.

Children samples will include 25 boys <10 years old and 25 girls <9 years old. B. Reference Interval of Patient Test Results

Reference Intervals

Ran e n /dL

C. Reference Range Transfer

Guidance provided by NCCLS allows reference range transfer where 2 out of 20 (10%) of samples fall outside the original reference range. It was found that all normal adult female samples were within range for reference range transfer of FT4. Adult female reference ranges are transferable. All normal adult male samples were within range for reference range transfer of FT4. Adult male reference ranges are transferable. There was one pre-pubertal reference sample that was outside the normal reference range, and another sample that was potentially out of range. Both samples initially produced high results and upon repeat one sample was still high outside of normal range, while the other was within normal range. The reference sample that repeated within range was not run a third time, which would be in accordance with sample repeat requirements, due to the fact that the pre-pubertal reference range passes with or without this sample being in the normal reference range. A total of 48 out of 50 pre-pubertal reference samples were within range for reference range transfer of FT4. Pre-pubertal reference ranges are transferable. Thus, the free thyroxine reference range rransfer acceptance criteria were met. Free Thyroxine Adult Reference Range Verification

Concentration (ng/dL) Concentration (ng/dL)

Adults FT4 Result Adults FT4 Result

Female 1 1.039 Male 1 1.474

Female 2 1.074 Male 2 1.566

Female 3 1.311 Male 3 1.631

Female 4 1.383 Male 4 1.210

Female 5 0.977 Male 5 1.520

Female 6 1.120 Male 6 1.226

Female 7 1.501 Male 7 1.417

Female 8 1.010 Male 8 1.432

Female 9 1.236 Male 9 1.538

Female 10 1.277 Male 10 1.430

Female 11 1.263 Male 1 1 1.292

Female 12 .317 Male 12 1.153

Female 13 .452 Male 13 1.268

Female 14 ( ).983 Male 14 .107

Female 15 .432 Male 15 .528

Female 16 .270 Male 16 .479

Female 17 .005 Male 17 .338

Female 18 .071 Male 18 .411

Female 19 .132 Male 19 .470

Female 20 1.414 Male 20 .597

Female 21 1.405 Male 21 1.660

Female 22 1.355 Male 22 1.312

Female 23 1.767 Male 23 1.150

Female 24 1.086 Male 24 1.188

Female 25 1.566 Male 25 1.780

Male and Female samples are from an in house draw and are considered to be a healthy reference population. Free thyroxine adult reference range is 0.8 to 2.3ng/dL.

Free Thyroxine Pre-pubertal Reference Range Verification Concentration (ng/dL) Concentration (ng/dL)

Children FT4 Result Children FT4 Result

Female 1 1.848 Male 1 1.845 Female 2 1.543 Male 2 1.740 Female 3 1.561 Male 3 1.569 Female 4 1.604 Male 4 1.753 Female 5 1.974 Male 5 1.849 Female 6 1.826 Male 6 2.090 Female 7 1.485 Male 7 1.624 Female 8 1.846 Male 8 1.704 Female 9 1.528 Male 9 2.047 Female 10 1.612 Male 10 2.754 RPT=2.474 Female 1 1 1.800 Male 1 1 1.765 Female 12 1.585 Male 12 2.54 RPT=I.799 Female 13 1.812 Male 13 2.167 Female 14 1.801 Male 14 .304 Female 15 1.471 Male 15 .656 Female 16 1.853 Male 16 .860 Female 17 1.617 Male 17 .063 Female 18 1.443 Male 18 .536 Female 19 1.619 Male 19 .494 Female 20 1.553 Male 20 .875 Female 21 1.827 Male 21 1.629 Female 22 1.956 Male 22 1.966 Female 23 1.549 Male 23 1.583 Female 24 1.820 Male 24 1.508 Female 25 1.886 Male 25 1.902

Pre-pubertal children samples were previously tested in Allergy screens and assumed as normal. Children ranging from ages 2-8 years for both males and females were selected. Free thyroxine pre-pubertal reference range is 0.8 to 2.2ng/dL RPT is the repeat value for the sample after being run a second time for verification of initial high result.

Standard Curve Fitting and Reproducibility

The reproducibility of the standard curve was evaluated by comparing the back- calculated concentrations to the theoretical concentration of the standard in 5 analytical runs. The calibrator concentrations for free thyroxine (ng/dL) were as follows: 0.1, 0.2, 0.5, 1.0,

2.5, 5.0, and 10.0.

The reproducibility of the standard curve was evaluated, using standards 2-7, by comparing the back-calculated concentrations to the actual concentration of the standard in five analytical runs. The curve was fit with a straight line with weighted 1/x fit, as established during method development. Free thyroxine calibration curves exhibited mean imprecision <15% for all concentrations between 0.2ng/dL and 10.0ng/dL. Correlation coefficients were greater than 0.98. Analytical Reportable Range

A. LLOQ (Lower Limit of Quantification)

The lower limit of quantification for free thyroxine using this assay was 0.2ng/dL as determined during evaluation of inaccuracy, imprecision and calibration curve reproducibility.

B. ULOQ (Upper Limit of Quantification) The upper limit of quantification using this assay was lO.Ong/dL for free thyroxine, as determined during evaluation of inaccuracy, imprecision and calibration curve reproducibility.

Dye Marker Analysis A. Dye Marker Correlation

Blue dextran was used as a visual dye marker to indicate membrane leakage during dialysis. Various concentrations of blue dextran (18mg/mL, 9mg/mL, and 4.5mg/mL in water) were added to the sample side of the dialysis plate, and the results were compared to the same samples that were run with no dye added to see if the dye had any effect on dialysis ofFT4

All concentrations of blue dextran tested correlated to the results obtained when no dye was used. The highest concentration of blue dextran tested, 18.0mg/mL, will be used as an indicator of membrane leakage during the dialysis step. This concentration of blue dextran was easily noted when membrane leakage occurred.

Free Thyroxine Standard Curve Fitting and Reproducibility

Std 2 Std 3 Std 4 Std 5 Std 6 Std 7

Actual Concentration (ng/dL)

Batch 0.200 0.500 1.000 2.500 5.000 10.000

0.231 0.489 0.960 2.51 1 4.824 - 10.642

1 0.190 0.480 0.989 2.536 5.023 9.524

0.181 0.518 0.976 2.501 4.728 10.478

2 0.190 0.516 1.106 2.623 4.881 9.701

0.484* 0.525 0.999 2.451 4.556 10.816

3 0.227 0.473 0.938 2.432 5.070 9.713

0.188 0.509 1.030 2.565 4.936 10.796

4 2.1 13* 0.498 0.980 2.580 5.007 9.1 1 1

0.207 0.508 0.992 2.531 5.061 9.878

5 0.184 0.549 0.923 2.412 5.228 9.927

Mean 0.200 0.507 0.989 2.514 4.931 10.059

Accuracy (%RE) -0.12 1.30 -1.07 0.57 -1.37 0.59 Precision (%RSD) 9.83 4.46 5.17 2.68 3.91 5.85

8 10 10 10 10 10

* Outliers excluded due to preparative error

Free Thyroxine Standard Curve Fitting and Reproducibility Continued

Batch K₀ (Y-intercept) K i (slope) Correlation Coefficient

1 0.0489 0.2840 0.9987

2 0.0457 0.2980 0.9988

3 0.0344 0.3070 0.9978

4 0.0268 0.31 10 0.9974

5 0.0408 0.2880 0.9995

Mean 0.0393 0.2976 0.9984 Precision (%RSD) NA 3.92 0.08 n 5 5 5

NA = Not Applicable.

Data calculated using Standards 2-7. Free Thyroxine Dye Marker Correlation

Concentration (ng/dL)

Blue Dextran Patient

QCl QC2 QC3 QC4 Concentration 3

Omg/mL Dye 1.23 1.90 2.49 7.00 0.93 Omg/mL Dye 1.09 1.75 2.71 7.06 N/A

Average

1.16 1.83 2.60 7.03 0.93 Concentration

4.5mg/mL Dye 1.11 1.87 2.53 7.71 0.94 4.5mg/mL Dye 1.14 1.59 2.69 7.53 0.88 Average

Average Bias (%)

1.13 1.73 2.61 7.62 0.91 Concentration Bias (%) -3.02 -5.23 0.33 8.45 -1.51 -0.20

9.0m g/mL Dye 1.14 1.70 2.69 7.09 0.94 9.0mg/mL Dye 1.06 1.70 2.74 6.55 0.99 Average

Average Bias (%)

1.10 1.70 2.72 6.82 0.97 Concentration Bias (%) -5.39 -6.90 4.48 -2.96 4.37 -1.28

18.0mg/mL Dye 1.14 1.90 2.79 6.95 0.99 18.0mg/mL Dye 1.16 1.58 2.85 7.45 1.02 Average

Average Bias (%)

1.15 1.74 7.20 1.00 Concentration 2.82 Bias (%) -0.95 -4.52 8.42 2.50 8.48 2.79

N/A = Not Applicable; only I duplicate run. Bias (%) = (Average concentration dye added - Average concentration no dye added) / Average concentration no dye added, expressed as a percentage.

Example 3

For dialysis of samples to remove free thyroxine or other free hormones from hormones that are bound to proteins in the sample, a 200 microliter (μL) sample and 10 μL of an 18 mg/mL dextran blue are added to one side of a 5 or 10 kilodalton molecular weight cut- off cellulose dialysis membrane in a 96-well equilibrium dialysis plate and capped. Then, 300 microliters of a dialyzing buffer (described in Example 2) is added to the other side of the plate and the wells are capped. The plate is placed vertically within a temperature controlled (37⁰C) rotating oven and rotated at 15 cycles per minute for 16 hours. The 96-well plate may then be removed from the rotating oven and the dialysate buffer side is uncapped. A sample of 200 μL may be removed for processing by either liquid liquid extraction using the isotope dilution LC-MS/MS method.

In some cases, a liquid extraction step is performed after the dialysis to remove residual salts and/or other additives which are used in the dialysis solution and/or remain from the sample, but that may interfere with the MS/MS analysis. The dialysate is extracted with 71.25:23.75:5 ethyl acetate:hexane:methanol. Alternatively, the dialysate is diluted with a solution of 1:1 methanol:water containing stable labeled iunternal standard and directly injected onto the LC-MS/MS system for analysis.

Example 4: 2D-LC-MS/MS Analysis for 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 25-Hydroxyvitamin D3 (Native) and 25-Hydroxyvitamin D2 (Supplemented) analysis was validated to 1 ng/mL using 200-μJL of sample. Optimum analytical specificity and sensitivity was generated using 2D LC using gradient separations in both LC dimensions, heart-cutting and chromato-focusing prior to MS/MS detection.

Abbreviations for Examples

ALQ: Above limit of quantification; BLQ: Below limit of quantification; CAP: College of American Pathologists; CLIA: Clinical Laboratory Improvement Act; CPS: Counts per scan;

CV: Coefficient of variance; ED: Equilibrium Dialysis; EDTA: Ethylenediamminotetraacetic acid; FT4: Free Thyroxine; IE: Injection Error; IS: Internal Standard; LC: Liquid

Chromatography; LLE: Liquid-Liquid Extraction; LLOQ: Lower Limit of Quantitation;

MARR: Mean average response ratio; MS/MS: Tandem MS/MS detection; N: Number of replicates; NA: Not Applicable; QC: Quality Control; R: Correlation Coefficient; RIA:

Radioimmunoassay; SD: Standard Deviation; ULOQ: Upper Limit of Quantitation

All documents referred to in this specification are herein incorporated by reference.

Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

THAT WHICH IS CLAIMED:

1. A method for determining the presence or amount of estradiol in a sample by tandem mass spectrometry, comprising: (a) generating a dehydrated precursor ion of the estradiol; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the presence or amount of one or more of the ions generated in step (a) or (b) or both, and relating the detected ions to the presence or amount of the estradiol in the sample.

2. The method of claim 1, wherein the sample is subjected to a purification step prior to ionization.

3. The method of claim 2, wherein the purification step comprises chromatography.

4. The method of claim 3, wherein the chromatography comprises high performance liquid chromatography (HPLC).

5. The method of claim 2, wherein the chromatography comprises extraction and analytical liquid chromatography.

6. The method of claim 1, further comprising at least one of liquid-liquid extraction of the sample or dilution of the sample prior to mass spectrometry.

7. The method of claim 1, wherein the precursor ion has a mass/charge ratio (m/z) of about 255.2 and the fragment ions comprise ions having a mass/charge ratio (m/z) of about

159.0 and 133.0.

8. The method of claim 1, wherein the sample further comprises estrone.

9. The method of claim 4, wherein the sample is heated at the MS/MS interface.

10. The method of claim 1, further comprising detection of estradiol over a range of from about 1 pg/ml to about 500 pg/mL.

11. The method of claim 9, further comprising detection of estrone over a range of from about 2.5 pg/mL to about 500 pg/mL.

12. A method for determining the presence or amount of free thyroxine in a plurality of samples by tandem mass spectrometry, comprising: (a) dialyzing the plurality of samples to separate the free thyroxine from the protein-bound thyroxine in the samples; (b) generating a precursor ion of the thyroxine in each sample; (b) generating one or more fragment ions of the thyroxine in each sample; and (c) detecting the presence or amount of one or more of the ions generated in step (b) or (c) or both, and relating the detected ions in each sample to the presence or amount of the free thyroxine in the plurality of samples.

13. The method of claim 12, wherein the samples are subjected to a purification step prior to ionization.

14. The method of claim 13, wherein the purification step comprises chromatography.

15. The method of claim 14, wherein the chromatography comprises high performance liquid chromatography (HPLC).

16. The method of claim 12, further comprising at least one of of liquid-liquid extraction or dilution of the plurality of samples prior to mass spectrometry.

17. The method of claim 12, further comprising detection of thyroxine over a range of from about 2.0 pg/mL to about 100 pg/mL.

18. The method of claim 12, wherein the dialysing step comprises the use of a buffer, and wherein the buffer comprises and sufficient salts such that the buffer is isotonic.

19. A method for determining the presence or amount of at least one biomarker of interest in a biological sample, the method comprising: providing a biological sample believed to contain at least one biomarker of interest; chromatographically separating the at least one biomarker of interest from other components in the sample; and analyzing the chromatographically separated at least one biomarker of interest by mass spectrometry to determine the presence or amount of the at least one biomarker of interest in the sample.

20. The method of claim 19, further comprising partially purifying the biomarker of interest prior to chromatography.

21. The method of claim 19, wherein the at least one biomarker comprises at least one of steroid hormone or a thyroid hormone.

22. The method of claim 19, wherein the at least one biomarker comprises at least one of estradiol and estrone.

23. The method of claim 22, wherein the step of mass spectrometry comprises: (a) generating a dehydrated precursor ion of the estradiol; (b) generating one or more fragment ions of the precursor ion; and (c) detecting the presence or amount of one or more of the ions generated in step (a) or (b) or both, and relating the detected ions to the presence or amount of the estradiol in the sample.

24. The method of claim 23, wherein the precursor ion has a mass/charge ratio (m/z) of about 255.2.

25. The method of claim 23, wherein the fragment ions comprise ions having a mass/charge ratio (m/z) of about 159.0 and 133.0.

26. The method of claim 23, further comprising detection of estradiol over a range of from about 1 pg/ml to about 500 pg/mL.

27. The method of claim 23, further comprising detection of estrone over a range of from about 2.5 pg/mL to about 500 pg/mL.

28. The method of claim 19, wherein at least one biomarker comprises free thyroxine.

29. The method of claim 28, wherein the method comprises dialyzing a plurality of samples to separate the free thyroxine from the protein-bound thyroxine in the samples and isolating the portion of the samples comprising the majority of the free thyroxine prior to chromatographically separating the free thyroxine from other components extracted from the pluralty of samples.

30. The method of claim 29, further comprising detection of thyroxine over a range of from about 2.0 pg/mL to about 100 pg/mL.

31. The method of claim 29, wherein the dialysing step comprises the use of a buffer, and wherein the buffer comprises and sufficient salts such that the buffer is isotonic.

32. The method of claim 19, wherein the biological sample comprises blood, serum, plasma, urine, or saliva.

33. The method of claim 19, wherein the chromatography comprises high performance liquid chromatography (HPLC).

34. The method of claim 19, wherein the chromatography comprises extraction and analytical liquid chromatography.

35. The method of claim 20, wherein the partial purification comprises liquid-liquid extraction.

36. The method of claim 19, further comprising diluting the sample into a solvent used for liquid chromatography or mass spectrometry.

37. The method of claim 19, wherein the tandem MS/MS spectrometer is operated in an atmospheric pressure chemical ionization (APCI) mode.

38. A system for determining the presence or amount of at least one biomarker of interest in a test sample, the system comprising: a station for providing a test sample suspected of containing one or more hormones or metabolites; a station for partially purifying the at least one biomarker of interest from other components in the sample; a station for chromatographically separating the at least one biomarker of interest from other components in the sample; and a station for analyzing the chromatographically separated one or more hormones or metabolites by mass spectrometry to determine the presence or amount of the one or more hormones or metabolites in the test sample.

39. The system of claim 38, further comprising a station for dialyzing a plurality of samples as a means to separate the hormone or metabolite from protein-bound hormone or metabolite.

40. The system of claim 38, wherein at least one of the stations is controlled by a computer.