BRPI0920028B1 - Process for determining the amount of non-derivatized dihydrotestosterone (DHT) in a sample - Google Patents

Description

(54) Title: PROCESS FOR DETERMINING THE QUANTITY OF NON-DERIVATIZED DIHYDROTESTOSTERONE (DHT) IN A SAMPLE (51) lnt.CI .: C12Q 1/02 (30) Unionist Priority: 10/06/2008 US 61 / 103,202,17 / 11/2008 US 12 / 272,663 (73) Holder (s): QUEST DIAGNOSTICS INVESTMENTS LLC (72) Inventor (s): AMIT GHOSHAL; NIGEL J. CLARKE; RICHARD REITZ (85) National Phase Start Date: 05/04/2011

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Invention Patent Descriptive Report for PROCESS

FOR DETERMINING THE AMOUNT OF NON-DERIVATIZED DHHYDROTESTOSTERONE (DHT) IN A

SAMPLE.

CROSS REFERENCE TO RELATED ORDERS

This patent application claims priority to US Patent Application No. 12 / 272,663, filed on November 17, 2008 and claims the benefit of US Provisional Patent Application No. 61 / 103,202 filed on October 6, 2008, of which each one is incorporated here as a reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the detection of dihydrotestosterone (DHT). In a particular aspect, the invention relates to methods for detecting dihydrotestosterone (DHT) by mass spectrometry. BACKGROUND OF THE INVENTION

The following description of the background of the invention is provided simply as an aid to understanding the invention and it is not permitted to describe or constitute the art prior to the invention.

Dihydrotestosterone (DHT) [(17B-hydroxy-5a-androstan-3-one)] is a steroid hormone with a molecular weight of 290.4 Daltons. DHT is a potent androgen synthesized by peripheral tissues from testosterone. Excessive DHT secretion can produce acne, hirsutism and virilization through conversion to testosterone. DHT is a causative agent in prostate hyperplasia and blood measurements can be used to assess compatibility and response to testosterone inhibitors for conversion to DHT.

Mass spectrometry methods for measuring DHT in a sample have been reported. See, for example, Chang, Y. and others, Analyst 2003,128: 363-8; Caruso, D. et al., Neurochem Int 2008, 52: 560-8; Wang, C. et al., Steroids 2008, XXX: XXX-XXX (doi: 10.1016 / j.steroids.2008.05.004); Zhao, M. et al., Steroids 2004, 69: 721-6; Janzen, N. et al., J Chroma B 2008, 861: 117-22; Licea-Perez, H. et al., Steroids 2008, 73: 601-10; Kashiwagi, B. et al., JAndrology 2005, 26: 586-91; Kashiwagi, B. and others,

06/12/2017, p. 8/31

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Urology 2005, 66: 218-23; Umera, M. et al., Cancer Sei 2007, 99: 81-86; and

Mohler et al., U.S. Patent Application No. 11 / 973,127 (filed on October 8, 2007).

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount of dihydrotestosterone (DHT) in a sample by mass spectrometry, including tandem mass spectrometry. Preferably, the methods of the invention do not include derivatization of DHT in a sample prior to analysis by mass spectrometry.

In one aspect, methods are provided for determining the amount of non-derivatized dihydrotestosterone (DHT) in a body fluid sample. Methods of this aspect include: (a) the purification of DHT in a sample of body fluid by solid phase extraction; (b) the ionization of DHT from the body fluid sample to produce one or more DHT ions detectable by mass spectrometry, in which the ions produced are selected from the group consisting of a precursor DHT ion with a mass ratio in relation to the charge of 291.10 ± 0.50 and one or more ions of DHT fragments selected from the group consisting of 255.20 ± 0.50 and 79.20 ± 0.50; and (c) detecting the amount of one or more DHT ions by mass spectrometry. Once the amount of one or more DHT ions is measured, the amount of DHT ion (s) is used to calculate the amount of non-derivatized DHT in the test sample. In some embodiments, mass spectrometry is tandem mass spectrometry. In some modalities, solid phase extraction and mass spectrometry analysis are performed online. In some modalities, solid phase extraction is performed in the form of high-turbulence liquid chromatography (HTLC). In some embodiments, the methods further include the purification of DHT in a body fluid sample before mass spectrometry with high performance liquid chromatography (HPLC); preferably with online processing. In some embodiments, the sample of body fluid is plasma or serum. In some modalities, the methods have

3/32 a limit of quantification within the range of 5 ng / dL up to and including 200 ng / dL. In some embodiments, the amount of one or more DHT ions detected by mass spectrometry is used to calculate the amount of non-derivatized DHT in a test sample by comparison with an internal standard; preferably 16, 16, 17-d ₃ dihydrotestosterone. The characteristics of the modalities listed above can be combined without limitation for use in the methods of the present invention.

In a second aspect, methods are provided for determining the amount of non-derivatized dihydrotestosterone (DHT) in a test sample by mass spectrometry. Methods of this aspect include: (a) the purification of DHT in a test sample using high-performance liquid chromatography (HTLC); (b) ionizing DHT from a test sample to produce one or more DHT ions detectable by mass spectrometry; and (c) detecting the amount of one or more DHT ions by mass spectrometry. In these methods, the amount of the measured DHT ion (s) is used to calculate the amount of DHT in the test sample. In some embodiments, mass spectrometry is tandem mass spectrometry. In some embodiments, the purification of DHT in a test sample comprises purification with high performance liquid chromatography (HPLC); preferably configured for online processing. In some embodiments, the test sample is a sample of body fluid; preferably plasma or serum. In some embodiments, DHT ions detectable by mass spectrometry include one or more ions selected from the group consisting of ions with a mass / charge ratio of 291.10 ± 0.50, 255.20 ± 0.50 and 79 , 20 ± 0.50. In some embodiments, the DHT ionization step includes the production of a precursor ion with a mass / charge ratio of 291.10 ± 0.50 and the production of one or more ions from fragments selected from the group consisting of ions with a mass / charge ratio of 255.20 ± 0.50 and 79.20 ± 0.50. In some modalities, the methods have a limit of quantification within the range of 5.0 ng / dL to 200 ng / dL, inclusive. In some embodiments, the amount of one or more ions of

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DHT detected by mass spectrometry is used to determine the amount of non-derivatized DHT in a test sample by comparison with an internal standard; preferably 16, 16, 17-d ₃ dihydrotestosterone. The characteristics of the modalities listed above can be combined without limitation for use in the methods of the present invention.

In a third aspect, methods are provided for determining the amount of non-derivatized dihydrotestosterone (DHT) in a sample of body fluid by tandem mass spectrometry. Methods of this aspect include: (a) the purification of DHT from a sample of body fluid by high-performance liquid chromatography (HTLC); (b) the production of a precursor ion of said DHT that has a mass / charge ratio of 291.10 ± 0.50; (c) the production of one or more fragment ions of a precursor ion, wherein at least one of said one or more fragment ions comprises a fragment ion selected from the group of fragment ions that have a mass / charge ratio 255.20 ± 0.50 and 79.20 ± 0.50; and (d) detecting the amount of one or more of said ions generated in step (b) or (c) or both. The amount of ions detected is used to calculate the amount of non-derivatized DHT in a sample of body fluid. In some embodiments, DHT purification from a test sample also includes high performance liquid chromatography (HPLC); preferably configured for online processing. In some embodiments, the sample of body fluid is plasma or serum. In some modalities, the methods have a limit of quantification within the range of 5.0 ng / dL to 200 ng / dL, inclusive. In some embodiments, the amount of one or more DHT ions detected by mass spectrometry is used to calculate the amount of non-derivatized DHT in the test sample by comparison with an internal standard; preferably 16, 16, 17-d ₃ dihydrotestosterone. The characteristics of the modalities listed above can be combined without limitation for use in the methods of the present invention.

The methods of the present invention involve the combination of

5/32 liquid chromatography with mass spectrometry. In preferred embodiments, liquid chromatography is HPLC. A preferred embodiment uses HPLC alone or in combination with one or more purification methods such as, for example, HTLC and / or protein precipitation and filtration, to purify DHT in samples. In another preferred embodiment, mass spectrometry is tandem mass spectrometry (MS / MS).

In certain preferred embodiments of the methods disclosed herein, mass spectrometry is performed in the mode of positive ions. Alternatively, mass spectrometry is performed in the negative ion mode. Various sources of ionization, including, for example, atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) can be used in the embodiments of the present invention. In certain preferred embodiments, DHT is measured using APCI in the positive ion mode.

In preferred embodiments, the DHT ions detectable in a mass spectrometer are selected from the group consisting of positive ions with a mass / charge ratio (m / z) of 291.10 ± 0.50, 255.20 ± 0, 50 and 79.20 ± 0.50. In particularly preferred embodiments, a DHT precursor ion has a m / z of 291.10 ± 0.50 and one or more fragment ions are selected from the group consisting of ions having a m / z of 255.20 ± 0.50 and 79.20 ± 0.50.

In preferred embodiments, an internal standard detectable separately in the sample is provided, the amount of which is also determined in the sample. In these modalities, all or a part of both endogenous DHT and the internal standard present in the sample is ionized to produce a large number of ions detectable in a mass spectrometer and one or more ions produced from each one are detected by mass spectrometry.

A preferred internal standard is 16, 16, 17-d3 dihydrotestosterone (16, 16, 17-d ₃ DHT). In preferred embodiments, the detectable ions of the internal standard in a mass spectrometer are selected from the group consisting of positive ions with m / z of 294.10 ± 0.50 and 258.20 ± 0.50. In particularly preferred embodiments, a precursor ion of the internal standard

6/32 has m / z of 294.10 ± 0.50; and an internal pattern fragment ion has a m / z of 258.20 ± 0.50.

In preferred embodiments, the presence or quantity of the DHT ion is related to the presence or quantity of DHT in a test sample by comparison with a reference such as 16, 16, 17-d ₃ dihydrotestosterone.

In certain preferred embodiments, the DHT limit of quantification (LOQ) is within the range of 5.0 ng / dL to 200 ng / dL, inclusive; preferably within the range of 5.0 ng / dL to 100 ng / dL, inclusive; preferably within the range of 5.0 ng / dL to 50 ng / dL, inclusive; preferably within the range of 5.0 ng / dL to 25 ng / dL, inclusive; preferably within the range of 5.0 ng / dL to 15 ng / dL, inclusive; preferably within the range of 5.0 ng / dL to 10 ng / dL, inclusive; preferably approximately 5.0 ng / dL.

As used here, unless otherwise stated, the singular forms one, one, o and a include reference in the plural. Thus, for example, a reference to a protein includes a large number of protein molecules.

As used here, derivatization means the reaction of two molecules to form a new molecule. Derivatization an androgen molecule, such as a DHT molecule, can be performed with various derivatization reagents well known in the art. See, for example, Kashiwagi, B. et al., J Andrology 2005, 26: 586-91 and Kashiwagi, B. et al., Urology 2005, 66: 218-23, which report the derivatization of DHT with fluoro-1 sulfonate. -methylpyridinium-P-tolulene before extraction. As used here, non-derivatized means not derivatized. Thus, dihydrotestosterone (DHT), without indication of derivatization, is non-derivatized DHT.

As used here, the term purification or purifying does not refer to the removal of all materials from the sample other than the analyte (s) of interest. Instead, purification refers to a procedure that enriches the amount of one or more analytes of interest in relation to

7/32 other components in the sample that may interfere with the detection of the analyte of interest. Purification of the sample by various means may allow the relative reduction of one or more interfering substances, for example, one or more substances that may or may not interfere with the detection of the parental or child ions of DHT selected by mass spectrometry. The relative reduction in how this term is used does not require that any substance, present with the analyte of interest in the material to be purified, be removed entirely by purification.

As used here, the term test sample refers to any sample that may contain DHT. As used herein, the term body fluid means any fluid that can be isolated from an individual's body. For example, body fluid can include blood, plasma, serum, bile, saliva, urine, tears, sweat and the like.

As used here, the term chromatography refers to a process in which a chemical mixture carried by a liquid or a gas is separated into components as a result of the differential distribution of chemical entities as they flow around or through a liquid phase stationary or solid.

As used here, the term liquid chromatography or LC means a process of selective delay of one or more components of a fluid solution as the fluid undergoes uniform percolation through a column of a finely divided substance or through capillary passages. The delay results from the distribution of the components of the mixture between one or more stationary phases and the mass fluid, (i.e., mobile phase), as the fluid moves in relation to the stationary phase (s) . Examples of liquid chromatography include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and high turbulence liquid chromatography (HTLC).

As used here, the term high performance liquid chromatography or HPLC refers to liquid chromatography in which the degree of separation is greatest by forcing the mobile phase under pressure along a stationary phase, typically a densely packed column.

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As used here, the term high-turbulence liquid chromatography or HTLC refers to a form of chromatography that uses the turbulent flow of material that will be analyzed through the compacted column as the basis for carrying out the separation. HTLC was applied in the preparation of samples containing two non-demonized drugs before analysis by mass spectrometry. See, for example, Zimmer et al., J Chromatogr A 854: 23-35 (1999); see also, U.S. Patent Nos. 5,968,367, 5,919,368, 5,795,469 and 5,772,874, which further explain HTLC. Those of ordinary skill in the art understand turbulent flow. When the fluid flows slowly and smoothly, the flow is called a laminar flow. For example, the fluid that moves along an HPLC column at low flow rates is laminar. In the laminar flow, the movement of the fluid particles is ordered with the particles that generally move in straight lines. At higher speeds, the inertia of the water overcomes the frictional forces of the fluid and results in turbulent flow. The fluid that is not in contact with the irregular limit exceeds that which is delayed by friction or deflected by an irregular surface. When a fluid flows in a turbulent form, it flows in swirls and fast turns (or vortices), with more drag than when the flow is laminar. Many references are available to assist in determining when the flow is laminar or turbulent (for example, Turbulent Flow Analysis: Measurement and Prediction, PS Bernard & JM Wallace, John Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

As used here, the term gas chromatography or GC refers to chromatography in which the sample mixture is vaporized and injected into a carrier gas stream (such as nitrogen or helium) that moves along a column that contains a phase stationary compound of a liquid or a particulate solid and is separated into its component compounds according to the affinity of the compounds for the stationary phase.

As used here, the term large particle column or extraction column refers to a column for chromatography that contains

9/32 an average particle diameter greater than approximately 50 pm. As used in this context, the term approximately means ± 10%.

As used here, the term analytical column refers to a column for chromatography that has sufficient chromatographic plates to perform a separation of materials in a sample that are eluted from the column sufficient to permit a determination of the presence or quantity of an analyte. Such columns are often distinguished from extraction columns, which have the general purpose of separating or extracting the retained material from the non-retained materials in order to obtain a purified sample for further analysis. As used in this context, the term approximately means ± 10%. In a preferred embodiment the analytical column contains particles approximately 4 pm in diameter.

As used here, the term online or online, for example, as used in an automated online way or online extraction refers to a procedure performed without the need for operator intervention. In contrast, the term disconnected as used here refers to a procedure that requires manual intervention by an operator. Thus, if the samples are subjected to precipitation and the supernatants are then loaded manually into an automatic injector, the precipitation and loading steps are disconnected from the subsequent steps. In several methods, one or more steps can be performed in an automated way online.

As used here, the term mass spectrometry or MS refers to an analytical technique for identifying compounds by their mass. MS refers to methods of filtration, detection and measurement of ions based on their ratio of mass to charge or m / z. MS technology generally includes (1) ionizing compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a ratio of mass to charge. The compounds can be ionized and detected by any suitable means. A mass spectrometer usually includes an ionizer and a detector

10/32 ions. In general, one or more molecules of interest are ionized and the ions are subsequently introduced into a mass spectrographic instrument in which, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent on the mass (m ) and the load (z). See, for example, U.S. Patent Nos. 6,204,500, entitled Mass Spectrometry From Surfaces; 6,107,623, entitled Methods and Apparatus for Tandem Mass Spectrometry: 6,268,144, entitled DNA Diagnostics Based On Mass Spectrometry; 6,124,137, entitled Surface-Enhanced Photolabile Attachment and Release For Desorption and Detection Of Analytes; Wright et al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; and Merchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

As used here, the term operating in the mode of negative ions refers to mass spectrometry methods in which negative ions are generated and detected. The term operating in the mode of positive ions as used here, refers to methods of mass spectrometry in which positive ions are generated and detected.

As used here, the term ionization or ionizing refers to the process of producing an analyte ion that has a net electrical charge equal to one or more electron units. Negative ions are those that have a net negative charge of one or more electron units, while positive ions are those that have a net positive charge of one or more electron units.

As used here, the term electron ionization or E1 refers to methods in which an analyte of interest in a gas or vapor phase interacts with an electron flow. The impact of electrons on the analyte produces ions from the analyte, which can then be subjected to a mass spectrometry technique.

As used here, the term chemical ionization or Cl refers to methods in which a reactive gas (eg, ammonia) is subjected to electron impact and the analyte ions are formed by the interaction of the reactant gas ions and the analyte molecules.

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As used here, the term rapid atom bombardment or FAB refers to methods in which a beam of high-energy atoms (often Xe or Ar) is impacted on a non-volatile sample, removing a substance from the absorber and ionizing the molecules contained in the sample. The test samples are dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol; 18-crown-6 crown ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine and triethanolamine. Choosing an appropriate matrix for a compound or sample is an empirical process.

As used here, the term ionization with matrix-assisted laser desorption or MALDI refers to methods in which a non-volatile sample is exposed to laser irradiation, which removes analyte from the absorbent and ionizes analytes in the sample through various ionization pathways, including photoionization, protonation, deprotonation and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates the desorption of the analyte molecules.

As used here, the term surface-enhanced laser desorption ionization or SELDI refers to another method in which a non-volatile sample is exposed to laser irradiation, which removes the analytes in the sample from the absorber and ionizes the sample through various pathways. ionization, including photoionization, protonation, deprotonation and decay of clusters. For SELDI, the sample is typically attached to a surface that preferably holds one or more analytes of interest. As with MALDI, this process can also employ an energy-absorbing material to facilitate ionization.

As used here, the term electrospray ionization or ESl, refers to methods in which a solution is passed along a short length of a capillary tube, until the end where a high positive or negative electrical potential is applied. The solution that reaches the end of the tube is vaporized (nebulized) in a jet or a spray of very small droplets of solution in the solvent vapor. This mist of droplets flows through an evaporation chamber, which is heated slightly to prevent condensation and evaporate the solvent. As the droplets get smaller, the density of the electrical surface charge increases until such time that the natural repulsion between similar charges causes the ions as well as the neutral molecules to be released.

As used here, the term chemical ionization with atmospheric pressure, or APCI, refers to methods of mass spectrometry that are similar to ESI; however, APCI produces ions through ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electrical discharge between the spray capillary and a counter electrode. Then the ions are typically extracted into the mass analyzer through the use of a set of differently pumped skimmer stages. A counterflow of dry, preheated N ₂ gas can be used to increase solvent removal. Ionization of the gas phase in APCI may be more efficient than in ESI for the analysis of polar species.

The term atmospheric pressure photoionization or APPI as used here refers to the form of mass spectrometry in which the mechanism for photoionization of the M molecule is the absorption of photons and the expulsion of electrons to form the molecular ion M +. Due to the fact that photon energy is typically just above the ionization potential, the molecular ion is less susceptible to dissociation. In many cases it may be possible to analyze the samples without the need for chromatography, thereby saving significant time and costs. 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 affinity for protons. This does not affect the accuracy of the quantification because the sum of M + and MH + is constant. Drug compounds in protic solvents are generally seen in the form of MH +, while non-polar compounds such as naphthalene or testosterone generally form M +. See, for example, Robb et al., Anal. Chem. 2000, 72 (15): 3653-3659.

As used herein, the term induced coupled plasma or ICP refers to methods in which a sample interacts with a gas

13/32 partially ionized at a sufficiently high temperature so that most elements are atomized and ionized.

As used here, the term field desorption 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 ions from the analyte.

As used here, the term desorption refers to the removal of an analyte from a surface and / or the entry of an analyte into a gas phase. Thermal desorption with laser desorption is a technique in which a sample containing the analyte is removed from the absorbent thermally into the gas phase by a laser pulse. The laser strikes the back of a 96-well plate specially produced with a metal base. The laser pulse heats the base and heating causes the sample to be transferred into the gas phase. The sample in the gas phase is then pulled into the mass spectrometer.

As used here, the term selective ion monitoring is a detection mode for a mass spectrometric instrument in which only ions within a relatively limited mass range, typically approximately one unit of mass, are detected.

As used here, multiple reaction mode, sometimes known as selected reaction monitoring, is a detection mode for a mass spectrometric instrument in which a precursor ion and one or more fragment ions are selectively detected.

As used here, the term limit of quantification or LOQ refers to the point at which the measures are quantitatively significant. The analyte response in this LOQ can be identified, separated and reproduced with a relative standard deviation (RSD%) of 20% and an accuracy of 80% to 120%.

As used here, the term limit of detection or LOD is the point at which the measured value is greater than the uncertainty associated with it.

The LOD is the point at which a value is beyond the uncertainty associated with its measurement and is defined as twice the RSD of the mean in the concentration

14/32 zero.

As used here, an amount of DHT in a body fluid sample generally refers to an absolute value that reflects the detectable DHT mass in the body fluid volume. However, an amount also considers a relative amount compared to another amount of DHT. For example, an amount of DHT in a body fluid can be an amount that is greater than a control or a normal level of DHT normally present.

The term approximately as used here in reference to quantitative measurements, not including the measurement of the mass of an ion, refers to the indicated value plus or minus 10%. Mass spectrometry instruments can vary slightly in determining the mass of a certain analyte. The term approximately in the context of the mass of an ion or the mass / charge ratio of an ion refers to +/- 0.50 atomic mass unit.

The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention and the claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a graphical representation of the variation coefficient of the tests for a blank and five standards used to determine the limit of quantification of the DHT test. The details are discussed in Example 5.

Figure 2 shows the linearity of DHT quantification in stock samples diluted in series using an LC-MS / MS assay. Details are described in Example 6.

Figures 3A and 3B show the correlation of DHT determination by an example of the HPLC-MS method of the present invention with DHT determination using a reference radioimmunoassay (RIA) method. The correlation shown in figure 3A was determined by linear regression. The correlation shown in figure 3B was determined by Deming's analysis. Details are described in Example 10.

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DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention are described for measuring the amount of DHT in a sample. More specifically, mass spectrometry methods are described for the detection and quantification of DHT in a test sample. The methods can use high-turbulence liquid chromatography (HTLC) to perform a purification of the selected analytes and combine this purification with mass spectrometry (MS) methods, thus providing a high-throughput test system for the detection and quantification of DHT in a test sample. The preferred embodiments are particularly well suited for application in large clinical laboratories for the automated DHT assay.

Test samples suitable for use in the methods of the present invention include any test sample that may contain the analyte of interest. In some preferred embodiments, a sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, a cell culture, an organ culture, etc. In certain preferred embodiments, samples are obtained from a mammalian animal, such as a dog, cat, horse etc. Particularly preferred mammalian animals are primates, more preferably male or female humans. Particularly preferred samples include body fluids such as blood, plasma, serum, saliva, cerebrospinal fluid or tissue samples. Such samples can be obtained, for example, from a patient; that is, a living person, male or female, who presents himself to a clinical setting for the diagnosis, prognosis or treatment of a disease or health condition. The test sample is preferably obtained from a patient, for example, serum or blood plasma. A sample volume of approximately 0.5 ml is preferred; however, samples of approximately 0.1 mL can be analyzed.

The present invention considers kits for a DHT quantification assay. A kit for a DHT quantification assay from this

The invention may include a kit comprising an internal standard, in quantities sufficient for at least one test. Typically, kits will also include instructions recorded in a tangible form (for example, contained on paper or an electronic medium) for the use of packaged reagents for use in a measurement assay for determining the amount of DHT.

Calibration and QC sets for use in the embodiments of the present invention can be primed using plasma or stripped serum (stripped of DHT): for example, serum stripped with charcoal and double delipidized. All sources of human or non-human stripped plasma or serum should be checked to ensure that they do not contain measurable amounts of DHT.

Sample Preparation for Mass Spectrometry

Test samples can be stored below room temperature. Test samples (including controls) stored below room temperature are first allowed to reach room temperature and are mixed with a mechanical vortex. An internal standard can be added to the test samples at this point.

The samples can then be prepared for mass spectrometry through liquid-liquid or solid phase extraction. Various methods can be used to enrich DHT in relation to other components in the sample (eg protein) before mass spectrometry, including, for example, liquid chromatography, filtration, centrifugation, thin layer chromatography (TLC), electrophoresis including capillary electrophoresis, affinity separations including immunoaffinity separations, extraction methods including extraction with ethyl acetate or methanol extraction and the use of chaotropic agents or any combination of the above or similar.

Protein precipitation is a method of preparing a test sample, especially a biological test sample, such as serum or plasma. Such protein purification methods are well known in the art, for example, Polson et al., Journal of Chromatografy B 2003,

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785: 263-275, describe protein precipitation techniques suitable for use in the methods of the present invention. Protein precipitation can be used to remove most of the protein from the sample leaving DHT in the supernatant. The samples can be centrifuged to separate the liquid supernatant from the precipitated proteins; alternatively the samples can be filtered to remove precipitated proteins. The resulting supernatant or filtrate can then be applied directly to the analysis by mass spectrometry; or alternatively to liquid chromatography and subsequent analysis by mass spectrometry. In certain embodiments, the use of protein precipitation, such as, for example, protein precipitation with formic acid, can avoid the need for HTLC or other online extraction before mass spectrometry or HPLC and mass spectrometry.

Consequently, in some embodiments, protein precipitation, alone or in combination with one or more purification methods, can be used for DHT purification before mass spectrometry. In these modalities, the methods may involve (1) carrying out a protein precipitation of the sample of interest; and (2) loading the supernatant directly onto the LC mass spectrometer without using online extraction or HTLC. Alternatively, the methods may involve (1) performing a protein precipitation of the sample of interest; and (2) loading the supernatant over an HTLC using online extraction for further purification before mass spectrometry.

A sample purification medium that can be used before mass spectrometry is liquid chromatography (LC). Certain liquid chromatography methods, including HPLC, are based on relatively slow laminar flow technology. Traditional HPLC analysis is based on column compaction where the laminar flow of the sample along the column is the basis for separating the analyte of interest from the sample. The person skilled in the art will understand that the separation in such columns is a diffusion process and will be able to select HPLC instruments and columns that are suitable for use with DHT. The chromatographic column typically includes a medium

18/32 (ie, a compaction material) to facilitate the separation of chemical groups (ie, fractionation). The medium can include tiny particles. The particles include a bonded surface that interacts with the various chemical groups to facilitate the separation of the chemical groups. A suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded or cyan bonded surface. Alkyl-bonded surfaces can include C-4, C-8, C-12 or C-18 bonded alkyl groups. In preferred embodiments, the column is a cyan column. The chromatographic column includes an entrance port for receiving a sample directly or indirectly from a solid phase extraction or an HTLC column and an exit port for the discharge of an effluent that includes the fractionated sample.

In one embodiment, the sample can be applied to the LC column at the inlet port, eluted with a solvent or a mixture of solvents and discharged at the outlet port. Different solvent modes can be selected for eluting the analyte (s) of interest. For example, liquid chromatography can be performed using a gradient mode, an isocratic mode or a polytype (i.e., mixed) mode. During chromatography, the separation of materials is carried out through variables such as the choice of eluent (also known as a mobile phase), the mode of elution, gradient conditions, temperature etc.

In certain embodiments, an analyte can be purified by applying a sample to a column under conditions where the analyte of interest is reversibly retained by the column's compaction material, while one or more other materials are not retained. In these embodiments, a first mobile phase condition can be used in which the analyte of interest is retained by the column and a second mobile phase condition can subsequently be used to remove the retained material from the column, once the non-retained materials have been washed . Alternatively, an analyte can be purified by applying a sample to a column under mobile phase conditions in which the analyte of interest is eluted at a differential rate compared to one or more other materials. Such procedures can enrich the quantity of one or more analytes of interest in relation to one or more other components of the sample.

In a preferred embodiment, HPLC is performed with a hydrophobic column chromatographic system. In certain preferred embodiments, a cyan analytical column (for example, a BetaBasic Cyano analytical column from Thermo Scientific, Inc. (particle size 5 pm, 50 x 2.1 mm) or equivalent) is used. In certain preferred embodiments, HTLC and / or HPLC are performed using 0.1% Grade HPLC aqueous formic acid and 100% methanol as the mobile phases.

Through careful selection of connecting valves and piping, two or more chromatography columns can be connected when necessary so that the material is passed from one to the next without the need for any manual steps. In preferred modalities, the selection of valves and piping is controlled by a pre-programmed computer to perform the necessary steps. More preferably, the chromatography system is also connected online to the detector system, for example, an MS system. Thus, an operator can place a sample tray on a self-sampler and the remaining operations are carried out under the control of a computer, resulting in the purification and analysis of all selected samples.

In some embodiments, HTLC can be used for the purification of DHT before mass spectrometry. In such modalities, samples can be extracted using an HTLC extraction cartridge that captures the analyte, then eluted and subjected to chromatography on a second HTLC column or on an HPLC analytical column before ionization. For example, the extraction of the samples with an HTLC extraction cartridge can be carried out with a compacted column with a large particle size (50 pm). The sample eluted from this column can then be transferred to an HPLC analytical column, such as a cyan analytical column, for further purification before mass spectrometry. Due to the fact that the steps involved in these procedures

20/32 chromatography can be linked in an automated manner, the need for operator involvement during analyte purification can be minimized. This feature can result in savings in time and costs and eliminate the opportunity for operator error.

Mass Spectrometry Detection and Quantification

In various embodiments, the DHT present in a test sample can be ionized using any method known to the person skilled in the art. Mass spectrometry is performed using a mass spectrometer, which includes an ion source for the ionization of the fractionated sample and for the creation of charged molecules for further analysis. For example, sample ionization can be performed through electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), bombardment rapid atomization (FAB), secondary liquid ionization (LSI), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasmaspray ionization, surface-enhanced laser desorption ionization ( SELDI), ionization of induced coupled plasma (ICP) and particle beams. The person skilled in the art will understand that the choice of the ionization method can be determined based on the analyte to be measured, the type of sample, the type of detector, the choice of the positive versus negative mode, etc.

DHT can be ionized in a positive or negative mode; In preferred embodiments, DHT is ionized by APCI in the positive mode. In related preferred embodiments, DHT ions are in a gaseous state and the inert collision gas is argon or nitrogen; preferably argon.

In mass spectrometry techniques, generally, after the sample has been ionized, positively charged or negatively charged ions created in this way can be analyzed to determine the ratio of mass to charge. Suitable analyzers for determining mass to load ratios include 21/32 quadripolar analyzers, ion capture analyzers and flight time analyzers. Ions can be detected using several detection modes. For example, the selected ions can be detected, that is, using a selective ion monitoring mode (SIM) or alternatively, the ions can be detected using a scanning mode, for example, monitoring multiple reactions (MRM) or monitoring selected reaction (SRM). Preferably, the ratio of mass to load is determined using a quadripolar analyzer. For example, in a quadripolar or quadripolar ion capture instrument, the ions in a field with oscillating radio frequency undergo a force proportional to the DC potential applied between the electrodes, the amplitude of the RF signal and the mass / charge ratio. Voltage and amplitude can be selected so that only ions that have a particular mass / charge ratio travel the length of the quadripole, while all other ions are deflected. Thus, quadripolar instruments can act both as a mass filter and as a mass detector for the ions injected into the instrument.

It is possible to increase the resolution of the MS technique through the use of tandem mass spectrometry or MS / MS. In this technique, a precursor ion (also called a parent ion) generated from a molecule of interest can be filtered on an MS instrument and the precursor ion is subsequently fragmented to produce one or more fragment ions (also called child ions). or product ions) which are then analyzed in a second MS procedure. Through careful selection of precursor ions, only the ions produced by certain analytes are passed to the fragmentation chamber, where collisions with atoms of an inert gas produce the fragment ions. Due to the fact that both precursor ions and fragments are produced in a way that can be reproduced under a certain set of ionization / fragmentation conditions, the MS / MS technique can provide an extremely powerful analytical tool. For example, the filtration / fragmentation combination can be used to eliminate interfering substances

22/32 and can be particularly useful in complex samples, such as biological samples.

The mass spectrometer typically provides the user with an ionic scan; that is, the relative abundance of each ion with a particular mass / charge over a certain range (for example, 100 to 1000 amu). The results of an analyte assay, i.e., a mass spectrum, can be related to the amount of the analyte in the original sample by various methods known in the art. For example, given that such sampling and analysis parameters are carefully controlled, the relative abundance of a certain ion can be compared with a table that converts that relative abundance into an absolute amount of the original molecule. Alternatively, molecular patterns can be run with the samples and a standard curve can be constructed based on the ions generated from these patterns. Using such a standard curve, the relative abundance of a certain ion can be converted into an absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the amount of DHT. The methods of producing and using such standard curves are well known in the art and a common expert is able to select an appropriate internal standard. For example, an isotope-labeled steroid can be used as an internal standard; in certain preferred embodiments the standard is 16, 16, 17-d ₃ dihydrotestosterone (16, 16, 17-d ₃ DHT). Various other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art.

One or more steps of the methods can be performed using automated machines. In certain embodiments, one or more purification steps are performed online and most preferably all purification and mass spectrometry steps can be performed online.

In certain embodiments, such as MS / MS, where precursor ions are isolated for further fragmentation, dissociation with collision activation is often used to generate fragment ions for

23/32 additional detection. In CAD, precursor ions gain energy through collisions with an inert gas and subsequently fragment through 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 vibration energy.

In particularly preferred embodiments, DHT is detected and / or quantified using MS / MS as follows. The samples are subjected to liquid chromatography, preferably HTLC; the liquid solvent flow from the chromatographic column enters the heated nebulization interface of an MS / MS analyzer; and the solvent / analyte mixture is converted to steam in the heated interface pipe. The analyte (eg DHT), contained in the nebulized solvent, is ionized by the interface's corona discharge needle, which applies a high voltage to the nebulized solvent / analyte mixture. The ions, for example, the precursor ions, pass through the orifice of the instrument and enter the first quadripole. Quadripoles 1 and 3 (Q1 and Q3) are mass filters, which allow the selection of ions (that is, the selection of precursor and fragment ions in Q1 and Q3, respectively) based on their mass ratio in relation to charge (m / z). Quadripole 2 (Q2) is the collision cell, where the ions are fragmented. The first quadripole of the mass spectrometer (Q1) selects molecules with the mass ratios in relation to the DHT charge. Precursor ions with the correct mass / charge ratios are allowed to pass into the collision chamber (Q2), while unwanted ions with any other mass / charge ratio collide with the sides of the quadripole and are eliminated. The precursor ions entering Q2 collide with neutral argon gas molecules and fragment. This process is called collision-activated dissociation (CAD). The generated fragment ions are passed into quadripole 3 (Q3), where the DHT fragment ions are selected while other ions are eliminated.

The methods can involve MS / MS performed in the mode of positive or negative ions; preferably in the mode of positive ions. Using standardized methods well known in the art, a common expert is able to

24/32 to identify one or more ions of fragments of a particular precursor ion of DHT that can be used for selection in quadripole 3 (Q3).

As the ions collide with the detector, they produce a pulse of electrons that are converted into a digital signal. The acquired data is relayed to a computer, which graphically represents the collected ions counts versus time. The resulting mass chromatograms are similar to the chromatograms generated in traditional HPLC methods. The areas under the peaks corresponding to particular ions or the amplitude of such peaks are measured and the area or amplitude is correlated to the quantity of the analyte of interest. In certain embodiments, the area under the curves or the peak amplitude for fragment ion (s) and / or precursor ions is measured to determine the amount of DHT. As previously described, the relative abundance of a certain ion can be converted into an absolute amount of the original analyte, for example, DHT, using standard calibration curves based on the peaks of one or more ions of an internal molecular standard, such as 16, 16 , 17-d ₃ dihydrotestosterone.

The following examples serve to illustrate the invention. These examples are in no way intended to limit the scope of the methods.

EXAMPLES

Example 1: Preparation of Serum and Reagent Samples

Plasma samples were prepared by collecting blood in a Vacutainer tube without additives and allowed to clot for approximately 30 minutes at room temperature. The samples were then centrifuged and the serum separated from the cells. Samples that exhibited evident hemolysis were excluded.

Three stock solutions were prepared with DHT (Sigma Chemical Company, Cat. No. A7755 or equivalent). A standard DHT stock solution of 1 mg / mL in methanol was prepared in a volumetric flask. A portion of the standard DHT stock solution was then diluted 1: 100 to prepare a standard DHT intermediate stock solution of 1,000,000 ng / dL in methanol. A portion of the standard es25 / 32 touch intermediate solution was used to prepare a second standard solution of 2,000 ng / dL intermediate stock in methanol. The second intermediate stock standard solution was used to prepare a 200 ng / dL DHT working standard in stripped serum.

16, 16, 17-d ₃ dihydrotestosterone (CDN, Cat. No. D-5079 or equivalent) was used to prepare an internal standard stock solution of 16, 16, 17-d3 dihydrotestosterone in deuterated methanol at 1.0 mg / mL, which was used to prepare an intermediate internal standard stock solution of 16, 16, 17-d3 dihydrotestosterone in deuterated methanol at 1,000,000 ng / dL. 1.0 mL of this intermediate stock solution was used to prepare a second intermediate internal stock solution at 1000 ng / dL of 16, 16, 17-d ₃ dihydrotestosterone in water. An internal standard working solution of 16, 16, 17-d ₃ dihydrotestosterone at 500 ng / dL was prepared by diluting 20 mL of the second intermediate internal standard stock solution with Dl water to volume in a volumetric flask of 200 mL.

Example 2: DHT extraction from samples using liquid chromatography

Ambient temperature standards, controls and patient samples were prepared for liquid chromatography (LC) by first mixing with a mechanical vortex.

300 pL of each standard, control and patient sample mixed with vortex was then transferred to a well of a 96-well plate. 300 pL of 20% formic acid and 100 pL of 16, 16, 17-d ₃ internal standard working solution of 500 ng / ml dihydrotestosterone were then added to each. The plates were then vortexed and incubated at room temperature for 30 minutes before being loaded into an autosampler drawer.

The samples were injected with an HTLC system

Cohesive Technologies Aria TLX-1 using Aria OS V 1.5 or newer software. A washing solution from the autosampler was prepared using 60% acetonitrile, 30% isopropanol and 10% acetone (v / v).

The HTLC system automatically injected 100 pL of the previously prepared samples 26/32 into a TurboFIow column (50 x 1.0 mm, 50 pm C-18 column from Cohesive Technologies) compacted with large particles. The samples were loaded at a high flow rate (5.0 mL / min, 0.1% formic acid loading reagent) to create turbulence within the extraction column. This turbulence ensured the optimal binding of DHT with the large particles in the column and the passage of residual protein and cell debris for disposal.

After loading, the flow direction was reversed and the sample was eluted into the analytical column (Thermo Scientific, BetaBasic Cyano column, 5 pm particle size, 50 x 2.1 mm). A binary HPLC gradient was applied to the analytical column, to separate DHT from other analytes contained in the sample. Mobile phase A was 0.1% formic acid and mobile phase B was 100% methanol. The HPLC gradient started with a 3% organic gradient that increased to 50% in approximately 4.75 minutes. The separate sample was then submitted to MS / MS for DHT quantification.

The specificity of DHT against similar analytes was determined for the following compounds (each at a concentration of 1000 ng / dL in stripped serum): testosterone, estriol, dehydroepiandrosterone (DHEA), estrone, pregnenolone, estradiol, androstenedione, 17-OH pregnenolone, corticosterone and aldosterone. No significant interference was observed for any of these compounds.

Example 3: DHT detection and quantification using MS / MS

MS / MS was performed using a Finnigan TSQ Quantum Ultra MS / MS system (Thermo Electron Corporation). The following software programs all from ThermoElectron were used in the Examples described here: Quantum Tune Master V 1.2 or later, Xcalibur V 1.4 SR1 or later, TSQ Quantum 1.4 or later and LCQuan V 2.0 with SP1 or later. The liquid solvent / analyte coming out of the analytical column flowed to the heated nebulizer interface of a Thermo Finnigan MS / MS analyzer. The solvent / analyte mixture was converted to steam in the heated interface pipe. The analytes in the nebulized solvent were ionized by APCI / 27.

The ions passed to the first quadripole (Q1), which selected ions with a mass to load ratio of 291.10 ± 0.50 m / z. The ions that entered Quadripole 2 (Q2) collided with argon gas to generate ion fragments, which were passed to quadripole 3 (Q3) for further selection. Simultaneously, the same process using isotope-diluted mass spectrometry was performed with an internal standard, 16, 16, 17-d3 dihydrotestosterone. The following mass transitions were used for detection and quantification during positive polarity validation.

Table 1. Mass Transitions to DHT (Positive Polarity)

Analyte Precursor ion (m / z) Ion Product (m / z) DHT 291.10 255.20 and 79.2 16, 16, 17-d ₃ dihydrotestosterone (internal standard) 294.10 258.2

Example 4: Precision and Accuracy Intraassay and Interassay Three groups of quality controls (QC) were prepared starting from human serum stripped with charcoal (Golden West Biologicals, 15 Temecula, CA) with the addition of DHT in a concentration of 25, 75 and 150 ng / dL, to cover the presumptive assay range that can be reported.

Ten aliquots from each of the three QC groups were analyzed in a single assay to determine the coefficient of variation (CV (%)) of a sample within an assay. The following values were determined:

Table 2. Intra-assay variation and accuracy

Level 1 (25 ng / dL) Level 1 (75 ng / dL) Level III (150 ng / dL) Mean (ng / dL) 26.8 73.83 145.23 Standard Deviation (ng / dL) 3.98 3.02 4.58 CV (%) 14.8% 4.09% 3.16% Accuracy (%) 107.2% 98.44% 96.82%

28/32

Ten aliquots from each of the three QC groups were analyzed over ten days to determine the coefficient of variation (CV (%)) between trials. The following values were determined:

Table 3. Variation and Inter-Assay Accuracy

Level 1 (25 ng / dL) Level 1 (75 ng / dL) Level III (150 ng / dL) Mean (ng / dL) 24.51 75.18 152.31 Standard Deviation (ng / dL) 2.47 3.32 5.81 CV (%) 10.06% 4.42% 3.82% Accuracy (%) 98.04% 100.24% 101.54%

Example 5: Analytical Sensitivity: Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOQ is the point where the measures become quantitatively significant. The analyte response in this LOQ can be identified, distinguished and reproduced with an accuracy of 20% and an accuracy of 80% to 120%.

LOQ was determined by analyzing samples of serum stripped of analyte with the addition of concentrations of 1.25, 2.5, 5.0, 10.0 and 20.0 ng / dL of DHT (five replicates at each level) then the CV determination. The results were plotted (shown in Figure 1) and the LOQ was determined from the curve to be 5.0 ng / dL.

LOD is the point at which a value is beyond the uncertainty associated with its measurement and is defined as two standard deviations from zero concentration. To determine the LOQ for the DHT assay, samples of white serum stripped with charcoal were run in ten replicates. The results of these tests were analyzed statistically with an average value of 1.0 ng / dL and a standard deviation of 0.5 ng / dL. Thus, the LOD for DHT was 2.0 ng / dL.

Example 6: Assay Reportable Range and Linearity

To establish the linearity of DHT detection in the assay, a blank specified as a zero standard and five serum standards with additions in concentrations ranging from 10 to 200 ng / dL were analyzed. The correlation value of the tested concentration range (0 to 200 ng / dL) was

29/32 greater than 0.995. A graph showing the linearity of the standard curve up to 200 ng / dL is shown in Figure 2.

Example 7: Matrix specificity

Matrix specificity was assessed by diluting 5 patient serum samples twice and four times with the following matrices: serum stripped of the analyte (serum stripped with charcoal, Cat. No. SP1070, Golden West Biologicals, Inc.) , normal human defibrinated serum (Cat. No. 1101-00, Biocell Labs, Carson, CA 90746 or equivalent) and deionized water (Dl). Two serum samples received the addition of the following concentrations of DHT: 92.4 ng / dL, 64.1 ng / dL. The sera that received additions were then diluted 2x and 4x with the previous matrices and were analyzed. The study indicated that all three matrices could be used to dilute samples with analyte values above the linear range. The results of this study are shown in Table 4.

Table 4. Specificity of the DHT_ Matrix

Factor Dilution Concentration Expected (ng / dL) Stripped Serum (ng / dL) Biocell Serum (ng / dL) Water Dl (ng / dL) Sample 1 ... 92.4 2x 46.2 47.7 50.8 49 4x 21.1 24.1 21.1 22.5 Sample 2 .. 64.1 2x 32.05 37.3 34.6 34.8 4x 16.03 19.1 18.9 16.4

Example 8: Recovery Studies

Quality control (QC) samples were used for recovery studies. Samples of low, medium and high QC received addition of DHT at a concentration of 112.5, 137.5 and 175 ng / dL, respectively.

A recovery study was carried out on these samples that received DHT addition (five tests at each concentration). The absolute recovery was calculated by dividing the detected concentration

30/32 DHT in the samples grouped by the expected DHT concentration in the samples. The average recoveries were 89.01%, 90.15% and 94.93%, respectively. All recoveries were acceptable, that is, they were within the range of 80% to 120%.

Example 9: Specimen Studies

The specimens were derived from sample collection tubes without additives (for serum), serum separator tubes (SST), tubes with EDTA or tubes with sodium heparin (36 samples, 18 from men and 18 from women). Four samples with EDTA for men and one for women, as well as one sample with heparin for women, were excluded as being unsuitable for analysis due to the fact that they suffer from strong hemolysis or are lipemic. The rest were tested for the ability to apply the present methods to various types of samples. Analysis of the data revealed that there was little difference between the levels of DHT detected in the various types of samples (see Tables 5 and 6).

Table 5. Comparison of Sample Types for DHT, Men

Sample Type Average DHT (ng / dL) Standard Deviation (ng / dL) Serum 34.1925 20.5794 SST 39.2492 17,1014 EDTA 37.4059 15.6782 Heparin 42,7082 15.0552

Table 6. Comparison of Sample Types for DHT, Women

Sample Type Average DHT (ng / dL) Standard Deviation (ng / dL) Serum 26.6482 14.6048 SST 24.3991 18.8417 EDTA 28.9525 12.8975 Heparin 23.0536 18.8463

Example 10: Comparison of HTLC-MS and RIA Studies

A comparative study was carried out using samples from 20 patients covering the reported range, analyzed using the present methods with a reference radioimmunoassay (RIA) method. The correlation31 / 32 was determined through linear regression (shown in figure 3A) and Deming analysis (shown in figure 3B). The correlation coefficient for the linear regression analysis was 0.88.

The contents of articles, patents and patent applications and all other documents and information available electronically mentioned or cited here, are incorporated here by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated as being incorporated as a reference. Applicants reserve the right to physically incorporate into this patent application any and all materials and information arising from any such articles, patents, patent applications or other physical and electronic documents.

The methods described here in an illustrative manner can be properly practiced in the absence of any element or elements, limitations or limitations, not specifically disclosed here. Thus, for example, the terms comprising, including, containing etc. should be read in an expansive manner and without limitation. In addition, the terms and expressions used here were used as terms of description and not of limitation and there is no intention in using such terms and expressions to exclude any equivalents of the displayed and described characteristics or parts of them. It is recognized that several modifications are possible within the scope of the claimed invention. Thus, it should be understood that although the present invention has been specifically disclosed by the preferred modalities and optional features, the modification and variation of the invention incorporated herein disclosed may be appealed by those skilled in the art and that such modifications and variations are considered to be within the scope of this invention.

The invention has been described here in a broad and generic way. Each of the more limited species and subgeneric groupings that fit within the generic disclosure is also part of the methods. This includes the generic description of the methods with a negative condition or limitation that removes any objective subject of the genre, regardless of whether or not the cut material is specifically mentioned here.

Other modalities are within the following claims. In addition, when characteristics or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also described here in terms of any individual member or subgroup of members of the Markush group.

Claims (26)

1. Process for determining the amount of non-derivatized dihydrotestosterone (DHT) in a sample of body fluid, characterized by the fact that it comprises: 5 a. the ionization of non-derivatized DHT, purified from the body fluid sample by solid phase extraction (SPE), under conditions suitable to produce one or more ions detectable by mass spectrometry; wherein said ions comprise one or more ions selected from the group consisting of ions with a mass / charge ratio of 291.10 ± 0.50, 255.20 ± 10 0.50 and 79.20 ± 0.50; and B. determining the amount of one or more ions by mass spectrometry and using the determined amount to calculate the amount of non-derivatized DHT in the body fluid sample, in which said SPE is performed in the form of high turbulence liquid chromatography 15 ( HTLC). 2. Process according to claim 1, characterized in that said mass spectrometry is tandem mass spectrometry. Process according to claim 1 or 2, characterized by the fact that said ionization comprises the production of a precursor ion with a mass / charge ratio of 291.10 ± 0.50 and the production of one or more ions from fragments selected from the group consisting of ions with a mass / charge ratio of 255.20 ± 0.50 and 79.20 ± 0.50. Process according to any one of claims 1 to 25 3, characterized by the fact that said SPE and mass spectrometry are carried out online. Process according to any of claims 1 to 4, characterized in that said purification further comprises purification with high performance liquid chromatography (HPLC). 30 6. Process according to claim 5, characterized by the fact that said SPE and said HPLC are linked for the online processing of the sample. Petition 870170076295, of 10/09/2017, p. 4/10 Process according to any one of claims 1 to 6, characterized in that said sample of body fluid is plasma or serum. Process according to any one of claims 1 to 5 7, characterized by the fact that said process has a limit of quantification within the range of 5.0 ng / dL up to and including 200 ng / dL. Process according to any one of claims 1 to 8, characterized in that the amount of the one or more ions determined by mass spectrometry is related to the presence or the 10 amount of non-derivatized DHT in the test sample by comparison with an internal standard. Process according to claim 9, characterized in that the internal standard comprises 16, 16, 17-d3 dihydrotestosterone. 11. Process for determining the amount of non-derivatized dihydrotestosterone (DHT) in a test sample by mass spectrometry, characterized by the fact that it comprises: The. ionization of non-derivatized DHT, purified from the test sample by high-swirl liquid chromatography (HTLC), under conditions suitable to produce one or more ions detectable by spectrometry of Mass; and B. determining the amount of one or more ions by mass spectrometry and using the determined amount to calculate the amount of non-derivatized DHT in the body fluid sample. Process according to claim 11, characterized by the fact that said mass spectrometry is tandem mass spectrometry. Process according to claim 11 or 12, characterized in that said purification further comprises purification with high performance liquid chromatography (HPLC). 30 Process according to any one of claims 12 to 13, characterized by the fact that said HTLC and said HPLC are configured for online processing. Petition 870170076295, of 10/09/2017, p. 5/10 Process according to any one of claims 11 to 14, characterized in that said test sample is a sample of body fluid. 16. Process according to any one of claims 11 to 5 15, characterized by the fact that said test sample is plasma or serum. 17. Process according to any one of claims 11 to 16, characterized by the fact that the one or more ions detectable by mass spectrometry comprise one or more ions selected from the group consisting of ions with a mass / charge ratio of 291.10 ± 0.50, 10 255.20 ± 0.50 and 79.20 ± 0.50. 18. Process according to any one of claims 11 to 17, characterized by the fact that said ionization comprises the production of a precursor ion with a mass / charge ratio of 291.10 ± 0.50 and one or more ions from fragments selected from the group consisting of ions with a 15 mass / load ratio of 255.20 ± 0.50 and 79.20 ± 0.50. 19. Process according to any one of claims 11 to 18, characterized by the fact that said process has a limit of quantification within the range of 5.0 ng / dL to 200 ng / dL, inclusive. 20. Process according to any one of claims 11 to 20 19, characterized by the fact that the quantity of one or more ions detected by mass spectrometry is related to the presence or quantity of non-derivatized DHT in the test sample by comparison with an internal standard. 21. Process according to claim 20, characterized by the fact that the internal standard comprises 16, 16, 17-d3 dihydrotestosterone. 22. Process for determining the amount of non-derivatized dihydrotestosterone (DHT) in a sample of body fluid by tandem mass spectrometry, characterized by the fact that it comprises: 30 a. the production of a non-derivatized DHT precursor ion, purified from a body fluid sample by high-turbulence liquid chromatography (HTLC), which has a mass / charge ratio of 291.10 ± 0.50; Petition 870170076295, of 10/09/2017, p. 6/10 B. the production of one or more fragment ions of said precursor ion, wherein at least one of said one or more fragment ions comprises a fragment ion selected from the group of ions that has a mass / charge ratio of 255.20 ± 0.50 and 79.20 ± 0.50; and C. determining the amount of one or more of said ions generated in step (b) or (c) or both and using the amount determined to calculate the amount of non-derivatized DHT in said test sample. 23. Process according to claim 22, characterized by the fact that said purification further comprises purification with 10 high performance liquid chromatography (HPLC). 24. Process according to claim 23, characterized by the fact that said HTLC and said HPLC are configured for online processing. 25. Process according to any of claims 22 to 15 24, characterized by the fact that said sample of body fluid is plasma or serum. 26. Process according to any of claims 22 to 25, characterized by the fact that said process has a limit of quantification within the range of 5.0 ng / dL to 200 ng / dL, inclusive. 27. Process according to any of claims 22 to 26, characterized by the fact that the amount of one or more ions determined by mass spectrometry is related to the presence or the amount of non-derivatized DHT in the test sample through comparison with an internal standard. 28. Process according to claim 27, characterized in that the internal standard comprises 16, 16, 17-d ₃ dihydrotestosterone. Petition 870170076295, of 10/09/2017, p. 7/10 1/3