WO2010136455A1 - Procédés et réactifs permettant un dosage quantitatif des métabolites présents dans des échantillons biologiques - Google Patents

Procédés et réactifs permettant un dosage quantitatif des métabolites présents dans des échantillons biologiques Download PDF

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WO2010136455A1
WO2010136455A1 PCT/EP2010/057162 EP2010057162W WO2010136455A1 WO 2010136455 A1 WO2010136455 A1 WO 2010136455A1 EP 2010057162 W EP2010057162 W EP 2010057162W WO 2010136455 A1 WO2010136455 A1 WO 2010136455A1
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sample
concentration
metabolite
test sample
isotopologue
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PCT/EP2010/057162
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Cesar Marquez Gonzalez
Javier CALVO MARTÍNEZ
Niels-Christian Reichardt
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Metabolic Renal Disease, S.L.
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Priority to EP10724355A priority Critical patent/EP2435832A1/fr
Publication of WO2010136455A1 publication Critical patent/WO2010136455A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • the invention relates to the field of diagnostic assays and, more in particular, to the field of diagnostics based on the determination of the concentration of metabolites in a sample from a subject under study, said concentration being indicative of the presence of a given condition.
  • LC/MS Liquid Chromatography coupled to Mass Spectrometry
  • LC/MS can be used for the identification of metabolites in biological samples as well as for the quantification of known metabolites.
  • Quantification can be relative, i.e. it only provides the ratio of the concentration of the metabolite in the test sample with respect to the concentration of the metabolite in a reference sample or absolute, i.e. it provides the concentration of metabolite in a sample.
  • Relative quantification is often carried out by in vivo labeling of metabolites with 2 H, 13 C, or 15 N, and comparing the labeled sample with a control sample that has natural isotopic abundances.
  • That strategy works fairly well for certain organisms such as yeast, bacteria, and some plants, but uniform incorporation of these isotopes into all metabolites for animals is quite difficult/expensive or impossible (e.g., metabolites from humans).
  • Another strategy for relative quantification is chemical labeling, which has proven to be useful for quantification in genomics (e.g., two color fluorescent dye labeling)(Lockhart DJ et al, Nature Biotechnol. 1996, 14, 1675-1680; and, Schena M et al, Science 1995, 270, 467-470) and proteomics (e.g., isotope-coded affinity tags)(Aebersold R et al., Nature 2003, 422, 198-207; Gygi SP et al., Nature Biotechnol. 1999, 17, 994-999).
  • genomics e.g., two color fluorescent dye labeling
  • proteomics e.g., isotope-coded affinity tags
  • Relative quantification by labeling has seen limited use for metabolomics due in part to the lack of a single functional group present in all metabolites to act as the target for the labeling chemistry. Moreover, relative quantification can also be carried out using isotopic labeling. This technique is also ill-suited for metabolomic studies for the same reasons as the chemical labeling, i.e. the lack of a single functional group present in all metabolites.
  • WO2007109292 describes a method for relative quantification of a metabolite wherein the test and reference samples are reacted with a reagent that differs only in its isotopic kit, thereby creating heavy and light versions of derivatized metabolites, which are easily distinguished by MS. Labeled metabolites co-elute from the LC-column and appear in the mass spectrum as pairs of peaks with a mass-shift equal to the difference in mass of the two isotopic labels. The ratio of peak intensities for each pair yields the relative concentration of each metabolite between the two samples.
  • WO0398182 describes a method for the determination of the relative amount of a protein in sample wherein both the reference samples and the test samples are metabolically labeled and combined in equal amounts. The relative amounts of the different metabolites can then be determined by analyzing the test sample, the reference sample and the mixture thereof by mass spectrometry followed by the determination of the intensity of the peaks corresponding to the atom added during metabolic labeling as well as of the isotopologues thereof.
  • relative quantification is only suitable when a reference sample is available or when detecting changes in concentration between two different time points, but does not provide absolute concentration values which are required when samples obtained from different places or time-points are to be compared.
  • Mass spectrometry is rapidly becoming a method of choice for analysis of proteins, peptides and other biological molecules.
  • Laser desorption/ionization mass spectrometry methods such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and surface enhanced laser desorption/ionization mass spectrometry (SELDIMS) procedures are very sensitive analytical methods and are probably the MS procedures most compatible with biological samples. Further, the ability of these procedures to generate high-mass ions at high efficiency from sub- picomole quantities of biological macromolecules makes these techniques extremely useful for macromolecule analysis and macromolecule identification in biological samples. Analysis of peptide analytes in crude biological samples, such as blood, plasma, or serum, however offers special problems for mass spectrometry analysis.
  • LIDI-MS Laser desorption/ionization methods of mass spectrometry
  • protein analytes in complex sample mixtures such as serum or urine
  • peak intensity of an analyte can vary based on the nature of the protein milieu in which it is found, variances in sample handling and variances in instrument performance.
  • One way to overcome these problems is to introduce normalization standards into a sample against which the analyte can be compared.
  • Another method is to construct a standard curve of the analyte on which the quantity of the analyte can be measured.
  • standard curves can suffer if the calibration series is not matched with the sample being tested.
  • the use of LDI-MS to quantify an analyte within a biological sample may involve certain difficulties.
  • the laser desorption process is a complex and poorly understood event that depends on the interaction of a laser ionization source and co-crystals consisting of both protein sample and desorption matrix. In the laser desorption event, ionization of any one analyte cannot occur independent of any other analyte exposed at the same time to the ionization source.
  • two samples containing an analyte as well as a number of other proteins, where the other proteins are vastly different in either concentration or composition are ionized the relative abundance of ions formed for the analyte in each of the samples may not be correlated with their concentration. Thus, the ability to quantify the analyte becomes problematic.
  • API atmospheric pressure ionization
  • APCI atmospheric pressure chemical ionization
  • ESI electrospray ionization
  • API sources permit the direct transportation of ionized analytes from liquid into gas-phase.
  • the coupling of chromatographic techniques helps to circumvent matrix effects like ion suppression due to competition in the ionization process between different analytes or due to the presence of surfactants and inorganic salts.
  • the methods of this invention provide a method of standardizing an assay using a calibration curve with absolute standards achieved by minimizing interference from non-analyte (i.e., background) proteins naturally associated with the biological sample while simultaneously providing a complex set of invariant internal standards. This results in a normalized analyte value that can be quantified directly from clinical or test samples by comparison to the calibration curve.
  • the invention relates to a method for determining the concentration of a metabolite (M) of known m/z in a test sample by mass spectrometry which comprises the steps of:
  • the invention relates to a method for the determination of the concentration of a metabolite (M) in a test sample by mass spectrometry comprising the steps of
  • the invention relates to a computer program or a computer-readable media containing means for carrying out a method as defined in the first and second aspects of the invention.
  • the invention relates to a kit for the determination of the concentration of an analyte in a test sample comprising
  • the processed chromatograms here presented have been smoothed twice by a mean method. They also have been aligned using an offset time within ⁇ 4sec.
  • the processed chromatograms here presented have been smoothed twice by a mean method. They also have been aligned using an offset time within ⁇ 4sec.
  • the processed chromatograms here presented have been smoothed twice by a mean method. They also have been aligned using an offset time within ⁇ 4sec.
  • the processed chromatograms here presented have been smoothed twice by a mean method. They also have been aligned using an offset time within ⁇ 4sec.
  • the authors of the present invention have found that the quantitative determination of an analyte in a sample can be improved by using a standard curve prepared from the same sample to which increasing and known concentrations of the analyte has been added. Since the standard curve samples and the test samples have a very similar composition, undesired artifacts due to differences between the standard curve samples and the test samples, such as ion suppression, are avoided.
  • the invention relates to a method (hereinafter, first method of the invention) for determining the concentration of a metabolite (M) of known m/z in a test sample by mass spectrometry which comprises the steps of:
  • metabolite refers to any analyte of interest that may be determined.
  • analytes can include, but are not limited to, proteins, peptides, nucleotides, oligonucleotides (both DNA or RNA), carbohydrates, lipids, steroids, amino acids and/or other small molecules with a molecular weight of less than 1500 Daltons.
  • the metabolite or metabolites can be natural or synthetic, endogenous or exogenous.
  • m/z is used herein interchangeably with mass/charge, mass/ionization ratio or mass-to-charge ratio) and refers to the ratio between the mass of an object and the charge of the same object.
  • the mass is customarily expressed in terms of atomic mass units (amu), also called Daltons (Da).
  • the charge is customarily expressed in terms of multiples of elementary charge, which is the amount of charge in an electron or a proton. Because the ion usually has a single charge, the m/z ratio value is usually coincident with the mass of the ion expressed in Daltons, or its molecular weight (MW).
  • the mass-to-charge ratio of a target metabolite can be determined using any conventional mass spectrometry technique.
  • a time-of- flight mass analyzer is a preferred mass analyzer for measuring the desorbed and ionized target, and even more preferably, the time-of- flight mass analyzer can be preceded by an ion reflector to correct for kinetic energy differences among ions of the same mass.
  • Another preferred enhancement of the time of flight mass analyzer is a short, controlled, delay between the desorption and ionization of the target and the application of the initial acceleration voltage by the mass analyzer.
  • the m/z of a test analyte is determined by preparing different samples having the same composition and which contain increasing amounts of the test analyte. These samples are then analyzed by mass spectrometry. Ideally, the mass spectra of the different samples are identical except for the region wherein the test analyte is found, which should show increasing intensities. The m/z of the peak which intensity increases as the concentration of analyte in the sample increases is then considered to be the m/z of interest for the test analyte.
  • sample is understood as any complex composition wherein the concentration of at least one of the components is unknown.
  • the method according to the invention can be applied to any type of samples, including chemical analysis samples, food samples, environmental samples and biological samples.
  • the determination is carried out in a biological sample.
  • biological sample as used herein, relates to any sample having a biological origin.
  • the biological sample is a fluid or an extract.
  • biological sample any solid or fluid sample obtained from, excreted by, or secreted by any living organism, including single-celled micro-organisms (such as bacteria and yeasts) and multicellular organisms (such as plants and animals, for instance a vertebrate or a mammal, and in particular a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated).
  • single-celled micro-organisms such as bacteria and yeasts
  • multicellular organisms such as plants and animals, for instance a vertebrate or a mammal, and in particular a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated.
  • a biological sample may be a biological fluid obtained from any location (such as blood, plasma, serum, urine, bile, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion), an exudate (such as fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (such as a normal joint or a joint affected by disease such as rheumatoid arthritis).
  • a biological sample can be obtained from any organ or tissue (including a biopsy or autopsy specimen) or may comprise cells (whether primary cells or cultured cells) or medium conditioned by any cell, tissue or organ. If desired, the biological sample is subjected to preliminary processing, including preliminary separation techniques.
  • cells or tissues can be extracted and subjected to subcellular fractionation for separate analysis of biomolecules in distinct subcellular fractions, e. g., proteins or drugs found in different parts of the cell.
  • a sample may be analyzed as subsets of the sample, e. g., bands from a gel.
  • the biological sample is blood serum or plasma.
  • step (i) of the first method of the invention the test sample is analyzed by mass spectrometry using standard mass spectrometry equipment.
  • mass spectrometry is applied to a number of reference samples of the same nature as the test sample to which known and different amounts of M have been previously added.
  • the biological sample can be analyzed as such or, alternatively, the metabolites may be first extracted from the sample prior to analysis and then the metabolite extract is then analyzed. If the metabolites are extracted prior to analysis, different extraction methods are available to the skilled person. The selection of one or other extraction method will depend on the class of metabolites/small molecules that are targeted from a particular analysis. Suitable extraction methods include "Extraction of free metabolite pools”, “Vapor Phase Extraction”, and “Total Metabolite Extraction”. The first type of extraction, “Extraction of free metabolite pools”, is mainly used in metabolomics research.
  • free intracellular metabolite pools are obtained from a biological sample through methanol-water extraction for polar metabolites, or chloroform extraction for non-polar metabolites.
  • the second type of extraction “Vapor Phase Extraction”, refers to the extraction of metabolites that are volatile at room temperature. The metabolites are expelled from the biological sample in the vapor phase. These metabolites are either measured directly by connecting the flask or reactor in which the vapors are generated to the analytical instrument or by absorbing first the vapors in charcoal/solvent and then analyzing the acquired solution.
  • Total Metabolite Extraction refers to the extraction of the free metabolite pools along with the metabolites that have been incorporated in cellular macromolecules, e.g. lipids, proteins etc.
  • the present invention provides extraction of a particular class of metabolites from macromolecules (e.g. amino acids from proteins or sugars from cell wall components).
  • the present invention also provides a combined high-throughput method which extracts all metabolites simultaneously.
  • the metabolite quantification can be carried out in the biological sample.
  • the sample may be prepared to enhance detectability of the markers.
  • a blood serum sample from the subject can be preferably fractionated by, e.g., Cibacron blue agarose chromatography and single stranded DNA affinity chromatography, anion exchange chromatography, affinity chromatography (e.g., with antibodies) and the like.
  • the method of fractionation depends on the type of detection method used. Any method that enriches for the metabolite of interest can be used.
  • preparation involves fractionation of the sample and collection of fractions determined to contain the biomarkers.
  • Methods of pre-fractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity chromatography, sequential extraction, gel electrophoresis and liquid chromatography.
  • the analytes also may be modified prior to detection. These methods are useful to simplify the sample for further analysis. For example, it can be useful to remove high abundance proteins, such as albumin, from blood before analysis.
  • a sample can be pre- fractionated by removing proteins that are present in a high quantity or that may interfere with the detection of markers in a sample.
  • Proteins in general may be removed by using conventional techniques such as precipitation using organic solvents such as methanol precipitation, ethanol, acetonitrile, acetone or combinations thereof, in particular, combination of methanol, acetone and acetonitrile, acid precipitation using, for example, trichloroacetic acid or perchloric acid, heat denaturation and any combination of organic solvent, acid and heat precipitation.
  • serum albumin or other proteins abundant in serum such as apo lipoproteins, glycoproteins, inmunoglobulins may obscure the analysis of markers since they are present in a high quantity. Thus, it may be sufficient to remove one or more of the above proteins albumin in order to detect the metabolites or minor proteins.
  • the blood serum sample can be pre-fractionated by removing serum albumin.
  • Serum albumin can be removed using a substrate that comprises adsorbents that specifically bind serum albumin.
  • a column which comprises, e.g., Cibacron blue agarose (which has a high affinity for serum albumin) or anti-serum albumin antibodies can be used.
  • a sample can be pre-fractionated by isolating proteins that have a specific characteristic, e.g. are glycosylated.
  • a blood serum sample can be fractionated by passing the sample over a lectin chromatography column (which has a high affinity for sugars).
  • affinity adsorbents exist which are suitable for pre-fractionating blood serum samples.
  • An example of one other type of affinity chromatography available to pre- fractionate a sample is a single stranded DNA spin column. These columns bind proteins which are basic or positively charged. Bound proteins are then eluted from the column using eluants containing denaturants or high pH.
  • a sample can be fractionated using a sequential extraction protocol.
  • sequential extraction a sample is exposed to a series of adsorbents to extract different types of biomolecules from a sample.
  • mass spectrometry means an analytical technique to identify unknown compounds including: (1) ionizing the compounds and potentially fractionating the compounds parent ion formed into daughter ions; and (2) detecting the charged compounds and calculating a mass-to-charge ratio (m/z).
  • the compounds may be ionized and detected by any suitable means.
  • a "mass spectrometer” includes means for ionizing compounds and detecting charged compounds.
  • the ionization source for the mass spectrometric analysis includes, but is not limited to, electron impact (EI), chemical ionization (CI), atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), sonic spray ionization, matrix-assisted laser- desorption ionization (MALDI), or the like.
  • EI electron impact
  • CI chemical ionization
  • APCI atmospheric pressure chemical ionization
  • ESI electrospray ionization
  • MALDI matrix-assisted laser- desorption ionization
  • ionization is carried out by electrospray ionization (ESI).
  • Mass analyzers suitable for separating and detecting the heavy- and light-labeled forms of the analyte end-products are suitable for use with the instant labeling reagents, such as, but not limited to, magnetic sector, double focusing, time-of- flight (TOF), linear ion trap, quadrupole (Q), triple quadrupole (QQQ), quadrupole time-of- flight (QTOF), ion- trap time-of- flight, Fourier transform spectrometers such as the ion cyclotron resonance (FT-ICR) and Orbitrap, and the like.
  • TOF time-of- flight
  • Q quadrupole
  • QQQQ triple quadrupole
  • QTOF quadrupole time-of- flight
  • ion- trap time-of- flight ion- trap time-of- flight
  • Fourier transform spectrometers such as the ion cyclotron resonance (FT-ICR) and Orbitrap, and the like.
  • the above mentioned ionization methods generally produce what is known in the art as a protonated molecule, meaning the addition of a proton or a hydrogen nucleus, [M+H] + where M signifies the molecule of interest, and H signifies the hydrogen ion, which is the same as a proton.
  • Some ionization methods will also produce analogous ions. Analogous ions may arise by the addition of an alkaline metal cation, rather than the proton discussed above. A typical species might be [M+Na] + or [M+K] + .
  • the analysis of the ionized molecules is similar irrespective of whether one is concerned with a protonated ion as discussed above or dealing with an added alkaline metal cation.
  • a proton adds one mass unit (typically called one Dalton), in case of the hydrogen ion (i.e., proton), 23 Daltons in case of sodium, or 39 Daltons in case of potassium.
  • These additional weights or masses are simply added to the molecular weight of the molecule of interest and the MS peak occurs at the point for the molecular weight of the molecule of interest plus the weight of the ion that has been added.
  • These ionization methods can also produce negative ions.
  • the most common molecular signal is the deprotonated molecule [M-H] " , in this case the mass is one Dalton lower than the molecular weight of the molecule of interest.
  • multiply charged ions are of the general identification type of [M+nH] n+ , where small n identifies the number of additional protons that have been added.
  • the reference samples are prepared by adding known and increasing amounts of the analyte which concentration is to be determined.
  • the skilled person will appreciate that the number of samples in the collection of reference samples is not limiting as long the reference values are sufficient to obtain statistically significant values.
  • the number of samples in the collection may be as low as two, preferably three, more preferably four, five, six, seven, eight, nine, ten or more.
  • test sample is plasma
  • reference samples are obtained by adding increasing amounts of the test analyte to either two or more aliquots of the same plasma that is to be determined or, alternatively, to plasma preparations which are obtained by pooling a plurality of plasma samples.
  • the number of samples that can be pooled in order to obtain a test sample will vary but it may contain at least 1, at least 10, at least 100, at least 200, at least 300, at least 400, at least 500 or more samples.
  • the reference samples have been supplemented with known and different concentrations of M so that each reference sample will contain a concentration of final M which will be the result of the addition of the unknown concentration of M in the sample plus the concentration of the added M.
  • the amounts of M added to the reference samples will be such that the final concentration in the reference sample will be as low as 5% above the estimated concentration for the endogenous M and as much as 10 times the estimated concentration for the endogenous M.
  • the reference samples of the collection are prepared so that they contain, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, 9.5, 10, 35, 40, 45 and/or 50 ⁇ M of M.
  • Step (ii) of the first method of the invention involves the determination of the peak intensity of M in the spectra obtained from the test sample and of the peak intensities of M and of at least one naturally-occurring isotopologue of M in the reference samples.
  • peak intensities relate to the number of incident ions able to trigger a pulse in a mass spectrometer detector during a limited time. Counts-per- second (cps) is the commonly used unit for peak intensity.
  • the term "naturally occurring isotopologue” refers to a species that differs from a specific compound only in the isotopic composition thereof.
  • the number and concentration of isotopologues of a given compound in a given sample will depend on the natural abundance of the isotopes as well as on the number of atoms in the compound that may be substituted by an isotope.
  • compounds having a single carbon atom will typically contain 1.1122% of the 13 C isotope, since this is the natural abundance of 13 C in nature.
  • the isotopologue resulting from the replacement of the single nitrogen atom 14 N by the 15 N isotope will typically appear in nature with an abundance of 0.3673% since this is the natural abundance Of 15 N.
  • Isotopologues are usually classified as “lighter” and “heavier” isotopologues.
  • heavier isotopologues would be molecules wherein positions containing 12 C or 14 N are occupied by 13 C or 15 N.
  • Suitable isotopologues that can be measured in the method of the present invention are those containing one or more 15 N, 14 C, 13 C, 13 N, 18 O and 34 S.
  • the isotopologue used in the method of the invention is a heavier isotopologue.
  • isotopologues may exist of the same compound. These isotopologues are usually referred to M+l, M+2, etc.
  • the M+l isotopologue is usually a family of isotopic isomers (isotopomers) wherein one of the positions comprises the isotope, having the M+l isotopologue family as many isotopomers as numbers of atoms in the molecule that may be substituted by the isotopic form. This situation may result in isotopologues pattern having two or more peaks.
  • the relative abundance of 12 C versus 13 C on the earth is 12 C at 98.9% and 13 C at 1.1% respectively, in any naturally occurring sample of carbon.
  • Each of these different carbon isotopes have identical chemical values and have weights that differ by one Dalton.
  • One peak represents the molecule containing all 12 C atoms, and the second peak at one Dalton higher representing the same chemical molecule, containing all 12 C atoms but one 13 C atom. Further, there will be yet another peak having about 61% of the height of the first peak, in which there will be two random 12 C atoms replaced by 13 C atoms, thus resulting in a mass two Daltons higher than the base isotope molecule. There are further isotopologues mass spectra peaks representing three 13 C substitutions and having about 22% of the height of the first 12 C peak, and so on. Thus, any compound containing carbon will always produce multiple mass spectra peaks, large organic molecules containing in 80 to 100 carbons will appear as several peaks separated by one m/z unit.
  • natural isotopic abundance refers to the level (or distribution) of one or more isotopologues found in a compound based upon the natural prevalence of an isotope or isotopes in nature.
  • a natural compound obtained from living plant matter can typically contain about 1.08 % 13 C relative to 12 C.
  • Step (iii) of the first method of the invention involves the determination of the concentration of M in the test sample by interpolating the peak intensity of M in the test sample within a calibration curve obtained from (a) the intensities of the peaks of M and of the at least one naturally-occurring isotopologue of M in the samples of the collection of reference samples and (b) the concentrations of M and of the at least one naturally- occurring isotopologue of M in said reference samples.
  • interpolation means the process of calculating a new point between two existing data points.
  • the interpolation process comprises comparing the peak intensity within the test sample with a data set which contains at least two pairs of peak intensity/concentration values obtained from the reference samples.
  • the skilled person will appreciate that many methods exist for the interpolation of a given peak intensity value within a correspondence table reflecting peak intensities as a function of metabolite concentration.
  • the interpolation can be carried out using methods such as piecewise constant interpolation (also known as nearest neighbour interpolation), linear interpolation, polynomial interpolation, spline interpolation, rational interpolation, trigonometric interpolation, bilinear interpolation, bicubic interpolation and the like.
  • the accuracy of the interpolation method will depend on the number of values included in the standard data set, although it is possible to carry out an interpolation with only a single pair of values.
  • the interpolation is carried out using a data set comprising at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven pairs of values.
  • the results of the interpolation applied to the peak intensities of the test sample will provide the concentration of the metabolite in said test sample.
  • the interpolation step is carried out using a calibration curve which contains the concentrations of M and those of at least one isotopologue in each of the reference samples in the X axis and the peak intensities associated with M and with the isotopologues for each of the reference samples in the Y axis.
  • the concentration of the first heavier isotopologue will be 1.08% of the concentration of the test analyte.
  • the calibration curve includes peak intensity values for each of the concentrations of M and for each of the concentrations of the at least one naturally occurring isotopologue.
  • the interpolation is carried out by linear regression analysis,
  • a proportionality constant (m) or slope is calculated as a function of the concentration of metabolite added to the sample.
  • m proportionality constant
  • C 0 the intensity signal in the test sample
  • I 1 the intensity signal in the reference sample i
  • X 1 the known concentration of metabolite added in reference sample i
  • I 1 m(C o + X 1 ) + I noise
  • I 1 m(C o + X 1 ) + I noise
  • I 1 - I 0 m(C o + X 1 ) + I noise - (mC o + I noise )
  • the proportionality constant m is, as expected, dependent on the X 1 , but not of C 0 or the noise level (I nO ise).
  • the lower limit for the reliability in the calculation of a metabolite concentration in the test sample is independent of the concentrations values used for the determination of C 0 .
  • the lower limit could be defined as the concentration of the naturally occurring lighter isotopologue in the test sample.
  • step (iii) of the method of the invention the concentration of C 0 in the test sample is calculated from the proportionality constant/slope m using the formula
  • the analysis may also be performed subsequent to a separation of the test sample.
  • a wide variety of techniques for separating the metabolites within the test sample are well known to those skilled in the art (see, for example, Laemmli, Nature 1970,227 : 680-685 ; Washburn et al. , Nat. Biotechnol. 2001, 19: 242-7; Schagger et al. , Anal. Biochem. 1991, 199:223-31) and may be employed according to the present invention.
  • the test sample may be fractionated on the basis of charge (e. g. , by chromatofocusing or isoelectric focusing), of electrophoretic mobility (e.g.
  • chromatography including LC, FPLC, and HPLC, on any suitable matrix (e. g., gel filtration chromatography, ion exchange chromatography, reverse phase chromatography).
  • a denaturing agent such as urea or sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • 2-mercaptoethanol or dithiothreitol 2-mercaptoethanol or dithiothreitol
  • chromatography including LC, FPLC, and HPLC, on any suitable matrix (e. g., gel filtration chromatography, ion exchange chromatography, reverse phase chromatography).
  • the separation is carried out using chromatography.
  • Chromatography in general, is a method for mixture component separation that relies on differences in the flowing behavior of the various components of a mixture/solution carried by a mobile phase through a support/column coated with a certain stationary phase. Specifically, some components bind strongly to the stationary phase and spend longer time in the support, while other components stay predominantly in the mobile phase and pass faster through the support.
  • the criterion based on which the various compounds are separated through the column is defined by the particular problem being investigated and imposed by the structure, composition and binding capacity of the stationary phase.
  • a stationary phase could be constructed such that the linear and low molecular weight molecules elute faster than the aromatic and high- molecular weight ones.
  • LC Liquid Chromatography
  • IC Ion Chromatography
  • SEC Size-Exclusion Chromatography
  • SFC Supercritical-Fluid Chromatography
  • TLC Thin-Layer Chromatography
  • CE Capillary Electrophoresis
  • GC cyclopentadioxane chromatography
  • LC cyclopentadioxane chromatography
  • HILIC hydrophilic interaction liquid chromatography
  • HPLC High-pressure liquid chromatography
  • HPLC techniques for fractionation of analytes include reverse-phase HPLC and normal phase HPLC.
  • Reverse-phase (RP) chromatography involves a nonpolar stationary phase and a polar mobile phase.
  • the stationary phase that is characterized by alkyl chains of specific length immobilized to a silica bead support.
  • Typical stationary phase for reversed phase HPLC include alkyl hydrocarbons such as octadecyl (C 18) is the most common stationary phase, but octyl (C8) and butyl (C4) are also used in some applications.
  • the most polar compounds elute first with the most nonpolar compounds eluting last.
  • the mobile phase is generally a binary mixture of water and a miscible polar organic solvent like methanol, acetonitrile or THF.
  • RP-HPLC is suitable for the separation and analysis of various types of compounds including without limitation biomolecules, (e.g., glycoconjugates, proteins, peptides, and nucleic acids, and, with mobile phase supplements, oligonucleotides).
  • biomolecules e.g., glycoconjugates, proteins, peptides, and nucleic acids, and, with mobile phase supplements, oligonucleotides.
  • Aqueous normal phase chromatography is a chromatographic technique wherein the stationary phase is polar and the mobile phase is nonpolar.
  • Typical stationary phases for normal phase chromatography are silica or organic moieties with cyano and amino functional groups.
  • the mobile phase consists of a very nonpolar solvent like hexane or heptane mixed with a slightly more polar solvent like isopropanol, ethyl acetate or chloroform. Retention increases as the amount of nonpolar solvent in the mobile phase increases.
  • the samples containing the metabolites are injected into a HPLC column, typically silica based C 18.
  • a HPLC column typically silica based C 18.
  • an aqueous medium is used to elute the inorganic salts, while the metabolites are eluted with a mixture of aqueous solvent (water) and organic solvent (acetonitrile, methanol, propanol).
  • the aqueous phase is generally HPLC grade water with 0.1% acid and the organic solvent phase is generally an HPLC grade acetonitrile or methanol with 0.1% acid.
  • the acid is used to improve the chromatographic peak shape and to provide a source of protons in reverse phase LC/MS.
  • the acids most commonly used are formic acid, trifluoro acetic acid, and acetic acid.
  • RP HPLC compounds are separated based on their hydrophobic character.
  • the test and the reference samples are fractionated prior to mass spectrometry analysis, a mass spectrum will be obtained for each of the fractions obtained during the chromatography.
  • the test compound elutes in two or more chromatographic fractions, known as retention time interval, so that the total intensity of the compound can be determined by integrating the peak intensity within retention time interval.
  • the peak intensities of M and/or of the at least one naturally- occurring isotopologue of M in each sample are determined in step (ii) by integration of the peak intensities within a retention time interval, wherein said interval is determined using a known analyte of known m/z present in a continuous mode within the retention time interval of M.
  • the integration of the peak areas over a retention time interval can only be carried out once the retention time interval in which the test compound elutes is known.
  • the retention time interval of the test compound will be that where the monitored intensity of the selected ion increases with the concentration of metabolite in the reference samples.
  • the time interval in which the integration is to be carried out is determined using the retention time of M and the naturally occurring isotopologue of M in the most concentrated of the reference samples. The most concentrated reference sample provides a clear intensity increment of the corresponding m/z exact value when compared to the test sample.
  • the integrated signal intensity of M or of M and the naturally occurring isotopologue of M are normalized by dividing them by the integrated signal intensity of a known analyte within the retention time interval of M.
  • the analyte is permanently injected during the entire experiment via a HPLC cross settled post-column and prior the ionization source. Two reasons justify the continuous injection of an exogenous analyte during the time expand of the quantification experiment. First, it is used as internal standard for the unambiguous determination of exact mass of the different metabolites. At the same time, the analyte peak intensity is used for the normalization of the peak intensity of the metabolite between the collected spectra. This way, the normalization precludes the effects of unexpected system instability.
  • the first method of the invention allows the quantification of a test analyte in a test sample. This determination is useful for diagnostic/prognostic purposes if it is known that the concentration of the analyte correlates with a given disease or condition.
  • the invention relates to a method (hereinafter, the second method of the invention) for the determination of the concentration of a metabolite (M) in a test sample by mass spectrometry comprising the steps of (i) performing mass spectrometry on the test sample and on at least one reference sample of the same nature than the test sample wherein said at least one reference sample contains a known concentration of M, (ii) determining the peak intensity of M in the test sample and of M in the at least one reference sample and
  • test sample is as defined previously in the context of the first method of the invention.
  • reference sample as understood in the context of the second method of the invention, relates to a sample wherein the concentration of the test analyte is known. This concentration of the analyte in the reference sample can be determined using any method known in the art but preferably, the concentration is determined using the first method of the invention.
  • Step (i) involves performing mass spectra on the test sample and on the reference sample.
  • the sample can be of any origin, more preferably a biological sample and, even more preferably, a biofluid and, even more preferably, urine or plasma.
  • the sample may be applied as such or may be fractionated prior to the mass spectrometry so as to separate the test analyte from other compounds present in the sample. In a preferred embodiment, separation is carried out by reverse phase liquid chromatography.
  • Step (ii) of the second method of the invention involves the determination of the total peak intensities of the test analyte in the test samples and in the reference sample from the mass spectra obtained from both samples. As explained above, this determination can be carried out on the peak corresponding to M or, if the sample has been fractionated prior to the mass spectrometric analysis, on an interval of retention times wherein substantially all the test compound elutes.
  • proportionality constant relates to a ratio of peak intensities which, ideally should not be dependent on the background levels of the test analyte in the test sample or to the noise level.
  • the concentration of the test analyte in the reference sample can be determined by the formula:
  • Ctest m ⁇ ref wherein C tes t is the concentration of the analyte in the test sample, C re f is the concentration corresponding to the analyte in the reference sample and m is the proportionality constant.
  • the mass spectrometry is carried out after fractionation of the test sample and of the reference sample.
  • the fractionation is carried out by reverse phase chromatography.
  • Computer implementation of different embodiments of the first and second method of the invention can be achieved using a computer program providing instructions in a computer readable form.
  • the computer would collect the data, analyze the data as in accordance with the methods described herein, and then provide a result of the analysis.
  • the invention relates to a computer program or a computer-readable media containing means for carrying out a method of the invention.
  • the computer implementation of the first method of the invention includes, without limitation, one or more of the following modules:
  • Control means for a mass spectrometer and, as the case may be, for separating the sample, so that mass spectra at a given m/z value and, as the case may be, at a given retention time, are collected.
  • This module includes instructions for controlling a mass spectrometer including ionization and detection conditions. If the method involves a first fractionation step, the module may comprise additionally control means for the fractionation means. In the particular case that the fractionation is carried out by reverse phase chromatography, the module includes instructions for controlling column pressure, mobile phase composition, frequency of fraction collection and the like.
  • the module comprises means for performing integration of the peak areas corresponding to the test analyte and to the isotopologue in a given set of spectra.
  • the module will preferably incorporate means for detecting the test analyte in a single peak or for integrating peaks of the same m/z value corresponding to a given retention time interval.
  • the time interval where the integration is to be carried out can be inputted manually or, alternatively, can be determined by the program based on the comparison of the different spectra at different retention times.
  • the module should preferably contain means for subtracting the background value from the peak intensities.
  • the computer implementation of the second method invention includes, without limitation, one or more of the following modules: Control means for a mass spectrometer and, as the case may be, for separating the sample, so that mass spectra at a given m/z value and, as the case may be, at a given retention time are collected,
  • Different types of computer language can be used to provide instructions in a computer readable form.
  • the computer program can be written using languages such as C, C++, Microsoft C#, Microsoft Visual Basic, FORTRAN, PERL, HTML, JAVA, S, UNIX or LINUX shell command languages such as C shell script, and different dialects of such languages.
  • "R” an S language dialect is an example of a dialect with attributes facilitating analyses like those presented here (see http://cran.us.r-project.org).
  • Computer programs for performing analysis techniques described herein can be run on a computer having sufficient memory and processing capability.
  • An example of a suitable computer is one having an Intel Pentium (g)-based processor of 200 MHZ or greater, with 64 MB or more main memory. Equivalent and superior computer systems are well known in the art.
  • Standard operating systems can be employed for different types of computers.
  • Examples of operating systems for an Intel Pentium (2)-based processor includes the Microsoft Windows TM family such as Windows NT, Windows XP, and Windows 2000 and LINUX.
  • Examples of operating systems for Apple computers include OSX, UNIX and LINUX operating systems.
  • Other computers and their operating systems are well known in the art.
  • the R language is used on an Intel- based computer with 4GB ram dual 866 MHz Pentium m processors running the LINUX-operating system or an IBM computer running the AIX operating system with an Intel-based computer running the Windows NT or XP operating system as an x- windows terminal.
  • the invention provides a kit for the determination of the concentration of an analyte in a test sample comprising
  • Component (i) corresponds to a reference sample of the same nature than the test sample.
  • the term "same nature" has been defined in detail in the context of the first method of the invention.
  • the test sample is serum
  • the reference sample is urine.
  • the reference sample corresponds to a normalized sample obtained by pooling samples from many different individuals.
  • Component (ii) contains the information needed for determining the concentration of the test metabolite in the test sample, namely, the concentration of test metabolite in the reference sample and the proportionality factor between the concentration of test metabolite and the peak intensity in the mass spectrum. These values are determined using any method known in the art but, preferably, they are determined using the first method of the invention.
  • the kit is suitable for use in a method wherein the sample is fractionated prior to its analysis by mass spectrometry, in which case the kit also contains information related to the retention time interval of the test analyte.
  • the reference sample is lyophilized. In this case, the sample is reconstituted prior to the determination.
  • the kit of the invention further comprises one or more components selected from the group of
  • Component (i) comprises a buffer solution suitable for reconstituting the reference sample in case it is provided in the lyophilized form or for diluting the reference and the test samples to working concentrations.
  • Suitable buffer systems may be found e.g., in Sambrook, J., et al, Molecular Cloning, A Laboratory Manual, 3rd edition, CSHL Press (2001) Cold Spring Harbor, N. Y.
  • Preferred buffer substances are Tris-(hydroxymethyl)- aminomethane (TRIS), 2-morpholinoethanesulfonic acid (MES) phosphate, N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), acetate, salts thereof, and other suitable substances.
  • Component (ii) relates to means for obtaining the test sample and may comprise a syringe, a micro catheter, an endoscope, a toothpick, a brush, a needle, a lancet, a swab, a cup, a spatula, a biopsy gun, a cotton bud and the like.
  • Component (iii) of the kit of the invention can also comprise tubes for sample collection.
  • the sample is blood and/or plasma, where the tubes for blood collection are operated manually or by vacuum.
  • the kit may contain anticoagulants, low molecular weight heparin, citrate, EDTA, Hirudin, Draculin and other anticoagulants.
  • the kit may contain clot activators such as factor X activators, and/or glass pearls or other structures to increase the surface thereby facilitating and enhancing the clotting process.
  • Component (iv) includes reagents useful for extracting the test metabolite from the biological sample.
  • Suitable reagents for this purpose include, without limitation, organic solvents and mixtures thereof with water, charcoal and the like.
  • Component (v) includes deproteinizing reagents such as perchloric acid (HClO 4 ), acetonitrile, trichloroacetic acid, sulfo salicylic acid and the like.
  • deproteinizing reagents such as perchloric acid (HClO 4 ), acetonitrile, trichloroacetic acid, sulfo salicylic acid and the like.
  • Component (vi) includes separation means for separating metabolites present in the reference sample.
  • Suitable separation means include without limitation, chromatographic columns, isoelectrofocusing and electrophoretic gels.
  • the package can comprise a box, and a wrapper enveloping the box.
  • the package can be hermetically sealed.
  • the package can be wrapped in shrink wrap.
  • Component (viii) comprises instructions as to how to use the kit.
  • the kit can further comprise information in electronic or paper form.
  • the information can comprise, for example, instructions for measuring the level of the analyte of interest.
  • Components (vii) and (viii) contain informative material than can be provided in printed form or, alternatively, can be provided in a computer readable media such as magnetic disks, tapes and the like), optical media (CD-ROM, DVD) and the like.
  • the media can additionally or alternatively contain Internet websites providing said instructions.
  • the invention relates to a kit for the determination of the concentration of an analyte in a test sample comprising
  • component (ii) of the kit further comprises information as to the retention time interval of the analyte.
  • the kit contains a reference sample is lyophilized.
  • the kit further comprising one or more of a component selected from the group of (i) a buffer solution for reconstituting the lyophilised reference sample,
  • Diagnostic analysis can be performed by measuring the presence or amount of a marker associated with a disease or disorder.
  • a marker can be based on a single or multiple analytes.
  • the determination of the concentration of a given analyte in a sample may be suitable for determining (1) the presence or level of a disease or disorder; (2) the possibility of contracting a disease or disorder, (3) the probability of response to a given therapy.
  • Different types of markers can be measured to determine whether an association exists including markers attributed to a causative agent, markers directly involved in disease and disorder, and/or markers reflecting a disease or disorder state.
  • the analytical techniques described herein are also suitable for the evaluation of compound pharmacology.
  • concentration of a given compound or of the metabolites thereof can be determined at different time points after the compound as been administered.
  • Metabolic studies include the determination by sampling of biological materials the absorption, distribution, metabolism, and excretion of a compound and its metabolic by-products. Such evaluations have a variety of different uses including identifying important therapeutic targets, prioritizing potentially therapeutic compounds, identifying toxic metabolites, identifying therapeutic metabolites, identifying increased or decreased production of analytes that may harm a cell or animal and identifying increased or decreased production of analytes providing a beneficial effect to a cell or organism.
  • Compound reverse pharmacology can be performed using compounds with known effects to determine new therapeutic targets or new uses for one or more known compounds. Such studies could involve the identification of biomarkers for an unintended effect, beneficial or not, of a therapeutic intervention.
  • the methods of the invention are also suitable for the evaluation of a disease treatment since the_expression state of biomarkers provides information on the health of a cell or animal. Changes in the biomarker levels can be used to select particular treatments and to monitor treatment efficacy. The changes can be, for example, with respect to an untreated subject, different subjects at different states of treatment, or a subject at different times during treatment.
  • Solutions in methanol of groups of five selected metabolites at 10 ⁇ M concentration are prepared.
  • the UPLC-MS experiment is designed to alternate the injection of samples of those solutions and methanol blanks. Each group is injected a total of three times.
  • the chromatographic separation is carried out on a reverse phase UPLC Acquity column BEH-C 18 100 ⁇ 2.1 mm from Waters Corporation.
  • the volume injection is 2.5 ⁇ L and the total run time is ten minutes using a flow rate of 100 ⁇ L/min.
  • the elution buffers are methanol (A) and a 999:1 (v/v) mixture of water and formic acid (B).
  • the column is eluted with a constant mixture A/B 95:5 in volume during 0.5 minutes, followed by a linear gradient during 1.0 minute to reach a mixture A/B 20:80 in volume. It follows another linear gradient during 3.5 minutes to reach an eluent containing exclusively B, which is passed during 2.5 minutes. Finally, a new gradient is applied during 0.2 minutes to return to a mixture A/B 95:5 in volume. This mixture ratio is kept for 2.3 minutes before the next injection.
  • Spectrometric method The injection into the electrospray ionization source takes place via the analyte probe.
  • a splitter-stainless steel cross with a inner bore of 0.15 mm from Valco (reference JV1X.5XCS6) is set up between the UPLC post-column capillary and the stainless steel capillary of the analyte probe to infuse 100 ⁇ L/min methanol and to reduce the incoming total volume from 200 to approximately 5 ⁇ L/min.
  • the spectra are recorded in a centroid data format mode.
  • the instrument operates in positive ion mode using W optics in a range of masses from 50 to 1000 Daltons.
  • the ionization is achieved using 523 K as capillary temperature and 373 K as source temperature.
  • the capillary voltage is set at 3000 V and the cone at 0 V.
  • the desolvation gas flow is fixed at 500 L/min and the cone gas flow at 50 L/min.
  • the collected data is analyzed using the implanted software MassLynx (Waters Corporation, Milford, MA). Once the metabolite is identified by its m/z ratio, the maximum in its chromatographic retention time is recorded. The chromatographic peak is integrated, and the total intensity collected. A proportionality factor obtained from the division of the total intensity by the concentration is calculated. Any particularity observed is also carefully annotated.
  • the metabolite is identified in a human plasma reference sample using the retention time and m/z ratio obtained in the first phase.
  • the human plasma used as reference sample is averaged human plasma obtained from 1.500 different healthy individuals, i.e., one single flask from Sigma Human Plasma
  • This plasma is processed before analysis to remove plasma proteins. This way, a sample of 100 ⁇ L of the stock solution is thawed in an ice bath and 900 ⁇ L of methanol (Sigma Chromasolv grade) are added. The mixture is vortexed during 2 min, centrifuged at 13000 rpm for 30 min and finally filtered using polypropylene 0.2 mm syringe filters from Teknokroma. The supernatant containing the targeted metabolites is then ready to be use as reference sample in the analysis protocol.
  • methanol Sigma Chromasolv grade
  • solutions in plasma of groups of five selected metabolites at eight different concentrations are prepared.
  • the concentration set consists in the addition of 0.1, 0.5, 1.0, 4.0, 6.0, 8.0 and 10.0 times the estimated concentration of metabolite to the plasma sample in a relation 1/1 in volume.
  • the UPLC-MS experiment is designed alternating the injection of all groups of samples and methanol blanks, defining a series. Each series is injected a total of three times.
  • the UPLC-MS experimental conditions are maintained as presented in the first phase.
  • the collected data is analyzed using an ad hoc tool after the *.raw files provided by MassLynx (Waters Coporation, Milford, MA) are converted into *.mzXML files using MassWolf (Seattle Proteome Centre/Institute for Systems Biology, Seattle, WA).
  • the metabolite m/z ratio is automatically identified and all the integrated intensities of it in the three series annotated.
  • the required calculations for the determination of the concentration of the metabolite in the human plasma sample are done by the software tool.
  • the calculus of a metabolite concentration in the test sample using MS signal intensities is achieved by linear regression. A calibration curve obtained measuring the respective MS intensity signals at different metabolite concentrations as explain above.
  • a metabolite concentration (C 0 ) in the test sample can be interpolated by linear regression measuring the change in the metabolite MS intensity signal (I 1 ) after adding different well-known amounts of the same metabolite (X 1 ) to the reference samples.
  • I 1 m(C o + X 1 ) + I noise
  • I 1 m(C o + X 1 ) + I noise
  • I 1 - I 0 m(C o + X 1 ) + I noise - (mC o + I noise )
  • the lower limit for the reliability in the calculation of a metabolite concentration in our client's plasma sample is independent of the concentrations values used for the determination of C 0 .
  • the entire MS methodology is based on the linear relation C ⁇ /(I). Based only on the interpolation, the lower limit will be defined by the concentration of the naturally occurring isotopologue.
  • the metabolites concentration in the test sample determined by linear regression can be validated using isotopically labeled internal standards.
  • Isotopically labeled metabolites are exogenous, meaning they are not naturally present in the human plasma. Most interesting, they possess the same physic-chemical properties of their natural equivalents in the plasma, and therefore their behavior is assumed to be identical to their endogenous equals. However, having a different mass they can be distinguished from the equivalent by their mass/charge ratio in a mass spectrometric spectrum.
  • the concentration of an isotopically labeled metabolite added to the test sample can be established.
  • the deviation from its well-known concentration determines the validity of the method.
  • C L Concentration of the labeled metabolite
  • C 0 Concentration of the test metabolite to validate
  • I L Intensity of the labeled metabolite
  • I 0 Intensity of the test metabolite to validate MDRenal performs three test experiments at different concentrations for certain number of metabolites in order to guarantee the reliability of our methodology.
  • This phase considers the determination of the metabolite concentrations in the reference sample.
  • the reference sample is averaged human plasma obtained from a large number of different healthy individuals, e.g., a mixture of four flasks from different lots of Sigma Human Plasma (reference P9523-5mL) containing plasma from 1.500 different individuals.
  • the plasma is obtained lyophilized from the provider.
  • LC-MS grade water Sigma Chromasolv grade
  • this plasma is processed before analysis to remove plasma proteins.
  • a sample of 100 ⁇ L of the stock solution is thaw in an ice bath and 900 ⁇ L of methanol (Sigma Chromasolv grade) are added.
  • the mixture is vortex during 2 min, centrifuge at 13000 rpm for 30 min and finally filtered using polypropylene 0.2 ⁇ m syringe filters from Teknokroma.
  • the supernatant containing the targeted metabolites is then ready to be use as reference sample in the analysis protocol.
  • the software provided in the kit will automatically calculate hundreds of metabolite concentrations from individual plasma samples. At the same time, the software will be able to produce a set of statistical studies and provide the user an overview of the metabolite functions within the metabolome.
  • a specific data set is generated consisting of mass/charge ratio, isotopic distribution, expected retention time when pre-fractioning of the sample is considered, concentration in reference sample, range of linearity, and didactic general information of every metabolite.
  • This data file includes the mass/charge ratio for metabolite and its heavier isotopologue, isotopic distribution, expected retention time when pre-fractioning of the sample is considered and concentration in the reference sample for each metabolite analyzed. It will also include the uncertainty in the concentration value and a range of linearity.
  • the information on every metabolite contained in the software encloses a graphical representation of its chemical structure according to the IUPAC Recommendations, the empirical formula, a text commenting the relevance of the metabolite, an average concentration range obtained from different sources (KEGG database, Human Metabolome database) and significant bibliography.
  • Spectral data processing The software tools used in the method of the invention are based in the same principles when selecting a spectral signals and adding their intensity in time. Herein it is defined the common steps in the data processing once the spectra have been converted to *.mzXML format.
  • a group of exogenous analytes (A) is added continuously post-column in all cases for providing a permanent set of signals to be used in the normalization of the spectra.
  • Deviation ⁇ M' accepted to recognize a peak as valid.
  • the software calculates the metabolite concentration in a test sample (TS) exploiting the linearity existing between the ionized metabolite signal intensity and its concentration in solution. It uses reference samples (RS) where well-known amounts of the same metabolite have been added, and takes advantage of the fact that that linearity also applies to their increments according to the equations
  • the experiment contains three different kinds of samples, i.e, test (TS), reference (RS), and blank samples (BS).
  • the experimental setup will consist in a series repeated three times. The series will follow the order:
  • a second software tool makes use of a reference sample (RS) where the metabolites concentration is known for determining the metabolites concentration in Customer Samples (CS). It is based on the comparison between MS intensity signals of the metabolites in CS and those of the metabolites in RS. Notice that the herein named
  • reference sample refers to the previously named “test sample” in this example. At this stage the concentration of the test analyte in this sample is already known, becoming the “reference sample”.
  • the experiment contains three different kinds of samples, i.e, reference (RS), customer (CS), and blank samples (BS).
  • RS reference
  • CS customer
  • BS blank samples
  • the experimental setup will consist in a series.
  • the series order is defined depending on the experimental requirements. For example, it could follow the order:
  • a stepwise description of the process is as follows: Getting Required Information
  • the metabolites concentration in every one of the n CS will be calculated by comparison of its I M CS with I M RS - AS the concentration in the reference sample (C RS ) is known and provided by MD Renal, the concentration of the metabolite in the Customer sample (Ccs) will be calculated straightforward as follows:
  • the software includes a processing tool for the calculation of the metabolite concentrations in a particular customer sample using internal standards. Comparing the results from this methodology with those obtained using a reference sample will test the reliability of the final product.
  • the equation calculates the concentration of a metabolite presents in a customer sample (CM CS) using the ratio of two MS signal intensities (Res) and comparing it to the corresponding one (R RS ) in RS.
  • the localization, identification and integration of metabolite signals require some data to be previously provided by MD Renal. They are summarized in the following points: 1. The time interval (t ls t 2 ) where the metabolite signal is expected.
  • Deviation ⁇ x accepted to recognize a peak as valid 9. Deviation ⁇ x accepted to recognize a peak as valid. 10. Deviation ⁇ y accepted to recognize a peak as valid.
  • test plasma extract An extract of 1 mL of the test plasma mixture (450 healthy subjects, 1.5 mL/subject) obtained from Banco de Sangre de Guip ⁇ zcoa and stored at 203 K was thawed in an ice bath. In order to precipitate the plasma proteins in the extract, methanol in a 9/1 (v/v) ratio was added. The resulting solution was stirred by means of a Vortex for two minutes and centrifuged at 13000 rpm for 30 minutes. The supernatant was then collected and filtered using 0.2 ⁇ m filters. With this process the test plasma extract (TPE) is ready to be used.
  • TPE test plasma extract
  • This extract was mixed with methanol and with a stock solution (SS) of the metabolites threonine in methanol (100 ⁇ M, 50 mL) as is described in Table 1.
  • SS stock solution
  • TS test plasma to be calibrated
  • RS reference samples
  • the concentration in the test to be calibrated (TS) is 20 times less than in relation to the untreated plasma.
  • Table 1 List of volumes ( ⁇ L) added and final concentration ( ⁇ M) of the metabolite threonine in the different samples.
  • a PEEK cross (Teknokroma, UP-P729) with an inner diameter of 0.020" and a dead volume of 2.736 ⁇ L was installed between the Waters Acquity UPLC used in the chromatography and the Waters LCT Premier XE analyte probe.
  • the cross allows incorporating a reference analyte (40 ⁇ M, 2.5 ⁇ L/min) to the complex mixture from the UPLC (250 ⁇ L/min) and reducing the inlet flow in the ionization source by means of electrospraying at 8.5 ⁇ L/min.
  • the LCT Premier XE was adjusted and calibrated to work in positive mode using a W arrangement for the flight tube and a mass range of 50 to 1000 Da.
  • the ionization was done using a voltage of 3000 V and a temperature of 523 K in the capillary.
  • the source was maintained at 373 K with a voltage in the entrance cone fixed at 15 V.
  • the nitrogen flow used as desolvation gas was adjusted at 500 L/min and the cone flow to 50 L/min.
  • This software was developed by Seattle Proteome Center (Institute for Systems Biology, 1441 North 34th Street, Seattle, WA 98103-8904) and released under GLPL (Lesser General Public License), that is, free software available to non-free software. It uses MassLynx libraries from Waters Corporation (Milford , MA) to convert *.raw directories into *.mzXML format files. In any case, any other software able to convert the *.raw data files from Waters to *.mzXML could be used.
  • Table 3 Parameters used in estimating the concentration of threonine. Notice that the analyte is used to normalize the peaks intensity between spectra. The analyte used in this experiment is rhodamine B in a final concentration of 12.5nM. The intensity values obtained for different concentrations of metabolite are shown in Table 4. The selected ion monitoring chromatograms of the different samples for the lighter isotopologue of threonine considered is shown in Figure 1 and for the heavier isotopologue in Figure 2. The mass spectrometric spectrum for this metabolite is shown in Figure 3.
  • test plasma mixture 450 healthy subjects, 1.5 mL/subject obtained from Banco de Sangre de Guipuzcoa and stored at 203 K was thawed in an ice bath.
  • TPE test plasma extract
  • This extract was mixed with methanol and with a stock solution (SS) of the metabolite tryptophan in methanol (100 ⁇ M, 50 mL) as is described in Table 6.
  • SS stock solution
  • RS reference samples
  • the chromatographic separation was carried out in a Waters Acquity system.
  • a BEH HILIC column (100x1 mm; 1.7 ⁇ m particle size) maintained at 313 K was used.
  • the total time per chromatogram was 5 minutes.
  • a PEEK cross (Teknokroma, UP-P729) with an inner diameter of 0.020" and a dead volume of 2.736 ⁇ L was installed between the Waters Acquity UPLC used in the chromatography and the Waters LCT Premier XE analyte probe.
  • the cross allows incorporating a reference analyte (40 ⁇ M, 2.5 ⁇ L/min) to the complex mixture from the UPLC (250 ⁇ L/min) and reducing the inlet flow in the ionization source by means of electrospraying at 8.5 ⁇ L/min.
  • the LCT Premier XE was adjusted and calibrated to work in positive mode using a W arrangement for the flight tube and a mass range of 50 to 1000 Da.
  • the ionization was done using a voltage of 3000 V and a temperature of 523 K in the capillary.
  • the source was maintained at 373 K with a voltage in the entrance cone fixed at 15 V.
  • the nitrogen flow used as desolvation gas was adjusted at 500 L/min and the cone flow to 50 L/min.
  • This software was developed by Seattle Proteome Center (Institute for Systems Biology, 1441 North 34th Street, Seattle, WA 98103-8904) and released under GLPL (Lesser General Public License), that is, free software available to non-free software. It uses MassLynx libraries from Waters Corporation (Milford , MA) to convert *.raw directories into *.mzXML format files. In any case, any other software able to convert the *.raw data files from Waters to *.mzXML could be used.
  • the intensity values obtained for different concentrations of metabolite are shown in Table 9.
  • the selected ion monitoring chromatograms of the different samples for the lighter isotopologue of tryptophan considered is shown in Figure 4 and for the heavier isotopologue in Figure 5.
  • the mass spectrometric spectrum for this metabolite is shown in Figure 6.
  • test plasma mixture 450 healthy subjects, 1.5 mL/subject obtained from Banco de Sangre de Guipuzcoa and stored at 203 K was thawed in an ice bath.
  • TPE test plasma extract
  • This extract was mixed with methanol and with a stock solution (SS) of the metabolite methionine in methanol (100 ⁇ M, 50 mL) as is described in Table 11.
  • SS stock solution
  • TS test plasma to be calibrated
  • RS reference samples
  • the concentration in the test to be calibrated (TS) is 20 times less than in relation to the untreated plasma.
  • the chromatographic separation was carried out in a Waters Acquity system.
  • a BEH HILIC column (100x1 mm; 1.7 ⁇ m particle size) maintained at 313 K was used.
  • the total time per chromatogram was 5 minutes.
  • a PEEK cross (Teknokroma, UP-P729) with an inner diameter of 0.020" and a dead volume of 2.736 ⁇ L was installed between the Waters Acquity UPLC used in the chromatography and the Waters LCT Premier XE analyte probe.
  • the cross allows incorporating a reference analyte (40 ⁇ M, 2.5 ⁇ L/min) to the complex mixture from the UPLC (250 ⁇ L/min) and reducing the inlet flow in the ionization source by means of electrospraying at 8.5 ⁇ L/min.
  • the LCT Premier XE was adjusted and calibrated to work in positive mode using a W arrangement for the flight tube and a mass range of 50 to 1000 Da.
  • the ionization was done using a voltage of 3000 V and a temperature of 523 K in the capillary.
  • the source was maintained at 373 K with a voltage in the entrance cone fixed at 15 V.
  • the nitrogen flow used as desolvation gas was adjusted at 500 L/min and the cone flow to 50 L/min.
  • test plasma mixture 450 healthy subjects, 1.5 mL/subject obtained from Banco de Sangre de Guipuzcoa and stored at 203 K was thawed in an ice bath.
  • TPE test plasma extract
  • This extract was mixed with methanol and with a stock solution (SS) of the metabolite cis-4-hydroxyproline in methanol (100 ⁇ M, 50 mL) as is described in Table 16.
  • SS stock solution
  • RS reference samples
  • the concentration in the test to be calibrated (TS) is 20 times less than in relation to the untreated plasma.
  • the chromatographic separation was carried out in a Waters Acquity system.
  • a BEH HILIC column (100x1 mm; 1.7 ⁇ m particle size) maintained at 313 K was used.
  • the total time per chromatogram was 5 minutes.
  • a PEEK cross (Teknokroma, UP-P729) with an inner diameter of 0.020" and a dead volume of 2.736 ⁇ L was installed between the Waters Acquity UPLC used in the chromatography and the Waters LCT Premier XE analyte probe.
  • the cross allows incorporating a reference analyte (40 ⁇ M, 2.5 ⁇ L/min) to the complex mixture from the UPLC (250 ⁇ L/min) and reducing the inlet flow in the ionization source by means of electrospraying at 8.5 ⁇ L/min.
  • the LCT Premier XE was adjusted and calibrated to work in positive mode using a W arrangement for the flight tube and a mass range of 50 to 1000 Da.
  • the ionization was done using a voltage of 3000 V and a temperature of 523 K in the capillary.
  • the source was maintained at 373 K with a voltage in the entrance cone fixed at 15 V.
  • the nitrogen flow used as desolvation gas was adjusted at 500 L/min and the cone flow to 50 L/min.
  • test plasma mixture 450 healthy subjects, 1.5 mL/subject obtained from Banco de Sangre de Guipuzcoa and stored at 203 K was thawed in an ice bath.
  • TPE test plasma extract
  • This extract was mixed with methanol and with a stock solution (SS) of the metabolite cis-4-hydroxyproline in methanol (100 ⁇ M, 50 mL) as is described in Table 21.
  • SS stock solution
  • RS reference samples
  • the concentration in the test to be calibrated (TS) is 20 times less than in relation to the untreated plasma.
  • Table 21 List of volumes ( ⁇ L) added and final concentration ( ⁇ M) of the metabolite caffeine in the different samples.
  • the chromatographic separation was carried out in a Waters Acquity system.
  • a BEH HILIC column (100x1 mm; 1.7 ⁇ m particle size) maintained at 313 K was used.
  • the total time per chromatogram was 5 minutes.
  • a PEEK cross (Teknokroma, UP-P729) with an inner diameter of 0.020" and a dead volume of 2.736 ⁇ L was installed between the Waters Acquity UPLC used in the chromatography and the Waters LCT Premier XE analyte probe.
  • the cross allows incorporating a reference analyte (40 ⁇ M, 2.5 ⁇ L/min) to the complex mixture from the UPLC (250 ⁇ L/min) and reducing the inlet flow in the ionization source by means of electrospraying at 8.5 ⁇ L/min.
  • the LCT Premier XE was adjusted and calibrated to work in positive mode using a W arrangement for the flight tube and a mass range of 50 to 1000 Da.
  • the ionization was done using a voltage of 3000 V and a temperature of 523 K in the capillary.
  • the source was maintained at 373 K with a voltage in the entrance cone fixed at 15 V.
  • the nitrogen flow used as desolvation gas was adjusted at 500 L/min and the cone flow to 50 L/min.
  • Table 23 Parameters used in estimating the concentration of caffeine. Notice that the analyte is used to normalize the peaks intensity between spectra. The analyte used in this experiment is rhodamine B in a final concentration of 12.5nM.

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Abstract

La présente invention concerne un procédé permettant de déterminer la concentration d'un échantillon biologique en un métabolite à étudier par spectrométrie de masse sans avoir à recourir à des analogues marqués du métabolite à étudier. Le procédé de l'invention implique l'utilisation d'une collection d'échantillons de référence contenant des quantités connues et croissantes du métabolite à étudier, ainsi que la détermination, dans ladite collection d'échantillons, des intensités obtenues en spectrométrie de masse concernant le métabolite à étudier et au moins un isotopologue naturel de celui-ci. L'invention concerne également des moyens informatiques et des nécessaires permettant la mise en œuvre de l'invention.
PCT/EP2010/057162 2009-05-25 2010-05-25 Procédés et réactifs permettant un dosage quantitatif des métabolites présents dans des échantillons biologiques WO2010136455A1 (fr)

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Cited By (6)

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WO2013086070A1 (fr) * 2011-12-07 2013-06-13 Glaxosmithkline Llc Procédés de détermination de la masse musculaire squelettique totale du corps
US9134319B2 (en) 2013-03-15 2015-09-15 The Regents Of The University Of California Method for replacing biomarkers of protein kinetics from tissue samples by biomarkers of protein kinetics from body fluids after isotopic labeling in vivo
US9720002B2 (en) 2004-02-20 2017-08-01 The Regents Of The University Of California Molecular flux rates through critical pathways measured by stable isotope labeling in vivo, as biomarkers of drug action and disease activity
WO2018007394A1 (fr) * 2016-07-08 2018-01-11 Basf Plant Science Company Gmbh Procédé destiné à l'étalonnage d'un échantillon biologique
US10386371B2 (en) 2011-09-08 2019-08-20 The Regents Of The University Of California Metabolic flux measurement, imaging and microscopy
WO2023119072A1 (fr) * 2021-12-21 2023-06-29 Dh Technologies Development Pte. Ltd. Étalonnage de spectromètre de masse à base d'isotopologues naturels

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9720002B2 (en) 2004-02-20 2017-08-01 The Regents Of The University Of California Molecular flux rates through critical pathways measured by stable isotope labeling in vivo, as biomarkers of drug action and disease activity
US9778268B2 (en) 2004-02-20 2017-10-03 The Regents Of The University Of California Molecular flux rates through critical pathways measured by stable isotope labeling in vivo, as biomarkers of drug action and disease activity
US10466253B2 (en) 2004-02-20 2019-11-05 The Regents Of The University Of California Molecular flux rates through critical pathways measured by stable isotope labeling in vivo, as biomarkers of drug action and disease activity
US10386371B2 (en) 2011-09-08 2019-08-20 The Regents Of The University Of California Metabolic flux measurement, imaging and microscopy
WO2013086070A1 (fr) * 2011-12-07 2013-06-13 Glaxosmithkline Llc Procédés de détermination de la masse musculaire squelettique totale du corps
US9737260B2 (en) 2011-12-07 2017-08-22 Glaxosmithkline Llc Methods for determining total body skeletal muscle mass
JP2018138914A (ja) * 2011-12-07 2018-09-06 グラクソスミスクライン エルエルシー 全身骨格筋量の定量方法
US9134319B2 (en) 2013-03-15 2015-09-15 The Regents Of The University Of California Method for replacing biomarkers of protein kinetics from tissue samples by biomarkers of protein kinetics from body fluids after isotopic labeling in vivo
WO2018007394A1 (fr) * 2016-07-08 2018-01-11 Basf Plant Science Company Gmbh Procédé destiné à l'étalonnage d'un échantillon biologique
WO2023119072A1 (fr) * 2021-12-21 2023-06-29 Dh Technologies Development Pte. Ltd. Étalonnage de spectromètre de masse à base d'isotopologues naturels

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