Improved quantification of testosterone in multiplexed samples by mass spectrometry

Abstract

A method for determining the amount of testosterone in each of several human samples using a single mass spectrometry assay includes the steps of: exposing each of the several human samples to a different derivatizing agent to produce testosterone derivatized in each of the several samples in a different manner; combining the several samples to form multiple samples; and quantifying the amount of testosterone in each sample by mass spectrometry.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 452,839, filed on March 17, 2023, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Testosterone is an anabolic steroid originally produced in the testes of men and mainly in the ovaries of women. The instability of testosterone is a major cause of hypogonadism in men, hirsutism and virilization in women, osteoporosis, and diabetes. The accurate and rapid measurement of testosterone is important for the diagnosis, prevention, and treatment of testosterone - related diseases in adults and children.

[0003] The accurate and rapid measurement of testosterone is important for the diagnosis, prevention, and treatment of testosterone - related diseases in adults and children.

Summary of the Invention

[0004] A method for high - throughput quantification of testosterone using at least two different derivatizing agents of different masses is provided.

[0005] In some embodiments, a method for determining the amount of testosterone in each of a plurality of human samples in a single mass spectrometry assay, comprising: derivatizing a first human sample with ethoxyamine chloride to produce ethoxyamine - derivatized testosterone in the first human sample; derivatizing a second human sample with methoxylamine chloride to produce methoxylamine - derivatized testosterone in the second human sample; combining the two samples to form a multiplex sample; and quantifying the amount of testosterone in each sample by mass spectrometry.

[0006] A method is provided for detecting the amount of testosterone in each of multiple patient samples within a single mass spectrometry assay. The method includes the steps of: processing each patient sample in a different manner to form multiple processed samples, wherein, as a result of the processing, the testosterone in each processed sample is distinguishable by mass spectrometry from the testosterone in the other processed samples; combining the processed samples to form multiple samples; exposing the multiple samples to an ionization source under conditions suitable for producing one or more ions detectable by mass spectrometry, wherein one or more ions produced from testosterone from each processed sample are different from one or more ions from testosterone in the other processed samples; detecting the amount of one or more ions from testosterone in each processed sample by mass spectrometry; and relating the amount of one or more ions from testosterone in each processed sample to the amount of testosterone in each patient sample.

[0007] In a particular embodiment, the method comprises determining the amount of testosterone in each of several human samples by a single mass spectrometry assay, the method comprising: i) exposing each of the several human samples to a different derivatizing agent to produce testosterone derivatized in each of the several samples in a different manner; ii) combining the several samples to form a multiple sample; and iii) quantifying the amount of testosterone in each sample by mass spectrometry.

[0008] In some embodiments, the method comprises determining the amount of testosterone in two human samples by a single mass spectrometry assay, the method comprising: i) exposing each of the two human samples to a different derivatizing agent to produce testosterone derivatized in each of the two samples in different ways; ii) combining the two samples to form a multiple sample; and iii) quantifying the amount of testosterone in each sample by mass spectrometry.

[0009] In certain embodiments, the derivatizing agent includes ethoxyamine or methoxyamine. In some embodiments, the derivatizing agent has advantages in reducing interference in the assay.

[0010] In some embodiments, the method is fully automated.

[0011] In some embodiments, the method is antibody-free.

[0012] In some embodiments, "purifying" includes extracting serum using solid-phase extraction (SPE). In some embodiments, SPE is anion exchange solid-phase extraction. In some embodiments, SPE is mixed-mode anion exchange solid-phase extraction. In some embodiments, the extracted sample is concentrated.

[0013] In some embodiments, "purification" includes liquid chromatography. In some embodiments, liquid chromatography includes high-performance liquid chromatography (HPLC). In some embodiments, liquid chromatography includes highly turbulent liquid chromatography (HTLC).

[0014] In further embodiments, ionization includes heated electrospray ionization (HESI). In any such embodiment, ionization includes ionization in positive mode. In some embodiments, ionization includes ionization in negative mode.

[0015] In some embodiments, ionization includes atmospheric pressure chemical ionization (APCI). In some embodiments, ionization includes ionization in positive mode. In some embodiments, ionization includes ionization in negative mode.

[0016] In some embodiments, the method includes measuring the amount of a precursor ion having a mass-to-charge ratio of 332.2 ± 0.5 for ethoxyamine-derivative testosterone. In some embodiments, the method includes measuring the amount of a precursor ion having a mass-to-charge ratio of 318.21 ± 0.5 for methoxylamine-derivative testosterone.

[0017] In some embodiments, the method includes measuring the amount of fragment ions having a mass-to-charge ratio of 140.02±0.5 or 152.02±0.5 for ethoxyamine-derivative testosterone. In some embodiments, the method includes measuring the amount of fragment ions having a mass-to-charge ratio of 126.07±0.5, 138.07±0.5, or 152.08±0.5 for methoxylamine-derivative testosterone.

[0018] In some embodiments, the method reduces or eliminates interference in multiplexed testosterone mass spectrometry assays observed in other procedures.

[0019] In some embodiments, the method further includes adding an internal standard. In some embodiments, the internal standard is labeled with an isotope.

[0020] In some embodiments, the method includes measuring the amount of an internal standard precursor ion having a mass-to-charge ratio of 335.2 ± 0.5 (ethoxyamine derivatized) or 321.21 ± 0.5 (methoxylamine derivatized). In some embodiments, the method includes measuring the amount of an internal standard fragment ion having a mass-to-charge ratio of 143.03 ± 0.5 (ethoxyamine derivatized) or 129.07 ± 0.5 (methoxylamine derivatized).

[0021] In certain embodiments, sample multiplexing is at least twice as fast as single assay or column multiplexing. In certain embodiments, sample multiplexing is at least three times faster than single assay. In certain embodiments, sample multiplexing is at least four times faster than single assay.

[0022] In certain embodiments, the limit of quantification of the method is 10 ng / dL or less. In some embodiments, the limit of quantification of the method is 5 ng / dL or less. In some embodiments, the limit of quantification of the method is 4 ng / dL or less. In some embodiments, the limit of quantification of the method is 3 ng / dL or less. In some embodiments, the limit of quantification of the method is 2 ng / dL or less. In some embodiments, the limit of quantification of the method is 1 ng / dL or less.

[0023] In some embodiments, the method includes linearity of quantification over a range between 2.5 ng / dL and 2,000 ng / dL. In some embodiments, the method includes linearity of quantification over a range between 1 ng / dL and 2,000 ng / dL.

[0024] In some embodiments, the method includes measurement inaccuracy (CV) at concentrations of 8 to 1,200 ng / dL, with concentrations of 1% to 11%, 1% to 9%, or 2% to 11%.

[0025] In some embodiments, the method includes a recovery rate of 90% to 110%, or between 95% and 105%.

[0026] In some embodiments, the method includes a Clinical Reportable Range (CRR) of up to 10,000 ng / dL.

[0027] In certain embodiments, the sample is a body fluid. In some embodiments, the sample is plasma or serum. In some embodiments, the sample is whole blood. In some embodiments, the sample is saliva or urine. In some embodiments, the sample is cerebrospinal fluid (CSF).

[0028] In some embodiments, the method can include adding an agent to the sample in an amount sufficient to remove proteins from the sample.

[0029] Suitable test samples include any test sample that can contain the analyte of interest. In some preferred embodiments, the sample is a biological sample, i.e., a sample obtained from any biological source such as an animal, cell culture, organ culture, etc. In certain preferred embodiments, the sample is obtained from a mammal such as a dog, cat, horse, etc. Particularly preferred mammals are primates, most preferably male or female humans. Particularly preferred samples include blood, plasma, serum, hair, muscle, urine, saliva, tears, cerebrospinal fluid, or other tissue samples. Such samples may be obtained, for example, from a patient, i.e., a living male or female human who has presented in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition. The test sample is preferably obtained from a patient, such as serum.

[0030] The above summary of the invention is non-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 Drawings

[0031] [Figure 1] Figure 1 shows the limit of quantitation and limit of detection for the assay. [Figure 2]Figure 2 shows that the mean bias for this comparison (SST E-Testo vs serum E-Testo) is acceptable (<TEa / 4). E-Testo refers to ethoxyamine-derivatized testosterone. [Figure 3] Figure 3 shows that the mean bias for this comparison (SST E-Testo vs serum E-Testo) is acceptable (<TEa / 4). [Figure 4] Figure 4 shows that the mean bias for this comparison (SST M-Testo vs serum M-Testo) is acceptable (<TEa / 4). M-Testo refers to methoxyamine-derivatized testosterone. [Figure 5] Figure 5 shows that the mean bias for this comparison (SST M-Testo vs serum M-Testo) is acceptable (<TEa / 4). [Figure 6] Figure 6 shows that ethoxyamine Testo SST is stable at refrigerated temperature (2 - 8°C) for 3 days.

Mode for Carrying Out the Invention

[0032] As used herein, unless otherwise indicated, the singular forms "a", "an", and "the" include references to the plural. Thus, for example, reference to "a protein" includes multiple protein molecules.

[0033] As used herein, the term "purify" or "purifying" does not mean removing all substances other than the desired analyte(s) from a sample. Instead, purification refers to procedures that enrich the amount of one or more desired analytes relative to other components in the sample that may interfere with the detection of the desired analyte. Samples are purified herein by various means that allow for the removal of one or more interfering substances, e.g., one or more substances that would interfere with the detection of the parent and daughter ions of the selected testosterone by mass spectrometry.

[0034] As used herein, the term “test sample” refers to any sample that may contain testosterone. As used herein, the term “body fluid” refers to any fluid that can be isolated from the body of an individual. Examples of “body fluid” include blood, plasma, serum, bile, saliva, urine, tears, sweat, etc.

[0035] As used herein, the term “derivativeization” means reacting two molecules to form a novel molecule. Derivatizing agents may include isothiocyanate groups, dinitrofluorophenyl groups, nitrophenoxycarbonyl groups, and / or phthalaldehyde groups.

[0036] As used herein, the term "chromatography" refers to the process by which a chemical mixture carried by a liquid or gas flows around or over a stationary liquid or solid phase, resulting in the separation of its components as a result of the specific distribution of chemical entities.

[0037] As used herein, the term “liquid chromatography” or “LC” means the process of selective delay of one or more components of a fluid solution as a fluid uniformly permeates through a column of granulated material or through a capillary channel. The delay is due to the distribution of components of the mixture between one or more stationary phases and a bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Examples of “liquid chromatography” include reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and highly turbulent liquid chromatography (HTLC).

[0038] As used herein, the term “high-performance liquid chromatography” or “HPLC” refers to liquid chromatography in which the degree of separation is increased by passing a mobile phase through a stationary phase, typically a densely packed column, under pressure.

[0039] As used herein, the term “highly turbulent liquid chromatography” or “HTLC” refers to a form of chromatography that utilizes the turbulence of a substance being assayed through a column packing material as the basic principle for performing separation. HTLC has been applied to the preparation of samples containing two unspecified drugs prior to analysis by mass spectrometry. See, for example, Zimmer et al., J. Chromatogr. A 854: 23-35 (1999); also see U.S. Patents 5,968,367, 5,919,368, 5,795,469, and 5,772,874, which further describe HTLC. Those skilled in the art will understand “turbulence.” When a fluid flows slowly and smoothly, this flow is called “laminar flow.” For example, a fluid moving at a low velocity through an HPLC column is laminar flow. In laminar flow, the movement of fluid particles is orderly, and particles generally move linearly. At higher speeds, the inertia of water overcomes the frictional forces of the fluid, resulting in turbulence. Fluid not in contact with irregular boundaries "overtakes" fluid that has been slowed down by friction or deviated by a heterogeneous surface. When fluid is flowing in a turbulent state, it flows in a swirling (or vortex) manner and has more "resistance" than when the flow is laminar. Numerous references are available to help determine when a fluid flow is laminar or turbulent (e.g., 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)).

[0040] As used herein, the term “gas chromatography” or “GC” refers to chromatography in which a sample mixture is vaporized and injected into a stream of a carrier gas (such as nitrogen or helium) moving through a column containing a stationary phase consisting of liquid or particulate solids, and separated into its constituent compounds according to their affinity for the stationary phase.

[0041] As used herein, the terms “large particle column” or “extraction column” refer to a chromatography column containing an average particle size greater than approximately 35 μm. In this context, the term “approximately” means ±10%. In a preferred embodiment, the column contains particles with a diameter of approximately 60 μm.

[0042] As used herein, the term “analytical column” refers to a chromatography column having sufficient chromatographic plates to result in the separation of substances in a sample that elute from the column, enabling the determination of the presence or amount of an analyte. Such columns are often distinguished from “extraction columns,” which have the general purpose of separating or extracting retained substances from unretained substances in order to obtain a purified sample for further analysis. As used in this context, the term “about” means ±10%. In a preferred embodiment, the analytical column contains particles with a diameter of about 4 μm.

[0043] As used herein, the terms "online" or "inline," for example in "online automated method" or "online extraction," refer to procedures performed without operator intervention. In contrast, the term "offline," as used herein, refers to procedures requiring manual operator intervention. Therefore, if a sample undergoes precipitation and the supernatant is then manually loaded into an autosampler, the precipitation and loading steps are offline from the subsequent steps. In various embodiments of the method, one or more steps may be performed in an online automated method.

[0044] As used herein, the term “mass spectrometry” or “MS” refers to an analytical technique for identifying compounds by their mass. MS refers to a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or “m / z”. MS techniques generally include (1) ionizing a compound to form a charged compound, and (2) detecting the molecular weight of the charged compound and calculating its mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. “Mass spectrometer” generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized, and the ions are then introduced into a mass spectrographic apparatus, where, due to a combination of magnetic and electric fields, the ions travel along paths in space that depend on their mass ("m") and charge ("z"). For example, see U.S. Patent No. 6,204,500, titled "Mass Spectrometry From Surfaces," U.S. Patent No. 6,107,623, titled "Methods and Apparatus for Tandem Mass Spectrometry," U.S. Patent No. 6,268,144, titled "DNA Diagnostics Based On Mass Spectrometry," U.S. Patent No. 6,124,137, titled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes," Wright et al., Prostate Cancer and Prostatic Diseases 2:264-76 (1999), and Merchant and Weinberger, Electrophoresis 21:1164-67 (2000).

[0045] As used herein, the term "operating in anion mode" refers to a mass spectrometry method in which anions are generated and detected. As used herein, the term "operating in cation mode" refers to a mass spectrometry method in which cations are generated and detected.

[0046] As used herein, the term “ionization” or “the act of ionizing” refers to the process of producing an analyte ion having a net charge equal to one or more electron units. Anions are ions having a net negative charge equal to one or more electron units, while cations are ions having a net positive charge equal to one or more electron units.

[0047] As used herein, the terms “electron ionization” or “EI” refer to a method by which an analyte of interest interacts with a flow of electrons in the gaseous or vapor phase. The electron impact on the analyte generates analyte ions, which can then be subjected to mass spectrometry.

[0048] As used herein, the term “chemical ionization” or “CI” refers to a method by which a reagent gas (e.g., ammonia) is subjected to electron bombardment, and analyte ions are formed through the interaction of reagent gas ions with analyte molecules.

[0049] As used herein, the term “fast atomic impact” or “FAB” refers to a method in which a beam of high-energy atoms (often Xe or Ar) is struck against a non-volatile sample, thereby desorbing and ionizing molecules contained in the sample. Test samples are dissolved in viscous liquid matrices, such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine. The selection of a suitable matrix for a compound or sample is an experimental process.

[0050] As used herein, the term “matrix-assisted laser desorption / ionization” or “MALDI” refers to a method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample through various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. In the case of MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates the desorption of analyte molecules.

[0051] As used herein, the term “surface-enhanced laser desorption / ionization” or “SELDI” refers to an alternative method in which a non-volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample through various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. In SELDI, the sample is typically bonded to a surface that preferentially holds one or more analytes of interest. Similar to MALDI, this process may also utilize energy-absorbing materials to facilitate ionization.

[0052] As used herein, the term “electrospray ionization” or “ESI” refers to a method in which a solution passes along a short capillary tube to which a high positive or negative potential is applied at the end of the capillary tube. The solution that reaches the end of the tube is vaporized (atomized) into a jet or spray of very small droplets of the solution in solvent vapor. This mist of droplets flows through an evaporation chamber that is slightly heated to prevent condensation and to evaporate the solvent. As the droplets become smaller, the electrical surface charge density increases, and eventually, natural repulsion between like charges causes the release of ions and neutral molecules.

[0053] As used herein, the term “atmospheric pressure chemical ionization” or “APCI” refers to a mass spectrometry similar to ESI; however, APCI generates ions by ion-molecular reactions occurring in a plasma at atmospheric pressure. The plasma is maintained by a discharge between a spray capillary and a counter electrode. The ions are then extracted into a mass spectrometer, typically by the use of a series of differentially pumped skimmer stages. Solvent removal can be improved by using countercurrent of dried and preheated N2 gas. For analyzing less polar species, gas-phase ionization in APCI may be more effective than ESI.

[0054] The term "atmospheric pressure photoionization" or "APPI," as used herein, refers to a form of mass spectrometry in which the mechanism for the photoionization of a molecule M is photon absorption and electron emission, forming a molecular ion M+. Since the photon energy is generally just above the ionization potential, molecular ions are not very easily dissociated. In many cases, it may be possible to analyze a sample without requiring chromatography, thus saving considerable time and expense. In the presence of water vapor or a protic solvent, the molecular ion can draw out H to form MH+. This tends to occur when M has a high proton affinity. Since the sum of M+ and MH+ is constant, this does not affect the accuracy of quantification. Drug compounds in protic solvents are usually observed as MH+, while nonpolar compounds, such as naphthalene or testosterone, usually form M+. See, for example, Robb, DB, Covey, TR and Bruins, AP (2000): Robb et al., Atmospheric pressure photoionization: An ionization method for liquid chromatography-mass spectrometry. Anal. Chem. 72(15): 3653-3659.

[0055] As used herein, the term “inductively coupled plasma” or “ICP” refers to a method by which a sample interacts with a partially ionized gas at a sufficiently high temperature such that most elements are atomized and ionized.

[0056] As used herein, the term “field desorption” refers to a method in which a non-volatile test sample is placed on an ionization surface and a strong electric field is used to generate analyte ions.

[0057] As used herein, the term “desorption” refers to the removal of an analyte from a surface and / or the entry of an analyte into the gas phase.

[0058] As used herein, the terms “limit of quantification,” “limit of quantitation,” or “LOQ” refer to the point at which the measured value becomes quantitatively meaningful. The response of the analyte at this LOQ is verifiable, individual, and reproducible with a precision of 20% and accuracy of 80% to 120%.

[0059] As used herein, the term “Limit of Detection” or “LOD” is the point at which a measured value exceeds the associated uncertainty. The LOD is arbitrarily defined as two standard deviations (SD) from zero concentration.

[0060] As used herein, the “amount” of testosterone in a body fluid sample generally refers to the absolute value reflecting the detectable mass of testosterone in the volume of body fluid. However, the amount also intends to refer to a relative amount compared to another amount of testosterone. For example, the amount of testosterone in body fluid may be above or below the control or normal levels of testosterone normally present.

[0061] The term "approximately" as used herein in relation to quantitative measurements that do not include ion mass measurements refers to ±10% of the indicated value. Mass spectrometers may vary slightly in determining the mass of a given analyte. The term "approximately" in the context of ion mass or ion mass / charge ratio refers to ±0.5 atomic mass units.

[0062] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) offers improved accuracy compared to previously used techniques for measuring testosterone. High-throughput LC-MS / MS is achieved through column multiplexing, where multiple samples are run in parallel. In this specification, the applicant's goal was to further increase throughput by combining at least two patient samples per LC-MS / MS run. The principle of “sample multiplexing” involves using at least two derivatizing agents of different masses. Patient samples are individually tagged with one or the other derivatizing agent. Results for each patient are identified by the mass of characteristic fragments of the derivatizing agent, thereby yielding two separate results in a single LC-MS / MS run.

[0063] In a particular embodiment, a method is provided for detecting the amount of testosterone in each of several patient samples within a single mass spectrometry assay. The method includes the steps of: processing each patient sample in a different manner to form several processed samples, such that, as a result of the processing, the testosterone in each processed sample is distinguishable by mass spectrometry from the testosterone in the other processed samples; combining the processed samples to form multiple samples; exposing the multiple samples to an ionization source under conditions suitable for producing one or more ions detectable by mass spectrometry, such that one or more ions produced from the testosterone in each processed sample are different from one or more ions from the testosterone in the other processed samples; detecting the amount of one or more ions from the testosterone in each processed sample by mass spectrometry; and relating the amount of one or more ions from the testosterone in each processed sample to the amount of testosterone in each patient sample.

[0064] In a particular embodiment, the method comprises determining the amount of testosterone in each of several human samples by a single mass spectrometry assay, the method comprising: i) exposing each of the several human samples to a different derivatizing agent to produce testosterone derivatized in each of the several samples in a different manner; ii) combining the several samples to form a multiple sample; and iii) quantifying the amount of testosterone in each sample by mass spectrometry.

[0065] In some embodiments, the method comprises determining the amount of testosterone in two human samples by a single mass spectrometry assay, the method comprising: i) exposing each of the two human samples to a different derivatizing agent to produce testosterone derivatized in each of the two samples in different ways; ii) combining the two samples to form a multiple sample; and iii) quantifying the amount of testosterone in each sample by mass spectrometry.

[0066] In certain embodiments, the derivatizing agent includes ethoxyamine or methoxyamine.

[0067] In some embodiments, the method is fully automated.

[0068] In some embodiments, the method is antibody-free.

[0069] In some embodiments, purification involves extracting serum using solid-phase extraction (SPE). In some embodiments, the SPE is anion exchange solid-phase extraction. In some embodiments, the SPE is mixed-mode anion exchange solid-phase extraction. In some embodiments, the extracted sample is concentrated.

[0070] Purification may include liquid chromatography. In some embodiments, liquid chromatography includes high-performance liquid chromatography (HPLC). In some embodiments, liquid chromatography includes highly turbulent liquid chromatography (HTLC).

[0071] Ionization may include heated electrospray ionization (HESI). In preferred embodiments, ionization includes ionization in positive mode. In some embodiments, ionization includes ionization in negative mode.

[0072] Ionization may include atmospheric pressure chemical ionization (APCI). In some embodiments, ionization includes ionization in positive mode. In some embodiments, ionization includes ionization in negative mode.

[0073] In some embodiments, the method includes measuring the amount of a precursor ion having a mass-to-charge ratio of 332.2 ± 0.5 for ethoxyamine-derivative testosterone. In some embodiments, the method includes measuring the amount of a precursor ion having a mass-to-charge ratio of 318.21 ± 0.5 for methoxylamine-derivative testosterone.

[0074] In some embodiments, the method includes measuring the amount of fragment ions having a mass-to-charge ratio of 140.02±0.5 or 152.02±0.5 for ethoxyamine-derivative testosterone. In some embodiments, the method includes measuring the amount of fragment ions having a mass-to-charge ratio of 126.07±0.5, 138.07±0.5, or 152.08±0.5 for methoxylamine-derivative testosterone.

[0075] The method may further include adding an internal standard. In some embodiments, the internal standard is labeled with an isotope. In some embodiments, the internal standard is testosterone-2,3,4- 13 It is C3.

[0076] The method may include measuring the amount of an internal standard precursor ion having a mass-to-charge ratio of 335.2 ± 0.5 (ethoxyamine derivatized) or 321.21 ± 0.5 (methoxylamine derivatized). The method may also include measuring the amount of an internal standard fragment ion having a mass-to-charge ratio of 143.03 ± 0.5 (ethoxyamine derivatized) or 129.07 ± 0.5 (methoxylamine derivatized).

[0077] In certain embodiments, sample multiplexing is at least twice as fast as single assay or column multiplexing. In certain embodiments, sample multiplexing is at least three times faster than single assay. In certain embodiments, sample multiplexing is at least four times faster than single assay.

[0078] In certain embodiments, the limit of quantification of the method is 10 ng / dL or less. In some embodiments, the limit of quantification of the method is 5 ng / dL or less. In some embodiments, the limit of quantification of the method is 4 ng / dL or less. In some embodiments, the limit of quantification of the method is 3 ng / dL or less. In some embodiments, the limit of quantification of the method is 2 ng / dL or less. In some embodiments, the limit of quantification of the method is 1 ng / dL or less.

[0079] In some embodiments, the method includes linearity of quantification from 1 ng / dL to 2,000 ng / dL.

[0080] In some embodiments, the method includes measurement inaccuracy (CV) at concentrations of 8 to 1,200 ng / dL, with concentrations of 1% to 11%, 1% to 9%, or 2% to 11%.

[0081] In some embodiments, the method includes a recovery rate of 90% to 110%, or between 95% and 105%.

[0082] In some embodiments, the method includes a clinically reportable range (CRR) of up to 10,000 ng / dL.

[0083] In certain embodiments, the sample is a body fluid. In some embodiments, the sample is plasma or serum. In some embodiments, the sample is whole blood. In some embodiments, the sample is saliva or urine. In some embodiments, the sample is cerebrospinal fluid (CSF).

[0084] In some embodiments, the method may include adding a drug to the sample in an amount sufficient to remove the protein from the sample.

[0085] A preferred test sample may include any test sample that may contain the analyte of interest. In some preferred embodiments, the sample is a biological sample, i.e., a sample obtained from any biological source such as an animal, cell culture, or organ culture. In certain preferred embodiments, the sample may be obtained from a mammal such as a dog, cat, or horse. Particularly preferred mammals are primates, most preferably male or female humans. Particularly preferred samples include blood, plasma, serum, hair, muscle, urine, saliva, tears, cerebrospinal fluid, or other tissue samples. Such samples may be obtained, for example, from a patient, i.e., a living male or female human who has appeared in a clinical setting for the diagnosis, prognosis, or treatment of a disease or condition. The test sample is preferably obtained from a patient, for example, from serum.

[0086] Sample preparation for mass spectrometry Methods that may be used to enrich testosterone compared to other components in a sample (e.g., proteins) include, for example, filtration, centrifugation, thin-layer chromatography (TLC), electrophoresis, e.g., capillary electrophoresis, affinity separation, e.g., immunoaffinity separation, extraction methods, e.g., ethyl acetate extraction and methanol extraction, and the use of chaotropic agents, or any combination of the above.

[0087] Protein precipitation is one preferred method for preparing test samples. Such protein purification methods are well known in the art, and for example, Polson et al., Journal of Chromatography B 785:263-275 (2003) describes a protein precipitation technique suitable for use in this method. Protein precipitation can be used to remove most of the protein from a sample, leaving testosterone in the supernatant. The sample may be centrifuged to separate the liquid supernatant from the precipitated protein. The resulting supernatant may then be subjected to liquid chromatography and subsequent mass spectrometry. In certain embodiments, the use of protein precipitation, e.g., acetonitrile protein precipitation, eliminates the need for high-turbulence liquid chromatography (HTLC) or other online extraction before HPLC and mass spectrometry. Thus, in such embodiments, the method includes (1) performing protein precipitation of the sample of interest, and (2) directly loading the supernatant into an HPLC-mass spectrometer without using online extraction or high-turbulence liquid chromatography (HTLC).

[0088] In some preferred embodiments, HPLC may be used alone or in combination with one or more purification methods to purify testosterone before mass spectrometry. In such embodiments, the sample is extracted using an HPLC extraction cartridge that captures the analyte, then eluted and chromatographed on a second HPLC column or on an HPLC column for pre-ionization analysis. Since the steps involved in these chromatographic procedures can be linked in an automated manner, the need for operator involvement during analyte purification can be minimized. This feature results in time and cost savings and eliminates opportunities for operator error.

[0089] For example, turbulence, such as that provided by HTLC columns and methods, is thought to improve mass transfer rates and thus enhance separation characteristics. HTLC columns separate components by high chromatographic flow rates through a packed column containing rigid particles. By utilizing high flow rates (e.g., 3-5 mL / min), turbulence is generated in the column, which causes nearly perfect interaction between the stationary phase and the target analyte(s). An advantage of using HTLC columns is that high molecular weight species are not retained under turbulent conditions, thus avoiding the accumulation of macromolecules associated with the biological fluid matrix. HTLC methods, which combine multiple separations in a single procedure, reduce the need for lengthy sample preparation and operate at significantly higher speeds. Such methods also achieve superior separation performance compared to laminar flow (HPLC) chromatography. HTLC allows for the direct injection of biological samples (plasma, urine, etc.). Direct injection is difficult to achieve with conventional chromatography because denatured proteins and other biological debris rapidly block the separation column. HTLC also allows for very small sample volumes of less than 1 mL, preferably less than 0.5 mL, preferably less than 0.2 mL, and preferably 0.1 mL.

[0090] Examples of HTLC applications for sample preparation prior to analysis by mass spectrometry are described elsewhere. See, for example, Zimmer et al., J. Chromatogr. A 854:23-35 (1999); also see U.S. Patents 5,968,367, 5,919,368, 5,795,469, and 5,772,874. In certain embodiments, the sample undergoes protein precipitation as described above before loading onto the HTLC column; in alternative preferred embodiments, the sample may be loaded directly onto the HTLC without protein precipitation. The HTLC extraction column is preferably a large particle column. In various embodiments, one or more steps of the method may be carried out in an online automated manner. For example, in one embodiment, steps (i) to (v) are carried out in an online automated manner. In another case, the ionization and detection steps are carried out online after steps (i) to (v).

[0091] Liquid chromatography (LC), including high-performance liquid chromatography (HPLC), relies on relatively slow laminar flow techniques. Conventional HPLC analysis relies on column packing materials, where the laminar flow of the sample through the column is the fundamental principle for separating the target analyte from the sample. Those skilled in the art understand that separation in such columns is a diffusion process. While HPLC has been successfully applied to the separation of compounds in biological samples, a considerable amount of sample preparation is required before separation and subsequent analysis by mass spectrometry (MS), making the technique labor-intensive. Furthermore, most HPLC systems do not fully utilize the capabilities of the mass spectrometer, and a single HPLC system can only be connected to a single MS instrument, resulting in long processing times for performing numerous assays.

[0092] Various methods for using HPLC for sample cleanup before mass spectrometry are described. For example, see Taylor et al., Therapeutic Drug Monitoring 22:608-12 (2000) and Salm et al., Clin. Therapeutics 22 Supl. B:B71-B85 (2000).

[0093] Those skilled in the art can select HPLC apparatus and columns suitable for use with testosterone. Chromatographic columns generally contain a medium (i.e., packing material) to facilitate the separation (i.e., fractionation) of chemical parts. The medium may contain fine particles. The particles contain binding surfaces that interact with various chemical parts to facilitate their separation. One suitable binding surface is a hydrophobic binding surface, such as an alkyl binding surface. Alkyl binding surfaces may contain C-4, C-8, C-12, or C-18 binding alkyl groups, preferably C-18 binding groups. Chromatographic columns include an injection port for receiving a sample and an exhaust port for discharging effluent containing the fractionated sample. In one embodiment, a sample (or a pre-purified sample) is applied to the column at the injection port, eluted with a solvent or solvent mixture, and discharged at the exhaust port. Various solvent modes may be selected to elute the desired analyte(s). For example, liquid chromatography may be performed using gradient mode, isocratic mode, or polymorphic (i.e., mixed) mode. During chromatography, the separation of materials is achieved by variable factors such as the selection of the eluent (also known as the "mobile phase"), the elution mode, gradient conditions, and temperature.

[0094] In certain embodiments, the analyte may be purified by applying the sample to a column under conditions in which the analyte of interest is reversibly retained by the column packing material, while one or more other substances are not retained. In these embodiments, a first mobile phase condition may be used in which the analyte of interest is retained by the column, and then a second mobile phase condition may be used in which the retained substances are removed from the column after the unretained substances have been washed out. Alternatively, the analyte may be purified by applying the sample to a column under mobile phase conditions in which the analyte of interest elutes at a different rate compared to one or more other substances. Such a procedure may enrich the amount of one or more analytes of interest compared to one or more other components of the sample.

[0095] In one preferred embodiment, HTLC may be followed by HPLC on a hydrophobic column chromatography system. In a particular preferred embodiment, a TurboFlow Cyclone P® polymer-based column from Cohesive Technologies (particle size 60 μm, column dimensions 50 × 1.0 mm, pore size 100 Å) is used. In a related preferred embodiment, a Synergi Polar-RP® ether-conjugated phenyl analytical column from Phenomenex Inc. with hydrophilic end capping (particle size 4 μm, column dimensions 150 × 2.0 mm, pore size 80 Å) is used. In a particular preferred embodiment, HTLC and HPLC are performed using HPLC-grade ultrapure water and 100% methanol as mobile phases.

[0096] Through careful selection of valves and connector piping, two or more chromatography columns can be connected as needed, allowing substances to pass from one column to the next without requiring any manual steps. In a preferred embodiment, the selection of valves and piping is controlled by a computer pre-programmed to perform the necessary steps. Most preferably, the chromatography system is also connected to a detector system, such as an MS system, in such an online manner. Thus, the operator can place the sample trays into the autosampler, and the rest of the work is performed under computer control, resulting in the purification and analysis of all selected samples.

[0097] In certain preferred embodiments, testosterone or its fragments in the sample may be purified before ionization. In particularly preferred embodiments, the chromatography is not gas chromatography.

[0098] Detection and quantification by mass spectrometry In various embodiments, testosterone or its fragments can be ionized by any method known to those skilled in the art. Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing the fractionated sample and producing charged molecules for further analysis. For example, sample ionization can be performed by electron ionization, chemical ionization, heated electrospray ionization (HESI), electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atomic bombardment (FAB), liquid secondary ionization (LSI), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, surface-enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP), and particle beam ionization. Those skilled in the art will understand that the selection of the ionization method can be determined based on the analyte to be measured, the type of sample, the type of detector, the selection of positive mode versus negative mode, etc.

[0099] In a preferred embodiment, testosterone or its fragments are ionized by heated electrospray ionization (HESI) in cation mode.

[0100] After the sample is ionized, the resulting positively or negatively charged ions can be analyzed to determine the mass-to-charge ratio. Suitable analyzers for determining the mass-to-charge ratio include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. Ions can be detected using several detection modes. For example, selected ions can be detected using selective ion monitoring mode (SIM), or ions can be detected using scanning modes, such as multiple reaction monitoring (MRM) or selective reaction monitoring (SRM). Preferably, the mass-to-charge ratio is determined using a quadrupole analyzer. For example, in a "quadrupole" or "quadrupole ion trap" apparatus, ions in an oscillatory radio frequency field experience a force proportional to the DC potential applied between the electrodes, the amplitude of the RF signal, and the mass / charge ratio. The voltage and amplitude can be selected so that only ions with a particular mass / charge ratio move along the length of the quadrupole, while all other ions are deflected. Therefore, a quadrupole-type device can act as both a "mass filter" and a "mass detector" for ions injected into the device.

[0101] The resolution of MS techniques can be enhanced by utilizing "tandem mass spectrometry" or "MS / MS." In this technique, precursor ions (also called parent ions) generated from the target molecule can be filtered within the MS instrument. The precursor ions are then fragmented, yielding one or more fragment ions (also called daughter ions or product ions), which are then analyzed in a second MS procedure. Careful selection of the precursor ions ensures that only ions generated by a particular analyte proceed to the fragmentation chamber, where fragment ions are generated through collisions with atoms of an inert gas. Since both precursor and fragment ions are reproducibly generated under a given set of ionization / fragmentation conditions, MS / MS techniques can provide an extremely powerful analytical tool. For example, the filtering / fragmentation combination can be used to eliminate interfering substances and may be particularly useful in complex samples, such as biological samples.

[0102] Mass spectrometers generally provide the user with an ion scan, i.e., the relative abundance of each ion with a specific mass / charge over a given range (e.g., 100–1000 amu). The results of an analytic assay, i.e., a mass spectrum, can be correlated with the amount of analytic in the original sample by many methods known in the art. For example, given that sampling and analytical parameters are carefully controlled, the relative abundance of a given ion can be compared to a table that converts its relative abundance to the absolute amount of the original molecule. Alternatively, a molecular standard can be run with the sample, and a standard curve can be constructed based on the ions generated from that standard. Using such a standard curve, the relative abundance of a given ion can be converted to the absolute amount of the original molecule. In a particular preferred embodiment, an internal standard is used to generate a standard curve for calculating the amount of testosterone. Methods for generating and using such standard curves are well known in the art, and those skilled in the art have the ability to select a suitable internal standard. For example, isotopes of testosterone can be used as internal standards. Many other methods for relating the amount of an ion to the amount of the original molecule are well known to those skilled in the art.

[0103] One or more steps of the method may be carried out using automated machinery. In certain embodiments, one or more purification steps may be carried out online, and more preferably, all purification and mass spectrometry steps may be carried out online.

[0104] In certain embodiments, such as MS / MS where a precursor ion is isolated for further fragmentation, collision-activated dissociation is often used to generate fragment ions for further detection. In CAD, the precursor ion gains energy through collisions with an inert gas and is then fragmented by a process called "monomolecular dissociation." Sufficient energy must be accumulated in the precursor ion so that certain bonds within the ion can be broken due to the increase in vibrational energy.

[0105] In a particularly preferred embodiment, testosterone is detected and / or quantified using MS / MS as follows: The sample is subjected to liquid chromatography, preferably HPLC, where the flow of liquid solvent from the chromatography column enters a heated nebulizer interface of the MS / MS analyzer, and the solvent / analyte mixture is converted to vapor in the heated tube of the interface. The analyte is ionized by a selected ionizer. Ions, e.g., precursor ions, pass through the opening of the apparatus and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters that allow selection of ions (i.e., "precursor" ions and "fragment" ions) based on their mass-to-charge ratio (m / z). Quadrupole 2 (Q2) is a collision cell where ions are fragmented. The first quadrupole (Q1) of the mass spectrometer selects molecules having the mass-to-charge ratio of testosterone. Precursor ions with the correct mass / charge ratio of testosterone are allowed to proceed to the collision chamber (Q2), while undesirable ions with any other mass / charge ratio collide with the sides of the quadrupole and are eliminated. Precursor ions entering Q2 collide with neutral argon gas molecules and fragment. This process is called collision-activated dissociation (CAD). The resulting fragment ions proceed to quadrupole 3 (Q3), where testosterone fragment ions are selected, while other ions are eliminated.

[0106] This method may include MS / MS performed in cation mode or anion mode. Using standard methods well known in the art, those skilled in the art have the ability to identify one or more fragment ions of specific testosterone precursors that can be used for selection in quadrupole 3 (Q3).

[0107] When an ion collides with a detector, it generates a pulse of electrons that is converted into a digital signal. The acquired data is sent to a computer, which plots the count of collected ions against time. The resulting mass chromatogram is similar to that produced by conventional HPLC. The area under the peak, or the amplitude of such a peak, corresponding to a particular ion is measured, and the area or amplitude is associated with the amount of the analyte of interest. In certain embodiments, the area under the curve or the amplitude of the peak for fragment ions and / or precursor ions is measured to determine the amount of testosterone. As described above, using a calibration standard curve based on the peaks of one or more ions of an internal molecular standard, the relative abundance of a given ion can be converted to the absolute amount of the original analyte. [Examples]

[0108] The following examples are helpful in illustrating the present invention. These examples are not intended to limit the scope of the method in any way.

[0109] Examples Example 1: Multiplexed testosterone quantification by mass spectrometry Healthy male and female donor samples were collected in serum isolation tubes (SSTs) and red-top tubes. Before protein precipitation, the serum was treated with stable isotope-labeled testosterone (e.g., testosterone-2,3,4-) as an internal standard (ISTD). 13The mixture was combined with C3). Testosterone in the supernatant was derivatized, enriched by solid-phase extraction, dried, and reconstituted. The sample was injected into an HTLC system for online extraction, transferred to an analytical column, and eluted using a two-component gradient. Testosterone derivatives and ISTD were detected and analyzed using a triple quadrupole mass spectrometer (Thermo) with HESI. Ionization and multiple reaction monitoring (MRM) scan parameters were optimized for maximizing ion permeability and for highly sensitive, specific, and stable quantification. MRM transitions were used as quantifiers and qualifiers for both testosterone derivatives. Ion suppression using T-column injection, sample stability by storage in SST tubes at 4°C, isobaric interference from dehydroepiandrosterone (DHEA), and carryover were also evaluated.

[0110] The samples to be analyzed were first divided into two sets, identified as Sample Plate A and Sample Plate B. Testosterone was then extracted from these samples using direct-in-plate protein precipitation and filtration in a 96-well plate format. This step was automated on a Hamilton STAR pipetting workstation by adding serum to each well of the 96-well protein precipitation plate. The Hamilton STAR then dispensed a precipitation reagent containing an internal standard (IS) [a stable isotope for testosterone] into each well. The fixed and stacked plates were vortexed and spun down in a centrifuge until all supernatant settled in the collection plate. Alternatively, direct protein precipitation may be performed by adding serum to a 96-deep-well plate and then adding the IS on top; the sample was then vortexed, centrifuged, and the clear supernatant transferred to a new 96-deep-well plate. Both sample plates were then derivatized independently.

[0111] Sample plate A is derivatized by the addition of methoxyamine hydrochloride, while sample plate B is derivatized by the addition of ethoxyamine hydrochloride. Both sample plates are incubated; the reaction is stopped by cooling. After derivatization, solid-phase extraction is performed automatically on a SPEware IP8 system connected to Hamilton STAR using a Strata C18 solid-phase extraction plate. The two derivatizations (sample plate A and sample plate B) are combined in this step. After elution of the analytes from the SPE plate to a deep-well collection plate, the samples are dried under nitrogen and reconstituted in a reconstitution solvent.

[0112] A 40 μL prepared sample is injected into a Thermo Fisher Scientific highly turbulent liquid chromatography (HTLC) system. The HTLC system can be logically divided into two functions: 1) solid-phase extraction using a large particle size (70 μm) packed column, and 2) conventional HPLC chromatography using a two-component gradient and a 2.5 μm reversed-phase analytical column.

[0113] In the solid-phase extraction mode of the HTLC system, the sample is first pumped at high flow rate through the Phenomenex extraction column using an HTLC loading pump. The high flow rate creates turbulence within the extraction column. This turbulence ensures optimal binding of differentially derivatized testosterone to larger particles in the column and the transfer of residual proteins and debris to waste. After this loading step, the flow is reversed to elute the sample from the extraction column and transfer it to a Phenomenex Synergi® Max-RP C12 with a TMS end-capping analysis column.

[0114] In the HTLC analysis mode, a two-component HPLC gradient is applied to the analytical column, resulting in the differential separation of derivatized testosterone from other analytes in the sample. The separated sample is then transferred to MS / MS, where it is differentially detected.

[0115] The MS is heated electrospray ionization (HESI), positive mode. A stream of liquid solvent from the HTLC enters the ThermoFisher LC-MS / MS analyzer, where a voltage is applied to the liquid held in the nozzle; a Taylor cone is formed and electrospray is initiated. As the liquid leaves the electrode tip, the solvent evaporates in flight. As ions pass through the opening, the vacuum draws the ions into the instrument, where the quadrupole isolates the analyte of interest. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowing ion selection based on their mass-to-charge ratio (m / z). Quadrupole 2 (Q2) is a collision cell, where ions are fragmented. The first quadrupole of the MS / MS (Q1) selects molecules with the mass-to-charge ratio of derivatized testosterone. Ions with this m / z are allowed to proceed to the collision chamber (Q2), while undesirable ions with any other m / z collide with the sides of the quadrupole and are destroyed. Ions entering Q2 collide with neutral gas molecules and fragment. This process is called collision-activated dissociation (CAD). The CAD gas used in this procedure is argon. The generated fragment ions proceed to quadrupole 3 (Q3), where the fragment ions of derivatized testosterone are selected, while other ions are selectively removed. The same process is performed for an internal standard.

[0116] Quantitative analysis is based on the unique parent-product transition. The following precursor-fragment pairs or "mass transitions" are used:

[0117] [Table 1]

[0118] The performance of a multiplexed derivatized testosterone assay was compared in parallel with results from sample waste accumulated from an existing testosterone assay (Salameh, 2010). 259 remaining specimen samples were assayed using the current method for testosterone detection by LC-MS / MS.

[0119] The intra-assay and inter-assay coefficients of variation (CV%) for total testosterone levels were less than 5% for both testosterone derivatives at 12, 80, 250, and 1,200 ng / dL. The limit of quantification (LOQ) for both derivatives was less than 1 ng / dL. Calibration linearity was confirmed between 2.5 and 2,000 ng / dL. Based on a comparison of their quantitative results, both agents were equivalent in the derivatization and recovery of testosterone from serum (R=0.991, bias=-3.8 ng / dL). No interference was observed in serum collected during SST using the new derivatizer. Serum samples collected in SST and red-top tubes from the same donor gave equivalent results (R=0.991, bias=0.2 ng / dL). Ion suppression was calculated to be less than 20% for both derivatives. The CV% of the ion ratio for both derivatives was less than 15% and was stable across the entire range. Testosterone levels remained stable during SST for 72 hours (at 4°C) without serum separation from the gel. No isobaric interference (due to DHEA) or carryover was observed.

[0120] Conclusion: The inventors have developed and validated an improved multiplexed HTLC-HESI-MS / MS testosterone assay using a novel derivatizing agent. The current method enables accurate, rapid, specific, and stable analysis of serum total testosterone. SST is an acceptable sample type for the current method. Precision, accuracy, linearity, ion suppression, and sample stability in SST were confirmed.

[0121] The content of any papers, patents, and patent applications, as well as all other documents and electronically available information, referenced or cited herein is incorporated herein in whole by reference to the same extent that individual publications are shown to be incorporated specifically and individually by reference. The applicant reserves the right to physically incorporate into this application any and all material and information derived from any such papers, patents, patent applications, or other physical and electronic documents.

[0122] The methods described herein exemplary can be suitably practiced in the absence of any one or more elements, one or more limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and non-restrictively. Furthermore, the terms and expressions used herein are used as descriptive terms, not restrictive terms, and in the use of such terms and expressions, there is no intention to exclude any equivalent or part thereof of the features presented and described. It is recognized that various modifications are possible within the scope of the claimed invention. Therefore, although the invention is specifically disclosed by preferred embodiments and optional features, modifications and alterations of the invention that are incorporated herein can be used by those skilled in the art, and such modifications and alterations should be understood to be within the scope of the invention.

[0123] The present invention is described extensively and comprehensively herein. Each of the narrower species and subgenus groupings included in the general disclosure also forms part of the method. This includes a general description of the method with conditions or negative limitations for removing any subject matter from a genus, whether or not the removed subject matter is specifically enumerated herein.

[0124] Other embodiments are included in the following claims. Furthermore, where any feature or aspect of the Method is described in relation to the Markush group, a person skilled in the art will recognize that the invention is thus described in relation to any individual member or subgroup of members of the Markush group.

Claims

1. A method for determining the amount of testosterone in each of several human samples using a single mass spectrometry assay, The first human sample is derivatized with ethoxyamine chloride to produce ethoxyamine-derivative testosterone in the first human sample. The second human sample is derivatized with methoxylamine chloride to produce methoxylamine-derivative testosterone in the second human sample. The steps include combining two samples to form a multiple sample, and A step to quantify the amount of testosterone in each sample by mass spectrometry. Methods that include...

2. The method according to claim 1, wherein the method further comprises liquid chromatography.

3. The method according to claim 2, wherein the liquid chromatography includes high-performance liquid chromatography (HPLC).

4. The method according to claim 2, wherein the liquid chromatography includes highly turbulent liquid chromatography (HTLC).

5. The method according to any one of claims 1 to 4, wherein the ionization includes heated electrospray ionization (HESI).

6. The method according to any one of claims 1 to 5, wherein the ionization is in a positive ion mode.

7. The method according to any one of claims 1 to 6, wherein the method comprises HTLC and HESI.

8. The method according to any one of claims 1 to 7, wherein the method comprises measuring the amount of one or more precursor ions, the one or more precursor ions comprising ions having a mass-to-charge ratio of 332.2 ± 0.5 or 318.21 ± 0.

5.

9. The method according to any one of claims 1 to 8, wherein the method comprises measuring the amount of one or more fragment ions, the one or more fragment ions comprising ions having a mass-to-charge ratio of 140.02±0.5, 152.02±0.5, 126.07±0.5, 138.07±0.5, or 152.08±0.

5.

10. The method according to any one of claims 1 to 9, further comprising adding an internal standard.

11. The method according to claim 10, wherein the internal standard is labeled with an isotope.

12. The method according to claim 10 or 11, wherein the method comprises measuring the amount of one or more internal standard precursor ions, the one or more internal standard precursor ions comprising ions having a mass-to-charge ratio of 335.2 ± 0.5 or 321.21 ± 0.

5.

13. The method according to any one of claims 10 to 12, wherein the method comprises measuring the amount of one or more internal standard fragment ions, the one or more internal standard fragment ions comprising ions having a mass-to-charge ratio of 143.03 ± 0.5 or 129.07 ± 0.

5.

14. The method according to any one of claims 1 to 13, wherein the quantitative limit of the method is 1 ng / dL or less.

15. The method according to any one of claims 1 to 14, wherein the method has linearity in quantitative determination of 1 ng / dL to 2,000 ng / dL.

16. The method according to any one of claims 1 to 15, wherein the method is fully automated.

17. The method according to any one of claims 1 to 16, wherein the method does not include an antibody.

18. The method according to any one of claims 1 to 17, wherein the human sample is serum.

19. The method according to any one of claims 1 to 17, wherein the human sample is plasma.

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