Determination of Testosterone in Multiplexed Patient Samples by Mass Spectrometry

Abstract

To provide methods for high throughput quantitation of testosterone utilizing at least two different derivatizing agents of different masses.SOLUTION: A method for determining the amount of testosterone in each of multiple human samples with a single mass spectrometric assay comprises: i) subjecting each of the multiple human samples to a different derivatizing agent to generate an individually derivatized testosterone in each of the multiple samples, where the derivatizing agents include hydroxylamine chloride and methoxylamine chloride; ii) combining the multiple samples to form a multiplexed sample; iii) purifying the multiple samples with solid phase extraction; and iv) quantifying the amount of testosterone in each sample by mass spectrometry.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - reference to related patent applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 644,351, filed Mar. 16, 2018, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Testosterone (4 - androstene - 17β - ol - 3 - one) is a steroid hormone and is the primary androgen in males. Major causes of testosterone decline in males include hypogonadotropic hypogonadism, testicular insufficiency, hyperprolactinemia, hypopituitarism, several types of liver and kidney diseases, and significant illnesses.

[0003] In females, if androgen levels are normal, they can provide a basis for estrogen production. When serum testosterone levels increase in females, it can suggest various conditions, particularly polycystic ovary syndrome and adrenal hyperplasia. Clinical symptoms of testosterone excess in females include infertility, hirsutism, amenorrhea, and obesity.

[0004] An accurate and efficient measurement of testosterone is required.

Summary of the Invention

[0005] Provided herein is a method for high - throughput quantification of testosterone using at least two different derivatizing agents of different masses.

[0006] In certain embodiments, provided herein is a method for detecting the amount of testosterone in each of a plurality of patient samples within a single mass spectrometry assay. The method comprises separately processing each patient sample to form a plurality of 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 a multiplexed sample; subjecting the multiplexed sample to an ion source under conditions suitable to generate one or more ions detectable by mass spectrometry, wherein one or more ions generated from the testosterone derived from each processed sample are different from one or more ions of testosterone derived from the other processed samples; detecting by mass spectrometry the amount of one or more ions obtained from the testosterone derived from each processed sample; and correlating the amount of one or more ions obtained from the testosterone derived from each processed sample with the amount of testosterone in each patient sample.

[0007] In certain embodiments, the method provided herein comprises determining the amount of testosterone in each of a plurality of human samples in a single mass spectrometry assay, the method comprising: i) subjecting each of the plurality of human samples to a different derivatizing agent to generate individually derivatized testosterone in each of the plurality of samples; ii) combining the plurality of samples to form a multiplexed sample; and iii) quantifying by mass spectrometry the amount of testosterone in each sample.

[0008] In some embodiments, the methods provided herein include determining the amount of testosterone in two human samples in a single mass spectrometry assay, the method comprising: i) subjecting each of the two human samples to a different derivatizing agent to produce derivatized testosterone individually in each of the two samples; ii) combining the two samples to form a multiplexed sample; and iii) quantifying the amount of testosterone in each sample by mass spectrometry.

[0009] In certain embodiments, the derivatizing agents provided herein include hydroxylamine or methoxyamine.

[0010] In some embodiments, the methods provided herein are fully automated.

[0011] In some embodiments, the methods provided herein are antibody-free methods.

[0012] In some embodiments, the purification provided herein includes extraction of 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 sample after extraction is concentrated.

[0013] In some embodiments, the purification provided herein includes liquid chromatography. In some embodiments, the liquid chromatography includes high performance liquid chromatography (HPLC). In some embodiments, the liquid chromatography includes high turbulence liquid chromatography (HTLC).

[0014] In preferred embodiments, ionization includes heated electrospray ionization (HESI). In preferred embodiments, 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 methods provided herein include measuring the amount of precursor ions having a mass-to-charge ratio of 304.18 ± 0.5 for hydroxylamine-derivatized testosterone. In some embodiments, the methods provided herein include measuring the amount of precursor ions having a mass-to-charge ratio of 318.21 ± 0.5 for methoxylamine-derivatized testosterone.

[0017] In some embodiments, the methods provided herein include measuring the amount of fragment ions having a mass-to-charge ratio of 112.05 ± 0.5 or 124.05 ± 0.5 for hydroxylamine-derivatized testosterone. In some embodiments, the methods provided herein include 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-derivatized testosterone.

[0018] In some embodiments, the methods provided herein further include adding an internal standard. In some embodiments, the internal standard is labeled with an isotope.

[0019] In some embodiments, the methods provided herein include measuring the amount of internal standard precursor ions having a mass-to-charge ratio of 307.19 ± 0.5 (in the case of hydroxylamine derivatization) or 321.21 ± 0.5 (in the case of methoxylamine derivatization), and / or product ions having a mass-to-charge ratio of 127.06 ± 0.5 (in the case of hydroxylamine derivatization) or 129.07 ± 0.5 (in the case of methoxylamine derivatization).

[0020] In certain embodiments, the multiplexing of samples provided herein is at least two times faster than the multiplexing of a single assay or column. In certain embodiments, the multiplexing of samples provided herein is at least three times faster than a single assay. In certain embodiments, the multiplexing of samples provided herein is at least four times faster than a single assay.

[0021] In certain embodiments, the limit of quantification of the method is less than or equal to 10 ng / dL. In some embodiments, the limit of quantification of the method is less than or equal to 5 ng / dL. In some embodiments, the limit of quantification of the method is less than or equal to 4 ng / dL. In some embodiments, the limit of quantification of the method is less than or equal to 3 ng / dL. In some embodiments, the limit of quantification of the method is less than or equal to 2 ng / dL. In some embodiments, the limit of quantification of the method is less than or equal to 1 ng / dL.

[0022] In some embodiments, the methods provided herein include linearity of quantification over a range of 2.5 ng / dL to 2,000 ng / dL. In some embodiments, the methods provided herein include linearity of quantification over a range of 1 ng / dL to 2,000 ng / dL.

[0023] In some embodiments, the methods provided herein include measurement inaccuracies (CV) of 1% to 11% or 1% to 9% or 2% to 11% at 8 to 1,200 ng / dL.

[0024] In some embodiments, the methods provided herein include a recovery rate of from 95% to 105%, out of 90% to 110%.

[0025] In some embodiments, the methods provided herein include a clinical reportable range (CRR) of up to 10,000 ng / dL.

[0026] 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).

[0027] In some embodiments, the method can include adding an agent to the sample in an amount sufficient to remove proteins from the sample.

[0028] Suitable test samples include any test sample that may contain the analyte of interest. In some preferred embodiments, the sample is a biological sample; that is, 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 can be obtained, for example, from a patient; that is, a survivor, male or female, presenting themselves at the clinical site for the diagnosis, prediction, or treatment of a disease or condition. The test sample is preferably obtained from a patient, such as serum.

[0029] As used herein, unless otherwise indicated, the singular forms "a", "an", and "the" include plural meanings. Thus, for example, when referring to "a protein", it includes multiple protein molecules.

[0030] As used herein, the term "purification" or "purifying" does not mean removing all substances other than the analyte of interest from a sample. Rather, purification means a procedure for enriching the amount of one or more analytes of interest compared to other components in the sample that may interfere with the detection of the analyte of interest. A sample is purified herein by various means that enable the removal of one or more interfering substances, for example, one or more substances that interfere with the detection of the selected parent and daughter ions of T by mass spectrometry.

[0031] As used herein, the term "test sample" refers to any sample that may contain T. As used herein, the term "body fluid" means any fluid that can be isolated from an individual's body. For example, "body fluids" can include blood, plasma, serum, bile, saliva, urine, tears, sweat, and the like.

[0032] As used herein, the term "derivatization" means reacting two molecules to form a new molecule. Derivatizing agents can include isothiocyanate groups, dinitrofluorophenyl groups, nitrophenoxycarbonyl groups, and / or phthalaldehyde groups, among others.

[0033] As used herein, the term "chromatography" means a process in which a chemical mixture transported by a liquid or gas separates into components as a result of the differential partitioning of chemical entities as it flows around or over a stationary liquid or solid phase.

[0034] As used herein, the term "liquid chromatography" or "LC" means a process in which one or more components of a fluid solution are selectively retarded as the fluid uniformly penetrates through a column composed of a fine substance or through a capillary passageway. The retardation results from the partitioning of the components in the mixture as the fluid moves relative to the stationary phase between one or more stationary phases and the bulk fluid (i.e., the mobile phase). Examples of "liquid chromatography" include reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and high-turbulence liquid chromatography (HTLC).

[0035] As used herein, the term "high-performance liquid chromatography" or "HPLC" means liquid chromatography in which the degree of separation is increased by applying force to the mobile phase under pressurized conditions through a stationary phase, typically a densely packed column.

[0036] As used herein, the term "high-turbulence liquid chromatography" or "HTLC" means a form of chromatography that utilizes the turbulence of the substance being assayed through a column packing material as the basic principle for carrying out the separation. HTLC has been applied to the preparation of samples containing two unnamed drugs prior to analysis by mass spectrometry. See, e.g., Zimmer et al., J. Chromatogr. A 854:23-35 (1999); see also U.S. Patent Nos. 5,968,367, 5,919,368, 5,795,469, and 5,772,874 for further description of HTLC. Those skilled in the art understand "turbulent flow". When a fluid flows slowly and smoothly, this flow is called "laminar flow". For example, a fluid moving at a low flow rate through an HPLC column is laminar flow. In laminar flow, the movement of particles in the fluid is regular, and the particles generally move linearly. At higher speeds, the inertial force of water exceeds the frictional force of the fluid, resulting in turbulent flow. A fluid that does not contact an irregular boundary "overtakes" a fluid that has been slowed down by friction or whose direction has been changed by an uneven surface. When the fluid flows in a turbulent state, it flows in a swirling (or vortical) manner and becomes more "resistant" than when the flow is laminar. Many reference materials are available to help determine whether the flow of a fluid is laminar or turbulent (e.g., Turbulent Flow Analysis: Measurement and Prediction, P.S. Bernard & J.M. Wallace, John Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

[0037] As used herein, the term "gas chromatography" or "GC" means a chromatography in which a sample mixture is vaporized and injected into a stream of carrier gas (nitrogen or helium) that moves through a column containing a stationary phase composed of a liquid or particulate solid, and is separated into its constituent compounds based on the affinity of the compounds for the stationary phase.

[0038] As used herein, the term "large particle column" or "extraction column" means a chromatography column containing an average particle diameter greater than about 35 μm. As used in this context, the term "about" means ±10%. In a preferred embodiment, the column contains particles having a diameter of about 60 μm.

[0039] As used herein, the term "analytical column" means a chromatography column having a sufficient chromatographic plate that enables sufficient separation to enable determination of the presence or amount of an analyte for substances in the sample eluting from the column. Such columns are often distinguished from "extraction columns" which have the general purpose of separating or extracting retained substances from non-retained 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 having a diameter of about 4 μm.

[0040] As used herein, terms such as "online automation mode" or "online extraction" when used as "online" or "in-line" mean procedures that are carried out without the need for operator intervention. In contrast, the term "offline" as used herein means a procedure that requires manual operator intervention. Thus, if a sample is precipitated and then the supernatant is manually loaded into an autosampler, the precipitation and loading steps are in an offline state from subsequent steps. In various embodiments of the method, one or more steps may be carried out in an online automation mode.

[0041] As used herein, the term "mass spectrometry" or "MS" means an analytical technique for identifying compounds by their mass. MS means 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 the mass-to-charge ratio. The compound can be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionization device and an ion detector. Generally, one or more target molecules are ionized and the ions are then introduced into a mass spectrometric device where a magnetic field and an electric field In combination, the ions follow a path in space that depends on their mass ("m") and charge ("z"). See, for example, U.S. Patent No. 6,204,500, entitled "Mass Spectrometry From Surfaces"; U.S. Patent No. 6,107,623, entitled "Methods And Apparatus for Tandem Mass Spectrometry"; U.S. Patent No. 6,268,144, entitled "DNA Diagnostics Based On Mass Spectrometry"; U.S. Patent No. 6,124,137, entitled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes"; Wright et al., Prostate Cancer and Prostatic Diseases, Vol. 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis, Vol. 21:1164-67 (2000).

[0042] As used herein, the term "operation in negative ion mode" means a mass spectrometry method in which negative ions are generated and detected. The term "operation in positive ion mode" means, as used herein, a mass spectrometry method in which positive ions are generated and detected.

[0043] As used herein, the term "ionization" or "ionizing" means a process of generating analyte ions having a net charge equal to one or more electron units. Negative ions are ions having a net negative charge of one or more electron units, while positive ions are ions having a net positive charge of one or more electron units.

[0044] As used herein, the term "electron ionization method" or "EI method" means a method in which an analyte in the gaseous phase or vapor phase interacts with a stream of electrons. The impact of electrons on the analyte produces analyte ions, which can then be the subject of mass spectrometry techniques.

[0045] As used herein, the term "chemical ionization method" or "CI method" means a method in which a reagent gas (e.g., ammonia) is subjected to an electron impact, and analyte ions are formed by the interaction of the reagent gas ions with analyte molecules.

[0046] As used herein, the term "fast atom bombardment method" or "FAB method" means a method in which a beam of high-energy atoms (usually Xe or Ar) impacts a non-volatile sample, desorbing and ionizing the molecules contained in the sample. The test sample is dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6-crown ether, 2-nitrophenyl octyl ether, sulfolane, diethanolamine, and triethanolamine, etc. The selection of a matrix suitable for a compound or sample is an experimentally based process.

[0047] As used herein, the term "matrix-assisted laser desorption ionization method" or "MALDI" means a method in which a non-volatile sample is exposed to laser irradiation that 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 that facilitates the desorption of analyte molecules.

[0048] As used herein, the term "surface-enhanced laser desorption ionization method" or "SELDI method" means that a non-volatile sample desorbs and ionizes analytes in the sample through various ionization pathways including photoionization, protonation, deprotonation, and cluster decay by It means another way of exposing to user irradiation. In SELDI, the sample generally binds to a surface that preferentially retains one or more target analytes. Similar to MALDI, this process can also utilize an energy-absorbing material to facilitate ionization.

[0049] As used herein, the term "electrospray ionization method" or "ESI method" means a method in which a solution passes along a short capillary tube and a positive or negative high potential is applied to the end of the capillary tube. When the solution reaches the end of the tube, it is vaporized (atomized) into a jet or spray consisting of very small droplets of the solution in the solvent vapor. This mist of droplets passes through an evaporation chamber slightly heated to prevent condensation and evaporate the solvent. As the droplets become smaller, the surface charge density increases, and eventually natural repulsion between like charges causes the release of ions and neutral molecules.

[0050] As used herein, the term "atmospheric pressure chemical ionization method" or "APCI method" means a mass spectrometry method similar to ESI, but APCI produces ions by ion-molecule reactions occurring in a plasma at atmospheric pressure. The plasma is maintained by a discharge between the spray capillary and the counter electrode. The ions are then generally extracted into the mass spectrometer by using a series of skimmer stages that are differentially pumped. Countercurrent flow of dry and preheated N 2 gas can be used to improve solvent removal. When analyzing less polar species, gas-phase ionization in APCI can be more effective than ESI.

[0051] As used herein, the term "atmospheric pressure photoionization method" or "APPI method" means a form of mass spectrometry in which the mechanism for photoionizing molecule M is photon absorption and electron emission to form molecular ion M+. Since the photon energy is generally just above the ionization potential, the molecular ion is not very likely to dissociate. In many cases, it is considered possible to analyze the sample without the need for chromatography, thus significantly reducing time and cost. In the presence of water vapor or a protic solvent, the molecular ion can abstract 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, etc., usually adopt the form of M+. See Robb, D.B., Covey, T.R. and Bruins, A.P. (2000): For example, see Robb et al., Atmospheric pressure photoionization: An ionization method for liquid chromatography - mass spectrometry. Anal. Chem. Vol. 72 (No. 15): pp. 3653 - 3659.

[0052] As used herein, the term "inductively coupled plasma method" or "ICP method" means a method in which the sample interacts with a partially ionized gas at a sufficiently high temperature such that most elements are atomized and ionized.

[0053] As used herein, the term "field desorption method" means 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.

[0054] As used herein, the term "desorption" means removing the analyte from the surface and / or introducing the analyte into the gas phase.

[0055] As used herein, the term "limit of quantification", "limit of quantitation", or "LOQ" means the point at which a measurement becomes quantitatively meaningful. The response of the analyte at this LOQ is distinguishable, discrete, and has reproducibility with a precision of 20% and an accuracy of 80% - 120%. As used herein, the term "limit of quantification", "limit of quantitation", or "LOQ" means the point at which a measurement becomes quantitatively meaningful. The response of the analyte at this LOQ is distinguishable, discrete, and has reproducibility with a precision of 20% and an accuracy of 80% - 120%. As used herein, the term "limit of quantification", "limit of quantitation", or "LOQ" means the point at which a measurement becomes quantitatively meaningful. The response of the analyte at this LOQ is distinguishable, discrete, and has reproducibility with a precision of 20% and an accuracy of 80% - 120%. As used herein, the term "limit of quantification", "limit of quantitation", or "LOQ" means the point at which a measurement becomes quantitatively meaningful. The response of the analyte at this LOQ is distinguishable, discrete, and has reproducibility with a precision of 20% and an accuracy of 80% - 120%.

[0056] As used herein, the term "limit of detection" or "LOD" is the point at which the measured value becomes greater than the uncertainty associated with it. The LOD is arbitrarily defined as 2 standard deviations (SD) from zero concentration.

[0057] As used herein, the "amount" of T in a body fluid sample generally means an absolute value that reflects the mass of T detectable within the volume of the body fluid. However, the amount also takes into account the relative amount compared to another amount of T. For example, the amount of T in a body fluid can be an amount that is above or below the control level or normal level of T that is normally present.

[0058] When used herein in connection with quantitative measurements, except for ion mass measurements, the term "about" means the numerical value shown ± 10%. A mass spectrometry device can hardly vary when determining the mass of a given analyte. In the context of the mass of an ion or the mass / charge ratio of an ion, the term "about" means ± 0.5 atomic mass units.

[0059] The above summary of the present invention is non - limiting, and other characteristics and advantages of the present invention will be apparent from the following detailed description of the invention and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0060]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0061] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) provides improved accuracy over the techniques previously used to measure testosterone (T). High-throughput LC-MS / MS is achieved by multiplexing columns on which multiple samples are run in parallel. Here, our 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 distinguished by the characteristic fragment masses of the derivatizing agent, thus yielding two separate results in one LC-MS / MS run. Yes.

[0062] In certain embodiments, provided herein is a method for detecting the amount of testosterone in each of a plurality of patient samples within one mass spectrometry assay. The method comprises processing each patient sample individually to form a plurality of processed samples, such that as a result of the processing, testosterone in each processed sample is distinguishable by mass spectrometry from testosterone in the other processed samples; combining the processed samples to form a multiplexed sample; subjecting the multiplexed sample to an ion source under conditions suitable to generate one or more ions detectable by mass spectrometry, wherein one or more ions generated from testosterone derived from each processed sample are different from one or more ions of testosterone derived from the other processed samples; detecting by mass spectrometry the amount of one or more ions obtained from testosterone derived from each processed sample; and correlating the amount of one or more ions obtained from testosterone derived from each processed sample with the amount of testosterone in each patient sample.

[0063] In certain embodiments, the methods provided herein include determining the amount of testosterone in each of a plurality of human samples in a single mass spectrometry assay, the method comprising: i) subjecting each of the plurality of human samples to a different derivatizing agent to generate individually derivatized testosterone in each of the plurality of samples; ii) combining the plurality of samples to form a multiplexed sample; and iii) quantifying the amount of testosterone in each sample by mass spectrometry.

[0064] In some embodiments, the methods provided herein include determining the amount of testosterone in two human samples in a single mass spectrometry assay, the method comprising: i) subjecting each of the two human samples to a different derivatizing agent to generate individually derivatized testosterone in each of the two samples; ii) combining the two samples to form a multiplexed sample; and iii) quantifying the amount of testosterone in each sample by mass spectrometry.

[0065] In certain embodiments, the derivatizing agents provided herein include hydroxylamine or methoxyamine.

[0066] In some embodiments, the methods provided herein are fully automated.

[0067] In some embodiments, the methods provided herein are antibody-free methods.

[0068] In some embodiments, the purification provided herein includes extraction of 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 sample after extraction is concentrated.

[0069] In some embodiments, the purification provided herein includes liquid chromatography. In some embodiments, the liquid chromatography includes high performance liquid chromatography (HPLC). In some embodiments, the liquid chromatography includes high turbulence liquid chromatography (HTLC).

[0070] In preferred embodiments, ionization includes heated electrospray ionization (HESI). In preferred embodiments, ionization includes ionization in positive mode. In some embodiments, ionization includes ionization in negative mode.

[0071] 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.

[0072] In some embodiments, the method provided herein includes measuring the amount of precursor ions having a mass-to-charge ratio of 304.18 ± 0.5 for hydroxylamine derivatized testosterone. In some embodiments, the method provided herein includes measuring the amount of precursor ions having a mass-to-charge ratio of 318.21 ± 0.5 for methoxylamine derivatized testosterone.

[0073] In some embodiments, the method provided herein includes measuring the amount of fragment ions having a mass-to-charge ratio of 112.05 ± 0.5 or 124.05 ± 0.5 for hydroxylamine derivatized testosterone. In some embodiments, the method provided herein 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 derivatized testosterone.

[0074] In some embodiments, the methods provided herein further comprise adding an internal standard. In some embodiments, the internal standard is labeled with an isotope.

[0075] In some embodiments, the methods provided herein comprise measuring the amount of internal standard precursor ions having a mass-to-charge ratio of 307.19 ± 0.5 (in the case of hydroxylamine derivatization) or 321.21 ± 0.5 (in the case of methoxylamine derivatization), and / or product ions having a mass-to-charge ratio of 127.06 ± 0.5 (in the case of hydroxylamine derivatization) or 129.07 ± 0.5 (in the case of methoxylamine derivatization).

[0076] In certain embodiments, the multiplexing of samples provided herein is at least 2-fold faster than the multiplexing of a single assay or column. In certain embodiments, the multiplexing of samples provided herein is at least 3-fold faster than a single assay. In certain embodiments, the multiplexing of samples provided herein is at least 4-fold faster than a single assay.

[0077] In certain embodiments, the limit of quantification of the method is less than or equal to 10 ng / dL. In some embodiments, the limit of quantification of the method is less than or equal to 5 ng / dL. In some embodiments, the limit of quantification of the method is less than or equal to 4 ng / dL. In some embodiments, the limit of quantification of the method is less than or equal to 3 ng / dL. In some embodiments, the limit of quantification of the method is less than or equal to 2 ng / dL. In some embodiments, the limit of quantification of the method is less than or equal to 1 ng / dL.

[0078] In some embodiments, the methods provided herein include linearity of quantification over a range of 1 ng / dL to 2,000 ng / dL.

[0079] In some embodiments, the methods provided herein are 8 - 1,200 ng / In dL, it includes measurement inaccuracies (CV) of 1% to 11% or 1% to 9% or 2% to 11%.

[0080] In some embodiments, the methods provided herein include recovery rates of 95% to 105% out of 90% to 110%.

[0081] In some embodiments, the methods provided herein include a clinical reportable range (CRR) of up to 10,000 ng / dL.

[0082] 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).

[0083] In some embodiments, the method can include adding an agent to the sample in an amount sufficient to remove proteins from the sample.

[0084] Suitable test samples include any test sample that can contain the analyte of interest. In some preferred embodiments, the sample is a biological sample; that is, a sample obtained from any biological source material, 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 can be obtained, for example, from a patient; that is, a survivor who presents themselves at the clinical site for the diagnosis, prediction, or treatment of a disease or condition, male or female. The test sample is preferably obtained from a patient and is, for example, serum.

[0085] Sample Preparation for Mass Spectrometry As a method that can be used to enrich T more than other components (e.g., proteins) in a sample, for example, it includes a filtration method, a centrifugation method, a thin layer chromatography (TLC) method, an electrophoresis method including capillary electrophoresis, an affinity separation method including an immunoaffinity separation method, an extraction method including an ethyl acetate extraction method and a methanol extraction method, and the use of chaotropic agents, or any combination of the above, etc.

[0086] The protein precipitation method is one preferred method for preparing a test sample. Such protein purification methods are well known in the art. For example, Polson et al., Journal of Chromatography B, Volume 785: pages 263 - 275 (2003) describes protein precipitation techniques suitable for use in this method. The protein precipitation method can be used to remove most proteins from the sample, leaving T in the supernatant. The sample can be centrifuged to separate the liquid supernatant from the precipitated protein. The resulting supernatant can then be applied to liquid chromatography and subsequent mass spectrometry analysis. In certain embodiments, if a protein precipitation method, such as an acetonitrile protein precipitation method, etc. is used, it is not necessary to perform high - turbulent liquid chromatography (HTLC) or other online extraction before performing HPLC and mass spectrometry. Thus, in such embodiments, the method involves (1) performing protein precipitation of the target sample; and (2) directly loading the supernatant onto an HPLC - mass spectrometer without using online extraction or high - turbulent liquid chromatography (HTLC).

[0087] In some preferred embodiments, HPLC can be used to purify T prior to mass spectrometry, either alone or in combination with one or more purification methods. In such embodiments, the sample can be extracted using an HPLC extraction cartridge that captures the analyte, then eluted, and chromatographed on a second HPLC column, or eluted on an analytical HPLC column prior to ionization. The steps involved in such chromatography procedures can be linked in an automated manner, so the need for operator involvement during the purification period of the analyte can be minimized. Such characteristics result in time and cost savings and can also eliminate the opportunity for operator error.

[0088] For example, the turbulent flow caused by HTLC columns and various methods may enhance the mass transfer rate and is considered to improve separation characteristics. HTLC columns separate components by the high flow rate of chromatography through a packed column containing rigid particles. By utilizing a high flow rate (e.g., 3 - 5 mL / min), turbulent flow is generated within the column, causing almost complete interaction between the stationary phase and the target analyte. An advantage of using HTLC columns is that high molecular weight species are not retained under turbulent flow conditions, thus avoiding the accumulation of high molecular polymers associated with biological fluid matrices. In the HTLC method that combines multiple separation methods in one procedure, the need for long - term sample preparation is reduced, and it operates extremely fast. Such a method also achieves separation performance superior to laminar flow (HPLC) chromatography. HTLC enables the direct injection of biological samples (plasma, urine, etc.). When injecting directly, it is difficult to achieve with conventional chromatography because denatured proteins and other biological debris rapidly block the separation column. Also, HTLC allows for a very small sample volume of less than 1 mL, preferably less than 0.5 mL, preferably less than 0.2 mL, and preferably 0.1 mL.

[0089] Examples of HTLC applied to sample preparation prior to analysis by mass spectrometry are described elsewhere. See, for example, Zimmer et al., J. Chromatogr. A 854:23-35 (1999); U.S. Patent Nos. 5,968,367; 5,919,368; 5,795,469; and 5,772,874. In certain embodiments of the method, the sample is subjected to protein precipitation as described above prior to loading onto the HTLC column; in alternative preferred embodiments, the sample can 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 can be performed in an on-line automated fashion. For example, in one embodiment, steps (i)-(v) are performed in an on-line automated fashion. In another case, the steps of ionization and detection are performed on-line after steps (i)-(v).

[0090] Liquid chromatography (LC), including high performance liquid chromatography (HPLC), is based on relatively slow laminar flow techniques. Conventional HPLC analysis methods rely on column packing materials where the laminar flow of the sample through the column forms the basis for separating the analyte of interest from the sample. Those skilled in the art understand that separation in such columns is a diffusion process. HPLC is applied without problems to the separation of compounds in biological samples, but a significant amount of sample preparation is required prior to separation and subsequent analysis by a mass spectrometer (MS), making this technique labor intensive. Further, most HPLC systems do not utilize a mass spectrometer to its maximum capacity and only allow one HPLC system to be connected to one MS device, resulting in a redundant time requirement for performing a large number of assays.

[0091] Various methods have been described for the use of HPLC for sample removal prior to mass spectrometry analysis. For example, Taylor et al., Therapeutic Dr See ug Monitoring, Vol. 22: 608-12 (2000); and Salm et al., Clin. Therapeutics, Vol. 22 Suppl. B: B71-B85 (2000).

[0092] One skilled in the art can select an HPLC apparatus and column suitable for use with T. A chromatographic column generally includes a medium (i.e., packing material) that facilitates the separation (i.e., fractionation) of chemical moieties. Examples of the medium can include fine particles. The particles have a binding surface that interacts with various chemical moieties to facilitate the separation of the chemical moieties. One suitable binding surface is a hydrophobic binding surface, such as an alkyl binding surface, etc. The alkyl binding surface can include a C-4, C-8, C-12, or C-18 bonded alkyl group, preferably a C-18 bonded group. The chromatographic column includes an injection port for receiving a sample and an outlet port for discharging an 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 from the outlet port. Different solvent modes can be selected to elute the analyte of interest. For example, liquid chromatography can be performed using a gradient mode, an isocratic mode, or a polymorphic (i.e., mixed) mode. During the chromatography period, the separation of materials is affected by variable factors, such as the selection of the eluent (also known as the "mobile phase"), the elution mode, the gradient conditions, the temperature, etc.

[0093] In certain embodiments, the analyte can be purified by applying the sample to a column under conditions such that the analyte of interest is reversibly retained by the column packing material while one or more other substances are not retained. In such embodiments, the conditions of the first mobile phase can be set such that the analyte of interest is retained by the column, and the conditions of the second mobile phase can then be set such that the retained substance is removed from the column after the non-retained substances have been eluted. Alternatively, the analyte can be purified by applying the sample to a column under mobile phase conditions such that the analyte of interest elutes at a different rate compared to one or more other substances. Such procedures can enrich the amount of one or more analytes of interest compared to one or more other components of the sample.

[0094] In one preferred embodiment, HTLC can follow HPLC on a hydrophobic column chromatography system. In certain preferred embodiments, a TurboFlow Cyclone P® polymer-based column (particle size 60 μm, column dimensions 50 × 1.0 mm, pore size 100 Å) from Cohesive Technologies is used. In related preferred embodiments, a Synergi Polar-RP® ether-bonded phenyl analytical column (particle size 4 μm, column dimensions 150 × 2.0 mm, pore size 80 Å) from Phenomenex Inc with hydrophilic end-capping is used. In certain preferred embodiments, HTLC and HPLC are performed using HPLC-grade ultrapure water and 100% methanol as the mobile phase.

[0095] By careful valve selection and connector piping work, two or more chromatography columns can be connected as needed so that the substance passes from one column to the next without any manual steps. In a preferred embodiment, the valve selection and piping work are managed by a computer pre-programmed to perform the necessary steps. Most preferably, the chromatography system is also connected in such an on-line manner to a detector system, such as an MS system. Thus, the operator only needs to place the sample tray in the autosampler, and the remaining work is carried out under computer control, resulting in the purification and analysis of all selected samples being completed.

[0096] In certain preferred embodiments, T or its fragments in the sample can be purified prior to ionization. In particularly preferred embodiments, the chromatography is not gas chromatography.

[0097] Detection and quantification by mass spectrometry In various embodiments, T or its fragments can be ionized by any method known to those skilled in the art. Mass spectrometry is performed using a mass spectrometer equipped with an ion source that ionizes the fractionated sample and produces charged molecules for further analysis. For example, ionization of the sample can be carried out 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 atom 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 choice of ionization method can be determined based on the analyte being measured, the type of sample, the type of detector, the choice of positive mode and negative mode, etc.

[0098] In a preferred embodiment, T or a fragment thereof is ionized by heated electrospray ionization (HESI) in positive ion mode.

[0099] After the sample is ionized, the positively charged or negatively charged ions thus generated 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. The ions can be detected using several detection modes. For example, selected ions can be detected using, for example, the selected ion monitoring mode (SIM), or alternatively, the ions can be detected using a scanning mode, such as multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). Preferably, the mass-to-charge ratio is determined using a quadrupole analyzer. For example, in a "quadrupole" or "quadrupole ion trap" device, a DC potential applied between the electrodes, the amplitude of the RF signal, and a force proportional to the mass / charge ratio are applied to the ions in an oscillating radio frequency field. The voltage and amplitude can be selected such that only ions having a specific mass / charge ratio move the length of the quadrupole while all other ions are deflected. Thus, a quadrupole device can act as both a "mass filter" and a "mass detector" for the ions injected into the device.

[0100] By using "tandem mass spectrometry" or "MS / MS", the resolution of MS technology can be enhanced. In this technique, precursor ions (also called parent ions) generated from the target molecule can be filtered within the MS apparatus, and the precursor ions are then fragmented and subsequently become one or more fragment ions (also called daughter ions or product ions) that are the subject of analysis in a second MS procedure. By carefully selecting the precursor ions, only the ions produced from a specific analyte pass through the fragmentation chamber, where they collide with atoms of an inert gas to produce fragment ions. Since both the precursor ions and the fragment ions are reproducibly produced under a series of predetermined conditions for ionization / fragmentation, the MS / MS technique can provide a very powerful analytical tool. For example, the combination of filtering / fragmentation can be used to remove interfering substances and can be particularly useful in complex samples, such as biological samples and the like.

[0101] A mass spectrometer generally provides the user with an ion scan; that is, the relative abundance of each ion having a specific mass / charge within a predetermined range (e.g., 100 - 1000 amu). The result of an analyte assay, i.e., the mass spectrum, can be associated with the amount of analyte in the original sample by a very large number of methods known in the art. For example, assuming that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion can be compared to a table that converts the relative abundance to the absolute amount of the original molecule. Alternatively, a molecular standard can be run with the sample, and a standard curve is constructed based on the ions generated from that standard. Such a standard curve can be used to convert the relative abundance of a given ion to the absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the amount of T. Methods for generating and using such standard curves are well known in the art, and one of ordinary skill in the art has the ability to select an appropriate internal standard. For example, an isotope of T is available as an internal standard. A very large number of other methods for associating the amount of ions with the amount of the original molecule are well known to those of ordinary skill in the art.

[0102] One or more steps of the method can be performed using automated machinery. In certain embodiments, one or more purification steps are performed online, and more preferably, all of the purification and mass spectrometry steps can be performed in an online fashion.

[0103] In certain embodiments, such as MS / MS where precursor ions are isolated for further fragmentation purposes, the collision activation dissociation method It is often used to generate fragment ions for further detection. In CAD, the precursor ion acquires energy through collisions with an inert gas and then fragments through a process called "unimolecular decomposition reaction". Sufficient energy must be accumulated within the precursor ion so that specific bonds within the ion can be broken due to the increased vibrational energy.

[0104] In a particularly preferred embodiment, T is detected and / or quantified as follows using MS / MS. The sample is processed by liquid chromatography, preferably HPLC, and the flow of the liquid solvent from the chromatographic column enters the heated nebulizer interface of the MS / MS analyzer, and the solvent / analyte mixture is converted to vapor within the heated tube of the interface. The analyte is ionized by a selected ionization device. Ions, such as precursor ions, pass through the aperture of the device and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters that enable the selection of ions (i.e., "precursor" 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 the molecule using the mass-to-charge ratio of T. Precursor ions having the correct mass / charge ratio of T can be introduced into the collision chamber (Q2), while any unwanted ions having other mass / charge ratios collide with the sides of the quadrupole and are removed. The precursor ions entering Q2 collide with neutral argon gas molecules and fragment. This process is called collision-activated dissociation (CAD). The generated fragment ions are introduced into quadrupole 3 (Q3), where the fragment ions of T are selected while other ions are removed.

[0105] The method may be related to MS / MS performed in either positive ion mode or negative ion mode. Using standard methods well known in the art, one of ordinary skill in the art has the ability to identify one or more fragment ions of a particular T's precursor ion available for selection at quadrupole 3 (Q3).

[0106] When the ion collides with the detector, the ion produces a pulse of electrons that is converted into a digital signal. The acquired data is transmitted to a computer that plots the count of the collected ions against time. The resulting mass chromatogram is similar to the chromatogram generated by conventional HPLC methods. The peak area under the curve 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 the fragment ion and / or precursor ion is measured to determine the amount of T. As described above, using a calibration standard curve based on the peak(s) derived from one or more ions of the internal molecular standard, the relative abundance of a given ion can be converted to the absolute amount of the original analyte.

[0107] The following examples serve to illustrate the present invention. These examples are not intended to limit the scope of the present method in any way.

Examples

[0108] [Example 1] Quantification of multiplexed testosterone by mass spectrometry Calibration standards and quality controls (QCs) were prepared by spiking testosterone into stripped serum. The QC levels were: QC1, 8 ng / dL; QC2, 80 ng / dL, QC3, 250 ng / dL; QC4, 1,200 ng / dL. Patient plasma or serum samples, as well as aliquots (200 μL) of the standards and QC samples, were processed based on the workflow shown in Figure 1. All pipetting steps were automated using a Hamilton Star liquid handling robot coupled with a Tecan IP8.

[0109] The sample to be analyzed was divided into two sets identified as sample plate A and sample plate B. An internal standard (IS) was added, and then (T) was extracted from the samples using in-plate protein precipitation and filtration directly. Both sample plates were derivatized independently. Sample plate A was derivatized using methoxyamine hydrochloride; sample plate B was derivatized using hydroxylamine hydrochloride. After derivatization, automated solid-phase extraction was performed by automation on an SPEware IP8 system linked to a Hamilton STAR using a Strata C18 solid-phase extraction plate. The two derivatives (sample plates A and B) were combined in this step. After elution, the samples were dried under nitrogen and reconstituted before injection.

[0110] Forty microliters of the sample was injected into a high-turbulence liquid chromatography (HTLC) system, and a TSQ Quantum Ultra MS / MS (Thermo Fisher Scientific) equipped with a heated electrospray ionization (HESI) source was used as the detector. Mass spectrometer data was acquired in positive ion mode. The total runtime was 6.5 minutes per injection including column equilibration. By using multiple channels, it was possible to analyze 2 samples every 1.5 minutes or 8 samples in 6.5 minutes. Quantification was based on specific parent-product transitions. The following precursor-fragment pairs or "mass transitions" were used:

[0111] For the hydroxylamine derivative and its (IS), 304.18 / 112.05, 124.05, and 307.19 / 127.06; and for the methoxyamine derivative and its IS, 318.21 / 126.07, 138.07, 152.08, and 321.21 / 129 / 07.

[0112] The performance of the multiplexed derivatization T assay was compared in a parallel test with the results obtained from sample waste accumulated from an existing T assay (Salameh, 2010). 259 Two hundred and fifty-nine residual sample samples were assayed by a state-of-the-art method for detecting T by LCMS / MS.

[0113] Column multiplexing enables the analysis of 4 samples every 6.5 minutes. Sample multiplexing doubled the throughput up to 8 samples in 6.5 minutes. Figure 2 compares the chromatographic profiles of single and multiplexed samples.

[0114] The method was linear up to 2,000 ng / dL (Figure 3), and the calibration curves showed consistency in reproducibility and linearity.

[0115] Analytical sensitivity: The limit of quantification was 1 ng / dL.

[0116] The inaccuracy (CV) of measurements at 8 - 1,200 ng / dL was 2.6% - 10.3% for the methoxyamine derivative and 1.7% - 8.7% for the hydroxylamine derivative (Figure 4).

[0117] The recovery rate of T in QC spiked with 2 - 4 in patient samples was 95% to 105% (Figure 4).

[0118] The recovery rate of T in 1:4 diluted patient samples was 95% - 109% (Figure 5); thus, the clinical reportable range (CRR) was extended up to 10,000 ng / dL for both the hydroxylamine and methoxyamine derivatives.

[0119] Regression analysis of T results was performed on the waste of 1,830 patients, both when run individually and when run using the multiplexed method, and a Deming's fitting of 4.79 + 1.03x was obtained in the concentration range of 3 - 4,862 ng / dL (Figure 6a).

[0120] Regression analysis was performed on 842 patient samples with T < 100 ng / dL, and Deming fitting of -1.51 + 1.09x was obtained (Figure 6b).

[0121] Conclusion: We developed and validated a fully automated "sample multiplexing" method that doubles the LC-MS / MS throughput without compromising assay accuracy and specificity.

[0122] The contents of the documents, patents, and patent applications described or cited in this specification, as well as all other documents and electronically available information, are hereby incorporated by reference in their entirety into this specification to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. The applicants reserve the right to physically incorporate into this application any and all materials and information derived from any such document, patent, patent application, or other physical and electronic documents.

[0123] The methods described herein by way of example can be suitably practiced without the presence of any one or more elements, one or more limitations not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," "containing," etc. should be construed in an expansive and non-limiting manner. Further, the terms and expressions used herein are used as terms of explanation and not of limitation, and there is no intention to exclude any equivalents or portions thereof of the features presented and described. It is recognized that various modifications are possible within the scope of the claimed invention. Accordingly, although the invention has been specifically disclosed by preferred embodiments and optional features, it is to be understood that modifications and variations of the invention incorporated herein disclosed may be practiced by those skilled in the art and that such modifications and variations are to be considered within the scope of the invention. employed are used as terms of explanation and not of limitation, and there is no intention to exclude any equivalents or portions thereof of the features presented and described. It is recognized that various modifications are possible within the scope of the claimed invention. Accordingly, the invention has been specifically disclosed by preferred embodiments and optional features but it is to be understood that modifications and changes to the invention incorporated herein disclosed may be practiced by those skilled in the art and that such modifications and changes are to be considered within the scope of the invention.

[0124] The present invention is described broadly and generically herein. Each of the narrower species and subgeneric groupings that fall within the scope of the general disclosure also forms part of the method. This includes a general description of the method with conditions or negative limitations that exclude any of the subject matter from the genus, whether or not the excised subject matter is specifically recited herein.

[0125] Other embodiments are within the scope of the following claims. Further, when the features or aspects of the method are described by a Markush group, one of ordinary skill in the art will recognize that the invention thereby describes any of the individual members or sub-groups of members of the Markush group.

Claims

1. 1. A method for determining the amount of testosterone in each of a plurality of human samples in a single mass spectrometry assay, comprising: i) subjecting each of a plurality of human samples to a different derivatization agent to produce individually derivatized testosterone in each of said plurality of samples, wherein said derivatization agents include hydroxylamine chloride and methoxylamine chloride; ii) combining said plurality of samples to form a multiplexed sample; iii) purifying said samples by solid phase extraction; and iv) quantitating the amount of testosterone in each sample by mass spectrometry. A method comprising:

2. The method of claim 1 further comprising liquid chromatography.

3. The method of claim 2 , wherein the liquid chromatography comprises high performance liquid chromatography.

4. The method of claim 2 , wherein the liquid chromatography comprises high turbulence liquid chromatography.

5. The method of claim 1 , wherein the mass spectrometry comprises heated electrospray ionization (HESI).

6. The method of claim 5 , wherein the HESI is in positive ion mode.

7. 10. The method of claim 1, comprising measuring the amount of one or more precursor ions having a mass-to-charge ratio selected from the group consisting of 304.18±0.5 and 318.21±0.

5.

8. 2. The method of claim 1, comprising measuring the amount of one or more fragment ions having a mass-to-charge ratio selected from the group consisting of 112.05±0.5, 124.05±0.5, 126.07±0.5, 138.07±0.5, and 152.08±0.

5.

9. The method of claim 1 further comprising adding an internal standard.

10. The method of claim 9 , wherein the internal standard is isotopically labeled.

11. 10. The method of claim 9, comprising measuring the amount of one or more internal standard precursor ions having a mass-to-charge ratio selected from the group consisting of 307.19±0.5 and 321.21±0.5, or one or more product ions having a mass-to-charge ratio selected from the group consisting of 127.06±0.5 and 129.07±0.

5.

12. 2. The method of claim 1, having linearity of quantification over the range of 1 ng / dL to 2,000 ng / dL.

13. The method of claim 1 , which is fully automated.

14. The method of claim 1 , which does not include an antibody.

15. The method of claim 1 , wherein the sample is serum.

16. The method of claim 1, wherein the method has a quantification limit of less than or equal to 1 ng / dL.

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