Method for quantifying adiponectin by mass spectrometry

Abstract

A method for determining the amount of adiponectin in a sample by mass spectrometry includes: (a) subjecting adiponectin from the sample to an ionization source under conditions suitable for generating one or more adiponectin ions detectable by mass spectrometry; (b) determining the amount of one or more adiponectin ions by mass spectrometry; and (c) determining the amount of adiponectin in the sample from the amount of one or more adiponectin ions determined in step (b).

Description

Technical Field

[0001] Cross - reference to related patent applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 456271, filed Mar. 31, 2023, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Adiponectin is a hormone and an adipokine protein mainly produced by adipose tissue, which has the effects of improving insulin resistance and anti - inflammation. Individuals with low adiponectin levels have three times the risk of developing metabolic syndrome and nine times the likelihood of developing type 2 diabetes. People who have two or more risk factors for metabolic syndrome and high adiponectin levels have half the likelihood of developing metabolic syndrome compared to those with low adiponectin levels. Thus, adiponectin has a significant effect on metabolic processes.

Summary of the Invention

Problems to be Solved by the Invention

[0003] There is a need for a reliable and accurate method to measure adiponectin levels.

Means for Solving the Problems

[0004] In one aspect, a method for measuring adiponectin levels in a patient is provided by determining the amount of adiponectin in a sample using mass spectrometry.

[0005] In some embodiments, a method is provided for determining the amount of adiponectin in a sample by mass spectrometry, comprising: (a) subjecting adiponectin derived from the sample to an ionization source under conditions suitable for generating one or more adiponectin ions detectable by mass spectrometry; (b) determining the amount of one or more adiponectin ions by mass spectrometry; and (c) determining the amount of adiponectin in the sample from the amount of one or more adiponectin ions determined in step (b).

[0006] In some embodiments, the amount of adiponectin in a sample is determined using the amount of one or more ions determined. In some embodiments, the amount of adiponectin in a sample is correlated with the amount of adiponectin in a patient.

[0007] In some embodiments, the sample is a serum sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a blood, saliva, or urine sample.

[0008] In some embodiments, ionization includes electrospray ionization (ESI). In some embodiments, ionization includes atmospheric pressure chemical ionization (APCI). In some embodiments, ionization is ionization in positive ion mode. In some embodiments, ionization is ESI in positive ion mode.

[0009] In some embodiments, one or more adiponectin ions include a precursor ion with a mass / charge ratio (m / z) of 421.23 ± 0.5.

[0010] In some embodiments, one or more adiponectin ions include fragment ions with a mass / charge ratio (m / z) of 314.18 ± 0.5. In some embodiments, one or more adiponectin fragment ions include ions with a mass / charge ratio (m / z) of 627.36 ± 0.5. In some embodiments, one or more adiponectin fragment ions include ions with a mass / charge ratio (m / z) of 530.30 ± 0.5.

[0011] In some embodiments, methods are provided herein that include the step of adding an internal standard to a sample. In some embodiments, an internal standard for adiponectin is added to the sample. In some embodiments, the internal standard is recombinant human adiponectin or adiponectin peptide. In some embodiments, one or more ions of the internal standard include a precursor ion with a mass / charge ratio (m / z) of 416.22 ± 0.5. In some embodiments, one or more ions of the internal standard include a fragment ion with a mass / charge ratio (m / z) of 319.19 ± 0.5. In some embodiments, one or more ions of the internal standard include a fragment with a mass / charge ratio (m / z) of 637.37 ± 0.5. In some embodiments, one or more ions of the internal standard include a fragment with a mass / charge ratio (m / z) of 540.31 ± 0.5.

[0012] In some embodiments, methods comprising the step of digestion with trypsin are provided herein. In some embodiments, methods comprising the step of precipitation with formic acid (for example, precipitation of deoxycholate with formic acid) are provided herein.

[0013] In some embodiments, methods are provided herein that include a step of purifying the sample before mass spectrometry. In some embodiments, the method includes a step of purifying the sample using liquid chromatography. In some embodiments, the liquid chromatography includes high-performance liquid chromatography (HPLC) or highly turbulent liquid chromatography (HTLC). In some embodiments, the method includes a step of subjecting the sample to solid-phase extraction (SPE).

[0014] In some embodiments, the limit of quantification is less than 2.0 μg / mL. In some embodiments, the limit of quantification is less than 1.0 μg / mL. In some embodiments, the limit of quantification is less than 0.9 μg / mL.

[0015] In some embodiments, the detection limit is less than 1.0 μg / mL. In some embodiments, the detection limit is less than 0.8 μg / mL. In some embodiments, the detection limit is less than 0.75 μg / mL.

[0016] In some embodiments, the mass spectrometry includes tandem mass spectrometry. In some embodiments, the mass spectrometry is high-resolution mass spectrometry. In some embodiments, the mass spectrometry is high-resolution / high-precision mass spectrometry.

[0017] In some embodiments, low levels of adiponectin (e.g., below the established reference ranges listed in Table A) correspond to an increased risk of metabolic syndrome. In some embodiments, low levels of adiponectin (e.g., below the established reference ranges listed in Table A) correspond to an increased risk of type 2 diabetes.

[0018] In some embodiments, the collision energy is in the range of approximately 5V to approximately 25V. In some embodiments, the collision energy is in the range of approximately 9V to approximately 21V. In some embodiments, the collision energy is approximately 9V. In some embodiments, the collision energy is approximately 21V.

[0019] In another embodiment, methods for diagnosing impaired blood glucose or insulin resistance syndrome in patients with diabetes and prediabetes are provided herein. In some embodiments, the methods for quantifying endogenous adiponectin provided herein are used to diagnose diabetes. In some embodiments, the methods for quantifying endogenous adiponectin provided herein are used to distinguish type 1 diabetes from type 2 diabetes. In some embodiments, the methods for quantifying endogenous adiponectin provided herein are used to assess the risk of diabetes in patients with prediabetes.

[0020] In another embodiment, a method for diagnosing or prognosing glycemic disorder or insulin resistance syndrome in patients with diabetes and prediabetes is provided herein, comprising the step of comparing relative amounts of adiponectin. In another embodiment, a method for diagnosing or prognosing glycemic disorder or insulin resistance syndrome in patients with diabetes and prediabetes is provided herein, comprising the step of determining the level of adiponectin. In some embodiments, the method comprises the step of determining the amount of adiponectin. In some embodiments, an abnormal or abnormal amount of adiponectin indicates glycemic disorder or insulin resistance syndrome. In some embodiments, a low level of adiponectin (e.g., below the established reference ranges listed in Table A) indicates glycemic disorder or insulin resistance syndrome.

[0021] The above summary of the present invention is not limiting, and other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0022] [Figure 1A] This graph shows that a comparison between the validated ELISA method and the LC / MS method resulted in a slope of 1.008 with an intercept of -0.167 (Figure 1A) and a difference plot between ELISA and LC / MS (Figure 1B). [Figure 1B]Same as above. [Figure 2A] Graph showing that all three levels of QC, low (Figure 2A), medium (Figure 2B), and high (Figure 2C), meet the total accuracy acceptance criteria of TEa / 3. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 3] Graph showing that the acceptance criteria were met across the AMR of 1.50 μg / mL to 50.00 μg / mL. [Figure 4A] Graph showing that carryover and pre-carryover did not exceed TEa / 4 for adiponectin and thus passed the acceptance criteria at LC3 (Figure 4A) and LC4 (Figure 4B). [Figure 4B] Same as above. [Figure 5] Graph showing that the LOB was calculated to be 0.419 μg / mL, the LOD was 0.740 μg / mL, and the LOQ was 0.831 μg / mL. Each of the low pools had an SD less than TEa / 3 and passed the acceptance criteria. [Figure 6] Graph showing that the average concentration was within TEa / 3 of the average concentration measured at t = 0 at all evaluation time points. [Figure 7] Graph showing that the average concentration was within TEa / 3 of the average concentration measured at t = 0 at all evaluation time points. [Figure 8] Graph showing that the average concentration was within TEa / 3 of the average concentration measured at t = 0 at all time points up to day 20. [Figure 9A] Graph showing that the average concentration was within TEa / 3 of the average concentration measured at t = 0 at all evaluation time points at -20 to -40 °C (Figure 9A) and -70 °C (Figure 9B). [Figure 9B] Same as above. [Figure 10] Graph showing that the average concentration was within TEa / 3 of the average concentration measured at t = 0 at all evaluated freeze-thaw cycles. [Figure 11A]These graphs show plate stability (cold stack) using adiponectin comparison (Figure 11A) and adiponectin difference plot (Figure 11B). [Figure 11B] Same as above. [Figure 12A] These graphs show hemolytic interference (Figure 12A) and hemolytic blood (Figure 12B). [Figure 12B] Same as above. [Figure 13A] These graphs show jaundice (bilirubin) interference (Figure 13A) and bilirubin (Figure 13B). [Figure 13B] Same as above. [Figure 14A] These graphs show the effects of lipemia (Intralipid) interference (Figure 14A) and Intralipid (Figure 14B). [Figure 14B] Same as above. [Modes for carrying out the invention]

[0023] As used herein, unless otherwise specified, the singular forms "a," "an," and "the" refer to multiple objects. Therefore, for example, a reference to "a protein" includes multiple protein molecules.

[0024] As used herein, the terms “purify,” “refine,” and “concentrate” do not mean the removal of all substances from the sample other than the analyte(s) of interest. Rather, these terms refer to a procedure that increases 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. Purification of a sample by various means allows for a relative reduction of one or more interfering substances, for example, one or more substances that may or may not interfere with the detection of selected parent or daughter ions by mass spectrometry. Relative reduction as used in this term does not require that substances present with the analyte of interest in the material to be purified be completely removed by purification.

[0025] As used herein, the terms “immunopurification” or “to immunopurify” mean a purification procedure that utilizes antibodies, including polyclonal or monoclonal antibodies, to concentrate one or more analytes. Immunopurification can be carried out using any of the immunopurification methods well known in the art. Often, immunopurification procedures utilize antibodies conjugated, attached to, or otherwise attached to a solid carrier, such as a column, well, tube, gel, capsule, particle, or similar. As used herein, immunopurification includes, without limitation, procedures often referred to in the art as immunoprecipitation, and procedures often referred to in the art as affinity chromatography or immunoaffinity chromatography.

[0026] As used herein, the term “immune particle” means a capsule, bead, gel particle, or similar having antibodies bound, conjugated, or otherwise bound to its surface (on and / or within the particle). In certain preferred embodiments, the immune particle is a cephalos or agarose bead. In preferred alternative embodiments, the immune particle includes glass, plastic, or silica beads or silica gel.

[0027] As used herein, the term "anti-adiponectin antibody" means any polyclonal or monoclonal antibody having affinity for adiponectin. In various embodiments, the specificity of an adiponectin antibody to chemical species other than adiponectin may vary. For example, in certain preferred embodiments, the anti-adiponectin antibody is specific to adiponectin and therefore has little to no affinity for chemical species other than adiponectin.

[0028] As used herein, the term “sample” means a sample that may contain the analyte of interest. As used herein, the term “body fluid” means a fluid that can be separated from an individual’s body. For example, “body fluid” may include blood, plasma, serum, bile, saliva, urine, tears, sweat, and the like. In a preferred embodiment, the sample includes a human body fluid sample, preferably plasma or serum.

[0029] As used herein, the terms “solid-phase extraction” or “SPE” mean a method of separating a mixture of chemical substances into its components as a result of the affinity of components dissolved or suspended in a solution (i.e., mobile phase) to a solid (i.e., solid phase) through which a solution flows or around. In some cases, as the mobile phase flows through or around the solid phase, undesirable components of the mobile phase may be retained by the solid phase, resulting in the purification of the analyte in the mobile phase. In other cases, the analyte may be retained by the solid phase, allowing undesirable components of the mobile phase to flow through or around the solid phase. In these cases, a second mobile phase is then used to elute the retained analyte from the solid phase for further processing or analysis. SPEs, including TFLCs, can function as single-mode or mixed-mode mechanisms. Mixed-mode mechanisms utilize ion exchange and hydrophobic retention in the same column; for example, the solid phase of a mixed-mode SPE column may exhibit strong anion exchange and hydrophobic retention, or strong cation exchange and hydrophobic retention.

[0030] Generally, the affinity of an SPE column packing material for the analyte may be due to one or more chemical or immunoaffinity interactions, among other diverse mechanisms. In some embodiments, SPE of adiponectin is performed without the use of an immunoaffinity column packing material. That is, in some embodiments, adiponectin is purified from the sample using an SPE column that is not an immunoaffinity column.

[0031] As used herein, the term "chromatography" means a method by which a mixture of chemicals carried by a liquid or gas flows around or over a stationary liquid or solid phase, resulting in the separation of the chemicals into their components as a result of the differential distribution of the chemicals.

[0032] As used herein, the terms “liquid chromatography” or “LC” mean a method by which one or more components of a fluid solution are selectively delayed as a fluid uniformly permeates a column or capillary passage of fine material. The delay is due to the distribution of the components of the mixture as the fluid moves relative to the stationary phase(s) between one or more stationary phases and a bulk fluid (i.e., mobile phase). Examples of “liquid chromatography” include reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), and turbulent liquid chromatography (TFLC) (sometimes known as high-turbulent liquid chromatography (HTLC) or high-process liquid chromatography).

[0033] As used herein, the terms “high-performance liquid chromatography” or “HPLC” (sometimes known as “high-pressure liquid chromatography”) mean liquid chromatography in which a mobile phase is forced under pressure through a stationary phase, generally a tightly packed column, to increase the degree of separation.

[0034] As used herein, the terms “turbulent liquid chromatography” or “TFLC” (sometimes known as high-turbulence liquid chromatography or high-process liquid chromatography) mean a form of chromatography that utilizes the turbulence of the substance being assayed passing through a column packing as the basis for separation. TFLC was applied to the preparation of a sample containing two unnamed drugs prior to analysis by mass spectrometry. See, for example, Zimmer et al., J Chromatogr, A854, pp. 23–35 (1999). See also U.S. Patents 5,968,367, 5,919,368, 5,795,469 and 5,772,874, which further describe TFLC. Those skilled in the art understand “turbulence.” When a fluid flows slowly and smoothly, that 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 motion of fluid particles is regular, and particles generally move substantially linearly. At higher speeds, the inertia of water overcomes the fluid's frictional forces, resulting in turbulence. A fluid not in contact with an irregular boundary will either be slowed down by friction or deflected by the non-flat surface, thus "overtaking" it. When a fluid is flowing turbulently, it flows in a swirling (or spiral) manner due to greater "resistance" than when the flow is laminar. Numerous references are available to help determine whether a fluid flow 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, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

[0035] As used herein, the terms “gas chromatography” or “GC” mean chromatography in which a sample mixture is evaporated and injected into a stream of carrier gas (such as nitrogen or helium) moving through a column containing a stationary phase consisting of a liquid or particulate solid, and the mixture is separated into its component compounds according to their affinity for the stationary phase.

[0036] As used herein, the terms “large particle column” or “extraction column” mean a chromatography column containing an average particle diameter greater than approximately 50 μm. In this context, the term “approximately” means ±10%.

[0037] As used herein, the term “analytical column” means a chromatographic column having a chromatographic plate sufficient to achieve separation of substances in a sample that elute from the column in a sufficient manner to enable the determination of the presence or amount of the 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 5 μm.

[0038] As used herein, the terms “online” and “inline” refer to procedures performed without operator intervention, as used, for example, in “online automated fashion” or “online extraction.” In contrast, the term “offline” refers to procedures that require manual operator intervention. Therefore, if a sample is subjected to 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 online and automated.

[0039] As used herein, the terms “mass spectrometry” or “MS” mean an analytical technique for identifying compounds by their mass. MS means a method of filtering, detecting, and measuring ions based on the mass-to-charge ratio or “m / z” of ions. MS techniques generally include the steps of (1) ionizing a compound to produce 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 appropriate means. A “mass spectrometer” generally includes an ionizer, a mass spectrometer, and an ion detector. Generally, one or more molecules of interest are ionized, and the ions are then introduced into a mass spectrometer, where a combination of magnetic and electric fields causes the ions to follow a path in space that depends on their mass ("m") and charge ("z"). For example, see U.S. Patent No. 6,204,500, entitled "Mass Spectrometry From Surfaces," Patent No. 6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry," Patent No. 6,268,144, entitled "DNA Diagnostics Based on Mass Spectrometry," Patent No. 6,124,137, entitled "Surface-Enhanced Photolabile Attachment And Release For Desoption And Detection Of Analytes," Wright et al., Prostate Cancer and Prostatic Diseases, 1999, Vol. 2, pp. 264-276, and Merchant and Weinberger, Electrophoresis, 2000, Vol. 21, pp. 1164-1167.

[0040] As used herein, "high-resolution / high-precision mass spectrometry" refers to mass spectrometry performed using a mass spectrometer capable of measuring the mass-to-charge ratio of charged species with sufficient precision and accuracy to identify unique chemical ions. Identification of unique chemical ions is possible when the individual isotopic peaks originating from that ion are readily identifiable. The specific resolution and mass accuracy required to identify unique chemical ions vary depending on the ion's mass and charge state.

[0041] In this specification, the terms "resolution" or "resolution (FWHM)" (in the art, "m / Δm") are used interchangeably. 50% The mass / charge ratio (also known as "FWHM") is the observed mass / charge ratio divided by the full width at half maximum (FWHM) of the mass peak at 50% of its maximum height. The effect of the difference in resolution is shown in Figures 1A-C, which show the theoretical mass spectra of ions with an m / z of approximately 1093. Figure 1A shows the theoretical mass spectrum from a mass spectrometer with a resolution of approximately 3000 (typical operating conditions for conventional quadrupole mass spectrometers). As can be seen in Figure 1A, individual isotope peaks are not distinguishable. By comparison, Figure 1B shows the theoretical mass spectrum from a mass spectrometer with a resolution of approximately 10,000, which has clearly distinguishable individual isotope peaks. Figure 1C shows the theoretical mass spectrum from a mass spectrometer with a resolution of approximately 12,000. At this maximum resolution, individual isotope peaks contribute less than 1% from the baseline.

[0042] As used herein, "unique chemical ion" in relation to mass spectrometry means a single ion having a single atomic structure. A single ion may be monovalent or polyvalent.

[0043] As used herein, the term “accuracy” (or “mass accuracy”) in relation to mass spectrometry means the potential deviation of the instrument response from the true m / z of the ion under consideration. Accuracy is generally expressed in parts per million (ppm). The effect of differences in mass accuracy is shown in Figures 2A–D, which illustrate the boundaries of the potential difference between the detected m / z and the actual m / z for the theoretical peak at m / z 1093.52094. Figure 2A shows the potential range of the detected m / z at an accuracy of 120 ppm. In contrast, Figure 2B shows the potential range of the detected m / z at an accuracy of 50 ppm. Figures 2C and 2D show even narrower potential ranges of the detected m / z at accuracys of 20 ppm and 10 ppm, respectively.

[0044] The high-resolution / high-precision mass spectrometry method of the present invention can be carried out in instruments capable of performing mass spectrometry at FWHM greater than or far exceeding 10,000, 15,000, 20,000, 25,000, 50,000, 100,000. Similarly, the method of the present invention can be carried out in instruments capable of performing mass spectrometry at accuracy of 50 ppm, 20 ppm, 15 ppm, 10 ppm, 5 ppm, less than 3 ppm, or far below. Instruments capable of these performance characteristics may incorporate certain orbitrap mass spectrometers, time-of-flight ("TOF") mass spectrometers, or Fourier transform ion cyclotron resonance mass spectrometers. In a preferred embodiment, the method is carried out by instruments including an orbitrap mass spectrometer or a TOF mass spectrometer.

[0045] The term "orbitrap" describes an ion trap consisting of an external barrel electrode and a coaxial internal electrode. Ions are injected tangentially into the electric field between the electrodes, and are captured as they orbit around the coaxial internal electrode. The electrostatic interaction between the ion and the electrode is balanced by centrifugal force. Because the ion orbits around the coaxial internal electrode, the orbital path of the captured ion oscillates along the axis of the central electrode at a harmonic frequency related to the ion's mass / charge ratio. Detection of the orbital frequency makes it possible to use an orbitrap as a mass spectrometer with high accuracy (only 1-2 ppm) and high resolution (FWHM) (up to approximately 200,000). Mass spectrometers based on orbitraps are described in detail in U.S. Patent No. 6,995,364, which is incorporated herein by reference. The use of orbitrap analyzers has been reported for the qualitative and quantitative analysis of various analytes. For example, see U.S. Patent Application Publication No. 2008 / 0118932 (filed November 9, 2007); Bredehoft et al., Rapid Commun. Mass Spectrom., 2008, Vol. 22: pp. 477-485; Le Breton et al., Rapid Commun. Mass Spectrom., 2008, Vol. 22: pp. 3130-36; Thevis et al., Mass Spectrom. Reviews, 2008, Vol. 27: pp. 35-50; Thomas et al., J. Mass Spectrom., 2008, Vol. 43: pp. 908-915; Schenk et al., BMC Medical Genomics, 2008, Vol. 1: p. 41; and Olsen et al., Nature Methods, 2007, Vol. 4: pp. 709-712.

[0046] As used herein, the term "operating in negative ion mode" means a mass spectrometry method that generates and detects negative ions. As used herein, the term "operating in positive ion mode" means a mass spectrometry method that generates and detects positive ions. In a preferred embodiment, mass spectrometry is performed in positive ion mode.

[0047] As used herein, the terms "ionization" or "ionization" mean a method of generating an analyte ion having a net charge equal to one or more electron units. A negative ion has a net negative charge of one or more electron units, while a positive ion has a net positive charge of one or more electron units.

[0048] As used herein, the terms “electron ionization” or “EI” mean a method by which the analyte in the gas or vapor phase interacts with a flow of electrons. Collisions between electrons and the analyte generate analyte ions that can then be subjected to mass spectrometry.

[0049] As used herein, the terms "chemical ionization" or "CI" mean a method in which a reagent gas (e.g., ammonia) is subjected to electron bombardment, and analyte ions are produced by the interaction of reagent gas ions with analyte molecules.

[0050] As used herein, the terms “fast atomic impact” or “FAB” refer to a method in which a beam of high-energy atoms (often Xe or Ar) collides with a non-volatile sample, causing molecules contained in the sample to desorb and ionize. The test sample is dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamine. The selection of a suitable matrix for a compound or sample is an empirical process.

[0051] As used herein, the terms “matrix-assisted laser desorption / ionization” or “MALDI” mean a method of exposing a non-volatile sample to laser irradiation that desorbs and ionizes the analyte in the sample through various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix that facilitates the desorption of the analyte molecules.

[0052] As used herein, the terms “surface-enhanced laser desorption / ionization” or “SELDI” refer to other methods of exposing a non-volatile sample to laser irradiation that desorbs and ionizes the analyte in the sample via various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For SELDI, the sample is generally bonded to a surface that preferentially holds one or more of the target analytes. Similar to MALDI, this method may also utilize energy-absorbing materials that facilitate ionization.

[0053] As used herein, the terms “electrospray ionization” or “ESI” refer to a method of passing a solution through a short capillary tube to which a high positive or negative potential is applied at its end. The solution that reaches the end of the tube evaporates (atoms) to form a jet or spray of very small droplets of the solution in the solvent vapor. This spray of droplets flows through an evaporation chamber. As the droplets become smaller, the surface charge density increases to a point in which ions and neutral molecules are released due to the natural repulsion between like-charged particles.

[0054] As used herein, the terms "atmospheric pressure chemical ionization" or "APCI" refer to a mass spectrometry method similar to ESI, but APCI generates ions through ion-molecular reactions occurring in an atmospheric pressure plasma. 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 using a pair of differential pumping skimmer stages. Solvent removal can be improved using counterflowing, dry, preheated N2 gas. Gas-phase ionization in APCI may be more effective than ESI for analyzing less polar species.

[0055] The terms "atmospheric pressure photoionization" or "APPI," as used herein, refer to a form of mass spectrometry in which the ionization mechanism of molecular M is the absorption of photons and emission of electrons, producing molecular ions M+. Since the photon energy is generally just above the ionization potential, molecular ions are not easily dissociated. In many cases, it is possible to analyze samples without the need for chromatography, which can save considerable time and cost. In the presence of water vapor or a protic solvent, molecular ions 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 usually form M+. See, for example, Robb et al., Anal. Chem., 2000, Vol. 72 (No. 15), pp. 3653-3659.

[0056] As used herein, the terms “inductively coupled plasma” or “ICP” mean 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.

[0057] As used herein, the term "field desorption" refers to a method of generating analyte ions by placing a non-volatile test sample on an ionization surface and using a strong electric field.

[0058] As used herein, the term "desorption" means the removal of the analyte from the surface and / or the entry of the analyte into the gas phase. Laser desorption and thermal desorption are techniques for thermally desorbing a sample containing the analyte into the gas phase using laser pulses. The laser is irradiated onto the back surface of a specially made 96-well plate with a metal base. The laser pulses heat the bottom, and the heat causes the sample to move into the gas phase. The gas phase sample is then drawn into a mass spectrometer.

[0059] As used herein, the term "selective ion monitoring" refers to a detection mode of a mass spectrometer in which only ions within a relatively narrow mass range, generally within a range of about 1 mass unit, are detected.

[0060] As used herein, the "multiple reaction mode," sometimes known as "selective reaction monitoring," is a detection mode of a mass spectrometer in which a precursor ion and one or more fragment ions are selectively detected.

[0061] As used herein, the terms “lower limit of quantification,” “lower limit of quantification,” or “LLOQ” refer to the point at which a measurement becomes quantitatively meaningful. The response of the analyte at this LOQ is identifiable, individual, and reproducible with a relative standard deviation (RSD%) of less than 20% and an accuracy of 85% to 115%.

[0062] As used herein, the term “Limit of Detection” or “LOD” refers to the point at which a measurement is greater than the uncertainty associated with it. The LOD is the point at which a value exceeds the uncertainty associated with that measurement and is defined as three times the mean RSD at zero concentration.

[0063] As used herein, the “amount” of an analyte in a bodily fluid sample generally refers to the absolute value that reflects the mass of the analyte detectable in the volume of the sample. However, the amount can also refer to a relative amount compared to the amounts of other analytes. For example, the amount of an analyte in a sample may be greater than the control or normal levels of the analyte normally present in the sample.

[0064] The term "approximately" as used herein in relation to quantitative measurements that do not involve the measurement of ion mass means plus or minus 10% of the indicated value. Mass spectrometers may vary slightly in determining the mass of a given analyte. The term "approximately" with respect to ion mass or mass / charge ratio means + / - 0.50 atomic mass units.

[0065] In one embodiment, a method for measuring adiponectin levels in a patient is provided herein, by determining the amount of adiponectin in a sample using mass spectrometry.

[0066] The method may be for determining the amount of adiponectin in a sample by mass spectrometry and includes: (a) subjecting adiponectin from the sample to an ionization source under conditions suitable for generating one or more adiponectin ions detectable by mass spectrometry; (b) determining the amount of one or more adiponectin ions by mass spectrometry; and (c) determining the amount of adiponectin in the sample from the amount of one or more adiponectin ions determined in step (b).

[0067] The amount of one or more ions determined can then be used to determine the amount of adiponectin in the sample. In some embodiments, the amount of adiponectin in the sample is correlated with the amount of adiponectin in the patient.

[0068] The sample may be a serum sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a blood, saliva, or urine sample.

[0069] Ionization may include electrospray ionization (ESI). In some embodiments, ionization includes atmospheric pressure chemical ionization (APCI). In some embodiments, ionization is in positive ion mode. In some embodiments, ionization is ESI in positive ion mode.

[0070] One or more adiponectin ions may include a precursor ion with a mass / charge ratio (m / z) of 421.23 ± 0.5. One or more adiponectin ions may also include a fragment ion with a mass / charge ratio (m / z) of 314.18 ± 0.5. In some embodiments, one or more adiponectin fragment ions may include an ion with a mass / charge ratio (m / z) of 627.36 ± 0.5. In some embodiments, one or more adiponectin fragment ions may include an ion with a mass / charge ratio (m / z) of 530.30 ± 0.5.

[0071] The methods provided herein may include the step of adding an internal standard to a sample. An internal standard for adiponectin can be added to the sample. In some embodiments, the internal standard is recombinant human adiponectin or adiponectin peptide. The method may also include the step of generating one or more ions of the internal standard that can be detected by mass spectrometry. One or more ions of the internal standard may include a precursor ion with a mass / charge ratio (m / z) of 416.22 ± 0.5. In some embodiments, one or more ions of the internal standard may include a fragment ion with a mass / charge ratio (m / z) of 319.19 ± 0.5. In some embodiments, one or more ions of the internal standard may include a fragment with a mass / charge ratio (m / z) of 637.37 ± 0.5. In some embodiments, one or more ions of the internal standard may include a fragment with a mass / charge ratio (m / z) of 540.31 ± 0.5.

[0072] The methods provided herein may include a step of digesting adiponectin in a sample. Digestion can be achieved using trypsin in some embodiments. Furthermore, the methods may include a step of subjecting a sample (e.g., a solution) containing adiponectin (and / or adiponectin digestion products) to precipitation (e.g., precipitating deoxycholate with formic acid). In some embodiments, a sample (e.g., a solution) containing adiponectin (and / or adiponectin digestion products) can be purified by precipitation (e.g., precipitation with formic acid).

[0073] The methods provided herein may involve purifying the sample before mass spectrometry. Such purification may be performed using liquid chromatography, which in some embodiments may be high-performance liquid chromatography (HPLC) or highly turbulent liquid chromatography (HTLC). The methods may also include a step of subjecting the sample to solid-phase extraction (SPE).

[0074] Various mass spectrometry techniques can be used, such as tandem mass spectrometry, high-resolution mass spectrometry, or high-resolution / high-precision mass spectrometry.

[0075] The method can yield a quantification limit of less than 2.0 μg / mL. This may include cases where the quantification limit of the method is less than 1.0 μg / mL, less than 0.9 μg / mL, less than 0.8 μg / mL, or less than 0.75 μg / mL, depending on the various embodiments.

[0076] Low levels of adiponectin may correspond to an increased risk of metabolic syndrome. For example, low levels of adiponectin may correspond to an increased risk of type 2 diabetes. As used herein, the term “low level” (e.g., “low level” of adiponectin) generally means a quantity below the established reference range of the analyte, determined from a cohort of seemingly healthy individuals. For example, without being bound by theory, the reference range for adiponectin can be considered to be the reference range shown in Table A, classified by the patient’s sex and body mass index (BMI).

[0077] [Table 1]

[0078] In some embodiments, the collision energy used in the method is approximately 5V to approximately 25V. This may include cases where the collision energy is approximately 9V to approximately 21V, approximately 9V, or approximately 21V.

[0079] In another embodiment, a method for diagnosing impaired blood glucose or insulin resistance syndrome in patients with diabetes and prediabetes is provided. Such a method of quantifying endogenous adiponectin can be used to diagnose diabetes or to distinguish type 1 diabetes from type 2 diabetes. In some embodiments, a method of quantifying endogenous adiponectin is used to assess the risk of diabetes in patients with prediabetes.

[0080] In another embodiment, a method is provided for diagnosing or determining the prognosis of impaired blood glucose or insulin resistance syndrome in patients with diabetes and prediabetes. Such a method may include a step of comparing relative amounts of adiponectin (for example, by comparing the amount of adiponectin in a patient with the corresponding reference listed in Table A).

[0081] In another embodiment, a method is provided for diagnosing or determining the prognosis of impaired blood glucose or insulin resistance syndrome in patients with diabetes and prediabetes. Such a method includes the step of determining the level of adiponectin in a sample derived from a subject. The method may also include the step of determining the amount of adiponectin. In some embodiments, abnormal or abnormal amounts of adiponectin indicate impaired blood glucose or insulin resistance syndrome. In other embodiments, low levels of adiponectin (e.g., below the established references listed in Table A) indicate impaired blood glucose or insulin resistance syndrome.

[0082] In a further embodiment, a kit for an adiponectin quantification assay is provided. The kit for an adiponectin quantification assay may include a kit comprising the compositions provided herein. For example, the kit may include packaging materials and a fixed amount of an isotope-labeled internal standard sufficient for at least one assay. Generally, it also includes instructions for use of the packaged reagent for the adiponectin quantification assay, recorded in tangible form (e.g., on paper or electronic media).

[0083] The calibration and QC (quality control) pools used in embodiments of the present invention are preferably prepared using a matrix similar to the sample matrix under the condition that adiponectin is essentially absent.

[0084] Preparation of samples for mass spectrometry In preparation for mass spectrometry, adiponectin can be concentrated compared to one or more other components in a sample by various methods known in the art, including, for example, immunocapsulation, liquid chromatography, filtration, centrifugation, thin-layer chromatography (TLC), electrophoresis including capillary electrophoresis, affinity separation including immunoaffinity separation, extraction methods including ethyl acetate or methanol extraction, and the use of chaotropic agents, or any combination of the above or similar methods.

[0085] One method of sample purification that can be used before mass spectrometry involves adding the sample to a solid-phase extraction (SPE) column under conditions where the analyte is reversibly retained by the column packing, but one or more other substances are not. In this technique, a first mobile phase condition can be used in which the analyte is retained by the column, and once the unretained substances have been washed away, a second mobile phase condition can then be used to remove the retained substances from the column.

[0086] In some embodiments, a packing material containing alkyl-bonded surfaces can be used to reversibly retain adiponectin in a sample on an SPE column. For example, in some embodiments, adiponectin can be concentrated before mass spectrometry using a C-8 online SPE column (e.g., Oasis HLB online SPE column / cartridge (2.1 mm x 20 mm) or equivalent from Phenomenex, Inc.). In some embodiments, the use of the SPE column is carried out using HPLC-grade 0.2% aqueous formic acid as a washing solution and 0.2% formic acid in acetonitrile as an elution solution.

[0087] In other embodiments, the method includes a step of immunopurifying adiponectin before mass spectrometry. The immunopurification step can be carried out using any immunopurification method known in the art. Often, immunopurification procedures utilize antibodies that are conjugated, immobilized, or otherwise bound to a solid carrier, such as a column, well, tube, capsule, particle, or the like. Generally, an immunopurification method includes (1) incubating a sample containing the analyte with an antibody so that the analyte binds to the antibody, (2) performing one or more washing steps, and (3) eluting the analyte from the antibody.

[0088] In some embodiments, the incubation step of immunopurification is carried out with free antibodies in solution, and then the antibodies are conjugated or ligated to a solid surface before the washing step. In some embodiments, this can be achieved using a primary antibody, which is an anti-adiponectin antibody, and a secondary antibody ligated to a solid surface that has affinity for the primary anti-adiponectin antibody. In alternative embodiments, the primary antibody is conjugated to the solid surface before the incubation step.

[0089] Suitable solid carriers include, without limitation, tubes, slides, columns, beads, capsules, particles, gels, and the like. In some preferred embodiments, the solid carrier is a multi-well plate, e.g., a 96-well plate, a 384-well plate, or the like. In some embodiments, the solid carrier is Sepharose or agarose beads or gel. There are many methods known in the art, e.g., covalent or non-covalent adsorption, affinity binding, ionic binding, and the like, that can be used to conjugate, link, immobilize, or couple antibodies (e.g., adiponectin antibodies or secondary antibodies) to a solid carrier. In some embodiments, antibodies can be coupled using CNBr, for example, antibodies can be coupled to CNBr-activated Sepharose. In other embodiments, antibodies are linked to the solid carrier via antibody-binding proteins, e.g., protein A, protein G, protein A / G, or protein L.

[0090] The washing step of immunopurification generally involves washing the solid carrier so that adiponectin remains bound to the anti-adiponectin antibody on the solid carrier. The elution step of immunopurification generally involves adding a solution that interferes with the binding of adiponectin to the anti-adiponectin antibody. Specific examples of elution solutions include organic solutions, salt solutions, and high or low pH solutions.

[0091] Another method for purifying a sample before mass spectrometry is liquid chromatography (LC). In liquid chromatography, an analyte can be purified by adding the sample to a chromatographic analysis column under mobile phase conditions in which the analyte elutes at a different rate than one or more other substances. Such a procedure can increase the amount of one or more analytes compared to one or more other components of the sample.

[0092] Certain methods of liquid chromatography, including HPLC, rely on relatively slow laminar flow techniques. Traditional HPLC analysis relies on column packing, where the laminar flow of the sample through the column is the basis for separating the analyte from the sample. Those skilled in the art will understand that separation in such columns is a partitioning process and can select LC, instruments, and columns, including HPLC, that are suitable for use with adiponectin. Chromatographic analysis columns generally contain a medium (i.e., packing material) to facilitate the separation (i.e., fractionation) of compound components. The medium may contain fine particles. The particles generally contain binding surfaces that interact with various compound components to facilitate their separation. One suitable binding surface is a hydrophobic binding surface, such as an alkyl or cyano bonding surface. Alkyl bonding surfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups. In some embodiments, the chromatographic analysis column is a monolithic C-18 column. The chromatographic analysis column includes an inlet for receiving the sample and an outlet for discharging elutes containing the fractionated sample. The sample can be supplied directly to the inlet or from an SPE column or TFLC column, such as an online SPE column. In some embodiments, an online filter is used on the SPE column to remove particles and phospholipids in the sample before it reaches the SPE and / or TFLC and / or HPLC column. and / or It can be used before an HPLC column.

[0093] In one embodiment, the sample can be added to the LC column at the inlet, eluted with a solvent or solvent mixture, and discharged at the outlet. Various solvent modes can be selected to elute the target analyte(s). For example, liquid chromatography can be performed using gradient mode, no-gradient mode, or polymorphic (i.e., mixed) mode. During chromatography, the separation of substances is affected by variables such as the eluent (also known as the "mobile phase"), elution mode, gradient conditions, and temperature selection.

[0094] Adiponectin in a sample can be purified by HPLC. This HPLC can be performed using a monolith C-18 column chromatography system, such as the Onyx monolith C-18 column (50 x 2.0 mm) from Phenomenex Inc., or an equivalent. In certain embodiments, the HPLC is performed using 0.2% aqueous formic acid for HPLC as solvent A and 0.2% formic acid in acetonitrile as solvent B.

[0095] With the appropriate selection of valves and fittings, two or more chromatography columns can be connected as needed, allowing substances to pass from one chromatography column to the next without the need for manual steps. The selection of valves and fittings may be controlled by a computer pre-programmed to perform the necessary steps. The chromatography system may also be connected online to a detection system, such as a mass spectrometry system. Thus, the operator can load the sample tray into the autosampler, and the remaining operations are performed under computer control, resulting in the purification and analysis of all selected samples.

[0096] In some embodiments, TFLC can be used for the purification of adiponectin prior to mass spectrometry. In such embodiments, the sample can be extracted using a TFLC column that captures the analyte. The analyte is then eluted and transferred online to an analytical HPLC column. For example, sample extraction can be achieved using a TFLC extraction cartridge with a large particle size (50 μm) packing material. The sample eluted from this column may be transferred online to an analytical HPLC column for further purification prior to mass spectrometry. Since the steps involved in these chromatographic procedures can be linked together in an automated manner, the need for operator involvement during the purification of the analyte can be minimized. This feature can result in time and cost savings and potentially eliminate opportunities for operator error.

[0097] In some embodiments, one or more of the above purification techniques can be used in parallel for the purification of adiponectin to enable simultaneous processing of multiple samples. In some embodiments, the purification techniques used exclude immunopurification techniques, such as immunoaffinity chromatography.

[0098] Detection and quantification of adiponectin by mass spectrometry Mass spectrometry is performed using a mass spectrometer that includes an ion source for ionizing the fractionated sample and generating charged molecules for further analysis. In various embodiments, adiponectin can be ionized by methods known to those skilled in the art. For example, adiponectin can be ionized by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), laser diode thermal desorption (LDTD), 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. The ionization method can be determined based on the analyte being measured, the type of sample, the type of detector, the selection of positive versus negative modes, etc. Adiponectin may be ionized in positive mode or in negative mode. In some embodiments, adiponectin is ionized in positive ion mode by ESI.

[0099] In mass spectrometry, the mass-to-charge ratio (m / z) can generally be determined by ionizing a sample and then analyzing the resulting positively or negatively charged ions. Various analyzers for determining m / z include quadrupole analyzers, ion trap analyzers, time-of-flight analyzers, Fourier transform ion cyclotron resonance mass spectrometers, and orbit trap analyzers. Some specific examples of ion trap methods are described by Bartolucci et al., Rapid Commun. Mass Spectrom., 2000, Vol. 14, pp. 967-963.

[0100] Ions can be detected using several detection modes. For example, selected ions can be detected using a selective ion monitoring mode (SIM), or alternatively, mass transitions resulting from collision-induced dissociation, i.e., neutral loss, can be monitored, such as multiple reaction monitoring (MRM) or selective reaction monitoring (SRM). In some embodiments, the mass-to-charge ratio is determined using a quadrupole analyzer. In a "quadrupole" or "quadrupole ion trap" instrument, ions in an oscillating high-frequency electric field are subjected to 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 traverse the quadrupole, while all other ions are deflected. Thus, a quadrupole instrument can function as both a "mass filter" and a "mass detector" for the ions injected into the instrument.

[0101] When ions collide with the detector, they produce pulses of electrons that are converted into digital signals. The acquired data is transferred to a computer, which plots the counts of the collected ions against time. The resulting mass chromatogram is similar to that obtained by traditional HPLC-MS. The area under the peak or the amplitude of such a peak corresponding to a specific ion can be measured and correlated with the amount of the analyte of interest. In certain embodiments, the amount of adiponectin is determined by measuring the area under the curve or amplitude of the peaks of fragment ions and / or precursor ions. The relative abundance of a given ion can be converted to the absolute amount of the initial analyte using a calibration standard curve based on the peaks of one or more ions of an internal or external molecular standard.

[0102] The resolution of MS techniques can be improved using specific mass spectrometers with "tandem mass spectrometry" or "MS / MS". In this technique, precursor ions (also called parent ions) obtained from the target molecule can be filtered by the MS instrument, and the precursor ions are then fragmented to produce one or more fragment ions (also called daughter ions or product ions) that are analyzed in a second MS procedure. Careful selection of precursor ions ensures that only ions produced by a specific analyte are passed through a fragmentation chamber, where fragment ions are generated by collisions with atoms of an inert gas. Since both precursor and fragment ions are reproducibly generated under a series of predetermined ionization / fragmentation conditions, MS / MS techniques can be an extremely powerful analytical tool. For example, the filtration / fragmentation combination can be used to remove interfering substances and may be particularly useful for complex samples such as biological samples. In certain embodiments, tandem mass spectrometry is performed using a mass spectrometer with multiple quadrupole analyzers (e.g., a triple quadrupole instrument).

[0103] In certain embodiments, MS / MS techniques are used to isolate precursor ions for subsequent fragmentation, and collision-activated dissociation (CAD) is used to generate fragment ions from the precursor ions for subsequent detection. In CAD, the precursor ion gains energy through collisions with an inert gas and then becomes a fragment through a process called "monomolecular decomposition." Sufficient energy must be accumulated in the precursor ion so that the increase in vibrational energy can break specific bonds within the ion.

[0104] In some embodiments, adiponectin in a sample is detected and / or quantified using MS / MS as follows: Adiponectin is concentrated in the sample by first subjecting it to SPE, then liquid chromatography, preferably HPLC, and the flow of liquid solvent from the chromatographic analysis column enters the heated nebulizer interface of the MS / MS analyzer, where the solvent / analyte mixture is converted into vapor in the heated charged tube of the interface. During these processes, the analyte (i.e., adiponectin) is ionized. The ions, e.g., precursor ions, pass through the opening of the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters that allow ion selection based on the mass-to-charge ratio (m / z) of the ions (i.e., selection of “precursor” and “fragment” ions in Q1 and Q3, respectively). Quadrupole 2 (Q2) is a collision cell into which the ions are fragmented. The first quadrupole (Q1) of the mass spectrometer selects molecules having the m / z of adiponectin ions. Precursor ions with the correct m / z are passed through the collision chamber (Q2), while unwanted ions with other m / z collide with the sides of the quadrupole and are removed. The precursor ions that enter Q2 collide with neutral gas molecules (e.g., argon molecules) and become fragments. The resulting fragment ions are passed through quadrupole 3 (Q3), where they are selected for detection.

[0105] The ionization of adiponectin can result in highly charged precursor ions (e.g., 4+, 5+, 6+ precursor ions). The ionization conditions, particularly the pH of the buffer used in electrospray technology, significantly affect the identity and quantity of the generated adiponectin precursor ions. This method can utilize either acidic or basic conditions, preferably acidic conditions.

[0106] This method may include MS / MS performed in positive or negative ion mode, preferably in positive ion mode. In certain embodiments, the electrospray buffer is acidic, and Q1 selects an adiponectin precursor ion with an m / z of approximately 421.23±0.5. One of these adiponectin precursor ions fragments to produce fragment ions with m / z approximately 637.37±0.5, 540.31±0.5, and / or 319.19±0.5. Therefore, in embodiments in which Q1 selects one or more adiponectin precursor ions selected from the group consisting of ions with an m / z of approximately 421.23±0.5, Q3 may select one or more fragment ions selected from the group of ions with m / z approximately 637.37±0.5, 540.31±0.5, and / or 319.19±0.5. In certain embodiments, the relative abundance of a single fragment ion derived from a single precursor ion can be measured. Alternatively, the relative abundance of two or more fragment ions derived from a single precursor ion can be measured. In these embodiments, the relative abundance of each fragment ion can be subjected to any known mathematical treatment to quantitatively evaluate the adiponectin originally present in the sample. In other embodiments, one or more fragment ions derived from two or more precursor ions can be measured and used as described above to qualitatively evaluate the adiponectin originally present in the sample.

[0107] Alternative modes of operation for the tandem mass spectrometer that can be used in certain embodiments include product ion scanning and precursor ion scanning. For a description of these operating modes, see, for example, E. Michael Thurman et al., Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues, Chapter 8 (edited by Amadeo R. Fernandez-Alba, Elsevier 2005) (387).

[0108] In other embodiments, a high-resolution / high-precision mass spectrometer can be used for the quantitative analysis of adiponectin by the method of the present invention. To achieve acceptable accuracy for the quantitative results, the mass spectrometer must be able to exhibit a resolution of 10,000 or higher (FWHM) with an accuracy of about 50 ppm or less for the target ion, preferably a resolution of 18,000 or higher (FWHM) with an accuracy of about 5 ppm or less, e.g., 20,000 or higher (FWHM) and an accuracy of about 3 ppm or less, e.g., 25,000 or higher (FWHM) and an accuracy of about 3 ppm or less. Three specific examples of analyzers that can exhibit the required level of performance for adiponectin ions are an orbitrap mass spectrometer, a specific TOF mass spectrometer, and a Fourier transform ion cyclotron resonance mass spectrometer.

[0109] Elements found in biologically active molecules such as carbon, oxygen, and nitrogen exist naturally in many isotopes. For example, most carbon is... 12 Although it exists as C, about 1% of all naturally occurring carbon is 13 It exists as C. Therefore, a portion of a naturally occurring molecule containing at least one carbon atom is at least one 13 This will result in the presence of a carbon atom. The inclusion of naturally occurring elemental isotopes in a molecule creates multiple molecular isotopes. The mass difference between molecular isotopes is at least one atomic mass unit (amu). This is because elemental isotopes differ by at least one neutron (mass of one neutron ≈ 1 amu). When molecular isotopes are ionized to a highly charged state, the mass difference between isotopes can be difficult to distinguish because detection in mass spectrometry is based on the mass-to-charge ratio (m / z). For example, two isotopes that are both ionized to the 5+ state and differ by 1 amu in mass will show a difference of only 0.2 in their m / z. High-resolution / high-precision mass spectrometry can distinguish isotopes of highly charged ions (such as ions with ±2, ±3, ±4, ±5 or higher charges).

[0110] Because elemental isotopes occur naturally, multiple isotopes generally exist for all molecular ions (each potentially producing a spectral peak that can be detected individually when analyzed with a sufficiently sensitive mass spectrometer). The m / z ratios and relative abundances of multiple isotopes collectively constitute the isotopic signature of the molecular ion. In some embodiments, the m / z ratios and relative abundances of two or more molecular isotopes can be used to confirm the identity of the molecular ion under consideration. In some embodiments, molecular ions are quantified using the mass spectrometry peaks of one or more isotopes. In some related embodiments, molecular ions are quantified using a single mass spectrometry peak of one isotope. In other related embodiments, molecular ions are quantified using multiple isotopic peaks. In these latter embodiments, the multiple isotopic peaks can be subjected to appropriate mathematical treatments. Some mathematical treatments are known in the art and include, but are not limited to, the sum of the areas under multiple peaks or the averaging of the responses by multiple peaks.

[0111] In some embodiments, the relative abundance of one or more ions is measured using a high-resolution / high-precision mass spectrometer to qualitatively assess the amount of adiponectin in the sample. In some embodiments, the one or more ions measured by high-resolution / high-precision mass spectrometry are highly charged adiponectin ions.

[0112] The use of high-resolution orbitrap analyzers for qualitative and quantitative analysis of various analytes has been reported. For example, see U.S. Patent Application Publication No. 2008 / 0118932 (filed November 9, 2007); Bredehoft et al., Rapid Commun. Mass Spectrom., 2008, Vol. 22: pp. 477-485; Le Breton et al., Rapid Commun. Mass Spectrom., 2008, Vol. 22: pp. 3130-36; Thevis et al., Mass Spectrom. Reviews, 2008, Vol. 27: pp. 35-50; Thomas et al., J. Mass Spectrom., 2008, Vol. 43: pp. 908-915; Schenk et al., BMC Medical Genomics, 2008, Vol. 1: p. 41; and Olsen et al., Nature Methods, 2007, Vol. 4: pp. 709-712.

[0113] The results of an analyte assay can be correlated to the amount of the analyte in the initial sample by many methods known in the art. For example, if sampling and analytical parameters are carefully controlled, the relative abundance of a given ion can be compared to a table that converts that relative abundance to the absolute amount of the initial molecule. Alternatively, external standards can be performed with the sample, and standard curves can be created based on the ions obtained from those standards. Using such standard curves, the relative abundance of a given ion can be converted to the absolute amount of the initial molecule. In certain preferred embodiments, a standard curve for calculating the amount of adiponectin is created using an internal standard. Methods for creating and using such standard curves are well known in the art, and those skilled in the art can select appropriate internal standards. For example, in preferred embodiments, one or more forms of isotope-labeled adiponectin can be used as internal standards. Many other methods for relating the amount of an ion to the amount of the initial molecule are well known to those skilled in the art.

[0114] As used herein, "isotope labeling" results in a mass shift of the labeled molecule compared to the unlabeled molecule when analyzed by mass spectrometry. Examples of suitable labeling include deuterium (2 H), 13 C and 15 Examples include N. One or more isotopic labels can be incorporated at one or more positions in the molecule, and one or more types of isotopic labels can be used on the same isotopically labeled molecule.

[0115] In other embodiments, adiponectin can be subjected to chemical treatment before mass spectrometry. For example, adiponectin can be treated with TCEP (tris(2-carboxyethyl)phosphine).

[0116] Adiponectin can then be subjected to any one or more of the purification steps described above for the purification of adiponectin. In a preferred embodiment, adiponectin is purified by HPLC before mass spectrometry.

[0117] After purification, adiponectin is then subjected to an ionization source.

[0118] In a preferred embodiment, adiponectin is ionized in positive mode by ESI.

[0119] One or more steps of the methods described above can be carried out using an automated apparatus. In certain embodiments, one or more purification steps can be carried out online, and more preferably, all purification and mass spectrometry steps can be carried out online.

[0120] The following examples serve to illustrate the present invention. These examples do not limit the scope of the method. [Examples]

[0121] Adiponectin LC / MS. A general method for the quantification of adiponectin in human serum samples by LC / MS. Calibration standards, quality controls (QC), and internal standards (IS) were prepared by reconstituting recombinant human adiponectin or peptide in MilliQ water and then diluted with treated serum. The concentrations of the calibrator and IS storage solution were established using independent laboratory amino acid analysis (AAA). Calibration standards, QC, and patient samples were denatured by heat, and IS was added to a sodium deoxycholate (DOC) solution, followed by digestion with TPCK (N-tosyl-L-phenylalanine chloromethyl ketone) trypsin. Digestion was stopped with formic acid, and DOC was precipitated. After centrifugation, the sample supernatant was transferred to a 96-well plate for injection into an Agilent StreamSelect LC system connected to an Agilent 6495C QqQ MS. Adiponectin was separated by chromatography using a Kinetex 2.6 μm C18 column (Phenomenex) and gradients of water and acetonitrile (both containing 0.1% formic acid). The analyte and IS were monitored by multiple reaction monitoring (MRM) using electrospray ionization in positive ion mode. The following ions were used for the analysis of adiponectin in this method.

[0122] [Table 2] [Examples]

[0123] Accuracy. A total of 98 samples were compared between validated ELISA (enzyme-linked immunosorbent assay) and MS Starscream (LC-MS / MS) under validation over five separate runs conducted over five days. Regressions were evaluated for accuracy at the maximum (37 μg / mL) and minimum (2 μg / mL) extremes of the population reference range.

[0124] A comparison between the validated ELISA method and the LC / MS method yielded a slope of 1.008 with an intercept of -0.167 (Table 1, Figure 1). Cut points 2, 15, and 37 μg / mL met the acceptance criteria with an average bias of ≤TEa / 4. Bias in the comparison of samples between the validated ELISA method and MS Starscream is acceptable (within TEa / 4).

[0125] Table 1. Comparison of validated ELISA and LC / MS methods. Tea (overall tolerance): 30.0% or 1.50 μg / mL

[0126] [Table 3] JPEG2026513260000005.jpg248117JPEG2026513260000006.jpg248117JPEG2026513260000007.jpg194150 [Examples]

[0127] Daily precision. QC pools (QCH, QCM, QCL) containing high, medium, and low concentrations of recombinant human adiponectin were prepared in bulk and stored at -80°C in fixed single-use volumes. For intra-assay precision, 20 replicates were prepared for each level and analyzed in the same batch on the same day. For inter-assay precision, 5 replicates for each level were evaluated over 20 separate runs.

[0128] The QC low substance had an average intrarun SD (standard deviation) of 0.38 μg / mL (intrarun CV (coefficient of variation) = 9.40%) and a total SD of 0.40 μg / mL (total CV = 9.94%) relative to the overall sigma = 3.76, resulting in an overall average of 4.01 μg / mL. The two samples highlighted in yellow and bold were considered outliers and were removed from the data calculation.

[0129] During QC, the substance had an average intrarun SD of 0.83 μg / mL (intrarun CV = 6.28%) and a total SD of 0.92 μg / mL (total CV = 6.90%) relative to the overall sigma of 4.35, resulting in an overall average of 13.28 μg / mL.

[0130] The QC high substance yielded an average intrarun SD of 1.29 μg / mL (intrarun CV = 5.78%) and a total SD of 1.43 μg / mL (total CV = 6.40%), relative to the overall sigma of 4.69, resulting in an overall average of 22.30 μg / mL.

[0131] All three levels of QC meet the overall accuracy acceptance criteria for TEa / 3 (Table 2, Figure 2).

[0132] [Table 4] JPEG2026513260000009.jpg237155

[0133] [Table 5] [Examples]

[0134] Daily accuracy Level 1 (low QC) resulted in an average of 4.53 μg / mL. The intrarun SD was 0.37 μg / mL (intrarun CV = 8.13%).

[0135] Level 2 (medium QC) resulted in an average of 14.02 μg / mL. The intrarun SD was 1.03 μg / mL (intrarun CV = 7.37%).

[0136] Level 3 (high QC) resulted in an average of 24.08 μg / mL. The intrarun SD was 1.56 μg / mL (intrarun CV = 6.48%).

[0137] All three levels of QC meet the overall accuracy acceptance criteria for TEa / 3 (Table 4).

[0138] [Table 6] [Examples]

[0139] Analytical Measurement Range (AMR). AMR validation was performed using two different lots of recombinant protein with target concentrations assigned by third-party amino acid analysis and serially diluted across the AMR range. The minimum and maximum samples had target values ​​within 1.5 Tea of ​​the lower and upper limits of the AMR, respectively. All samples were measured in triple replicates.

[0140] The acceptance criteria for adiponectin is that the mean of three repeated measurements at each level recovers to within TEa / 4 (7.5% or 0.375 μg / mL) between 1.50 μg / mL and 50.00 μg / mL.

[0141] Acceptance criteria were met across AMR levels ranging from 1.50 μg / mL to 50.00 μg / mL (Table 5, Figure 3). Table 5. AMR and Acceptance Criteria TEa: 30% or 1.50 μg / mL

[0142] [Table 7] [Examples]

[0143] Carried over. The following procedure was used to inject the samples three times separately: low concentration, low concentration, high concentration, high concentration, low concentration, low concentration, low concentration (the low-concentration substance was QC low, and the high-concentration substance was calibrator 1).

[0144] The acceptance criterion is that the carryover from high-concentration samples to low-concentration samples does not exceed TEa / 4 for each reported parameter.

[0145] Both before and after the carryover period, the TEa / 4 for adiponectin did not exceed 4, and therefore the acceptance criteria were met (Table 6, Figure 4).

[0146] Table 6. Carried over. LC3 TEa: 30.0% or 1.50 μg / mL

[0147] [Table 8] LC4 TEa 30% or 1.50 μg / mL

[0148] [Table 9] [Examples]

[0149] Analytical sensitivity. The limit of quantification (LOQ), limit of blank (LOB), and limit of detection (LOD) were validated by analyzing 25 matrix blank replicates and 25 replicates at the lower limit of AMR. Samples for the lower limit tests were generated by spiking treated serum recombinant human adiponectin to obtain concentrations at each parameter.

[0150] The acceptance criteria is that the concentration in the low pool is (LL AMR + 1.5 * The TE is less than TEa) and the low-pool imprecision is less than TEa / 3 for SD.

[0151] The calculated LOB was 0.419 μg / mL, the LOD was 0.740 μg / mL, and the LOQ was 0.831 μg / mL. Each of the low pools had an SD of less than TEa / 3 and passed the acceptance criteria (Table 7, Figure 5). Table 7. Analytical sensitivity TEa 30% or 1.50 μg / mL

[0152] [Table 10] [Examples]

[0153] Sample stability. Stability was evaluated by measuring adiponectin levels over time in at least seven newly collected serum samples from a single donor under room temperature (18-25°C), refrigeration (2-8°C), freezing (-20°C), and ultra-freezing (-70°C) temperature conditions.

[0154] The acceptance criterion is that the mean recovery value of the evaluated samples does not deviate from TEa / 3 (whichever is greater, 10% or 0.5 μg / mL) from day 0.

[0155] Room temperature (18~25℃) Samples were assayed at 0, 24, 48, and 96 hours.

[0156] The average concentration was within TEa / 3 of the average concentration measured at t=0 at all evaluation time points (Table 8, Figure 6). Adiponectin is stable at room temperature for up to 96 hours. Table 8. Sample stability (room temperature) TEa: 30% or 1.50 μg / mL

[0157] [Table 11]

[0158] Refrigerated (2-8℃) Samples were assayed on days 0, 2, 4, 6, and 20.

[0159] The average concentration was within TEa / 3 of the average concentration measured at t=0 at all evaluation time points (Table 9, Figure 7). Adiponectin remains stable up to 20 days at refrigerated temperatures. Table 9. Sample stability (refrigerated) TEa: 30% or 1.50 μg / mL

[0160] [Table 12]

[0161] Freezing (-20℃) Samples were assayed on days 0, 6, 13, 20, and 27.

[0162] The average concentration remained within TEa / 3 of the average concentration measured at t=0 at all time points up to day 20 (Table 10, Figure 8). Adiponectin is stable up to day 20 at freezing temperatures. Table 10. Sample stability (freezing) TEa: 30% or 1.50 μg / mL

[0163] [Table 13]

[0164] Ultra-freezing (below -70℃) Samples were assayed on days 0 and 27.

[0165] The average concentration was within TEa / 3 of the average concentration measured at t=0 at all evaluation time points (Table 11, Figure 9). Adiponectin remains stable up to day 27 at ultra-freezing temperatures. Table 11. Sample stability (ultra-freezing) TEa: 30% or 1.50 μg / mL

[0166] [Table 14]

[0167] Freeze-thaw stability The samples were frozen and thawed 0, 1, 2, 3, 4, and 5 times.

[0168] The average concentration was within TEa / 3 of the average concentration measured at t=0 in all evaluated freeze-thaw cycles (Table 12, Figure 10). Adiponectin remains stable for up to 5 freeze-thaw cycles. Table 12. Sample stability (freeze-thaw cycle) TEa: 30% or 1.50 μg / mL

[0169] [Table 15]

[0170] Autosampler Stability Forty-seven samples were processed and assayed on days 0 and 6, and the stability of the prepared samples was evaluated using an instrumental autosampler. Supporting data for this study are shown in Table 13 and Figure 11. All reported parameters remained within acceptance criteria throughout the study period. Table 13. Autosampler Stability TEa: 30% or 1.50 μg / mL

[0171] [Table 16] JPEG2026513260000022.jpg222102JPEG2026513260000023.jpg49131

[0172] Processed samples stored in the autosampler for this assay are stable for 6 days. [Examples]

[0173] analysis specificity - Interfering substances. The acceptance criterion in interfering substance studies is that the mean of the sample results does not deviate by more than TEa / 3 from the measurement value before the change.

[0174] hemolysis Hemolytic red blood cells were spiked into a single donor serum pool to concentrations of 0, 10, 40, 100, 200, and 500 mg / dL, and the tests were run in a quadruple sequence. Samples were assayed at each level, and the results are presented in Table 14 and Figure 12. All reported parameters remained within acceptance criteria, except for the maximum concentration of interfering substances tested. Macroscopically hemolytic samples were unacceptable for testing. Table 14. Hemolysis TEa: 30% or 1.50 μg / mL

[0175] [Table 17] Jaundice (bilirubin) Bilirubin was spiked into a single donor serum pool to concentrations of 0, 0.2, 2, and 5 mg / dL, and a quadruple run was performed. Samples were assayed at each level, and the results are presented in Table 15 and Figure 13. All reported parameters remained within acceptance criteria up to the maximum tested concentration of interfering substances. Table 15. Bilirubin TEa: 30% or 1.50 μg / mL

[0176] [Table 18]

[0177] Lipidemia (Intralipid) Intralipid was spiked into a single-donor serum pool to concentrations of 0, 100, 250, and 500 mg / dL, and a quadruple run was performed. Samples were assayed at each level, and the results are presented in Table 16 and Figure 14. All reported parameters remained within acceptance criteria, except for the maximum concentration of interfering substances tested. Samples with macroscopic lipemia were unacceptable for testing. Table 16. Intralipid TEa: 30% or 1.50 μg / mL

[0178] [Table 19] [Examples]

[0179] Reportable range. The reportable range for this assay is 1.50–50.00 μg / mL. [Examples]

[0180] Reference interval. The reference range in this assay (shown in the table below) will be adopted from a comparison with a currently validated ELISA assay, as a split sample comparison between the candidate LC-MS method and the validated ELISA method, compared across the reference interval within TEa / 4.

[0181] [Table 20]

[0182] The content of all papers, patents, and patent applications, as well as all other documents and electronically available information referred to or cited herein, is incorporated herein by reference in the same manner as each individual publication is incorporated specifically and individually by reference. The applicants reserve the right to physically incorporate into this application any and all material and information from any such papers, patents, patent applications, or other physical and electronic documents.

[0183] The embodiments described herein as illustrative can be adequately implemented in the absence of any element or element(s), limitation or limitation(s) not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein are for illustrative purposes only, not limitation, and the use of such terms and expressions does not preclude any equivalents or parts thereof of the indicated and described features, although it is acknowledged that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “essentially consisting of” is understood to include the elements specifically described and any additional elements that do not substantially affect the fundamental and novel characteristics of the claimed technology. The phrase “consisting of” excludes elements not explicitly stated.

[0184] This disclosure should not be limited by the specific embodiments described in this application. Many modifications and variations may be made without departing from the spirit and scope, as will be obvious to those skilled in the art. In addition to those enumerated herein, functionally equivalent methods and compositions within the scope of this disclosure will be obvious to those skilled in the art from the foregoing description. Such modifications and variations fall within the scope of the appended claims. This disclosure should be limited only by the appended claims, along with the entire scope of equivalents to which such claims are entitled. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, or compositions, and that these are naturally subject to change. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not limiting.

[0185] Furthermore, if any feature or aspect of the present disclosure is described by a Markush group, a person skilled in the art will understand that the present disclosure may also be described by any individual component or subgroup of components of the Markush group.

[0186] As will be understood by those skilled in the art, for any and all purposes, and especially by providing written explanations, all scopes disclosed herein also encompass any and all possible partial scopes and combinations thereof. The scopes enumerated can be readily recognized as sufficient to describe and enable the same scope divided at least into two, three, four, five, ten, etc. As a non-limiting example, each scope discussed herein can easily be divided into a lower third, a middle third, an upper third, etc. Again, as will be understood by those skilled in the art, all phrases, e.g., “maximum,” “at least,” “greater than,” “less than,” and similar ones, include the number described and mean a scope that can subsequently be divided into partial scopes, as discussed above. Finally, as will be understood by those skilled in the art, a scope includes each individual component.

[0187] All publications, patent applications, granted patents, and other documents referenced herein are incorporated herein by reference in such a way that each individual publication, patent application, granted patent, or other document is incorporated specifically and individually by reference as a whole. Definitions contained in the text incorporated by reference are excluded to the extent that they contradict the definitions in this disclosure.

[0188] While specific embodiments have been shown and described, it should be understood that modifications and changes can be made therein without departing from the broader art as defined in the following claims.

Claims

1. A method for determining the amount of adiponectin in a sample by mass spectrometry, (a) A step of applying adiponectin derived from a sample to an ionization source under conditions suitable for generating one or more adiponectin ions detectable by mass spectrometry, (b) A step of determining the amount of one or more adiponectin ions by mass spectrometry, (c) A step of determining the amount of adiponectin in the sample from the amount of one or more adiponectin ions determined in step (b), and Methods that include...

2. The method according to claim 1, wherein the sample comprises plasma or serum.

3. The method according to claim 1 or 2, wherein the ionization source is an electrospray (ESI) ionization source.

4. The method according to any one of claims 1 to 3, wherein the ionization is in positive mode.

5. The method according to any one of claims 1 to 4, wherein one or more adiponectin ions include ions with a mass / charge ratio (m / z) of 314.18 ± 0.

5.

6. The method according to any one of claims 1 to 4, wherein one or more adiponectin ions include ions having a mass / charge ratio (m / z) of 627.36 ± 0.

5.

7. The method according to any one of claims 1 to 4, wherein the one or more adiponectin ions include ions having a mass / charge ratio (m / z) of 530.30 ± 0.

5.

8. The method according to any one of claims 1 to 7, wherein an internal standard for adiponectin is added to the sample.

9. The method according to claim 8, wherein the internal standard is recombinant human adiponectin or adiponectin peptide.

10. The method according to claim 8, comprising the step of generating one or more ions of an internal standard detectable by mass spectrometry.

11. The method according to claim 10, wherein one or more ions of the internal standard include an ion having a mass / charge ratio (m / z) of 319.19 ± 0.

5.

12. The method according to claim 10, wherein one or more ions of the internal standard include an ion having a mass / charge ratio (m / z) of 637.37 ± 0.

5.

13. The method according to claim 10, wherein one or more ions of the internal standard include an ion having a mass / charge ratio (m / z) of 540.31 ± 0.

5.

14. The method according to any one of claims 1 to 13, further comprising the step of digestion with trypsin.

15. The method according to any one of claims 1 to 14, further comprising the step of precipitating with formic acid.

16. The method according to any one of claims 1 to 15, further comprising the step of purifying the sample before mass spectrometry.

17. The method according to claim 16, wherein the purification step includes subjecting the sample to liquid chromatography.

18. The method according to claim 17, wherein the liquid chromatography includes high-performance liquid chromatography (HPLC).

19. The method according to any one of claims 1 to 18, wherein the mass spectrometry is tandem mass spectrometry.

20. The method according to any one of claims 1 to 19, wherein low levels of adiponectin correspond to an increased risk of metabolic syndrome.

21. The method according to any one of claims 1 to 19, wherein low levels of adiponectin correspond to an increased risk of type 2 diabetes.

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