Analytical method using in-sample calibration curves by multi-isotopologue reaction monitoring

Abstract

The present disclosure provides several methods in LC-MS / MS analysis: (1) a method of LC-MS / MS analysis technique for determining the analyte concentration of a sample, wherein an in-sample calibration curve (ISCC) is used instead of an external calibration curve by monitoring multi-isotopologue transitions of a stable isotope-labeled (SIL) analyte added to each sample in multi-isotopologue reaction monitoring (MIRM) mode via MS / MS; (2) a method of LC-MS / MS analysis for determining the analyte concentration of a sample, wherein a single sample multi-point external calibration curve (OSMECC) is used instead of a multi-sample external calibration curve; and (3) a method of LC-MS / MS analysis for determining the analyte concentration of a sample having an analyte concentration exceeding an assay ULOQ, wherein isotopic sample dilution is used instead of physically diluting the sample during sample preparation based on calculating the isotopic abundance of the monitored MIRM channel.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 775,318, filed on December 4, 2018. The entire contents of U.S. Provisional Application No. 62 / 775,318 are hereby incorporated herein by reference.

[0003] References to sequence listings submitted electronically via EFS-WEB

[0004] This application includes a sequence listing submitted electronically via EFS-Web (name: "3338_148PC01_SL_ST25.txt"; size: 844 bytes; creation date: December 3, 2019), which is hereby incorporated by reference in its entirety. Background Art

[0005] With the recent rapid development of translational medicine research, quantitative determination of biomarkers in preclinical and clinical studies and quantitative proteomics have played an even more important role in molecular and cell biology research, drug discovery and development, such as identifying and validating potential biomarkers, facilitating patient stratification and dose selection, serving as surrogate endpoints, and establishing PK / PD relationships at the site of action. It is well known that the use of external calibration curves prepared in the same biological matrix as the real study samples, and the use of stable isotope-labeled (SIL) analytes as assay internal standards in both the external calibration curves and the real study samples, are the key to developing accurate and robust liquid chromatography-tandem mass spectrometry (LC-MS / MS) assays for quantitative determination of small molecule drugs and biotherapeutics in biological matrices.

[0006] However, for biomarker measurements in preclinical and clinical studies, the use of external calibration curves and reliable reference standards in a reliable matrix is ​​not possible in many cases due to the endogenous nature of the biomarkers. The preparation of external calibration curves with SIL surrogate analytes in a reliable matrix is ​​one of the alternatives and can provide the same assay performance as that of an assay using a reliable analyte in a reliable matrix, because the SIL surrogate analyte has the "same" physicochemical properties as the reliable analyte with respect to sample extraction, chromatographic separation, MS ionization, fragmentation and detection. However, this approach is not easy to use because another form of the SIL analyte is required as an assay internal standard, and the acquisition cost of two forms of the SIL analyte is extremely high and very time-consuming, especially for the analysis of proteins. Therefore, it is necessary to develop other methods for quantitative LC-MS / MS analysis of biomarkers, drugs or their metabolites in the preclinical or clinical stages of drug development. Summary of the invention

[0007] The present disclosure relates to an LC-MS / MS technique for quantifying the concentration of at least one analyte in a sample, the method comprising adding one or more known amounts of one or more stable isotope labeled (SIL) analytes to a sample containing at least one analyte to construct one or more in-sample calibration curves (ISCC) by multi-isotopologue reaction monitoring (MIRM) of each added SIL analyte, wherein MIRM of the SIL analyte refers to multiple reaction monitoring of multi-isotopic transitions of the SIL analyte; wherein the ISCC for each analyte is constructed in the sample based on the relationship between the calculated theoretical isotopic abundance (analyte concentration equivalent) in the MIRM transition and the measured tandem mass spectrometry (MS / MS) peak area in the corresponding MIRM transition; wherein the concentration of at least one analyte in the sample is quantified using the established ISCC and the measured peak area of ​​the analyte from a liquid chromatography-tandem mass spectrometry (LC-MS / MS) process, and wherein the tandem mass spectrometer is operated in a multiple reaction monitoring mode.

[0008] In some aspects, the analyte, the SIL analyte, and the naturally occurring isotopologues of the SIL analyte are ionized in a mass spectrometer to produce protonated (or deprotonated) parent ions of the analyte, the SIL analyte, and the naturally occurring isotopologues of the SIL analyte. In some aspects, the parent ions of the analyte, the parent ions of the SIL analyte, and the naturally occurring isotopologues of the SIL analyte in the mass spectrometer are fragmented at the same cleavage site to produce neutral losses and daughter ions;

[0009] In some aspects, transitions of the analyte from parent ions to daughter ions are monitored in a mass spectrometer, and the peak area of ​​the analyte transitions from parent ions to daughter ions is measured. In some aspects, selected multiple transitions from parent ions of the SIL analyte and parent ions of naturally occurring isotopologues of the SIL analyte to daughter ions of the SIL analyte and daughter ions of naturally occurring isotopologues of the SIL analyte are monitored in a mass spectrometer ("multi-isotopologue reaction monitoring" or "MIRM");

[0010] In some aspects, the peak area of ​​each MIRM transition is measured, wherein the MIRM transition includes a selected transition from a parent ion of the SIL analyte and a parent ion of a naturally occurring isotopologue of the SIL analyte to a daughter ion of the SIL analyte and a daughter ion of a naturally occurring isotopologue of the SIL analyte.

[0011] In some aspects, an in-sample calibration curve is generated based on the relationship between the peak areas measured in MIRM transitions of the SIL analyte and naturally occurring isotopologues of the SIL analyte and the analyte concentration equivalent for each MIRM transition.

[0012] In some aspects, the analyte concentration equivalent for each MIRM transition is calculated from the theoretical isotopic abundance of the corresponding MIRM transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte, wherein the theoretical isotopic abundance is calculated using the method disclosed in Analytical Chemistry, 2012, 84(11), 4844-4850, wherein the method is calculated based on the neutral loss of the SIL analyte and the isotopic distribution of the daughter ions. In other aspects, for a unit resolution triple quadrupole mass spectrometer, the SIL analyte and the naturally occurring isotopologue of the SIL analyte are calculated from (p+Z p +α) / Z p to (d+Z d +β) / Z d The theoretical isotopic abundance of each MIRM transition (m / z) is calculated based on formula (I):

[0013] (p+Z p +α) / Z p →(d+Z d +β) / Z d The isotopic abundance in the MIRM transition of

[0014] [Mass is (d+Z d +β)) product ions]*

[0015] [Relative isotopic distribution of neutral losses of mass n+(α-β)] (I)

[0016] where m / z is the mass-to-charge ratio

[0017] p is the monoisotopic mass of the parent molecule of the SIL analyte

[0018] Z p is the charge of the parent ion

[0019] d is the monoisotopic mass of the subfragment of the SIL analyte

[0020] Z d is the charge of the daughter ion

[0021] n is the neutrally lost monoisotopic mass of the SIL analyte

[0022] p=d+n

[0023] α and β are integers, which are the number of additional neutrons in the parent ion and the daughter ion, respectively, α ≥ 0, β ≥ 0 and α ≥ β

[0024] Z p and Z d is an integer

[0025] In some aspects, the isotope abundance calculator is located at worldwideweb.sisweb.com / mstools / isotope.html (accessed on November 10, 2019). In some aspects, the maximum analyte concentration equivalent (the "upper limit of quantification" or "ULOQ" of ISCC) is calculated based on formula (II):

[0026] (M / V)*(M 分析物 / M SIL分析物 )ng / mL (II)

[0027] Where M (ng) is the total amount of SIL analyte added to the sample;

[0028] V is the sample volume before adding the SIL analyte (mL);

[0029] M 分析物 is the molecular weight of the analyte;

[0030] M SIL分析物 is the molecular weight of the SIL analyte.

[0031] In some aspects, one or more other analyte concentration equivalents in a MIRM transition are calculated based on formula (III):

[0032] I a *ULOQ(ng / ml) (III)

[0033] Among them I a is the calculated theoretical isotopic abundance of the MIRM transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte.

[0034] The method of the present invention can effectively use the corresponding SIL analyte to detect or quantify the analyte as a protein. In some aspects, the analyte is a protein or a peptide. In some aspects, the SIL analyte is a stable isotope labeled protein or peptide. In some aspects, the parent ion of the analyte and the parent of the SIL analyte comprise at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids or at least about 20 amino acids.

[0035] In some aspects, the parent ion of the analyte and the parent ion of the SIL analyte comprise an amino acid sequence of between 4 and 20 amino acids, between 4 and 15 amino acids, between 5 and 15 amino acids, between 4 and 14 amino acids, between 5 and 14 amino acids, between 5 and 13 amino acids, between 5 and 12 amino acids, between 6 and 15 amino acids, between 6 and 14 amino acids, between 6 and 13 amino acids, between 6 and 12 amino acids, between 6 and 11 amino acids, between 6 and 10 amino acids, between 6 and 9 amino acids, between 6 and 8 amino acids, between 7 and 15 amino acids, between 7 and 14 amino acids, between 7 and 13 amino acids, between 7 and 12 amino acids, between 7 and 11 amino acids, between 7 and 10 amino acids, or between 7 and 9 amino acids. In some aspects, the analyte is an antibody. In other aspects, the analyte is a fusion protein. In some aspects, the analyte is a fusion protein comprising a protein and a heterologous portion. In other aspects, the analyte is an Fc fusion protein. In some aspects, the analyte is PD-1, PD-L1, CD73, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD73 antibody, or any combination thereof.

[0036] The inventive method can also effectively detect or quantify analytes as small molecules. In some aspects, analyte is a small molecule. In some aspects, the SIL analyte is a stable isotope labeled small molecule. In some aspects, the molar mass of the small molecule is at least about 100 g / mol, at least about 200 g / mol, at least about 300 g / mol, at least about 400 g / mol, at least about 500 g / mol, at least about 600 g / mol, at least about 700 g / mol, at least about 800 g / mol, at least about 900 g / mol, at least about 1000 g / mol, at least about 1100 g / mol, at least about 1200 g / mol, at least about 1300 g / mol, at least about 1400 g / mol, at least about 1500 g / mol, at least about 1600 g / mol, at least about 1700 g / mol, at least about 1800 g / mol, at least about 1900 g / mol or at least about 2000 g / mol.

[0037] The methods of the present invention involve the use of isotopically labeled SIL analytes. In some aspects, the SIL analyte contains at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 stable isotope labels.

[0038] In some aspects, the SIL analyte contains about 3 to about 20 isotopic labels, about 3 to about 19 isotopic labels, about 3 to about 15 isotopic labels, about 3 to about 10 isotopic labels, about 3 to about 8 isotopic labels, about 3 to about 7 isotopic labels, about 3 to about 6 isotopic labels, about 4 to about 15 isotopic labels, about 4 to about 10 isotopic labels, about 4 to about 8 isotopic labels, about 4 to about 7 isotopic labels, about 4 to about 6 isotopic labels, about or about 11 to about 13 isotope labels.

[0039] The selection of MIRM transitions for measurement is an important element of the methods of the present invention to ensure robust and accurate assay performance. In some aspects, the relative peak area of ​​each measurement in the MIRM transitions of the SIL analyte has less than 15% deviation compared to the theoretical isotopic abundance calculated in the corresponding MIRM transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte.

[0040] In some aspects, at least one measured relative peak area in a MIRM transition has a deviation of less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% compared to the theoretical isotopic abundance calculated in the corresponding MIRM transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte.

[0041] In some aspects, the number of MIRM transitions is at least two, at least three, at least four, at least five, at least six, at least seven, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20. In some aspects, the number of MIRM transitions is between 4 and 15.

[0042] In some aspects, the analyte concentration equivalents of the highest MIRM transition and the lowest MIRM transition of the SIL analyte are at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, or at least about 2000 times different.

[0043] In some aspects, the calculated theoretical isotopic abundances of two selected MIRM transitions differ by at least 0.01%, differ by at least 0.05%, differ by at least 0.1%, differ by at least 0.5%, differ by at least 1%, differ by at least 1.5%, differ by at least 2%, differ by at least 2.5%, differ by at least 3%, differ by at least 3.5%, differ by at least 4%, differ by at least 4.5%, differ by at least 5%, differ by at least 5.5%, differ by at least 6%, differ by at least 6.5%, differ by at least 7%, differ by at least 7.5%, differ by at least 8%, differ by at least 8.5%, differ by at least 9%, differ by at least 9.5%, differ by at least 10%, differ by at least 20%, differ by at least 30%, differ by at least 40%, or differ by at least about 50%.

[0044] In some aspects, the SIL analyte contains less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% unlabeled analyte.

[0045] In some aspects, the marker is 2 H. 13 C. 15 N. 33 S. 34 S. 36 S. 17 O or 18 O.

[0046] The inventive method relates to a mass spectrum, wherein an ion source is used to ionize an analyte (molecule). In some aspects, one or more protonated or deprotonated molecules are singly charged, doubly charged, triply charged or more highly charged. In some aspects, the mass spectrometer is a triple quadrupole mass spectrometer comprising Q1, Q2 and Q3. In some aspects, the resolution for Q1 and Q3 is unit resolution. In other aspects, the resolution for Q1 and Q3 is higher than unit resolution. In other aspects, the resolution for Q1 and Q3 is different. In other aspects, the resolution for Q1 is higher than the unit resolution of Q3.

[0047] In some aspects, an in-sample calibration curve (ISCC) composition (ie, a known amount of a SIL-analyte) is added prior to or during sample preparation.

[0048] The methods of the present invention are very efficient and improve the total instrument time required, especially when an external calibration curve does not need to be run on the instrument. In some aspects, the methods reduce the total instrument run time. In some aspects, no external calibration curve is used. In some aspects, the analyte is a biomarker. In some aspects, the analyte is a metabolite. In some aspects, the analyte is a small molecule drug. In some aspects, the analyte is a peptide. In some aspects, the analyte is a protein.

[0049] The methods of the present invention can be effectively used to detect or quantify analytes from a variety of sources, including biological sources. In some aspects, the sample is serum, tissue, biopsy tissue, formalin-fixed paraffin-embedded (FFPE), plasma, saliva, cerebrospinal fluid, tears, urine, synovial fluid, dried blood spots, or any combination thereof.

[0050] In some aspects, the analyte is CD73 or a portion thereof. In some aspects, the SIL analyte is V[Ile( 13 C6, 15 N)]YPAVEGR (SEQ ID NO: 1). In some aspects, the analyte is PD-1 or a portion thereof. In some aspects, the SIL analyte is LAAFPED [Arg ( 13 C6, 15 N4)] (SEQ ID NO: 2). In some aspects, the analyte is PD-L1 or a portion thereof. In some aspects, the SIL analyte is LQDAG[Val( 13 C5, 15 N)]YR (SEQ ID NO: 3). In some aspects, the analyte is daclatasvir. In some aspects, the SIL analyte is the SIL analyte is 13 C2 15 N4-Daclatasvir.

[0051] The present method discloses a composition comprising an in-sample calibration curve (ISCC), wherein the ISCC is constructed by multi-isotopologue reaction monitoring (MIRM) of stable isotope-labeled analytes.

[0052] The present invention also discloses a method for quantifying the concentration of at least one analyte in a research sample, the method comprising adding one or more known amounts of one or more analytes to a blank matrix sample to construct one or more single sample multi-point external calibration curves (OSMECC) by multi-isotopologue reaction monitoring (MIRM) of each analyte added, wherein the MIRM of the analyte refers to the multi-reaction monitoring of the multi-isotope transitions of the analyte; wherein the OSMECC of each analyte is constructed in the blank matrix sample based on the relationship between the theoretical isotopic abundance (analyte concentration equivalent) calculated in the MIRM transition and the tandem mass spectrometry (MS / MS) peak area (or peak area ratio, if an internal standard is used in the determination) measured in the corresponding MIRM transition; wherein the concentration of at least one analyte in the research sample is quantified using the established OSMECC and the measured peak area of ​​the analyte from a liquid chromatography-tandem mass spectrometry (LC-MS / MS) process (or peak area ratio, if an internal standard is used in the determination); wherein the peak area ratio of the analyte is the peak area of ​​the analyte divided by the peak area of ​​the internal standard, and wherein the tandem mass spectrometer is operated in a multiple reaction monitoring mode.

[0053] In some aspects, the analyte concentration equivalent for each MIRM transition of the analyte is calculated from the theoretical isotopic abundance of the corresponding MIRM transition of the analyte or a naturally occurring isotopologue of the analyte, wherein the theoretical isotopic abundance is calculated using the method disclosed in Analytical Chemistry, 2012, 84(11), 4844-4850, wherein the method is calculated based on neutral loss of the analyte and the isotopic distribution of the product ions.

[0054] In some aspects, for a unit resolution triple quadrupole mass spectrometer, the theoretical isotopic abundances for each MIRM transition (m / z) of the analyte and the naturally occurring isotopologues of the analyte from (p+Zp+α) / Zp to (d+Zd+β) / Zd are calculated based on formula (I):

[0055] (p+Z p +α) / Z p →(d+Z d +β) / Z d The isotopic abundance in the MIRM transition of

[0056] [Mass is (d+Z d +β)) product ions]*

[0057] [Relative isotopic distribution of neutral losses of mass n+(α-β)] (I),

[0058] where m / z is the mass-to-charge ratio,

[0059] p is the monoisotopic mass of the parent molecule of the analyte,

[0060] Z p is the charge of the parent ion,

[0061] d is the monoisotopic mass of the subfragment of the analyte,

[0062] Z d is the charge of the daughter ion,

[0063] n is the monoisotopic mass of the neutral loss of the analyte,

[0064] p=d+n,

[0065] α and β are integers, α ≥ 0, β ≥ 0 and α ≥ β, and

[0066] Z p and Z d is an integer

[0067] In some aspects, the isotope distribution calculator is located at worldwideweb.sisweb.com / mstools / isotope.html (accessed on November 10, 2019). In some aspects, the maximum analyte concentration equivalent (the "upper limit of quantitation" or "ULOQ" of ISCC) is calculated based on formula (II):

[0068] (M / V)*ng / mL (IV)

[0069] Where M (ng) is the total amount of analyte added to the sample;

[0070] V is the sample volume before adding the analyte (mL);

[0071] In some aspects, one or more other analyte concentration equivalents in a MIRM transition are calculated based on formula (III):

[0072] Ia*ULOQ(ng / mL) (III)

[0073] where Ia is the calculated theoretical isotopic abundance of the MIRM transition of the analyte or a naturally occurring isotopologue of the analyte.

[0074] In some aspects, the analyte is a protein or peptide. In some aspects, the parent ion of the analyte comprises at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, or at least about 20 amino acids. In some aspects, the parent ion of the analyte comprises an amino acid sequence of between 4 and 20 amino acids, between 4 and 15 amino acids, between 5 and 15 amino acids, between 4 and 14 amino acids, between 5 and 14 amino acids, between 5 and 13 amino acids, between 5 and 12 amino acids, between 6 and 15 amino acids, between 6 and 14 amino acids, between 6 and 13 amino acids, between 6 and 12 amino acids, between 6 and 11 amino acids, between 6 and 10 amino acids, between 6 and 9 amino acids, between 6 and 8 amino acids, between 7 and 15 amino acids, between 7 and 14 amino acids, between 7 and 13 amino acids, between 7 and 12 amino acids, between 7 and 11 amino acids, between 7 and 10 amino acids, or between 7 and 9 amino acids. In some aspects, the analyte is an antibody. In some aspects, the analyte is a fusion protein. In some aspects, the analyte is PD-1, PD-L1, CD73, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CD73 antibody, or any combination thereof. In some aspects, the analyte is a small molecule.

[0075] In some aspects, the molar mass of the small molecule is at least about 100 g / mol, at least about 200 g / mol, at least about 300 g / mol, at least about 400 g / mol, at least about 500 g / mol, at least about 600 g / mol, at least about 700 g / mol, at least about 800 g / mol, at least about 900 g / mol, at least about 1000 g / mol, at least about 1100 g / mol, at least about 1200 g / mol, at least about 1300 g / mol, at least about 1400 g / mol, at least about 1500 g / mol, at least about 1600 g / mol, at least about 1700 g / mol, at least about 1800 g / mol, at least about 1900 g / mol, or at least about 2000 g / mol. In some aspects, the number of MIRM transitions is at least two, at least three, at least four, at least five, at least six, at least seven, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20. In some aspects, the number of MIRM transitions is between 2 and 20. In some aspects, the analyte concentration equivalents of the highest MIRM and the lowest MIRM are at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, or at least about 2000-fold different. In some aspects, the calculated theoretical isotopic abundances of two selected MIRM transitions differ by at least 0.01%, differ by at least 0.05%, differ by at least 0.1%, differ by at least 0.5%, differ by at least 1%, differ by at least 1.5%, differ by at least 2%, differ by at least 2.5%, differ by at least 3%, differ by at least 3.5%, differ by at least 4%, differ by at least 4.5%, differ by at least 5%, differ by at least 5.5%, differ by at least 6%, differ by at least 6.5%, differ by at least 7%, differ by at least 7.5%, differ by at least 8%, differ by at least 8.5%, differ by at least 9%, differ by at least 9.5%, differ by at least 10%, differ by at least 20%, differ by at least 30%, differ by at least 40%, or differ by at least about 50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 shows a scheme diagram of the MIRM-ISCC-MS / MS method using a surrogate peptide of PD-L1 as an example. A certain amount of SIL analyte was added based on the expected concentration of the analyte to generate an appropriate calibration curve. Line 1 shows SIL analyte MIRM channel 1 (m / z: 464.2→686.4), isotopic abundance 100%: SIL analyte concentration of 100 ng / mL (analyte concentration equivalent to 99.4 ng / mL); Line 2 shows SIL analyte MIRM channel 2 (m / z: 464.7→687.4), isotopic abundance 30.0%: SIL analyte concentration of 30.0 ng / mL (analyte concentration equivalent to 29.8 ng / mL). / mL of analyte concentration equivalent); line 3 shows the SIL analyte MIRM channel 3 (m / z: 465.2→688.4), isotopic abundance 6.63%: SIL analyte concentration of 6.63ng / mL (analyte concentration equivalent of 6.59ng / mL); and line 4 shows the analyte selected reaction monitoring (SRM) channel (m / z: 461.2→680.4), measured concentration: 56.8ng / mL.

[0077] Figures 2A and 2B show representative chromatograms of ten MIRM channels of SIL CD73 peptide and one selected reaction monitoring (SRM) channel of CD73 peptide. These ten MIRM channels were used to construct ISCC for quantitative analysis of CD73. Figure 2A is a zoomed-out view; Figure 2B is an enlarged view.

[0078] Figure 3 shows the LC-MS / MS bioanalysis workflow for single sample multipoint external calibration curve (OSMECC) and isotopic sample dilution.

[0079] Figures 4A and 4B. Figure 4A shows a summary of the MRM and MIRM transitions monitored for the multiple sample external calibration curve, the single sample multipoint external calibration curve (OSMECC), the in-sample calibration curve (ISCC), and the isotopic sample dilutions used to measure daclatasvir. Figure 4B shows the calibration curve performance of the two multiple sample external calibration curves and two single sample multipoint external calibration curves used, as well as the two ISCCs.

[0080] Fig. 5A and Fig. 5B. Fig. 5A shows the accuracy and precision data of QC samples quantitatively using multi-sample external calibration curve, single sample multi-point external calibration curve and ISCC. Test concentrations are within the calibration curve range (1, 3, 40, 500 and 800 ng / mL) of QC samples and concentrations beyond the calibration curve range (5000 and 20000 ng / mL) of QC samples. However, during sample preparation, 5000 and 20000 ng / mL of QC samples were physically diluted (100 times and 200 times, respectively) to the calibration curve range. Fig. 5B shows the accuracy and precision data of QC samples quantitatively diluted using isotope samples. Only test concentrations beyond the calibration curve range (5000, 20000 and 50000 ng / mL) of QC samples. DETAILED DESCRIPTION

[0081] The present disclosure provides a highly effective method for quantifying the concentration of an analyte by LC-MS / MS. Specifically, the method uses an in-sample calibration curve (ISCC) that uses a stable labeled isotope (SIL) analyte to measure the concentration of an analyte in a sample. The present disclosure is characterized in that an ISCC can be used to replace an external calibration curve, thereby shortening the total LC-MS / MS instrument run time. In addition, because the ISCC is present inside the sample itself, the method of the present invention eliminates the need to prepare an external calibration curve using a reliable biological matrix and simplifies the quantitative LC-MS / MS bioanalysis process.

[0082] As shown in the working examples, the method can effectively quantify the concentration of the analyte by monitoring isotope transitions through MIRM to generate a concentration curve. In certain aspects, the present disclosure provides a method of adding a SIL analyte to a sample containing an analyte, wherein the analyte can be quantified by ISCC generated by "multi-isotopologue reaction monitoring" or "MIRM" from the SIL analyte. In certain aspects, the present disclosure provides a method for analyzing protein analytes by using stable isotope-labeled proteins or protein fragments. In some aspects, the present disclosure provides a method for quantifying antibody concentrations. In other aspects, the present disclosure provides a method for analyzing small molecules using stable isotope-labeled small molecule variants.

[0083] I. Terminology

[0084] Where used herein, the term "and / or" is considered to be a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0085] It is to be understood that wherever aspects are described herein with the language "comprising," other similar aspects described with the phrases "consisting of" and / or "consisting essentially of" are also provided.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide a general dictionary of many of the terms used in the present disclosure to the skilled person.

[0087] Units, prefixes and symbols are expressed in a form acceptable to the International System of Units (SI). Numerical ranges include numbers that limit the ranges. The headings provided herein are not limitations on the various aspects of the disclosure, which can be obtained by reference to the specification as a whole. Therefore, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0088] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any stated or listed component.

[0089] The term "about" or "consisting essentially of..." refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" or "consisting essentially of..." can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "consisting essentially of..." can mean a range of up to 10%. In addition, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times of a value. When a particular value or composition is provided in the application and claims, unless otherwise stated, it should be assumed that the meaning of "about" or "consisting essentially of..." is within an acceptable error range for that particular value or composition.

[0090] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the stated range and fractions of the stated value (such as tenths and hundredths of an integer) where appropriate.

[0091] As used herein, "biomarker" is virtually any detectable compound present in or derived from a biological sample, such as a protein, peptide, proteoglycan, glycoprotein, lipoprotein, carbohydrate, lipid, nucleic acid (e.g., DNA, such as cDNA or amplified DNA, or RNA, such as mRNA), organic or inorganic chemical, natural or synthetic polymer, small molecule (e.g., metabolite), or a distinctive molecule or distinctive fragment of any of the foregoing; or any other characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention or its indications.

[0092] As used herein, "metabolite" refers to any intermediate or product of metabolism. The presence of a drug metabolite is a reliable indicator that a person is using the "parent" drug of that metabolite. A metabolite can be a biomarker.

[0093] As used herein, "metabolic profile" refers to any defined set of values ​​that can be used for quantitative results of metabolites compared to a reference value or profile derived from another sample or group of samples.

[0094] As used herein, the term "isotopologue" refers to a composition that differs from its parent composition in that at least one atom has a different number of neutrons.

[0095] As used herein, the term "analyte" is used in its broadest sense to include any molecule or protein (natural or recombinant) present in a mixture that needs to be analyzed or quantified. Such analytes include, but are not limited to, small molecules, enzymes, hormones, growth factors, cytokines, peptides, immunoglobulins (e.g., antibodies), and / or any fusion proteins.

[0096] As used herein, the terms "isotope" and "isotopologue" are used interchangeably and refer to a molecule having a different isotopic composition as compared to a parent molecule.

[0097] As used herein, the term "analyte equivalent" or "analyte concentration equivalent" refers to the calculated concentration of the SIL analyte used to construct an in-sample calibration curve (ISCC) after adjusting for the mass difference between the SIL analyte and the analyte. The ISCC calibration curve is constructed based on parallel measurements of the MIRM of the SIL analyte to determine the peak area of ​​each MIRM. Due to the mass difference between the SIL analyte and the unlabeled analyte, the calculated concentration curve must be adjusted to account for these slight mass differences while also adjusting for isotopic abundance. A representative ISCC concentration curve can be seen in Figure 1, in which the SIL analyte concentration is adjusted to account for differences in mass and isotopic abundance. The sample adjustment for calculating the analyte equivalent of the highest concentration (upper limit of quantitation "ULOQ") of the concentration curve is represented as point 1 in Figure 1 and is calculated based on formula (II):

[0098] (M / V)*(M 分析物 / M SIL分析物 )ng / mL (II)

[0099] Where M (ng) is the total amount of SIL analyte added to the sample;

[0100] V is the sample volume before adding the SIL analyte (mL);

[0101] M 分析物 is the molecular weight of the analyte;

[0102] M SIL分析物 is the molecular weight of the SIL analyte.

[0103] For example, if the SIL analyte is spiked at a known concentration of 100 ng / mL and the isotopic abundance of the MIRM is 100%, then the analyte concentration equivalent will be 99.4 ng / mL, represented as data point 1 in Figure 1 and is also the ULOQ. The sample adjustments for the remaining analyte equivalent concentrations of the concentration curve are represented as points 2 and 3 in Figure 1 and are calculated based on formula (III):

[0104] I a*ULOQ(ng / ml) (III)

[0105] Among them I a is the calculated theoretical isotopic abundance of the MIRM transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte.

[0106] As used herein, the term "parent ion" or "precursor ion" refers to a charged molecular moiety that can dissociate to form fragments (one or more fragments can be charged) and one or more neutral species. The parent ion can be a molecular ion or a charged fragment of a molecular ion.

[0107] As used herein, the term "daughter ion" refers to the charged product of a reaction of a particular parent (precursor) ion. Typically, such ions have a direct relationship to a particular precursor ion, and may refer to a unique state of the precursor ion. The reaction need not involve fragmentation, but may, for example, involve a change in the number of charges carried. Thus, a fragment ion is a daughter ion, but not all daughter ions are fragment ions.

[0108] As used herein, the term "neutral loss" refers to the mass of neutral charge that is lost during the reaction of a particular parent (precursor) ion during operation of a mass spectrometer.

[0109] As used herein, the term "non-peptide molecule" is intended to be its most extensive meaning, and may include small molecules and small molecule drugs. "Small molecules" or "small molecule drugs" are widely used herein to refer to organic, inorganic or organometallic compounds that are generally less than about 1000-2000g / mol molecular weight, but for the purposes of the present invention, this characterization is not intended to be restrictive. "Small molecules" may also refer to non-peptide, non-oligomeric organic compounds synthesized in a laboratory or found in nature. Examples of "small molecules" include, but are not limited to, paclitaxel, clopidogrel and apixaban. Other examples of small molecules include dapagliflozin, saxagliptin, tamsavir, ledipasvir, sofosbuvir and rosuvastatin.

[0110] The term "chromatography" refers to any kind of technique for separating a molecule (e.g., an antibody) from other molecules (e.g., contaminants) present in a mixture. Typically, molecules are separated from other molecules (e.g., contaminants) as a result of differences in the rate at which individual molecules in a mixture migrate through a fixed medium under the influence of a mobile phase or during binding and elution. The terms "matrix" or "chromatographic matrix" are used interchangeably herein and refer to any kind of adsorbent, resin, or solid phase that separates molecules from other molecules present in a mixture during separation. Non-limiting examples include particulate, monolithic, or fibrous resins and membranes that can be placed in a column or cartridge. Examples of materials for forming matrices include polysaccharides (e.g., agarose and cellulose); and other mechanically stable matrices such as silica (e.g., controlled pore glass), poly(styrenedivinyl) benzene, polyacrylamide, ceramic particles, and derivatives of any of the above. Examples of typical matrix types suitable for the methods of the present disclosure are cation exchange resins, affinity resins, anion exchange resins, or mixed mode resins. "Ligands" are functional groups attached to a chromatographic matrix and that determine the binding properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, sulfophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations of the above). Some preferred ligands that can be used in this article include, but are not limited to, strong cation exchange groups, such as sulfopropyl, sulfonic acid; strong anion exchange groups, such as trimethylammonium chloride; weak cation exchange groups, such as carboxylic acids; weak anion exchange groups, such as N5N diethylamino or DEAE; hydrophobic interaction groups, such as phenyl, butyl, propyl, hexyl; and affinity groups, such as protein A, protein G, and protein L. In order to make it easier to understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning set forth below. Additional definitions are set forth throughout this application.

[0111] The term "affinity chromatography" refers to a protein separation technique in which a molecule (e.g., a molecule or antibody containing an Fc region) specifically binds to a ligand that is specific to the molecule. Such a ligand is commonly referred to as a biospecific ligand. In some aspects, a biospecific ligand (e.g., protein A or its functional variant) is covalently attached to a chromatographic matrix material and can be close to the molecule in solution when the solution contacts the chromatographic matrix. During the chromatographic step, the molecule generally retains its specific binding affinity to the biospecific ligand, while other solutes and / or proteins in the mixture will not bind significantly or specifically to the ligand. The binding between the molecule and the immobilized ligand allows contaminating proteins or protein impurities to pass through the chromatographic matrix while the molecule remains specifically bound to the immobilized ligand on the solid phase material. The specifically bound molecule is then removed from the immobilized ligand in an active form under suitable conditions (e.g., low pH, high pH, ​​high salt, competitive ligands, etc.), and is passed through a chromatographic column with an elution buffer, free of contaminating proteins or protein impurities that were allowed to pass through the column earlier. Any component can be used as a ligand for purifying its corresponding specific binding protein (e.g., antibody). However, in various methods according to the present disclosure, protein A is used as a ligand for a target protein containing an Fc region. One of ordinary skill in the art can easily determine the conditions for eluting a biospecific ligand (e.g., protein A) from a target protein (e.g., a protein containing an Fc region). In some aspects, protein G or protein L or a functional variant thereof can be used as a biospecific ligand. In some aspects, a biospecific ligand (e.g., protein A) is used to bind to a protein containing an Fc region in the pH range of 5-9, and the biospecific ligand / target protein conjugate is washed or re-equilibrated, followed by elution with a buffer having a pH of about 4 or less containing at least one salt.

[0112] The terms "purify", "separate" or "isolate" as used interchangeably herein refer to increasing the purity of a molecule from a composition or sample comprising the molecule and one or more impurities. Typically, the purity of a molecule is increased by removing (completely or partially) at least one impurity from the composition.

[0113] The term "chromatography column" or "column" in connection with chromatography as used herein refers to a container, usually in the form of a cylinder or hollow column, which is filled with a chromatography matrix or resin. A chromatography matrix or resin is a material that provides physical and / or chemical properties for purification.

[0114] The terms "ion exchange" and "ion exchange chromatography" refer to a chromatographic process in which an ionizable solute of interest (e.g., a molecule in a mixture) interacts with an oppositely charged ligand linked (e.g., by covalent attachment) to a solid phase ion exchange material under appropriate conditions of pH and conductivity, such that the solute of interest interacts nonspecifically with the charged compound more or less than with solute impurities or contaminants in the mixture. Contaminating solutes in the mixture can be washed from a column of ion exchange material, or bound to or excluded from the resin faster or slower than the solute of interest. "Ion exchange chromatography" explicitly includes cation exchange (CEX), anion exchange (AEX), and mixed mode chromatography.

[0115] "Cation exchange resin" or "cation exchange membrane" refers to a negatively charged solid phase and has free cations for exchange with cations in an aqueous solution passing over or through the solid phase. Any negatively charged ligand attached to a solid phase suitable for forming a cation exchange resin may be used, such as carboxylates, sulfonates, and other ligands as described below. Commercially available cation exchange resins include, but are not limited to, for example, those with the following groups: sulfonate-based groups (e.g., MonoS, MiniS, Source 15S and 30S, SP from GE Healthcare) Fast Stream, SP High performance from Tosoh SP-650S and SP-650M from BioRad High S, ceramic HyperD S from Pall Technologies, M and LS SP and Spherodex LS SP); sulfoethyl-based groups (e.g., SE, Applied Biosystems S-10 and S-20); sulfopropyl-based groups (e.g., TSK gels SP 5PW and SP-5PW-HR from Tosoh, HS-20, HS 50 and XS); sulfoisobutyl-based groups (e.g., EMD SO3 - ); sulfoethyl-based groups (e.g., SE52, SE53, and Express-Ion S from Whatman); carboxymethyl-based groups (e.g., CM from GE Healthcare); Fast Flow, Hydrocell CM from Biochrom Labs Inc., CM, Ceramic HyperD CM from Pall Technologies, M CM, LS CM, Matrx from Millipore C500 and C200, CM52, CM32, CM23 and Express-Ion C from Whatman, and CM-650S, CM-650M and CM-650C); sulfonic acid and carboxylic acid based groups (e.g., Carboxyl-Sulfon); carboxylic acid based groups (e.g., WP CBX from JT Baker, MAC-3 from Sigma-Aldrich Weak cation exchanger, Weak cation exchangers and Weak cation exchangers, and from EMD EMD COO--); sulfonic acid-based groups (e.g., Hydrocell SP from Biochrom Labs Inc., Fine mesh strong acid cationic resin, UNOsphere S, WP Sulfonic from JTBaker, S membrane from Sigma-Aldrich Strong cation exchanger, Strong cations and strong cation exchangers); or orthophosphate-based groups (e.g., P11 from Whatman).

[0116] Other cation exchange resins include Poros HS, Poros XS, carboxymethyl cellulose, BAKERBOND ABXTM, sulfopropyl immobilized on agarose and sulfonyl immobilized on agarose, MonoS, MiniS, Source 15S, 30S, SPSEPHAROSE TM CM SEPHAROSE TM、BAKERBOND Carboxy-Sulfon、WP CBX、WP Sulfonic、HydrocellCM、Hydrocel SP、UNOsphere S、Macro-Prep High S、Macro-Prep CM、Ceramic HyperD S、Ceramic HyperD CM、Ceramic HyperD Z、Trisacryl M CM、Trisacryl LS CM、Trisacryl M SP、Trisacryl LS SP、Spherodex LS SP、DOWEX Fine Mesh Strong Acid Cationic Resin、DOWEX MAC-3、MatrexCellufine C500、Matrex Cellufine C200、Fractogel EMD SO3-、Fractogel EMD SE、Fractogel EMD COO-、Amberlite Weak and Strong Cation Exchangers、Diaion Weak and Strong Cation Exchangers、TSK Gel SP-5PW-HR、TSK Gel SP-5PW、Toyopearl CM(650S, 650M, 650C), Toyopearl SP(650S, 650M, 650C), CM(23, 32, 52), SE(52, 53), P11, Express-Ion C or Express-Ion S.

[0117] "Anion exchange resin" or "anion exchange membrane" refers to a positively charged solid phase to which one or more positively charged ligands are attached. Any positively charged ligand, such as a quaternary amino group, attached to a solid phase suitable for forming an anion exchange resin may be used. Commercially available anion exchange resins include DEAE cellulose from Applied Biosystems, PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Sartorius Q, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX Fast flow, Q High performance, QAE and FAST Q (GE Healthcare), WP PEI, WP DEAM, WP QUAT from JTBaker, Hydrocell DEAE and HydrocellQA from Biochrom Labs Inc., UNOsphere Q from Biorad, DEAE and High Q, Ceramic HyperD Q, Ceramic HyperD DEAE, M and LS DEAE, Spherodex LS DEAE from Pall Technologies, QMA LS, QMA M and Q, from Dow Liquid Separations Fine mesh strong base type I and II anion resins and MONOSPHER E 77, weak base anion, from Millipore Q film, Matrex A200, A500, Q500 and Q800 from EMD EMD TMAE, EMD DEAE and EMD DMAE from Sigma-Aldrich Weak and strong anion exchangers type I and II, Weak and strong anion exchangers type I and II, Weak and strong anion exchangers type I and II, TSK Gel Q and DEAE 5PW and 5PW-HR from Tosoh, SuperQ-650S, 650M, and 650C, QAE-550C and 650S, DEAE-650M and 650C, QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D, or Express-Ion Q from Whatman, and Q (Sartorius Corporation, New York, USA).

[0118] Other anion exchange resins include POROS HQ, Q SEPHAROSE TM Fast Flow, DEAE SEPHAROSE TM Fast flow, Q. ANX SEPHAROSE TM 4 Fast Flow (High Sub), Q SEPHAROSE TM XL, Q SEPHAROSE TM Big beads, DEAE Sephadex A-25, DEAE Sephadex A-50, QAE Sephadex A-25, QAE Sephadex A-50, QSEPHAROSE TMHigh performance, Q SEPHAROSE TM XL, Source 15Q, Source 30Q, Resourse Q, Capto Q, Capto DEAE, Mono Q, Toyopearl Super Q, Toyopearl DEAE, Toyopearl QAE, Toyopearl Q, Toyopearl GigaCap Q, TS gel SuperQ, TS gel DEAE, Fractogel EMD TMAE, Fractogel EMDTMAE HiCap, Fractogel EMD DEAE, Fractogel EMD DMAE, Macroprep High Q, Macro-prep-DEAE, Unosphere Q, Nuvia Q, PORGS PI, DEAE Ceramic HyperD or Q Ceramic HyperD.

[0119] "Mass spectrometry" ("MS" or "mass-spec") is an analytical technique for measuring the mass-to-charge ratio of ions. This can be achieved by ionizing a sample and separating particles of different masses, and recording their relative abundance by measuring the intensity of the ion flow. A typical mass spectrometer contains three parts: an ion source, a mass analyzer, and a detector system. The ion source is the part of the mass spectrometer that ionizes the substance being analyzed (the analyte). The ions are then transported to the mass analyzer by a magnetic or electric field, which separates the ions according to their mass-to-charge ratio (m / z). Many mass spectrometers use two or more mass analyzers to perform tandem mass spectrometry (MS / MS). The detector records the charge induced or the current generated when the ions pass through or hit a surface. The mass spectrum is the result of measuring the signal generated in the detector when the m / z ions are scanned with a mass analyzer.

[0120] As used herein, the term "in-sample calibration curve (ISCC)" refers to a calibration curve that exists in the sample matrix itself, rather than an external calibration curve as, for example, is traditionally used to perform LC-MS / MS analysis.

[0121] As used herein, the term "SIL analyte" or "stable isotope labeled analyte" refers to a compound that is an analyte or fragment thereof that has been modified to contain an isotope at one or more positions. The most commonly used labeling techniques use 13 C or 15 N is used as a stable isotope, and the compound may be labeled at one or more positions. Other suitable stable isotopes include 2 H. 33 S. 34 S.36 S. 17 O or 18 O.

[0122] The term "multi-isotopologue reaction monitoring" or MIRM refers to the following process, by which a mass spectrometer is tuned to monitor specific multiple reaction monitoring (MRM) transitions from parent ions and their isotopologue ions (parent ions with different neutrons) to their product ions with the same cleavage site, also referred to as "MIRM channels" or "MIRM transitions". More generally, when a mass spectrometer is used to measure a single ion alone, the process is also referred to as selected reaction monitoring (SRM). When multiple reactions (wherein multiple product ions are produced from one or more precursor ions) are measured by SRM, the process is referred to as multiple reaction monitoring (MRM) in the art. In an SRM experiment on a triple quadrupole mass spectrometer, the first quadrupole (Q1) is set to pass only ions (precursor ions) with a specified m / z of the expected chemical species in the sample. The second quadrupole (i.e., Q2 or collision cell) is used to fragment the ions passing through Q1. The third quadrupole (Q3) is set to allow only ions (fragment ions) with a specified m / z corresponding to the expected fragmentation products of the expected chemical species to pass to the detector. When multiple SRM experiments are run, the process is called multiple reaction monitoring ("MRM").

[0123] As used herein, the term "MIRM transition" or alternatively parent-daughter ion transition pair "PDITP" refers to the m / z value pair monitored. In simple terms, for the parent ion P(p+1.00783*Z p The monoisotopic mass, Z p is the charge of the parent ion, and the mass of the hydrogen monoisotope is 1.00783Da) and the daughter ion D(d+1.00783*Z d The monoisotopic mass, Z d is the charge number of the product ion) and the neutral loss N (monoisotopic mass of n), the most abundant (100%) MIRM channel (m / z) is shown below:

[0124] (p+1.00783*Z p ) / Z p →(d+1.00783*Z d ) / Z d

[0125] For a unit resolution triple quadrupole mass spectrometer using the most commonly used charge states (singly, doubly, and triply charged ions), this MIRM channel (m / z) can be simplified to:

[0126] (p+Z p ) / Z p →(d+Z d) / Z d

[0127] The isotopic abundance in adjacent MIRM channels (m / z) is

[0128] (p+Z p +α) / Z p →(d+Z d +β) / Z d

[0129] It can be calculated as:

[0130] (p+Z p +α) / Z p →(d+Z d +β) / Z d Isotope abundance in adjacent MIRM channels =

[0131] [Mass is (d+Z d +β)) product ions]*

[0132] [Relative isotopic distribution of neutral losses of mass n+(α-β)]

[0133] Where: (1) p = d + n

[0134] (2) α and β are integers, which are the number of additional neutrons in the parent ion and the daughter ion, respectively, α ≥ 0, β ≥ 0 and α ≥ β

[0135] (3) Z p and Z d is an integer

[0136] (4) The isotopic distribution of a molecule can be found using an online calculator (worldwideweb.sis web.com / mstools / isotope.html, accessed on November 10, 2019)

[0137] (5) The relative isotopic distribution of the product ions and neutral losses with masses d (α=0) and n (α-β=0) is 100%.

[0138] By using different combinations of α and β, (p+Z p +α) / Z p →(d+Z d +β) / Z d Isotope abundances in different adjacent MIRM channels (m / z).

[0139] Various aspects of the disclosure are described in more detail in the following subsections.

[0140] II. Quantitative Methods

[0141] The present disclosure relates to a multi-isotopologue reaction monitoring-in-sample curve calibration-LC-MS / MS (MIRM-ISCC-LC-MS / MS) method that allows each individual sample to be measured instantly and accurately without the use of an external calibration curve, thereby eliminating the need to use a reliable biological matrix, simplifying the quantitative LC-MS / MS bioanalysis process, and significantly reducing instrument time. Although the MIRM-ISCC-LC-MS / MS method can be applied to routine pharmacokinetic (PK) sample analysis in drug discovery and development, this method is particularly useful for the following situations: in situations where reliable matrices are difficult to obtain (such as biomarker measurements and quantitative proteomics), in situations where low throughput and long turnaround time are major issues that hinder the use of LC-MS / MS technology (such as clinical diagnostics in clinical diagnostic laboratories), and in situations where the preparation of calibration curves is cumbersome (such as fresh frozen and FFPE tissue analysis). In addition, ISCC can also be used as an external calibration curve by spiking a known amount of unlabeled analyte in a blank matrix, and therefore, an external calibration curve can be constructed in only one sample, thereby eliminating the need to prepare multiple samples for external calibration curves.

[0142] Typically, the most abundant MIRM channel of an analyte is monitored in an LC-MS / MS assay for quantitative analysis. Due to the naturally occurring isotopes of an element, in addition to the MS / MS response observed in its most abundant MIRM channel, the isotopic abundances in the isotopic MIRM channels adjacent to the most abundant MIRM channel can also be accurately calculated and measured by LC-MS / MS. In simple terms, for the parent ion P(p+1.00783*Z p The monoisotopic mass, Z p is the charge of the parent ion, and the mass of the hydrogen monoisotope is 1.00783Da) and the daughter ion D(d+1.00783*Z d The monoisotopic mass, Z d is the charge number of the product ion) and the neutral loss N (monoisotopic mass of n), the most abundant (100%) MIRM channel (m / z) is shown below:

[0143] (p+1.00783*Z p ) / Z p →(d+1.00783*Z d ) / Z d

[0144] For a unit resolution triple quadrupole mass spectrometer using the most commonly used charge states (singly, doubly, and triply charged ions), this MIRM channel (m / z) can be simplified to:

[0145] (p+Z p ) / Z p →(d+Z d ) / Z d

[0146] The isotopic abundance in adjacent MIRM channels (m / z) is

[0147] (p+Z p +α) / Z p →(d+Z d +β) / Z d

[0148] It can be calculated as:

[0149] (p+Z p +α) / Z p →(d+Z d +β) / Z d Isotope abundance in adjacent MIRM channels =

[0150] [Mass is (d+Z d +β)) product ions]*

[0151] [Relative isotopic distribution of neutral losses of mass n+(α-β)]

[0152] Where: (1) p = d + n

[0153] (2) α and β are integers, which are the number of additional neutrons in the parent ion and the daughter ion, respectively, α ≥ 0, β ≥ 0 and α ≥ β

[0154] (3) Z p and Z d is an integer

[0155] (4) The isotopic distribution of a molecule can be found using an online calculator (worldwideweb.sis web.com / mstools / isotope.html, accessed on November 10, 2019)

[0156] (5) The relative isotopic distribution of the product ions and neutral losses with masses d (α = 0) and n (α-β = 0) is 100%

[0157] By using different combinations of α and β, (p+Z p +α) / Z p →(d+Z d +β) / Z disotopic abundance in different adjacent MIRM channels (m / z). The isotopic abundance from the most abundant MIRM channel (α=0 and β=0) to the least abundant MIRM channel can reach up to 5 to 6 orders of magnitude. In theory, because these isotopic abundances come from the combination of different isotopes of product ions and neutral losses, and they have the "same" physicochemical properties, there should be a linear relationship between the MS / MS response (peak area) and the calculated theoretical isotopic abundance in all adjacent MIRM channels over the entire range. However, this linear relationship is limited by the detection limit of the mass spectrometer and the linear range of most mass spectrometers. In order to maintain this linear relationship, the same mass spectrometer parameters, such as sampling time, collision energy (CE), and declustering voltage (DP), need to be set for all MIRM channels for SIL analytes and SRM channels for analytes.

[0158] The present method shows a completely new approach that uses the isotopic abundance in the multi-isotopologue reaction monitoring (MIRM) channel to construct an in-sample calibration curve (ISCC) in each study sample for quantitative LC-MS / MS bioanalysis. The MIRM-ISCC-LC-MS / MS method has a variety of potential applications, such as for quantitative analysis of small molecules, peptides, proteins, biomarkers, quantitative proteomics, clinical diagnostic laboratories and other fields.

[0159] Absolute quantification using the isotope dilution principle in LC-MS proteomics is achieved by spiking a known amount of SIL peptides (AQUA method) or proteins (PSAQ method) into each study sample. The peptide (or protein) concentration of the study sample can be calculated using the ratio between the peak area of ​​the unlabeled peptide (or protein) and the peak area of ​​the labeled peptide (or protein). However, this quantitative method is based on only one calibration point and the following assumption: there is a linear relationship through the origin between the MS response (or reaction ratio) and the corresponding concentration.

[0160] Recently, to address this issue, Chiva et al. (66th ASMS Conference on Mass Spectrometry and Allied Topics, June 3-7, 2018, San Diego, California, p ThP481) reported that an accurate and robust assay was developed by spiking a calibration curve into each study sample for absolute quantification of targeted peptides in formalin-fixed paraffin-embedded (FFPE) samples. This calibration curve was prepared in advance using five different SIL peptide analytes (for each SIL peptide analyte, the total labeling positions were 10, 16, 23, 33, and 39, respectively) at different concentration levels from 2 to 200 fmol. However, since the analysis of a targeted peptide requires multiple differently labeled peptide analytes with up to 30 to 40 labeling positions, this method is extremely costly and time-consuming, especially for quantitative proteomics of multiple targeted peptides.

[0161] Methods useful in the present disclosure relate to detecting the presence and / or quantifying the concentration of at least one analyte in a sample, the method comprising adding one or more known amounts of one or more stable isotope labeled (SIL) analytes to a sample containing at least one analyte to construct one or more in-sample calibration curves (ISCC) by multi-isotopologue reaction monitoring (MIRM) of each added SIL analyte, wherein MIRM of the SIL analyte refers to multiple reaction monitoring of multi-isotopic transitions of the SIL analyte; wherein the ISCC for each analyte is constructed in the sample based on the relationship between the calculated theoretical isotopic abundance (analyte concentration equivalent) in the MIRM transition and the measured tandem mass spectrometry (MS / MS) peak area in the corresponding MIRM transition; wherein the concentration of at least one analyte in the sample is quantified using an established ICSS and the measured peak area of ​​the analyte from a liquid chromatography-tandem mass spectrometry (LC-MS / MS) process, and wherein the tandem mass spectrometer is operated in a multiple reaction monitoring mode.

[0162] In some aspects, the analyte, the SIL analyte, and the naturally occurring isotopologues of the SIL analyte are ionized in a mass spectrometer to produce protonated (or deprotonated) parent ions of the analyte, the SIL analyte, and the naturally occurring isotopologues of the SIL analyte. In some aspects, the parent ions of the analyte, the parent ions of the SIL analyte, and the naturally occurring isotopologues of the SIL analyte in the mass spectrometer are fragmented at the same cleavage site to produce neutral losses and daughter ions.

[0163] In some aspects, transitions of the analyte from parent ions to daughter ions are monitored in a mass spectrometer, and the peak area of ​​the analyte transitions from parent ions to daughter ions is measured. In some aspects, selected multiple transitions from parent ions of the SIL analyte and parent ions of naturally occurring isotopologues of the SIL analyte to daughter ions of the SIL analyte and daughter ions of naturally occurring isotopologues of the SIL analyte are monitored in a mass spectrometer ("multi-isotopologue reaction monitoring" or "MIRM");

[0164] In some aspects, the peak area of ​​each MIRM transition is measured, wherein the MIRM transition includes a selected transition from a parent ion of the SIL analyte and a parent ion of a naturally occurring isotopologue of the SIL analyte to a daughter ion of the SIL analyte and a daughter ion of a naturally occurring isotopologue of the SIL analyte.

[0165] In some aspects, an in-sample calibration curve is generated based on the relationship between the peak areas measured in MIRM transitions of the SIL analyte and naturally occurring isotopologues of the SIL analyte and the analyte concentration equivalent for each MIRM transition.

[0166] In some aspects, the analyte concentration equivalent for each MIRM transition is calculated from the theoretical isotopic abundance of the corresponding MIRM transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte, wherein the theoretical isotopic abundance is calculated using the method disclosed in Analytical Chemistry, 2012, 84(11), 4844-4850, wherein the method is calculated based on the neutral loss of the SIL analyte and the isotopic distribution of the daughter ions. In other aspects, the SIL analyte and the naturally occurring isotopologue of the SIL analyte are calculated from (p+Z p +α) / Z p to (d+Z d +β) / Z d The theoretical isotopic abundance of each MIRM transition (m / z) is calculated based on formula (I):

[0167] (p+Z p +α) / Z p →(d+Z d +β) / Z d The isotopic abundance in the MIRM transition of

[0168] [Mass is (d+Z d +β)) product ions]*

[0169] [Relative isotopic distribution of neutral losses of mass n+(α-β)] (I)

[0170] where m / z is the mass-to-charge ratio

[0171] p is the monoisotopic mass of the parent molecule of the SIL analyte

[0172] Z p is the charge of the parent ion

[0173] d is the monoisotopic mass of the subfragment of the SIL analyte

[0174] Z d is the charge of the daughter ion

[0175] n is the neutrally lost monoisotopic mass of the SIL analyte

[0176] p=d+n

[0177] α and β are integers, which are the number of additional neutrons in the parent ion and the daughter ion, respectively, α ≥ 0, β ≥ 0 and α ≥ β

[0178] Z p and Z d is an integer

[0179] In some aspects, an isotope abundance calculator can be found at worldwideweb.sisweb.com / mstools / isotope.html (accessed on November 10, 2019). In some aspects, the maximum analyte concentration equivalent (the "upper limit of quantitation" or "ULOQ" of the ISCC) is calculated based on formula (II):

[0180] (M / V)*(M 分析物 / M SIL分析物 )ng / mL (II)

[0181] Where M (ng) is the total amount of SIL analyte added to the sample;

[0182] V is the sample volume before adding the SIL analyte (mL);

[0183] M 分析物 is the molecular weight of the analyte;

[0184] M SIL分析物 is the molecular weight of the SIL analyte.

[0185] In some aspects, one or more other analyte concentration equivalents in a MIRM transition are calculated based on formula (III):

[0186] I a *ULOQ(ng / ml) (III)

[0187] Among them I ais the calculated theoretical isotopic abundance of the MIRM transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte.

[0188] Methods useful in the present disclosure relate to the use of a stable isotope labeled (SIL) analyte, which is added to a sample containing an analyte. This SIL analyte can then be monitored by MIRM techniques to construct a concentration calibration curve to quantify the concentration of the analyte present in the sample. In some aspects, the analyte is a protein or peptide and the SIL analyte is a stable isotope labeled protein or peptide. In some aspects, the parent ion of the SIL analyte comprises at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, or at least about 20 amino acids, at least about 2 In some embodiments, the present invention comprises at least about 1 amino acid, at least about 22 amino acids, at least about 23 amino acids, at least about 24 amino acids, at least about 25 amino acids, at least about 26 amino acids, at least about 27 amino acids, at least about 28 amino acids, at least about 29 amino acids, at least about 30 amino acids, at least about 31 amino acids, at least about 32 amino acids, at least about 33 amino acids, at least about 34 amino acids, at least about 35 amino acids, at least about 36 amino acids, at least about 37 amino acids, at least about 38 amino acids, at least about 39 amino acids, or at least about 40 amino acids.

[0189] In some aspects, the parent ion of the SIL analyte comprises an amino acid sequence of between 4 and 40 amino acids, between 4 and 35 amino acids, between 5 and 35 amino acids, between 4 and 34 amino acids, between 5 and 34 amino acids, between 5 and 33 amino acids, between 5 and 32 amino acids, between 6 and 35 amino acids, between 6 and 34 amino acids, between 6 and 33 amino acids, between 6 and 32 amino acids, between 6 and 31 amino acids, between 6 and 30 amino acids, between 6 and 29 amino acids, between 6 and 28 amino acids, between 7 and 35 amino acids, between 7 and 34 amino acids, between 7 and 33 amino acids, between 7 and 32 amino acids, between 7 and 31 amino acids, between 7 and 30 amino acids, or between 7 and 29 amino acids. In some aspects, the parent ion of the analyte or SIL analyte comprises an amino acid sequence of between 7 and 11 amino acids. In some aspects, the parent ion of the SIL analyte comprises an amino acid sequence of between 8 and 11 amino acids. In some aspects, the parent ion of the SIL analyte comprises an amino acid sequence of between 8 and 10 amino acids. In some aspects, the parent ion of the SIL analyte comprises an amino acid sequence of between 8 and 9 amino acids. In some aspects, the parent ion of the SIL analyte comprises an amino acid sequence of between 6 and 9 amino acids. In some aspects, the parent ion of the SIL analyte comprises an amino acid sequence of between 6 and 10 amino acids.

[0190] In some aspects, the parent ion of the SIL analyte comprises an amino acid sequence of between 4 and 30 amino acids, between 4 and 25 amino acids, between 5 and 25 amino acids, between 4 and 24 amino acids, between 5 and 24 amino acids, between 5 and 23 amino acids, between 5 and 22 amino acids, between 6 and 25 amino acids, between 6 and 24 amino acids, between 6 and 23 amino acids, between 6 and 22 amino acids, between 6 and 21 amino acids, between 6 and 20 amino acids, between 7 and 25 amino acids, between 7 and 24 amino acids, between 7 and 23 amino acids, between 7 and 22 amino acids, between 7 and 21 amino acids, or between 7 and 20 amino acids.

[0191] In some aspects, the parent ion of the SIL analyte comprises an amino acid sequence of between 4 and 20 amino acids, between 4 and 15 amino acids, between 5 and 15 amino acids, between 4 and 14 amino acids, between 5 and 14 amino acids, between 5 and 13 amino acids, between 5 and 12 amino acids, between 6 and 15 amino acids, between 6 and 14 amino acids, between 6 and 13 amino acids, between 6 and 12 amino acids, between 6 and 11 amino acids, between 6 and 10 amino acids, between 6 and 9 amino acids, between 6 and 8 amino acids, between 7 and 15 amino acids, between 7 and 14 amino acids, between 7 and 13 amino acids, between 7 and 12 amino acids, between 7 and 11 amino acids, between 7 and 10 amino acids, or between 7 and 9 amino acids.

[0192] In some aspects, the SIL analyte is a stable isotope labeled protein or peptide. In some aspects, the parent ion of the SIL analyte comprises at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, or at least about 20 amino acids.

[0193] In some aspects, the analyte is an antibody. In other aspects, the analyte is a fusion protein. In some aspects, the analyte is a fusion protein comprising a protein and a heterologous portion. In other aspects, the analyte is an Fc fusion protein. In some aspects, the analyte is PD-1, PD-L1, CD73, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD73 antibody, or any combination thereof. In some aspects, the analyte is an anti-GITR antibody, an anti-CXCR4 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, an anti-IL8 antibody, or any combination thereof.

[0194] The methods of the present invention can effectively use the corresponding SIL analytes to detect or quantify analytes that are proteins. In some aspects, the analyte is an antibody. In some aspects, the analyte is CD73 or an anti-CD73 antibody or a fragment thereof. In some aspects, the analyte is PD-1 or an anti-PD-1 antibody (such as nivolumab) or a fragment thereof. In some aspects, the analyte is PD-L1 or an anti-PD-L1 antibody (such as ipilimumab) or a fragment thereof. In some aspects, the analyte is an anti-OX40 (also known as CD134, TNFRSF4, ACT35 and / or TXGP1L) antibody (e.g., BMS986178 or MDX-1803) or a fragment thereof. In some aspects, the analyte is Ulopumab or a fragment thereof. In some aspects, the analyte is BMS-986156 or a fragment thereof. In some aspects, the analyte is BMS-986016 or a fragment thereof. In some aspects, the analyte is BMS-986207 or a fragment thereof. In some aspects, the analyte is BMS-986253 or a fragment thereof. In some aspects, the analyte is BMS-986258 or a fragment thereof.

[0195] In some aspects, the analyte is an anti-PD-1 antibody. In some aspects, the anti-PD-1 antibody is selected from nivolumab (also known as 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as ), lamulimab and MK-3475; see WO 2008 / 156712), PDR001 (Novartis; see WO 2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012 / 145493), cemiplimab (Regeneron; also known as REGN-2810; see WO 2015 / 112800), JS001 (TAIZHOU JUNSHIPHARMA; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; see WO 2015 / 35606 and US 2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015 / 085847; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO 2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics, see WO 2017 / 19846), and IBI308 (Innovent; see WO 2017 / 19846). 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540).

[0196] In some aspects, the analyte is an anti-PD-L1 antibody. In some aspects, the anti-PD-L1 antibody is selected from BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Pat. No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; also known as MPDL3280A, RG7446; see US 8,217,149; see also Herbst et al. (2013) J Clin Oncol 31(Suppl):3000), durvalumab (AstraZeneca; also known as IMFINZI TM , MEDI-4736; see WO 2011 / 066389), Avelumab (Pfizer; also known as MSB-0010718C; see WO 2013 / 079174), STI-1014 (Sorrento; see WO 2013 / 181634), CX-072 (Cytomx; see WO 2016 / 149201), KN035 (3D Med / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (Eli Lilly Co.; see, e.g., WO 2017 / 034916), and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: Abstract 4606 (April 2016)).

[0197] In some aspects, the analyte is CD73 or a portion thereof. In some aspects, the SIL analyte is V[Ile( 13 C6, 15 N)]YPAVEGR (SEQ ID NO: 1). In some aspects, the analyte is PD-1 or a portion thereof. In some aspects, the SIL analyte is LAAFPED [Arg ( 13 C6, 15 N4)] (SEQ ID NO: 2). In some aspects, the analyte is PD-L1 or a portion thereof. In some aspects, the SIL analyte is LQDAG[Val( 13 C5, 15 N)]YR (SEQ ID NO: 3). In some aspects, the analyte is daclatasvir. In some aspects, the SIL analyte is the SIL analyte is 13 C2 15 N4-Daclatasvir.

[0198] In some aspects, the analyte is a non-peptide molecule. In some aspects, the molecular weight of the analyte is at least 100 g / mol, at least 200 g / mol, at least 300 g / mol, at least 400 g / mol, at least 500 g / mol, at least 600 g / mol, at least 700 g / mol, at least 800 g / mol, at least 900 g / mol, at least 1000 g / mol, at least 1100 g / mol, at least 1200 g / mol, at least 1300 g / mol, at least 1400 g / mol, at least 1500 g / mol, at least 1600 g / mol, at least 1700 g / mol, at least 1800 g / mol, at least 1900 g / mol or at least 2000 g / mol.

[0199] In some respects, analyte is an antibacterial or antiviral agent. In some respects, analyte is a medicament against hepatitis B, hepatitis C, HIV, syphilis or any combination thereof. In other respects, analyte with anti-HCV activity effectively inhibits those of the function of the target selected from: HCV metalloproteinase, HCV serine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCV entrance, HCV assembly, HCV exit, HCV NS5A protein and IMPDH and / or cyclosporin analogs and / or nucleoside analogs, and the analyte is used to treat HCV or Flaviviridae infection.

[0200] Analytes that can be used in the present disclosure (as selective HCV serine protease inhibitors) include peptide compounds disclosed in the following documents: WO / 1999 / 007733, WO / 2005 / 007681, WO / 2005 / 028502, WO / 2005 / 035525, WO / 2005 / 037860, WO / 2005 / 077969, WO / 2006 / 039488, WO / 2007 / 022459, WO / 2008 / 106058, WO 2008 / 106139, WO / 2000 / 009558, WO / 2000 / 009543, WO / 1999 / 064442, WO / 1999 / 007733, WO / 1999 / 07734, WO / 1999 / 050230 and WO / 1998 / 017679. NS5B polymerase inhibitors have also shown activity. These medicaments include but are not limited to other inhibitors of HCV RNA dependent RNA polymerase, such as, for example, WO01 / 90121 (A2) or U.S. Patent number 6,348,587B1 or WO 01 / 60315 or WO 01 / 32153 described nucleoside type polymerase inhibitors, or non-nucleoside inhibitors, such as the benzimidazole polymerase inhibitors described in EP 162196A1 or WO 02 / 04425.

[0201] In addition to the combination of pegylated alpha-interferon and ribavirin, other combinations of compounds that selectively inhibit HCV viral replication and can be used to treat HCV infected patients are also desirable. In particular, agents that effectively inhibit the function of the NS5A protein that can be combined with those agents that effectively inhibit other viral targets are desirable. The HCV NS5A protein is described, for example, in the following documents: Tan, S.-L.; Katzel, MG Virology (2001) 284, 1-12; and Park, K.-J.; Choi, S.-H, J. Biological Chemistry (2003). Related patent publications describing the synthesis of HCV NS5A inhibitors are: US 2009 / 0202478; US 2009 / 0202483; WO 2009 / 020828; WO 2009 / 020825; WO 2009 / 102318; WO 2009 / 102325; WO 2009 / 102694; WO 2008 / 144380; WO 2008 / 021927; WO 2008 / 021928; WO 2008 / 021936; WO 2006 / 133326; WO 2004 / 014852; WO 2008 / 070447; WO 2009 / 034390; WO 2006 / 079833; WO WO 2007 / 031791; WO 2007 / 070556; WO 2007 / 070600; WO 2008 / 064218; WO 2008 / 154601; WO 2007 / 082554; WO 2008 / 048589; WO 2010 / 017401; WO 2010 / 065668; WO 2010 / 065674; WO 2010 / 065681, the contents of each of which are expressly incorporated herein by reference.

[0202] In some aspects, the analyte is a small molecule. In some aspects, the analyte is paclitaxel, clopidogrel, apixaban, dapagliflozin, saxagliptin, tamsavir, ledipasvir, sofosbuvir or rosuvastatin. In some aspects, the analyte is paclitaxel. In some aspects, the analyte is clopidogrel. In some aspects, the analyte is apixaban. In some aspects, the analyte is dapagliflozin. In some aspects, the analyte is saxagliptin. In some aspects, the analyte is tamsavir. In some aspects, the analyte is ledipasvir. In some aspects, the analyte is sofosbuvir. In some aspects, the analyte is rosuvastatin.

[0203] In other aspects, the analyte is a nucleic acid molecule, eg, DNA, RNA (eg, mRNA). In some aspects, the nucleic acid molecule is at least about 10 nucleic acids, at least about 15 nucleic acids, at least about 20 nucleic acids, at least about 25 nucleic acids, at least about 30 nucleic acids, at least about 40 nucleic acids, at least about 50 nucleic acids, at least about 100 nucleic acids, at least about 200 nucleic acids, at least about 300 nucleic acids, at least about 400 nucleic acids, at least about 500 nucleic acids, at least about 600 nucleic acids, at least about 700 nucleic acids, at least about 800 nucleic acids, at least about 900 nucleic acids, at least about 1000 nucleic acids, at least about 1200 nucleic acids, at least about 1400 nucleic acids, at least about 1600 nucleic acids, at least about 1800 nucleic acids, at least about 2000 nucleic acids, at least about 2200 nucleic acids, at least about 2400 nucleic acids, at least about 2600 nucleic acids, at least about 2800 nucleic acids, or at least about 3000 nucleic acids.

[0204] In some aspects, the analyte is an antisense oligonucleotide. In other aspects, the analyte is a siRNA or miRNA. In other aspects, the analyte is a gene therapy vector or plasmid.

[0205] In some aspects, the SIL analyte contains at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20 isotopic labels, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, or at least about 40 isotopic labels.

[0206] The selection of MIRM transitions for measurement is an important element of the methods of the present invention to ensure robust and accurate assay performance. In some aspects, the relative peak area of ​​each measurement in the MIRM transition has less than 15% deviation compared to the theoretical isotopic abundance calculated in the corresponding MIRM transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte.

[0207] In some aspects, at least one measured relative peak area in a MIRM transition has less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09 ... 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.009%, less than 0.008%, less than 0.007%, less than 0.006%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, less than 0.009%, less than 0.008%, less than 0.007%, less than 0.006%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, or less than 0.0001% deviation. In some aspects, the relative peak areas of all measurements in the MIRM transitions have less than 14% deviation compared to the theoretical isotopic abundance calculated in the corresponding MIRM transitions of the SIL analyte or the naturally occurring isotopologue of the SIL analyte. In some aspects, the relative peak areas of all measurements in the MIRM transitions have less than 13% deviation compared to the theoretical isotopic abundance calculated in the corresponding MIRM transitions of the SIL analyte or the naturally occurring isotopologue of the SIL analyte. In some aspects, the relative peak areas of all measurements in the MIRM transitions have less than 12% deviation compared to the theoretical isotopic abundance calculated in the corresponding MIRM transitions of the SIL analyte or the naturally occurring isotopologue of the SIL analyte. In some aspects, the relative peak areas of all measurements in the MIRM transitions have less than 11% deviation compared to the theoretical isotopic abundance calculated in the corresponding MIRM transitions of the SIL analyte or the naturally occurring isotopologue of the SIL analyte. In some aspects, the relative peak areas of all measurements in the MIRM transitions have less than 10% deviation from the theoretical isotopic abundance calculated in the corresponding MIRM transitions of the SIL analyte or a naturally occurring isotopologue of the SIL analyte. In some aspects, the relative peak areas of all measurements in the MIRM transitions have less than 9% deviation from the theoretical isotopic abundance calculated in the corresponding MIRM transitions of the SIL analyte or a naturally occurring isotopologue of the SIL analyte.In some aspects, the relative peak areas of all measurements in the MIRM transitions have less than 8% deviation from the theoretical isotopic abundance calculated in the corresponding MIRM transitions of the SIL analyte or a naturally occurring isotopologue of the SIL analyte. In some aspects, the relative peak areas of all measurements in the MIRM transitions have less than 7% deviation from the theoretical isotopic abundance calculated in the corresponding MIRM transitions of the SIL analyte or a naturally occurring isotopologue of the SIL analyte.

[0208] In some aspects, the number of MIRM transitions is at least two, at least three, at least four, at least five, at least six, at least seven, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20. In some aspects, the number of MIRM transitions is between 4 and 15, between 4 and 14, between 5 and 13, between 5 and 12, between 6 and 12, between 6 and 11, between 7 and 11, between 7 and 10, between 8 and 10, or between 8 and 9. In some aspects, the number of MIRM transitions is between 4 and 10, between 4 and 9, between 5 and 9, between 6 and 9, between 6 and 8, or between 7 and 8. In some aspects, the number of MIRM transitions is 6. In some aspects, the number of MIRM transitions is 7. In some aspects, the number of MIRM transitions is 10. In some aspects, the number of MIRM transitions is fifteen.

[0209] In some aspects, the analyte concentration equivalent of the highest MIRM channel and the lowest MIRM channel is at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about 2400, at least about 2500, at least about 2600, at least about 2700, at least about 2800, at least about 4700, at least about 4800, at least about 4900, at least about 5000, at least about 5100, at least about 5200, at least about 5300, at least about 5400, at least about 5500, at least about 5600, at least about 5700, at least about 5800, at least about 5900, at least about 4000, at least about 4100, at least about 4200, at least about 4300, at least about 4400, at least about 4500, at least about 4600, at least about 4700, at least about 4800, at least about 4900, at least about 5000, at least about 5100, at least about 5200, at least about 5300, at least about 5400, at least about 5500, at least about 5600, at least about 5700, at least about 5800, at least about 5900, or at least about 6000-fold difference.

[0210] In some aspects, the calculated theoretical isotopic abundances of two selected MIRM transitions differ by at least 0.01%, differ by at least 0.05%, differ by at least 0.1%, differ by at least 0.5%, differ by at least 1%, differ by at least 1.5%, differ by at least 2%, differ by at least 2.5%, differ by at least 3%, differ by at least 3.5%, differ by at least 4%, differ by at least 4.5%, differ by at least 5%, differ by at least 5.5%, differ by at least 6%, differ by at least 6.5%, differ by at least 7%, differ by at least 7.5%, differ by at least 8%, differ by at least 8.5%, differ by at least 9%, differ by at least 9.5%, differ by at least 10%, differ by at least 20%, differ by at least 30%, differ by at least 40%, or differ by at least about 50%.

[0211] In some aspects, the SIL analyte contains trace amounts of unlabeled analyte. In some aspects, the SIL analyte contains less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% unlabeled analyte.

[0212] In some aspects, the SIL analyte is labeled with one or more stable isotopes at one or more positions. In some aspects, the stable isotope labeling is 2 H. 13 C. 15 N. 33 S. 34 S. 36 S. 17 O or 18 O. In some aspects, the stable isotope label is 13 C and / or 15 N. In some aspects, the stable isotope label is 13 C. In some aspects, the stable isotope label is 15 N.

[0213] Typically, for experiments on triple quadrupole mass spectrometers, the first quadrupole (Q1) is set to pass only through ions (precursor ions) with specified m / z of expected chemical species in the sample. The second quadrupole (i.e., Q2 or collision cell) is used to fragment the ions passing through Q1. The third quadrupole (Q3) is set to pass only ions (fragment ions) with specified m / z corresponding to the expected fragmentation products of the expected chemical species to the detector. In some aspects, the sample is ionized in a mass spectrometer to generate one or more protonated or deprotonated molecular ions. In some aspects, one or more protonated or deprotonated molecules are singly charged, doubly charged, triply charged or more highly charged. In some aspects, the mass spectrometer is a triple quadrupole mass spectrometer. In some aspects, the resolution for Q1 and Q3 is unit resolution. In other aspects, the resolution for Q1 and Q3 is different. In other aspects, the resolution for Q1 is higher than the unit resolution of Q3.

[0214] The utility of separation by high performance liquid chromatography has been demonstrated in a wide variety of applications, including analysis and purification of molecules in the low to high molecular weight range. In liquid chromatography, there are severe limitations, particularly due to the time required for analysis. The method of the present invention is very effective and improves the total instrument time required, particularly where an external calibration curve need not be run on the instrument. In some aspects, the method reduces the total instrument run time. In some aspects, an external calibration curve is not used. In some aspects, the analyte is a biomarker. In some aspects, the analyte is a metabolite.

[0215] The inventive method can be effectively used to detect or quantify analytes from a variety of sources (including biological sources). In some aspects, the sample is serum, tissue, biopsy tissue, formalin-fixed paraffin-embedded (FFPE), plasma, saliva, cerebrospinal fluid, tears, urine, synovial fluid, dried blood spots, or any combination thereof. In some aspects, the sample is serum. In some aspects, the sample is tissue. In some aspects, the sample is biopsy tissue. In some aspects, the sample is formalin-fixed paraffin-embedded (FFPE). In some aspects, the sample is plasma. In some aspects, the sample is saliva. In some aspects, the sample is cerebrospinal fluid. In some aspects, the sample is tears. In some aspects, the sample is urine. In some aspects, the sample is synovial fluid. In some aspects, the sample is dried blood spots.

[0216] The method of the present invention can also be used to construct a liquid chromatography-mass spectrometry system, the system comprising: a liquid chromatography comprising at least one liquid chromatography column capable of separating an analyte from a biological matrix, a sample comprising an analyte of interest, at least one stable isotope-labeled analyte added to the sample, and a mass spectrometer capable of ionizing, fragmenting, and detecting one or more protonated or deprotonated parent ions and daughter ions specific to the analyte and the stable isotope-labeled analyte. Typically, chromatography is any type of technique for separating a molecule (e.g., an antibody) from other molecules (e.g., a pollutant) present in a mixture.

[0217] Liquid chromatography (LC) is a perfect analytical technique for separating the components of fluid mixtures for subsequent analysis and / or identification, wherein a stationary phase material is filled into a column, a channel or a tube based on a microfluidic chip, and the stationary phase material is usually a fine solid or gel, such as a small particle with a diameter of several microns. Small particle size provides a large surface area that can be modified with a variety of chemical substances, thereby producing a stationary phase. Based on column size and particle size, a liquid eluent is pumped through a liquid chromatography column ("LC column") at a desired flow rate. This liquid eluent is sometimes referred to as a mobile phase. Before the LC column, the sample to be analyzed is introduced (e.g., injected) into the flow of the mobile phase with a small volume. The migration rate of the analyte in the sample is affected by the specific chemical and / or physical interaction with the stationary phase when it crosses the length of the column. The time when a specific analyte is eluted or leaves from the end of the column is referred to as a retention time or elution time, and may be a feature of the reasonable identification of a given analyte, especially when combined with other analytical features (such as the accurate mass of a given analyte). The separated components can be passed from the liquid chromatography column to other types of analytical instruments for further analysis, for example, liquid chromatography-mass spectrometry (LC / MS or LC / MS / MS) to chromatographically separate the compounds before introducing them into the ion source of a mass spectrometer.

[0218] Mass spectrometry ("MS" or "mass-spec") is an analytical technique for measuring the mass-to-charge ratio of ions. This can be achieved by ionizing a sample and separating particles of different masses, and recording their relative abundance by measuring the intensity of the ion flow. A typical mass spectrometer contains three parts: an ion source, a mass analyzer, and a detector system. The ion source is the part of the mass spectrometer that ionizes the substance being analyzed (the analyte). The ions are then transported to the mass analyzer by a magnetic or electric field, which separates the ions according to their mass-to-charge ratio (m / z). Many mass spectrometers use two or more mass analyzers for tandem mass spectrometry (MS / MS). The detector records the charge induced or the current generated when the ions pass through or hit a surface. The mass spectrum is the result of measuring the signal generated in the detector when the m / z ions are scanned with a mass analyzer.

[0219] The present method discloses a composition comprising an intra-sample calibration curve (ISCC), wherein the ISCC comprises a multi-isotopologue reaction monitoring (MIRM) of a stable isotope-labeled analyte. The present method of generating an ISCC comprising a MIRM of a stable isotope-labeled analyte can be used to analyze biomarkers. The biomarker can be, for example, isolated from a biological sample, directly measured in a biological sample, or detected in a biological sample or determined to be in a biological sample. The biomarker can be functional, partially functional, or non-functional. If the biomarker is a protein or fragment thereof, it can be sequenced and its encoding gene can be cloned using well-established techniques. The present method can also be used to develop and / or validate biomarkers for regulatory acceptance as surrogate endpoints. A surrogate endpoint is a biomarker accepted by a regulatory agency as a surrogate for a clinical endpoint and is intended to be used as a surrogate for a clinically meaningful endpoint. Before a surrogate endpoint can be accepted, there must be sufficient evidence to show that it can be relied upon to predict or correlate with clinical benefit.

[0220] The methods of the present disclosure can also be used to analyze biomarkers, metabolites or metabolic profiles. Analysis of biomarkers or metabolites represents a sensitive measure of biological states in health or disease. The altered metabolic fingerprint (unique to each individual) provides a novel approach to better understand systems biology, detect or identify the potential risks of various diseases, and ultimately help achieve the goal of personalized medicine (i.e., one or more correct drugs are used for the right person at the right time with the right dose). Metabolite profiles as used in the present invention should be understood as any defined set of values ​​for the quantitative results of metabolites that can be compared with reference values ​​or profiles derived from another sample or sample group. For example, the metabolite profile of a sample from a sick patient may be significantly different from the metabolite profile of a sample from a similarly matched healthy patient. By comparing the metabolite profile with a reference or standard profile, the metabolite profile can help predict the susceptibility of a subject to a disorder.

[0221] The methods of the present invention also involve recommending or selecting the best treatment regimen and / or the best drug selection, combination and dosage for a particular patient. The peak concentration of the drug after each dose can be measured. The trough concentration of the drug after each dose can also be measured. The dosing interval (by time), including the variation of this time, can be optimized based on information discerned from the analysis of biomarkers or metabolites.

[0222] The present disclosure also includes a liquid chromatography-mass spectrometry system used by the methods of the present invention described herein. In some aspects, the LC-MS / MS comprises:

[0223] (i) a liquid chromatography system comprising at least one liquid chromatography column capable of separating an analyte from a biological matrix;

[0224] (ii) a sample containing an analyte of interest;

[0225] (iii) at least one stable isotope-labeled analyte added to the sample; and

[0226] (iv) a mass spectrometer capable of ionizing, fragmenting and detecting one or more protonated (or deprotonated) parent and daughter ions specific for the analyte and the stable isotope-labeled analyte.

[0227] In other aspects, the disclosure includes a composition comprising an in-sample calibration curve (ISCC), wherein the ISCC comprises a stable isotope-labeled analyte.

[0228] The present disclosure also relates to the preparation of a single sample multi-point external calibration curve (OSMECC). This method does not use the isotope (SIL) analyte of stable labeling, but includes the analyte of known amount being spiked into a blank matrix sample. Blank matrix is ​​a matrix type that does not contain the target analyte. By spiked into a blank matrix sample by a known amount of analyte, the single sample multi-point external calibration curve in the blank matrix sample can be established based on the relationship between the MS / MS peak area (or peak area ratio, if internal standard is used in the determination) measured in the theoretical isotopic abundance (analyte concentration equivalent) of calculation and the corresponding MIRM channel of the analyte. This single sample multi-point external calibration curve can be used for bioanalysis based on quantitative LC-MS / MS in the same manner as traditional multi-sample external calibration curve. This method is used as an alternative method to eliminate the need for preparing traditional multi-sample external calibration curves in LC-MS / MS quantitative analysis.

[0229] Since the isotopic abundance in each MIRM channel can be accurately calculated and measured, isotopic sample dilution can be achieved by monitoring only one or a few MIRM channels of the analyte in addition to the most abundant MIRM channel of the study sample. While samples with concentrations within the range of the assay calibration curve are quantified using the most abundant MIRM channel (isotopic abundance of 100%), samples with concentrations exceeding the assay upper limit of quantitation (ULOQ) can be quantified using less abundant MIRM channels (isotopic abundance of IA%), resulting in an isotope dilution factor (IDF) of 100% / IA%. This approach is used as an alternative to eliminate the need to physically dilute the study sample in LC-MS / MS quantitative analysis.

[0230] In some aspects, the present disclosure relates to a method for quantifying the concentration of at least one analyte in a study sample, the method comprising adding one or more known amounts of one or more analytes to a blank matrix sample to construct one or more single sample multi-point external calibration curves (OSMECC) by multi-isotopologue reaction monitoring (MIRM) of each analyte added, wherein the MIRM of the analyte refers to multiple reaction monitoring of multi-isotopic transitions of the analyte; wherein the OSMECC for each analyte is based on the calculated theoretical isotopic abundance (analyte concentration equivalent) in the MIRM transition and the corresponding A relationship between the measured tandem mass spectrometry (MS / MS) peak areas (or peak area ratios, if an internal standard is used in the assay) in the MIRM transitions is constructed in a blank matrix sample; wherein the concentration of at least one analyte in the study sample is quantified using an established OSMECC and the measured peak area (or peak area ratio, if an internal standard is used in the assay) of the analyte from a liquid chromatography-tandem mass spectrometry (LC-MS / MS) process, wherein the peak area ratio of the analyte is the peak area of ​​the analyte divided by the peak area of ​​the internal standard, and wherein the tandem mass spectrometer is operated in multiple reaction monitoring mode.

[0231] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting.The contents of all references cited throughout this application are expressly incorporated herein by reference.

[0232] Example

[0233] Example 1

[0234] Preparation of SIL analytes for MIRM-ISCC-LC-MS / MS analysis

[0235] Formic acid (SupraPur grade) was purchased from EMD Chemicals (Gibbstown, NJ, USA). HPLC grade methanol and acetonitrile were purchased from JTBaker (Phillipsburg, NJ, USA). LC grade ammonium bicarbonate and phosphate buffered saline with 0.05% Tween (PBST) were purchased from Sigma-Aldrich (St. Louis, MO, USA). M-280 streptavidin was purchased from Invitrogen (Carlsbad, CA, USA). Sequencing grade modified trypsin was purchased from Promega Corporation (Madison, WI, USA). All unlabeled and labeled surrogate peptides of cluster of differentiation 73 (CD73): VIYPAVEGR (SEQ ID NO: 1) and V[Ile ( 13 C6, 15 N)]YPAVEGR (SEQ ID NO: 1); programmed cell death protein 1 (PD-1): LAAFPEDR (SEQ ID NO: 2) and LAAFPED [Arg ( 13 C6, 15 N4)] (SEQ ID NO: 2); and programmed death ligand 1 (PD-L1): LQDAGVYR (SEQ ID NO: 3) and LQDAG[Val( 13 C5, 15 N)]YR (SEQ ID NO:3) was purchased from Genscript (Piscataway, NJ, USA). Deionized water was generated using a NANOpure Diamond ultrapure water system from Barnstead International (Dubuque, IA, USA). Recombinant human CD73 (61,084 Da), anti-human CD73 monoclonal antibody (mAb), small molecule drug daclatasvir and SIL drug, 13 C2 15 N4-Daclatasvir.

[0236] The LC-MS / MS system used was a triple quadrupole 6500 mass spectrometer (AB Sciex, Foster City, CA) coupled to a Nexera UPLC system (Shimadzu, Columbia, MD). The UPLC system consisted of two LC-30AD pumps, a SIL-30ACMP autosampler, and a CTO-30AS column heater. Separation was achieved on an Acquity HSS T3 analytical column (2.1 mm x 50 mm, particle size 1.8 μm) (Waters, Milford, MA) using a gradient elution with a mobile phase of 0.01% formic acid in water (A) and 0.01% formic acid in acetonitrile (B). LC-MS / MS data were acquired using the ELISA software (1.6.2).

[0237] Selection of MIRM channels for monitoring

[0238] FIG1 shows a general MIRM-ISCC-LC-MS / MS method using the quantitative analysis of the programmed death ligand 1 (PD-L1) peptide LQDAGVYR (SEQ ID NO: 3) as an example. By adding a known amount of the SIL analyte LQDAG[Val( 13 C5, 15 N)]YR (SEQ ID NO: 3) (20 μL of 500 ng / mL = 10 ng) was spiked into each 100 μL study sample, and the theoretical isotopic abundance calculated in the MIRM channel of this labeled analyte can be converted to the isotopic concentration (or analyte concentration equivalent) of the SIL analyte in the corresponding MIRM channel. Therefore, the ISCC between the theoretical isotopic concentration (or analyte concentration equivalent) and the measured MS / MS response can be established in each study sample, and the analyte concentration of the sample can be calculated instantly based on the established calibration curve and the peak area of ​​the analyte.

[0239] MIRM-ISCC-LC-MS / MS quantification of endogenous CD73 in human and monkey serum

[0240] CD73 is a 70kDa protein and is highly expressed in many tumors. Quantitative analysis of CD73 in human and monkey serum is needed to assist in dose selection and provide pharmacodynamic information for anti-CD73 preclinical and clinical drug development. Initially, an immunocapture LC-MS / MS assay using a traditional external calibration curve was developed and validated by serial dilution of a recombinant CD73 reference standard (61,084Da) in a surrogate matrix. Immunocapture of CD73 was performed using an anti-CD73 mAb, followed by denaturation, trypsin digestion, and LC-MS / MS analysis. The surrogate peptide monitored in the LC-MS / MS assay (unique to both human and monkey CD73) is VIYPAVEGR (SEQ ID NO: 1), which has a peak from a doubly charged parent ion to a y6 ion (502.3 ++ →628.3 + ) SRM transition (m / z). After trypsin digestion, a 10 μL volume of SIL was used to replace the peptide V[Ile( 13 C6, 15N)]YPAVEGR (SEQ ID NO: 1) was added to each sample at a concentration of 100 ng / mL. Because the initial serum sample volume for this assay was 100 μL, this was equivalent to 10 ng / mL ([10 μL·100 ng / mL] / 100 μL) of SIL peptide in the initial serum sample, or 604.792 ng / mL of recombinant CD73 (10 ng / mL·[61,084 Da / 1010 Da]). In this experiment, this labeled peptide was used not only as an assay internal standard with an external calibration curve, but also as the ISCC for each individual study sample. This allows side-by-side comparisons of CD73 concentrations measured with an external calibration curve and ISCC.

[0241] Before using the external calibration curve and ISCC method for quantification of CD73 in human and monkey serum, it is necessary to calculate and measure the labeled surrogate peptide V[Ile( 13 C6, 15 N)] isotopic abundance in the MIRM channel of YPAVEGR (SEQ ID NO: 1) to confirm that the selected MIRM channel is reliable and accurate to establish ISCC without unexpected interferences.

[0242] The fragmentation of peptides in a triple quadrupole mass spectrometer can be easily resolved using online tools such as Skyline (MacCoss Lab, Department of Genome Sciences, UW). In this case, since the y6 ion is monitored as a product ion, the product ion and neutral loss are determined to be C 26 H 46 N9O9 + and 13 C6 15 NC 14 H 29 N2O4. The product ions (C 26 H 46 N9O9 + ) and neutral loss ( 13 C6 15 NC 14 H 29 N2O4) and are listed in Table 1. Calculate V[Ile( 13 C6, 15The isotopic abundances of the most abundant MIRM channel (100% abundance) and adjacent MIRM channels of V[ILE( 13 C6, 15 The results (peak area) measured with a Sciex API 6500 mass spectrometer in the corresponding MIRM channel of N)]YPAVEGR (SEQ ID NO: 1) are shown in Table 2. From the most abundant to the least abundant MIRM channel, the abundance covers more than 6,000 times. If necessary, lower isotopic abundances can also be calculated and used, however, in many cases, it will exceed the linear range of the triple quadrupole mass spectrometer. As shown in Table 2, the percentage difference between the measured results and the calculated theoretical isotopic abundances is within 13.5%, indicating that the results measured in the absence of any interference (such as interference from isotopic impurities and endogenous matrix) are accurate and reliable, and therefore, these MIRM channels can be selected for MIRM-ISCC-LC-MS / MS absolute quantitative analysis.

[0243] Using the theoretical isotopic abundance calculated in the MIRM channels of the SIL peptide, the concentrations of SIL peptide isotopes spiked in the selected ten MIRM channels to be used for ISCC (and CD73 protein concentration equivalents) were calculated and listed in the two right columns of Table 2. ISCCs were constructed in each investigated sample using these concentrations (x-axis) and the MS / MS peak areas measured in the corresponding MIRM channels (y-axis). Calibration curve regression and concentration calculations were performed using in-house developed software. All ISCCs were weighted (1 / x 2 ) Least squares linear regression. For sample No. 1 in human plasma (all samples were extracted and analyzed in triplicate), the ISCC performance, linear curves (intercept and linear slope), and calculated concentrations for the first three injections are shown in Tables 3 and 4, respectively. As shown in Table 3, excellent ISCC performance was observed for these three replicates. Representative chromatograms of ten MIRM channels for ISCC and one SRM channel for analyte from the second injection are shown in Figures 2A and 2B.

[0244] The results of the quantitative analysis of endogenous CD73 levels in human and monkey serum using the external calibration curve and the ISCC method are listed in Table 5. Overall, the CD73 concentrations measured by the ISCC method were approximately 11% to 17% lower than those measured using the external calibration curve. This was caused by the 86.0% recovery of the immunocapture and digestion because the immunocapture and digestion losses of the studied samples were tracked and compensated by the external calibration curve. However, this was not feasible for the ISCC method because the SIL peptide was spiked after digestion. Therefore, the concentrations measured by the ISCC method should be adjusted according to the 86.0% recovery, and the adjusted concentrations matched very well with the concentrations measured using the external calibration curve, as shown in Table 5.

[0245] It is not necessary to include all promising MIRM channels in the final assay. The selected MIRM channels used for quantification of CD73 in human serum are documented in Table 2. There are several considerations in selecting the MIRM channels to be used in a sample analysis run:

[0246] The ISCC curve range is defined by the isotopic abundance range of the selected MIRM channels. Therefore, appropriate MIRM channels should be selected to cover the expected concentration range. In this work, the selected MIRM channels covered an approximately 1,600-fold curve range to cover the expected increase in CD73 after drug administration.

[0247] Any MIRM channel with a large %Dev (>15%) between the calculated and measured isotopic abundances should not be selected, as a large %Dev generally indicates the presence of potential interferences in the MIRM channel, including interferences from isotopic impurities and endogenous matrix. Therefore, multiple matrix batches should be tested for MIRM channel selection.

[0248] Among multiple MIRM channels with close isotopic abundances, only one MIRM channel should be selected. In this embodiment, as shown in Table 2, MIRM channels 4 and 8 are not selected because MIRM channels 4 and 5, 7 and 8, respectively, have very close isotopic abundances.

[0249] For demonstration purposes only, a total of ten MIRM channels were used in this example. Using fewer MIRM channels (four to five MIRM channels for a 1,000-fold curve) did not affect data quality.

[0250] Table 1

[0251] Stable isotope labeled peptide V[Ile( 13 C6, 15 Neutral loss of YPAVEGR (SEQ ID NO: 1) 13 C6 15 NC 14 H29 N2O4) and daughter ions ([C 26 H 46 N9O9] + ) isotope distribution

[0252]

[0253]

[0254] Note: Precursor ions: [ 13 C6 15 NC 40 H 76 N 11 O 13 ] ++ , with a double charge (Z p =2)

[0255]

[0256]

[0257]

[0258]

[0259] Example 2

[0260] MIRM-ISCC-LC-MS / MS Quantification of Surrogate Peptides of Protein Biomarkers PD-1, PD-L1, and CD73 in Digested Human Colon Homogenates

[0261] The above MIRM-ISCC-LC-MS / MS method can be easily applied to quantitative proteomics by spiking known amounts of multiple SIL surrogate peptides into digested samples for absolute quantitative proteomics of multiple peptide targets. Similar to the AQUA method, ISCC quantification is also based on the concentration of the SIL surrogate peptide spiked into the sample. However, because only one calibration point is used for each target peptide in the AQUA method, the quantitative accuracy may be severely impaired, especially when the concentration of the target peptide is much higher or much lower than the concentration of the spiked SIL surrogate peptide. On the other hand, the MIRM-ISCC-LC-MS / MS method can provide a comprehensive calibration curve range of 3 to 4 orders of magnitude for each target peptide, and the quantitative accuracy can be guaranteed within the complete curve range.

[0262] In this example, three surrogate peptides, LAAFPEDR (SEQ ID NO: 2) for PD-1, LQDAGVYR (SEQ ID NO: 3) for PD-L1, and VIYPAVEGR (SEQ ID NO: 1) for CD73, were mixed and spiked into completely trypsinized human colon tissue homogenate at concentrations of 1.00, 10.0, and 50.0 ng / mL, respectively. A volume of 100 μL of the prepared sample was used in the assay. 10 ng (20 μL 500 ng / mL) of each SIL peptide (i.e., LAAFPED [Arg ( 13 C6, 15 N4)](SEQ ID NO:2), LQDAG[Val( 13 C5, 15 N)]YR (SEQ ID NO: 3) and V[Ile ( 13 C6, 15 A mixture of 10% methanol and 90% water of YPAVEGR (SEQ ID NO: 1) was added to the prepared samples for MIRM-ISCC-LC-MS / MS analysis. The concentration of each SIL peptide in the sample was 100 ng / mL (10 ng / 100 μL). Table 6 shows the MIRM channels, their isotopic concentrations, and analyte concentration equivalents used for MIRM-ISCC-LC-MS / MS quantitative analysis of these three peptides. All ISCC curves were weighted (1 / x 2 ) Least squares linear regression. Excellent ISCC curve performance was confirmed by very accurate predicted concentrations of all calibration points (within 10.0% of nominal concentration, data not shown). The measured concentrations of the three peptides are listed in Table 7 and the accuracy of the MIRM-ISCC-LC-MS / MS measurements was confirmed by all samples tested.

[0263] Table 6

[0264] MIRM channels and their isotopic concentrations for MIRM-ISCC-LC-MS / MS quantification of LAAFPEDR (SEQ ID NO: 2), LQDAGVYR (SEQ ID NO: 3), and VIYPAVEGR (SEQ ID NO: 1) in digested human colon homogenate

[0265]

[0266]

[0267] a: ISCC LAAFPEDR (SEQ ID NO: 2) concentration equivalent = ISCC SIL-LAAF PEDR (SEQ ID NO: 2) isotope concentration * (918 LAAFPEDR (SEQ ID NO: 2) molecular weight / 928 SIL-LAAFPEDR (SEQ ID NO: 2) molecular weight)

[0268] b : ISCC LQDAGVYR (SEQ ID NO: 3) concentration equivalent = ISCC SIL-LQD AGVYR (SEQ ID NO: 3) isotope concentration * (921 LQDAGVYR (SEQ ID NO: 3) molecular weight / 927 SIL-LQDAGVYR (SEQ ID NO: 3) molecular weight)

[0269] c :ISCC VIYPAVEGR (SEQ ID NO: 1) concentration equivalent = ISCC SIL-VIYP AVEGR (SEQ ID NO: 1) isotope concentration * (1003 VIYPAVEGR (SEQ ID NO: 1) molecular weight / 1010 SIL-VIYPAVEGR (SEQ ID NO: 1) molecular weight)

[0270] Table 7. Quantitative analysis of surrogate peptides LAAFPEDR (SEQ ID NO: 2), LQDAGVYR (SEQ ID NO: 3) and VIYPAVEGR (SEQ ID NO: 1) in fully digested colon tissue homogenate using MIRM-ISCC-LC-MS / MS

[0271]

[0272] A potential problem with the MIRM-ISCC-LC-MS / MS approach for targeted quantitative proteomics of multiple peptides is that the total number of MIRM channels that need to be monitored in an LC-MS / MS run may be too many for a triple quadrupole mass spectrometer to handle. This problem can be mitigated by using scheduled MIRM based on different retention time windows for each target peptide and using fewer MIRM channels in each ISCC. The results show that accurate and reliable quantification can still be achieved by using 3 to 4 MIRM channels in a multiplexed manner for a concentration range of 2 to 3 orders of magnitude.

[0273] Example 3

[0274] Analysis of the small molecule drug daclatasvir in human and rat plasma using MIRM-ICSS-LC-MS / MS

[0275] After determining product ions and neutral losses, the same MIRM-ISCC-LC-MS / MS workflow can also be used to measure small molecule analytes, including small molecule drugs and biomarkers. An example of the immediate quantification of the small molecule drug daclatasvir in human and rat plasma using the MIRM-ISCC-LC-MS / MS approach is shown here.

[0276] 100 μL of human and rat plasma samples with 10, 100, 500, and 1,000 ng / mL daclatasvir were mixed with 20 μL of 5,000 ng / mL SIL 13 C2 15 N4-daclatasvir was mixed in human and rat plasma. 13 C2 15 The equivalent concentration of N4-daclatasvir was 1,000 ng / mL ([20 μL x 5,000 ng / mL] / 100 μL = 1,000 ng / mL). The samples were extracted using liquid-liquid extraction (H. Jiang et al., Journal of Chromatography A 2012, 1245, 117-121) and injected for MIRM-ISCC-LC-MS / MS analysis. Table 8 shows the MIRM channels and their isotope concentrations used in ISCC for the quantification of daclatasvir. All ISCCs were performed using weighted (1 / x 2 ) least squares linear regression was constructed. The predicted concentrations of all calibration points were well within the acceptance criteria of the adjusted LC-MS / MS bioanalysis (data not shown). Table 9 shows the measurement results of daclatasvir in human and rat plasma, indicating that the MIRM-ISCC-LC-MS / MS analysis of daclatasvir is accurate.

[0277] Table 8. MIRM channels and their isotopic concentrations used for MIRM-ISCC-LC-MS / MS quantitative analysis of daclatasvir

[0278]

[0279]

[0280] a :ISCC daclatasvir concentration equivalent = ISCC SIL-daclatasvir isotope concentration * (739 daclatasvir molecular weight / 745 SIL-daclatasvir molecular weight)

[0281] Table 9. Quantitative analysis of daclatasvir in human and rat plasma using MIRM-ISCC-LC-MS / MS

[0282]

[0283] Additional considerations for MIRM-ISCC-LC-MS / MS assays

[0284] There are several other considerations for successful MIRM-ISCC-LC-MS / MS assay development and sample analysis. The selection of appropriate SIL analytes is one of the important factors in developing reliable and robust quantitative MIRM-ISCC-LC-MS / MS assays. Since the isotopic abundance of this labeled analyte in the selected MIRM channel will be used as a calibration curve for the quantitative analysis of the analyte, the labeled analyte should be designed to avoid isotopic interference from the analyte, because this interference may reduce the accuracy of the assay. The rule of thumb is that four to six labels are required to avoid interference from most small molecule compounds and peptide analytes with 6 to 12 amino acids. The impurities (the amount of unlabeled analytes) in the SIL analyte should be low enough to avoid interference from the SIL analyte to the analyte, because for the ISCC method, a large amount of SIL analytes are required in each study sample to define the upper limit of quantitative determination (ULOQ). In addition, the labeling impurities (the amount of the labeled analyte with less or more labeled positions than the SIL analyte) should also be low enough to avoid interference with the isotopic abundance in the MIRM channel of the SIL analyte.

[0285] Although deuterium labeling is very cost-effective and readily available, deuterium-labeled analytes should be avoided in ISCC methods because deuterium-labeled analytes are easily separated from unlabeled analytes and, more importantly, hydrogen-deuterium exchange reactions may easily occur on exchangeable protons and deuterons, making it impossible to accurately calculate isotopic abundances in MIRM channels.

[0286] The use of a properly calibrated triple quadrupole mass spectrometer is another factor in the success of the MIRM-ISCC-LC-MS / MS method. For singly and doubly charged parent ions, the unit resolution (full width at half maximum - FWHH = 0.7 mass units) of both Q1 and Q3 is high enough to generate accurate MS / MS reactions close to the calculated theoretical isotopic abundance in the corresponding MIRM channel. If necessary, a higher resolution in Q1 (FWHH = 0.5 mass units) can improve measurement accuracy at the expense of a certain loss of instrument sensitivity. Our test results show that using a higher resolution in Q3 (FWHH = 0.5 mass units) does not help improve measurement accuracy.

[0287] Because the isotopic spacing (1 Da for Zp = 1; 0.5 Da for Zp = 2; 0.33 Da for Zp = 3; etc.) gradually decreases with increasing charge number (Zp), it is expected that accurate measurement of isotopic abundance in MIRM channels with triply (and higher) charged precursor ions may be challenging even with a resolution of FWHH ≤ 0.5 mass units. In this work, the isotopic abundance in MIRM channels with triply (or higher) charged precursor ions was not tested because triply (or higher) charged precursor ions with high MS / MS responses were not available.

[0288] During assay qualification and sample analysis, the performance of an established ISCC should be very similar between samples, since the ISCC is constructed using naturally occurring isotopic abundances in the selected MIRM channels, without human and instrument manipulation. Any unexpected severe deviation in one MIRM channel for several samples usually indicates endogenous interference from those matrix batches, and excluding this point has no significant impact on data accuracy. On the other hand, any unexpected severe deviation in most MIRM channels for many samples indicates a failure of the MS instrument calibration.

[0289] In the MIRM-ISCC-LC-MS / MS method, no additional assay internal standard needs to be added because the ISCC is in each study sample, and therefore all changes after the SIL analyte is spiked into the study sample (including changes from extraction, injection, ionization, fragmentation, and detection, etc.) are tracked and compensated by the ISCC itself. Therefore, the assay performance can be further improved by spiking the SIL analyte as early as possible during sample preparation, as in the analysis of the small molecule drug daclatasvir in Example 3, where the SIL daclatasvir was spiked into the sample at the beginning of sample preparation. However, for protein analysis using immunocapture, the labeled peptide can only be spiked after immunocapture and will not track and compensate for any changes during immunocapture and trypsin digestion, as in the analysis of the CD73 protein in Example 1. This problem can be solved by spiking the SIL protein with a labeled surrogate peptide moiety at the beginning of sample preparation.

[0290] Because the ISCC is localized in each individual sample and there is currently no commercial software that can establish the ISCC using peak areas from multiple MIRM channels, in this work an in-house developed software was used to generate the ISCC by a weighted least squares regression algorithm and to calculate sample concentrations in batches. The widespread application of the MIRM-ISCC-LC-MS / MS method relies on the development of commercial software by large mass spectrometer companies.

[0291] Example 4

[0292] Quantitative LC-MS / MS bioanalysis using MIRM technology with single sample multipoint external calibration curves and isotopic sample dilution

[0293] The LC-MS / MS system used was a triple quadrupole 6500 mass spectrometer (AB Sciex, Foster City, CA) coupled to a Nexera UPLC system (Shimadzu, Columbia, MD). The UPLC system consisted of two LC-30AD pumps, a SIL-30ACMP autosampler, and a CTO-30AS column heater. Separation was achieved on an Acquity HSS T3 analytical column (2.1 mm×50 mm, particle size 1.8 μm) (Waters, Milford, MA) using a gradient elution with a mobile phase of 10 mM ammonium acetate in water / acetonitrile (90 / 10) containing 0.1% formic acid and 10 mM ammonium acetate in water / acetonitrile (10 / 90) containing 0.1% formic acid. The flow rate was 0.8 mL / min. LC-MS / MS data were acquired using Analyst software (1.6.2) (AB Sciex, Foster City, CA).

[0294] Sample preparation for LC-MS / MS quantification of daclatasvir using multi-sample external calibration, single-sample multi-point external calibration, and ISCC. 13 C2 15 All stock solutions of N4-daclatasvir were prepared in acetonitrile / DMSO (1 / 1, v / v). 20000 ng / mL of daclatasvir was prepared by appropriately diluting the 0.5 mg / mL stock solution with human plasma. A multi-sample external calibration curve with daclatasvir concentrations of 1000, 800, 500, 100, 20, 4, 2, and 1 ng / mL was prepared by serial dilution of 20000 ng / mL daclatasvir in human plasma. QC samples of 20000, 5000, 800, 500, 40, 3, and 1 ng / mL daclatasvir were prepared by serial dilution of 0.5 mg / mL daclatasvir stock solution.

[0295] A concentration of 5000 ng / mL of daclatasvir was prepared in methanol / water (10 / 90) by appropriate dilution of 0.5 mg / mL daclatasvir stock solution, and this solution was used to establish a single sample multi-point external calibration curve. 13 C2 15 N4-daclatasvir stock solution was used to prepare 5040.6 ng / mL 13 C2 15N4-daclatasvir, and this solution was used to establish ISCC in each sample and was also used as a stable isotope labeled internal standard (SIL-IS) in the assay for both the multiple sample external calibration curve and the single sample multi-point external calibration curve methods.

[0296] Aliquots of 100 μL plasma samples from 1 to 1000 ng / mL (six replicates of QC samples at 1, 3, 40, 500 and 800 ng / mL) for two multi-sample external calibration curves and two single-sample multi-point external calibration curves (two blank plasma samples to be spiked with known amounts of daclatasvir) were transferred to 96-well plates. The 5000 and 20000 ng / mL QC samples were diluted 100-fold and 200-fold, respectively, and 100 μL of both the diluted and undiluted QC samples of 5000 and 20000 ng / mL (six replicates each) were transferred to 96-well plates. 5000 ng / mL daclatasvir in 10% methanol and 90% water in a volume of 20 μL was added to two blank human plasma samples to construct two single-sample multi-point external calibration curves, and 20 μL of 10% methanol and 90% water was added to the other samples as makeup. 5040.6ng / mL in 20 μL of 10% methanol and 90% water 13 C2 15 Aliquots of N4-daclatasvir were added to each plasma sample in a 96-well plate. For multi-sample external calibration curves and single-sample multi-point external calibration curves, use 13 C2 15 N4-daclatasvir was used as the SIL-IS. The ISCC for each sample was constructed using N4-daclatasvir as the SIL analyte.

[0297] The samples were extracted using a Janus micro-liquid handler (PerkinElmer, Waltham, MA) using liquid-liquid extraction (H. Jiang et al., Journal of Chromatography A 2012, 1245, 117-121). A 50 μL volume of 1 M ammonium bicarbonate buffer was added to each sample followed by 600 μL of MTBE. The 96-well plate was vortexed for 5 min and 400 μL of supernatant was transferred to a clean 96-well plate and evaporated to dryness at 50° C. The samples were reconstituted with 100 μL of 10% methanol in water for LC-MS / MS analysis.

[0298] A summary of the monitored MRM and MIRM transitions for each of the multiple sample external calibration curve, the single sample multipoint external calibration curve (OSMECC), and the in-sample calibration curve (ISCC) is shown in Figure 4A. The SRM transitions and corresponding isotope dilution factors (IDFs) used for the isotopic sample dilutions are also shown in Figure 4A. The calibration curve performance of the two multiple sample external calibration curves and the two single sample multipoint external calibration curves and the two ISCCs used is shown in Figure 4B.

[0299] The accuracy and precision data of QC samples using multi-sample external calibration curves, single-sample multi-point external calibration curves and ISCC are shown in FIG5A . The QC samples of 5000 and 20000 ng / mL were physically diluted 100 times and 200 times using two-step dilution, respectively. In this example, three different types of calibration curves were used to calculate the concentrations of the same QC sample set. Accurate measurements of QC samples at all concentration levels using these three types of calibration curves confirmed that both single-sample multi-point calibration curves and ISCC can transmit bioanalytical data with the same accuracy level as traditional multi-sample external calibration curves. Therefore, they can be used in LC-MS / MS bioanalytical assays that traditionally use multi-sample external calibration curves.

[0300] In order to eliminate the sample dilution step for samples with concentrations higher than the determination ULOQ, the isotope sample dilution method using MIRM technology was evaluated with all three types of calibration curves using QC samples of 5000, 20000 and 50000 ng / mL. As shown in Figure 5B, accurate measurement of QC samples of 5000 and 20000 ng / mL was achieved using three different calibration curves and up to 1040 times of isotope dilution factors (IDF). Under the IDF of 1695 and 4386, only ISCC provided accurate measurement of QC samples of 20000 ng / mL. In addition, the IDF of 1695 and 4386 was evaluated for ISCC by using QC samples with a concentration of 50000 ng / mL, and accurate measurement was achieved for both IDFs. Therefore, the isotope sample dilution method can be used in LC-MS / MS bioanalysis to eliminate the physical sample dilution step.

[0301] ***

[0302] Throughout this application, various publications are referred to in parentheses by author name and date or by patent number or patent publication number. The disclosures of these publications are hereby incorporated by reference in their entirety into this application to more fully describe the state of the art as of the date of the disclosure as described and claimed herein, as known to the skilled artisan. However, citation of a reference herein should not be construed as an admission that this reference is prior art to the disclosure. Sequence Listing <110> Bristol-Myers Squibb <120> Analytical method using in-sample calibration curves by multi-isotopologue reaction monitoring <130> 3338.148PC01 <150> US 62 / 775,318 <151> 2018-12-04 <160> 3 <170> PatentIn Version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 1 Val Ile Tyr Pro Ala Val Glu Gly Arg 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 2 Leu Ala Ala Phe Pro Glu Asp Arg 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 3 Leu Gln Asp Ala Gly Val Tyr Arg 1 5

Claims

1. A method for quantifying the concentration of at least one analyte in a sample, the method comprising adding one or more known amounts of one or more stable isotope labeled (SIL) analytes to a sample containing at least one analyte to construct one or more in-sample calibration curves (ISCCs) by multi-isotopologue reaction monitoring (MIRM) of each added SIL analyte, wherein MIRM of a SIL analyte refers to multiple reaction monitoring of multiple isotopic transitions of the SIL analyte; wherein the ISCC for each analyte is constructed in the sample based on a relationship between a calculated theoretical isotopic abundance measured in MIRM transitions as analyte concentration equivalents and a tandem mass spectrometry (MS / MS) peak area measured in the corresponding MIRM transition; wherein the concentration of at least one analyte in the sample is quantified using an established ISCC and a measured peak area of ​​the analyte from a liquid chromatography-tandem mass spectrometry (LC-MS / MS) process, and wherein a tandem mass spectrometer is operated in a multiple reaction monitoring mode; wherein the SIL analyte and the naturally occurring isotopologues of the SIL analyte are (p+Z p +α) / Z p to (d+Z d +β) / Z d The theoretical isotopic abundance of each MIRM transition measured by mass-to-charge ratio is calculated based on formula (I): (p+Z p +α) / Z p →(d+Z d +β) / Z d The isotopic abundance in the MIRM transition = [mass (d + Z d +β)]*[Relative isotopic distribution of neutral loss with mass n+(α-β)] (I) Where p is the monoisotopic mass of the parent molecule of the SIL analyte; Z p is the charge number of the parent ion; d is the monoisotopic mass of the subfragment of the SIL analyte; Z d is the charge of the daughter ion; n is the monoisotopic mass of the neutral loss of the SIL analyte; p = d + n; α and β are integers which are the number of additional neutrons on the parent and daughter ions, respectively, α≥0, β≥0 and α≥β; and Z p and Z d is an integer.

2. The method according to claim 1, wherein (i) ionizing the analyte, the SIL analyte, and naturally occurring isotopologues of the SIL analyte in the mass spectrometer to produce protonated or deprotonated parent ions of the analyte, the SIL analyte, and naturally occurring isotopologues of the SIL analyte; (ii) fragmenting the parent ion of the analyte, the parent ion of the SIL analyte, and the parent ion of the naturally occurring isotopologue of the SIL analyte in the mass spectrometer at the same cleavage site to produce neutral losses and product ions; (iii) monitoring the transition of the analyte from a parent ion to a daughter ion in the mass spectrometer; (iv) measuring the peak area of ​​the analyte transition from the parent ion to the daughter ion; (v) monitoring in the mass spectrometer selected multiple transitions from parent ions of the SIL analyte and parent ions of naturally occurring isotopologues of the SIL analyte to daughter ions of the SIL analyte and daughter ions of naturally occurring isotopologues of the SIL analyte; and / or (vi) measuring the peak area of ​​each MIRM transition, wherein the MIRM transition comprises a selected transition from a parent ion of the SIL analyte and a parent ion of a naturally occurring isotopologue of the SIL analyte to a daughter ion of the SIL analyte and a daughter ion of a naturally occurring isotopologue of the SIL analyte.

3. The method of claim 2, further generating an in-sample calibration curve based on the relationship between the peak areas measured in the MIRM transitions of the SIL analyte and the naturally occurring isotopologues of the SIL analyte and the analyte concentration equivalents for each MIRM transition.

4. The method according to claim 1, wherein the upper limit of quantification (ULOQ) of ISCC is calculated based on formula (II): (M / V)*(M 分析物 / M SIL分析物 )ng / mL (II) wherein M (ng) is the total amount of the SIL analyte added to the sample; V is the sample volume (mL) before adding the SIL analyte; M 分析物 is the molecular weight of the analyte; and M SIL分析物 is the molecular weight of the SIL analyte, The upper limit of quantitation is equivalent to the highest analyte concentration.

5. The method of claim 4, wherein one or more other analyte concentration equivalents in the MIRM transition are calculated based on formula (III): I a *ULOQ(ng / mL) (III) Among them I a is the calculated theoretical isotopic abundance of the MIRM transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte. The method of claim 1 , wherein the analyte is a protein or a peptide.

7. The method of claim 1, wherein the SIL analyte is a stable isotope labeled protein or peptide.

8. The method of claim 7, wherein the parent ion of the SIL analyte comprises at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, or at least 20 amino acids.

9. The method of claim 7, wherein the parent ion of the SIL analyte comprises an amino acid sequence of between 4 and 20 amino acids, between 4 and 15 amino acids, between 5 and 15 amino acids, between 4 and 14 amino acids, between 5 and 14 amino acids, between 5 and 13 amino acids, between 5 and 12 amino acids, between 6 and 15 amino acids, between 6 and 14 amino acids, between 6 and 13 amino acids, between 6 and 12 amino acids, between 6 and 11 amino acids, between 6 and 10 amino acids, between 6 and 9 amino acids, between 6 and 8 amino acids, between 7 and 15 amino acids, between 7 and 14 amino acids, between 7 and 13 amino acids, between 7 and 12 amino acids, between 7 and 11 amino acids, between 7 and 10 amino acids, or between 7 and 9 amino acids.

10. The method of claim 1, wherein the analyte is an antibody.

11. The method of claim 1, wherein the analyte is a fusion protein.

12. The method of claim 1, wherein the analyte is PD-1, PD-L1, CD73, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD73 antibody, or any combination thereof.

13. The method of claim 1, wherein the analyte is a small molecule.

14. The method of claim 13, wherein the small molecule has a molar mass of at least 100 g / mol, at least 200 g / mol, at least 300 g / mol, at least 400 g / mol, at least 500 g / mol, at least 600 g / mol, at least 700 g / mol, at least 800 g / mol, at least 900 g / mol, at least 1000 g / mol, at least 1100 g / mol, at least 1200 g / mol, at least 1300 g / mol, at least 1400 g / mol, at least 1500 g / mol, at least 1600 g / mol, at least 1700 g / mol, at least 1800 g / mol, at least 1900 g / mol, or at least 2000 g / mol.

15. The method of claim 1, wherein the SIL analyte contains at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 stable isotope labels.

16. The method of claim 15, wherein the SIL analyte contains 3 to 20 isotopic labels, 3 to 19 isotopic labels, 3 to 15 isotopic labels, 3 to 10 isotopic labels, 3 to 8 isotopic labels, 3 to 7 isotopic labels, 3 to 6 isotopic labels, 4 to 15 isotopic labels, 4 to 10 isotopic labels, 4 to 8 isotopic labels, 4 to 7 isotopic labels, 4 to 6 isotopic labels. isotope labels, 5 to 8 isotope labels, 5 to 7 isotope labels, 6 to 10 isotope labels, 6 to 8 isotope labels, 7 to 16 isotope labels, 7 to 16 isotope labels, 8 to 16 isotope labels, 8 to 15 isotope labels, 9 to 15 isotope labels, 9 to 14 isotope labels, 10 to 14 isotope labels, 10 to 13 isotope labels or 11 to 13 isotope labels.

17. The method of claim 2, wherein each measured relative peak area in a MIRM transition has less than 15% deviation compared to the theoretical isotopic abundance calculated in the corresponding MIRM transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte.

18. The method of claim 17, wherein at least one measured relative peak area in a MIRM transition has a deviation of less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% compared to the theoretical isotopic abundance calculated in the corresponding MIRM transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte.

19. The method of claim 18, wherein at least one measured relative peak area in a MIRM transition has a deviation of between 1% and 15% compared to the theoretical isotopic abundance calculated in the corresponding transition of the SIL analyte or a naturally occurring isotopologue of the SIL analyte.

20. The method of claim 19, wherein the number of MIRM transitions is at least two, at least three, at least four, at least five, at least six, at least seven, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20.

21. The method of claim 20, wherein the number of the MIRM transitions is between 2 and 20.

22. The method of claim 2, wherein the analyte concentration equivalents of the highest MIRM and the lowest MIRM are at least 10, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000-fold different.

23. The method of claim 1, wherein the calculated theoretical isotopic abundances of two selected MIRM transitions differ by at least 0.01%, by at least 0.05%, by at least 0.1%, by at least 0.5%, by at least 1%, by at least 1.5%, by at least 2%, by at least 2.5%, by at least 3%, by at least 3.5%, by at least 4%, by at least 4.5%, by at least 5%, by at least 5.5%, by at least 6%, by at least 6.5%, by at least 7%, by at least 7.5%, by at least 8%, by at least 8.5%, by at least 9%, by at least 9.5%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, or by at least 50%.

24. The method of claim 1, wherein the SIL analyte contains less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1% unlabeled analyte.

25. The method of claim 1, wherein the marker is 2 H. 13 C. 15 N. 33 S. 34 S. 36 S. 17 O or 18 O.

26. The method of claim 2, wherein one or more of the protonated or deprotonated molecular ions are singly, doubly, triply, or more highly charged.

27. The method of claim 1, wherein the mass spectrometer is a triple quadrupole mass spectrometer comprising Q1, Q2, and Q3.

28. The method of claim 27, wherein the resolution for Q1 and Q3 is unity resolution.

29. The method of claim 27, wherein the resolutions for Q1 and Q3 are different.

30. The method of claim 27, wherein the resolution for Q1 is higher than the unit resolution of Q3.

31. The method of claim 1, wherein an in-sample calibration curve (ISCC) composition is added prior to or during sample preparation.

32. The method of claim 1 which reduces overall instrument run time.

33. The method of claim 1, wherein no external calibration curve is used.

34. The method of claim 1, wherein the analyte is a biomarker.

35. The method of claim 1, wherein the analyte is a metabolite.

36. The method of claim 1, wherein the sample is serum, tissue, formalin-fixed paraffin-embedded sample, plasma, saliva, cerebrospinal fluid, tears, urine, synovial fluid, dried blood spots, or any combination thereof.

37. The method of claim 36, wherein the tissue is a biopsy tissue.

38. The method of any one of claims 1 to 9 and 15 to 37, wherein the analyte is CD73 or a portion thereof.

39. The method of claim 1, wherein the SIL analyte is a SIL peptide, and the SIL peptide is V[Ile( 13 C6, 15 N)]YPAVEGR, which is also known as SEQ ID NO:

1.

40. The method of any one of claims 1 to 9 and 15 to 37, wherein the analyte is PD-1 or a portion thereof.

41. The method of claim 1, wherein the SIL analyte is a SIL peptide, and the SIL peptide is LAAFPED[Arg( 13 C6, 15 N4)], which is also referred to as SEQ ID NO:

2.

42. The method of any one of claims 1 to 9 and 15 to 37, wherein the analyte is PD-L1 or a portion thereof.

43. The method of claim 1, wherein the SIL analyte is a peptide, the peptide being LQDAG[Val( 13 C5, 15 N)]YR, which is also known as SEQ ID NO:

3.

44. The method of any one of claims 1 to 5 and 13 to 37, wherein the analyte is daclatasvir.

45. The method of claim 44, wherein the SIL analyte is 13 C2 15 N4-Daclatasvir.

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