Novel high performance liquid chromatography-tandem mass spectrometry analysis method for detecting aspartatide in human plasma

Abstract

The invention discloses a novel high performance liquid chromatography-tandem mass spectrometry analysis method for detecting aspartatide in human plasma, teriparatide is used as an internal standard substance to be detected, a solid phase extraction method is adopted to extract aspartatide to be detected and internal standard teriparatide from a plasma sample, and a standard curve is linear in a range of 0.010-1.00 ng / mL. During the verification of the method, selectivity, matrix effect, recovery, sensitivity, linearity, accuracy and precision, stability and dilution reliability are assessed to meet standards. The method disclosed by the invention has specificity on determination of the to-be-detected substance, i.e., the aspartatide and the internal standard teriparatide in the human plasma, and a standard curve is linear in a range of 0.010-1.00 ng / mL. The quantitative lower limit of methodology is 0.01 ng / mL. The method has the advantages of good accuracy, reliability, high sensitivity, high specificity and reproducibility.

Description

Technical Field

[0001] The present invention relates to a method for detecting abalotriptide, and in particular to a novel high performance liquid phase-tandem mass spectrometry analysis method for detecting abalotriptide in human plasma. Background Art

[0002] Abalotriptide is an analog of parathyroid hormone protein, which can selectively activate the signaling pathway of parathyroid hormone type 1 receptor, promote bone formation, increase bone density, and is used to treat postmenopausal women at risk of osteoporosis. Detecting the concentration of abalotriptide in human plasma is of great significance for studying the metabolic laws of drugs and establishing the relationship between drug concentration and drug efficacy.

[0003] In the past, radioimmunoassay was used to measure the concentration of abalotriptyline in human plasma. While radioimmunoassay has the advantage of high sensitivity, it also has many disadvantages, which leads to great limitations in its use.

[0004] Disadvantages of radioimmunoassay:

[0005] 1. The reagents used are radioactive and produce radioactive contamination, which makes it difficult for conventional laboratories to use them;

[0006] 2. The detection principle is antigen-antibody immune reaction, which requires the preparation of highly specific antibody reagents, and the preparation process is complicated and time-consuming;

[0007] 3. The half-life of the reagent is short and the cost of use is high;

[0008] 4. Poor specificity, easy to produce cross-reactions, and difficult to distinguish between the analyte and structural analogs;

[0009] 5. The linear range is narrow, and the sample needs to be diluted before testing, which is easy to cause detection errors;

[0010] Existing technology:

[0011] (Clinical Drug Investigation (2021) 41: 277–285 https: / / doi.org / 10.1007 / s40261-021-01008-7) disclosed the radioimmunoassay of abalotriptyline, but it has low sensitivity, pollutes the environment, and requires labeling with radioactive elements (125 iodine) and special antibody reagents (which must not cross-react with natural PTH or PTHrP, so it is difficult to prepare and obtain). The preparation and acquisition of reagents are somewhat difficult.

[0012] Therefore, a better abalotriptyline detection method is urgently needed in the market. Summary of the invention

[0013] Purpose of the invention: In view of the shortcomings of existing detection technologies, the present invention provides a new high performance liquid chromatography-tandem mass spectrometry analysis method for detecting abalotriptyline in human plasma that is accurate, reliable, highly sensitive, highly specific and reproducible.

[0014] Technical solution: The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting abaloparatide in human plasma of the present invention uses teriparatide as the internal standard analyte, and adopts solid phase extraction to extract the analyte abaloparatide and the internal standard teriparatide from the plasma sample, and the standard curve is linear in the range of 0.010 to 1.00 ng / mL.

[0015] 1. Reference standard substances used in testing

[0016] Reference Standard Internal Standard Abalotriptyline Standard Teriparatide

[0017] 2. The volumes of reagents prepared during the test can be adjusted proportionally as needed.

[0018] 1) MPA: 0.1% formic acid aqueous solution

[0019] Preparation method: Take 1000mL of ultrapure water in a 1000mL reagent bottle, add 1.0ml of formic acid, and mix well. Store at room temperature. The validity period is within two weeks from the date of preparation.

[0020] 2) MPB: acetonitrile solution containing 0.1% formic acid

[0021] Preparation method: Take 1000mL of acetonitrile in a 1000mL reagent bottle, add 1.0ml formic acid, and mix well. Store at room temperature. The validity period is within two weeks from the date of preparation.

[0022] 3) Needle Wash: 50% methanol aqueous solution containing 0.1% formic acid

[0023] Preparation method: Take 500mL methanol and 500mL ultrapure water and add them into a 1000mL reagent bottle, add 1.0mL formic acid and mix well. Store at room temperature, the validity period is one month from the date of preparation.

[0024] name describe Mass spectrometry API7500,AppliedBiosystems / Sciex Liquid chromatography LC-40, Shimadzu Autosampler RackChangerII sample rack switching device SIL-20ACHT Autosampler Data acquisition software SciexOSsoftware Data processing software Microsoft Office 2013 or other versions <![CDATA[WatsonLIMS TM 7.5SP1,Thermo]]>

[0025] 3. Instruments and Equipment

[0026] 4. Liquid chromatography conditions

[0027]

[0028]

[0029] 5. Mass spectrometry conditions

[0030]

[0031]

[0032] 6. Preparation of analyte stock solution

[0033] Accurately weigh the abalotriptide reference standard substance, and after conversion by the correction factor, add an appropriate amount of DMSO to prepare an abalotriptide stock solution with a concentration of 1.00 mg / mL, and store it in a refrigerator at -10 to -30°C until use.

[0034] 7. Preparation of internal standard stock solution and working solution

[0035] Accurately weigh the teriparatide reference standard substance, and after conversion by correction factor, add an appropriate amount of DMSO to prepare a teriparatide stock solution with a concentration of 1.00 mg / mL, and store it in a refrigerator at -10 to -30°C for use. Take 10 μL of the teriparatide stock solution and add it to 1000 mL of methanol water containing 0.1% human plasma (50:50, v / v).

[0036] 8. Preparation of standard curve samples and quality control samples

[0037] Roche cOmplete was added to human plasma at a dilution of 1:99. TM Protease inhibitors, the final protease inhibitor concentration in plasma is 0.2 mg / mL.

[0038] The standard curve contained 8 different concentration points by serial dilution using human plasma, and the concentrations of abalotriptyline were 0.010, 0.020, 0.050, 0.100, 0.200, 0.400, 0.800 and 1.00 ng / mL, respectively.

[0039] The quality control samples contained 4 different concentration points by serial dilution using human plasma, and the concentrations of abalotriptyline were 0.010 (LLOQ QC), 0.030 (LQC), 0.400 (MQC) and 0.750 (HQC) ng / mL.

[0040] For the analytical batches of precision and accuracy of methodological validation, personnel and instrument validation, each accuracy and precision analytical batch contains quality control samples at 4 concentration levels, with at least 5 replicates for each concentration. For other analytical batches and sample analysis of methodological validation, quality control samples include at least quality control samples at three concentration levels: LQC, MQC and HQC, with at least 2 replicates for each concentration.

[0041] 9. Sample processing method

[0042]

[0043]

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1) For the first time, a high performance liquid chromatography-tandem mass spectrometry method for abalotriptyline in human plasma was established;

[0046] 2) This method overcomes the disadvantage of poor sensitivity of the HPLC-MS / MS method and achieves extremely high sensitivity. The sensitivity of the present invention is 10pg / ml, while the sensitivity of the prior art is 20pg / ml, which is doubled. The quantitative lower limit of the methodology is 0.01ng / mL, which meets the needs of clinical trial detection;

[0047] 3) Through simple solid phase extraction treatment, interfering substances in human plasma can be removed, the analyte can be enriched, and the matrix effect can be improved;

[0048] 4) Protease inhibitors were added to the plasma samples to stabilize the analyte;

[0049] 5) The methodology used standard substances to prepare the standard curve and the results were corrected by internal standards, so the test results had good accuracy;

[0050] 6) Tandem mass spectrometry as a detector has high sensitivity, specificity and reproducibility, and is the gold standard for detecting the blood concentration of peptide drugs such as abalotriptyline;

[0051] 7) This method can detect one sample in less than 5 minutes, which can meet the high-throughput requirements of biological sample detection in clinical research;

[0052] 8) This HPLC-MS / MS method does not require additional preparation of antibody reagents and is simple to operate;

[0053] 9) This method does not require radioactive reagents, protecting the environment from being contaminated by radioactive substances;

[0054] 10) No antibody reagents or radioactive reagents are required, the specificity and reproducibility are better, and the linear range is wider;

[0055] 11) The matrix for preparing the standard curve is plasma with added protease inhibitors to protect abalotriptyline from being decomposed by endogenous proteases in human plasma. Standard products are used to prepare the standard curve, and teriparatide is used as an internal standard for result correction. The test results have good accuracy and reproducibility;

[0056] 12) The solid phase extraction method was used to purify and enrich abalotriptyline, which effectively removed interferences, improved matrix effects, enriched the analyte and increased the sensitivity of detection;

[0057] 13) The loading, washing and elution solvents of solid phase extraction have been fully optimized to achieve satisfactory extraction recovery;

[0058] 14) The liquid phase conditions are optimized to separate the analyte abalotriptide from the interfering peaks, thereby improving the sensitivity of the detection; the mass spectrometry conditions are optimized to achieve the best instrument response signal and a good signal-to-noise ratio;

[0059] 15) When preparing the working solution, a small amount of human plasma is added to prevent nonspecific adsorption;

[0060] 16) Use low adsorption plates to effectively reduce the nonspecific adsorption of abalotriptyline. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is the Q1 scan of abalotriptide;

[0062] Figure 2 This is the Q3 scan of abalotriptide;

[0063] Figure 3 This is the double blank sample chromatogram of abalotriptyline in human plasma;

[0064] Figure 4 is the single blank sample chromatogram of abalotriptyline in human plasma;

[0065] Figure 5 The chromatogram of the sample with the lower limit of quantification (LLOQ) of abalotriptyline in human plasma. DETAILED DESCRIPTION

[0066] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0067] (I) The detection method of this embodiment is as follows:

[0068] 1. Reference standard substances used in testing

[0069] Reference Standard Internal Standard Abalotriptyline Standard Teriparatide

[0070] 2. The volumes of reagents prepared during the test can be adjusted proportionally as needed.

[0071] 4) MPA: Aqueous solution containing 0.1% formic acid

[0072] Preparation method: Take 1000mL of ultrapure water in a 1000mL reagent bottle, add 1.0ml of formic acid, and mix well. Store at room temperature. The validity period is within two weeks from the date of preparation.

[0073] 5) MPB: acetonitrile solution containing 0.1% formic acid

[0074] Preparation method: Take 1000mL of acetonitrile in a 1000mL reagent bottle, add 1.0ml formic acid, and mix well. Store at room temperature. The validity period is within two weeks from the date of preparation.

[0075] 6) Needle Wash: 50% methanol aqueous solution containing 0.1% formic acid

[0076] Preparation method: Take 500mL methanol and 500mL ultrapure water and add them into a 1000mL reagent bottle, add 1.0mL formic acid and mix well. Store at room temperature, the validity period is one month from the date of preparation.

[0077] 3. Instruments and Equipment

[0078]

[0079]

[0080] 4. Liquid chromatography conditions

[0081]

[0082] 5. Mass spectrometry conditions

[0083]

[0084]

[0085]

[0086] 6. Preparation of analyte stock solution

[0087] Accurately weigh the abalotriptide reference standard substance, and after conversion by the correction factor, add an appropriate amount of DMSO to prepare an abalotriptide stock solution with a concentration of 1.00 mg / mL, and store it in a refrigerator at -10 to -30°C until use.

[0088] 7. Preparation of internal standard stock solution and working solution

[0089] Accurately weigh the teriparatide reference standard material, after conversion by correction factor, add appropriate amount of DMSO to prepare a teriparatide stock solution with a concentration of 1.00 mg / mL, and store it in a refrigerator at -10 to -30°C for use. Take 10 μL of the teriparatide stock solution and add it to 1000 mL of methanol water containing 0.1% human plasma (50:50, v / v).

[0090] 8. Preparation of standard curve samples and quality control samples

[0091] Roche cOmplete was added to human plasma at a dilution of 1:99. TMProtease inhibitors, the final protease inhibitor concentration in plasma is 0.2 mg / mL.

[0092] The standard curve contained 8 different concentration points by serial dilution using human plasma, and the concentrations of abalotriptyline were 0.010, 0.020, 0.050, 0.100, 0.200, 0.400, 0.800 and 1.00 ng / mL, respectively.

[0093] The quality control samples contained 4 different concentration points by serial dilution using human plasma, and the concentrations of abalotriptyline were 0.010 (LLOQ QC), 0.030 (LQC), 0.400 (MQC) and 0.750 (HQC) ng / mL.

[0094] For the analytical batches of precision and accuracy of methodological validation, personnel and instrument validation, each accuracy and precision analytical batch contains quality control samples at 4 concentration levels, with at least 5 replicates for each concentration. For other analytical batches and sample analysis of methodological validation, quality control samples include at least quality control samples at three concentration levels: LQC, MQC and HQC, with at least 2 replicates for each concentration.

[0095] 9. Sample processing method

[0096] 1. Take the tubes in a row and add samples as follows: for double blank samples (Double Blank) and zero concentration samples (Zero Blank), add 300 μL of mixed blank matrix; for standard curve samples, quality control samples, test samples, and system suitability samples, add 300 μL of the corresponding blank matrix sample;

[0097] 2. For zero blank samples, zero concentration samples, standard curve samples, quality control samples, samples to be tested, and system suitability samples, add 10.0uL of internal standard working solution (10.0ng / mL); for double blank samples, add 10.0uL of internal standard preparation solvent (50% methanol water containing 0.1% human plasma), and centrifuge at 3000g for 1min;

[0098] 3. Add 200 μL of methanol / water (40 / 60; v / v) solution to each well;

[0099] 4. Oscillate at 2500 rpm for 5 min;

[0100] 5. Centrifuge at 3500g for 5 min at 4°C;

[0101] 6. Take a new 96-well plate, take 450uL of the supernatant from step 5 above, and then add 550uL of water;

[0102] 7. Oscillate at 800 rpm for 10 min;

[0103] 8. Take the Oasis HLB uElution solid phase extraction plate and perform solid phase extraction:

[0104] a) → Activation: 200uL methanol activation;

[0105] b) →Balance: 200uL ultrapure water balance;

[0106] c) → Sample loading: Pipette 800 μL of the diluted supernatant onto the extraction plate, transfer twice, 400 μL each time, and adjust the pressure valve to allow the sample to pass slowly through the solid phase extraction plate;

[0107] d) → Cleaning 1: Clean once with 250uL ultrapure water;

[0108] e) → Cleaning 2: Clean once with 250uL methanol / water (10 / 90, v / v);

[0109] f) → Elution: 25.0uL acetonitrile / trifluoroacetic acid / water (75 / 1 / 25, w / w / v), eluted three times) + added to each well;

[0110] 50.0uL ultrapure water mixed evenly, this step uses a 96-well low adsorption plate for receiving;

[0111] g) → Centrifuge at 3500 g for 1 min at 4°C for the detection of abalotriptyline.

[0112] Use LC-MS / MS instrument to perform detection and analysis according to “4. Liquid chromatography conditions” and “5. Mass spectrometry conditions”. The injection volume of each sample is 5 μL, and the chromatogram is recorded.

[0113] A series of theoretical concentration values ​​of abalotriptide in the standard curve samples were used as the horizontal coordinates, and the ratio of the peak areas of abalotriptide and the internal standard teriparatide was used as the vertical coordinate. The weighted least square method was used for regression calculation, and the linear regression equation obtained was the 0.010-1.00 ng / mL standard curve. The ratio of the peak areas of abalotriptide and the internal standard teriparatide in the unknown plasma sample was substituted into the equation to obtain the blood concentration of abalotriptide in the unknown sample.

[0114] (II) Methodological Validation

[0115] (1) Methods:

[0116]

[0117]

[0118]

[0119]

[0120] (2) Results:

[0121] 1. Selectivity

[0122] The maximum interference effect of blank plasma, one hemolytic matrix and one hyperlipidemic matrix prepared by each individual at the retention time of the analyte was 0.0%, which was less than 20.0% of the average peak area of ​​the analyte in the LLOQ standard curve samples that met the acceptance criteria. The maximum interference effect at the retention time of the internal standard was 0.0%, which was less than 5.0% of the average peak area of ​​the internal standard in all standard curve samples and quality control samples that met the acceptance criteria.

[0123] Selectivity investigation of individuals from different sources, hemolytic matrices and high-fat matrices

[0124]

[0125] The interference of the analyte on the internal standard, the interference effect at the internal standard retention time in the ULOQ of abalotriptyline without internal standard samples was 0.0%, and the peak area of ​​the interference peak was less than 5.0% of the average peak area of ​​the internal standard in all standard curve samples and quality control samples that met the acceptance criteria.

[0126] Interference of the analyte on the internal standard

[0127]

[0128] The interference of the internal standard on the analyte was 0.0% at the retention time of the analyte in the zero concentration sample in the analytical batch, and the peak area of ​​the interference peak was less than 20.0% of the average peak area of ​​the analyte in the LLOQ standard curve samples that met the acceptance criteria in the analytical batch.

[0129] Interference of internal standard on analytes

[0130]

[0131] 2. Matrix Effect

[0132] Matrix effects for normal matrices as well as matrix effects for hyperlipidemic and hemolytic matrices met the acceptance criteria.

[0133] Normal individual matrix (LQC concentration)

[0134]

[0135]

[0136] Note: “-” is not applicable.

[0137] Normal individual matrix (HQC concentration)

[0138]

[0139]

[0140] Note: “-” is not applicable.

[0141] 3. Hemolytic matrix

[0142]

[0143] Note: “-” is not applicable.

[0144] 4. High-fat matrix

[0145]

[0146] Note: “-” is not applicable

[0147] 5. Standard curve

[0148] The standard curve results meet the acceptance criteria

[0149]

[0150] The standard curve results meet the acceptance criteria

[0151]

[0152]

[0153] 6. Accuracy and precision

[0154] Accuracy and precision experiments should be conducted on at least three independent analytical batches and at least 2 days. Accuracy and precision experimental quality control samples should be freshly prepared on the same day. Each accuracy and precision analytical batch contains quality control verification samples at 5 concentration levels, namely 0.010 (LLOQ QC), 0.030 (LQC), 0.400 (MQC) and 0.750 (HQC) ng / mL, with at least 5 replicates for each concentration.

[0155] The precision of the method was expressed as the percentage of the coefficient of variation (CV% = [(standard deviation / mean) × 100%]); the accuracy of the method was expressed as the percentage of the relative error (RE% = [(mean observed concentration-nominal concentration) / nominal concentration] × 100%).

[0156] The intra- and inter-batch accuracy and precision met the acceptance criteria for method validation.

[0157] Intra- and inter-assay accuracy and precision results.

[0158]

[0159]

[0160] 7. Extraction recovery rate

[0161] The extraction recoveries of the analytes met the acceptable standards.

[0162]

[0163] The dilution reliability sample (5.00 ng / mL) was diluted 10-fold with pooled human plasma and the results met the acceptance criteria.

[0164]

[0165]

[0166] The plasma samples remained stable after being frozen and thawed 5 times at -10 to -30°C (thawed at room temperature) and after being frozen and thawed 5 times at -60 to -90°C (thawed at room temperature).

[0167]

[0168] The plasma samples were placed at -10 to -30°C for 135 days and at -60 to -90°C for 135 days, and the stability met the acceptable standards.

[0169]

[0170] The purpose of this study was to validate the methodology for the quantitative determination of abalotriptyline concentrations in human plasma by liquid chromatography-mass spectrometry (LC-MS / MS) to support clinical studies of abalotriptyline. During method validation, selectivity, matrix effects, recovery, sensitivity, linearity, accuracy and precision, stability, and dilution robustness were evaluated.

[0171] In this methodological validation experiment, a liquid chromatography tandem mass spectrometry (LC-MS / MS) method was established for the determination of abaloparatide concentration in human plasma. Teriparatide was used as the internal standard analyte (IS) in this method, and solid phase extraction was used to extract the analyte abaloparatide and the internal standard teriparatide from plasma samples. The method was specific for the determination of the analyte abaloparatide and the internal standard teriparatide in human plasma, and the standard curve was linear in the range of 0.010-1.00 ng / mL. The plasma samples remained stable after 5 freeze-thaw cycles at -10 to -30°C (thawed at room temperature) and 5 freeze-thaw cycles at -60 to -90°C (thawed at room temperature). The plasma samples were stored at -10 to -30°C for 135 days and at -60 to -90°C for 135 days, and the stability met the acceptance criteria.

[0172] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any technician in the relevant technical field, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification to the technical solution and technical content disclosed in the present invention, which does not depart from the content of the technical solution of the present invention and still falls within the protection scope of the present invention.

Claims

1. A novel high performance liquid chromatography-tandem mass spectrometry method for detecting abalotriptyline in human plasma, characterized in that: Teriparatide was used as the internal standard analyte, and solid phase extraction was used to extract abaloparatide and internal standard teriparatide from plasma samples. The standard curve was linear in the range of 0.010-1.00 ng / mL.

2. The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting abalotriptyline in human plasma according to claim 1, characterized in that: The instruments used for testing are: Mass spectrometry: API 7500, Applied Biosystems / Sciex; Liquid chromatography: LC-40, Shimadzu.

3. The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting abalotriptyline in human plasma according to claim 1, characterized in that: The liquid chromatography conditions for detection are:

4. The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting abalotriptyline in human plasma according to claim 3, characterized in that: The mass spectrometry conditions for detection are:

5. The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting abalotriptyline in human plasma according to claim 4, characterized in that: Ion pairs and parameters:

6. The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting abalotriptyline in human plasma according to claim 1, characterized in that: Preparation of analyte stock solution: Weigh the abalotriptyline reference standard substance, add DMSO, and prepare abalotriptyline stock solution with a concentration of 1.00 mg / mL.

7. The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting abalotriptyline in human plasma according to claim 1, characterized in that: Preparation of internal standard stock solution and working solution: Take the teriparatide reference standard substance, add DMSO, and prepare a teriparatide stock solution with a concentration of 1.00 mg / mL; take 10 μL of the teriparatide stock solution, add 1000 mL of methanol water containing 0.1% human plasma to obtain a working solution.

8. The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting abalotriptyline in human plasma according to claim 1, characterized in that: Sample processing method: (1) Take a row of tubes and add samples as follows: For double blank samples and zero concentration samples, add 300 μL mixed blank matrix, for standard curve samples, quality control samples, test samples, system suitability samples samples, add 300 μL of the corresponding blank matrix sample; (2) For zero blank samples, zero concentration samples, standard curve samples, quality control samples, and samples to be tested, For the system suitability sample, add 10.0uL of internal standard working solution (10.0ng / mL). For the double blank sample, Add 10.0uL of internal standard preparation solvent (50% methanol water containing 0.1% human plasma) and centrifuge; (3) Add 200 μL of methanol / water (40 / 60; v / v) solution to each well; (4) Oscillation; (5) Centrifugation; (6) Take a 96-well plate, take 450uL of the supernatant from step 5, and then add 550uL of water; (7) Oscillation; (8) Take the Oasis HLB uElution solid phase extraction plate and perform solid phase extraction: a) Activation: 200uL methanol activation; b) Balance: 200uL ultrapure water balance; c) Loading: Pipette 800 μL of the diluted supernatant onto the extraction plate in two transfers, each time 400μL, adjust the pressure valve to allow the sample to slowly pass through the solid phase extraction plate; d) Cleaning: e) Elution: 25.0uL acetonitrile / trifluoroacetic acid / water (75 / 1 / 25, w / w / v, eluted three times) + add to each well 50.0uL ultrapure water mixed evenly, this step uses a 96-well low adsorption plate for receiving; f) Centrifugation for detection of abalotriptyline.

9. The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting abalotriptyline in human plasma according to claim 1, characterized in that: The preparation method of the standard curve is: Weigh the abalotriptide reference standard substance, add DMSO after correction factor conversion, and prepare abalotriptide stock solution with a concentration of 1.00 mg / mL. Add Roche cOmplete TM Protease inhibitors, the final protease inhibitor concentration in plasma was 0.2 mg / mL; The stock solution of abalotriptide was serially diluted using the above human plasma containing protease inhibitors to obtain a standard curve containing 8 different concentration values, and the concentrations of the abalotriptide standard curve were 0.010, 0.020, 0.050, 0.100, 0.200, 0.400, 0.800 and 1.00 ng / mL, respectively; Take 300 μL of the plasma sample to be tested, and perform LC-MS / MS analysis together with the standard curve, quality control and blank control samples. Record the chromatogram, use a series of theoretical concentration values ​​of abalotriptide in the standard curve sample as the abscissa, and use the ratio of the peak area of ​​abalotriptide and the internal standard teriparatide as the ordinate, and use the weighted least squares method to perform regression calculation. The linear regression equation obtained is the 0.010-1.00 ng / mL standard curve.

10. The novel high performance liquid phase-tandem mass spectrometry analysis method for detecting abalotriptyline in human plasma according to claim 9, characterized in that: The calculation method for the result of abalotriptide is as follows: the ratio of the peak areas of abalotriptide and the internal standard teriparatide in the unknown plasma sample is substituted into the equation to obtain the blood concentration of abalotriptide in the unknown sample.

Images