CN111796035A

CN111796035A - LC-MS/MS detection method for quantitatively analyzing human plasma vildagliptin concentration

Info

Publication number: CN111796035A
Application number: CN202010584694.3A
Authority: CN
Inventors: 谭志荣; 王医成; 彭静波; 饶泰; 陈尧; 周宏灏; 贺康
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2020-06-24
Filing date: 2020-06-24
Publication date: 2020-10-20

Abstract

The invention discloses an LC-MS/MS detection method for quantitatively analyzing human plasma vildagliptin concentration, which belongs to the technical field of drug analysis and comprises the following steps: (1) sample preparation: taking human blank plasma as a blank matrix, adding vildagliptin standard working solution, and preparing 8 known vildagliptin standard samples with different concentrations; adding vildagliptin standard solution into human plasma serving as a blank matrix to prepare low, medium and high vildagliptin quality control samples with known concentrations; (2) sample treatment: comprises a human plasma sample to be detected, a standard sample and a quality control sample; (3) sample detection: taking 2 mu l of the sample obtained in the step (2) for automatic sample introduction, and carrying out high performance liquid chromatography tandem mass spectrometry detection; (4) standard curve preparation and quantitative analysis. The method has the advantages of rapid and accurate measurement of the plasma concentration of vildagliptin, simple and easy pretreatment operation, high sensitivity, accurate quantification and wide application.

Description

LC-MS/MS detection method for quantitatively analyzing human plasma vildagliptin concentration

Technical Field

The invention belongs to the technical field of drug analysis, relates to an LC-MS/MS detection method for quantitatively analyzing human plasma vildagliptin concentration, and particularly relates to a method for detecting the vildagliptin concentration in human plasma by applying a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology.

Background

Vildagliptin (vildagliptin) is a selective, competitive, reversible dipeptidyl peptidase-4 (DPP-4) inhibitor, developed by nova pharmaceutical limited, switzerland under the chemical name of 1- [ [ (3-hydroxy-1-adamantyl) amino ] acetyl ] -2-cyano- (S) -tetrahydropyrrolidine. The action mechanism of the enzyme is to inhibit the activity of the enzyme by combining with DPP-4 to form a DPP-4 complex, improve the concentration of glucagon-like peptide-1 (GLP-1), promote insulin production of islet B cells and reduce the concentration of glucagon, thereby reducing blood sugar without obvious influence on body weight. Currently, there are over 132 countries in use around the world.

The method for detecting the concentration of vildagliptin in plasma at home and abroad mainly comprises a High Performance Liquid Chromatography (HPLC), a Capillary Electrophoresis (CE) and Ultraviolet (UV) or (PDA) photodiode array detector, a gas chromatography-mass spectrometry combined method (GC/MS) and a liquid chromatography-mass spectrometry combined method (LC-MS/MS). Of these methods, HPLC method based on UV detector and DAD detector and CE method are mainly used for analyzing the content of vildagliptin in pharmaceutical preparations, and the sensitivity and selectivity are low [16-22 ]. The GC/MS method requires a derivatization step prior to sample introduction, making the analysis cumbersome and time consuming. Another disadvantage of these analytical methods is the long run time, which is not well validated against international standards.

Disclosure of Invention

The invention aims to provide an LC-MS/MS detection method for quantitatively analyzing human plasma vildagliptin concentration, and establishes an analysis method based on a high performance liquid chromatography-mass spectrometry combined technology (HPLC-MS/MS) for quantitatively detecting vildagliptin in human plasma. The method has the advantages of rapid and accurate measurement of the plasma concentration of vildagliptin, simple and easy pretreatment operation, high sensitivity, accurate quantification and wide application.

The specific technical scheme is as follows:

an LC-MS/MS detection method for quantitatively analyzing human plasma vildagliptin concentration comprises the following steps:

(1) sample preparation:

a. preparation of a standard sample: precisely weighing 10.68mg of vildagliptin reference substance into a 10ml volumetric flask, respectively washing the tube wall and the weighed paper residue into the volumetric flask by using 4ml and 3ml of methanol solutions, uniformly mixing and dissolving, adding methanol to a constant volume, and reversely and uniformly mixing to prepare vildagliptin standard stock solution A with the concentration of 1.068 mg/ml. Using methanol: diluting with an aqueous solution (50: 50, V/V) to prepare a standard curve working solution of vildagliptin, wherein the concentrations are respectively as follows: 22.25ng/ml, 44.5ng/ml, 89.0ng/ml, 178.0ng/ml, 534.0ng/ml, 1186.67ng/ml, 3560.0ng/ml and 10680.0 ng/ml.

And (3) taking human blank plasma as a blank matrix, adding vildagliptin standard working solution, and preparing 8 known vildagliptin standard samples with different concentrations.

b. Preparing a quality control sample: accurately weighing 10.70mg of vildagliptin reference substance into a 10ml volumetric flask, respectively washing the tube wall and the weighed paper residue into the volumetric flask by using 4ml and 3ml of methanol solutions, uniformly mixing and dissolving, adding methanol to a constant volume, and reversely and uniformly mixing to prepare vildagliptin standard stock solution B with the concentration of 1.070 mg/ml. Using methanol: diluting an aqueous solution (50: 50, V/V) to prepare a quality control working solution of vildagliptin, wherein the concentrations are respectively as follows: 44.583ng/ml, 535.0ng/ml and 8560.0 ng/ml.

Adding vildagliptin standard solution into human plasma serving as a blank matrix to prepare low, medium and high vildagliptin quality control samples with known concentrations;

(2) sample treatment: the method comprises a human plasma sample to be detected, a standard sample and a quality control sample, and the processing methods are as follows:

a plasma sample 100 mu l to 2ml of EP tube is taken, 1.0ml of precipitant acetonitrile containing 3.15ng/ml of internal standard is added, the mixture is automatically shaken for 5min on an oscillator, after centrifugation (15000r/min) for 10min, 50 mu l of supernatant is absorbed to another clean 2ml of EP tube, 300 mu l of 5mM ammonium formate aqueous solution and acetonitrile mixed solution (1: 1) is added, the mixture is automatically shaken for 2min on the oscillator, after centrifugation (15000r/min) for 5min, the supernatant is transferred to a sample inlet bottle.

(3) Sample detection: taking 2 mu l of the sample obtained in the step (2) for automatic sample introduction, and carrying out high performance liquid chromatography tandem mass spectrometry detection;

(4) standard curve preparation and quantitative analysis: after the step (3) is finished, taking the ratio (Y) of the areas of the vildagliptin and the internal standard peak as a vertical coordinate, taking the concentration (X) of the vildagliptin in the plasma sample as a horizontal coordinate, and adopting a weighted least square method (weight factor l/X)²) And performing linear regression to prepare a standard curve equation of the vildagliptin, and quantifying the quality control sample and the human plasma sample to be detected through the standard curve.

Further, in the step (1), the series concentration of the vildagliptin standard sample is 1.113ng/ml, 2.225ng/ml, 4.45ng/ml, 8.90ng/ml, 26.70ng/ml, 59.33ng/ml, 178.0ng/ml and 534.0 ng/ml.

Further, in the step (1), the low, medium and high concentrations of the vildagliptin quality control sample are 2.229ng/ml, 26.75ng/ml and 428.0ng/ml respectively.

Further, in the step (2), a stable isotope label is adopted¹³C₅-¹⁵N-vildagliptin is used as an internal standard. The preparation process comprises the following steps: precise weighing isotope¹³C₅-¹⁵Placing 3.15mg of N-vildagliptin reference substance in a 10ml volumetric flask, respectively washing the tube wall and the weighed paper residue in the volumetric flask with 4ml and 3ml of methanol solution, uniformly mixing and dissolving, adding methanol to a constant volume, reversely mixing and preparing into the product with the concentration of 0.315mg/ml¹³C₅-¹⁵And (3) N-vildagliptin standard stock solution C. Using methanol: working solution of internal standard is prepared by diluting with water solution (50: 50, V/V), and working solution (3.15ng/ml) is prepared by diluting with acetonitrile.

Further, the assay in step (3) determines the concentration of vildagliptin in the plasma sample by gradient elution reverse phase chromatography with mobile phase A of 5mM ammonium formate in water, mobile phase B of acetonitrile, chromatographic column of Hyperpurity C18 column (150 mM. times.2.1 mM, 5 μm, Thermo Hypersil) at a flow rate of 0.5ml/min and column temperature of 40 ℃.

Still further, the gradient elution procedure was: 0.0-0.5min, 70% of mobile phase B, 0.5-2.0min, gradually increasing the content of the mobile phase B from 70% to 90%, 2.0-3.5min, 90% of the mobile phase B, 3.5-4.0min, gradually decreasing the content of the mobile phase B from 90% to 70%, 4.0-6.0min, and 70% of the mobile phase B.

Further, the mass spectrum conditions in step (3) are as follows: by adopting an electrospray ionization source (ESI) and a positive ion multi-reaction monitoring mode (MRM), the mass-to-charge ratios (m/z) of parent ions/daughter ions of vildagliptin and an internal standard thereof are respectively as follows: 304.3/154.2 and 310.3/160.3, declustering voltage 50V and 80V, collision energy 23eV and 24eV, source firing voltage: 5500V, source temperature: at 500 ℃.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention uses stable isotopic labels¹³C₅-¹⁵The N-vildagliptin is used as an internal standard, so that the influence of the matrix effect of the plasma sample can be effectively corrected, and the accuracy of the detection result is ensured.

2. The method adopts the MRM mode of monitoring the parent ions and the daughter ions for detection, effectively reduces the interference of complex components in the plasma sample by monitoring the characteristic parent ions and the characteristic daughter ions, and improves the specificity and the sensitivity of the method.

3. The invention has short detection time of a single sample, the whole detection time is 6min, and the higher performance liquid chromatography is obviously shortened. Meanwhile, an automatic sample introduction system is used, so that sample introduction automation and high-flux detection can be realized.

4. The invention has high sensitivity, and the lowest limit of quantitation of 2 mul of autoinjection is 1.113ng/ml by using 100 mul of plasma.

5. The invention has high precision and accuracy, the accuracy of quality control samples with different concentrations is between-0.23% and 8.18%, and the precision is between 1.62% and 8.46%.

6. The invention has high sample treatment and extraction recovery rate, the total recovery rate is 86.35 percent, and the coefficient of variation of the recovery rates among different concentrations is 2.85 percent.

7. The pretreatment of the sample is a protein precipitation method, the process is simple, the sample dosage is small, only 100 mu L of blood plasma is needed, and the used reagent is also a conventional chemical reagent. Therefore, the invention has low analysis cost and convenient operation.

Drawings

FIG. 1 is a representative standard graph;

FIG. 2 is a blank plasma chromatogram in which A is vildagliptin and B is an internal standard;

FIG. 3 is a chromatogram of a reference substance added to blank plasma, wherein A is vildagliptin and B is an internal standard;

fig. 4 is a chromatogram of a vildagliptin reference substance added into blank plasma, wherein a is vildagliptin, and B is an internal standard;

FIG. 5 is a chromatogram of a reference substance obtained by adding an internal standard and vildagliptin to blank plasma, wherein A is vildagliptin and B is the internal standard;

FIG. 6 is a chromatogram of a human plasma sample to be tested, wherein A is vildagliptin and B is an internal standard.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and examples.

1. Sample preparation:

And (3) taking 95 ul of human plasma as a blank matrix, adding 5 ul of vildagliptin standard solution, and preparing 8 known vildagliptin standard samples with different concentrations, wherein the concentrations are 1.113ng/ml, 2.225ng/ml, 4.45ng/ml, 8.90ng/ml, 26.70ng/ml, 59.33ng/ml, 178.0ng/ml and 534.0ng/ml respectively.

And taking 95 mu l of human plasma as a blank matrix, adding 5 mu l of vildagliptin standard solution, and preparing three vildagliptin quality control samples with known concentrations, namely 2.229ng/ml, 26.75ng/ml and 428.0 ng/ml.

c. Preparing an internal standard solution: precise weighing isotope¹³C₅-¹⁵Placing 3.15mg of N-vildagliptin reference substance in a 10ml volumetric flask, respectively washing the tube wall and the weighed paper residue in the volumetric flask with 4ml and 3ml of methanol solution, uniformly mixing and dissolving, adding methanol to a constant volume, reversely mixing and preparing into the product with the concentration of 0.315mg/ml¹³C₅-¹⁵And (3) N-vildagliptin standard stock solution C. Using methanol: working solution of internal standard is prepared by diluting with water solution (50: 50, V/V), and working solution (3.15ng/ml) is prepared by diluting with acetonitrile.

2. Sample treatment:

the human plasma sample, the standard sample and the quality control sample to be detected all adopt the following processing methods:

3. Sample detection: taking 2 mu l of the sample obtained in the step (2) for automatic sample introduction, and carrying out high performance liquid chromatography tandem mass spectrometry detection;

a. liquid phase conditions:

the instrument comprises the following steps: nexera UHPLC/HPLC high performance liquid chromatograph, Shimadzu Japan;

mobile phase A: 5mM ammonium formate;

mobile phase B: acetonitrile;

elution mode: a gradient; as shown in table 1.

TABLE 1

Time min	0.0	0.5	2.0	3.5	4.0	6.0
							B％	70％	70％	90％	90％	70％	70％

Flow rate: 500 mu L/min;

sample introduction volume: 2.0 μ l;

sample injector temperature: 15 ℃;

a chromatographic column: hypurity C18(150 mm. times.2.1 mm, 5 μm, Thermo Hypersil);

column oven: 40 ℃;

b. mass spectrum conditions:

the instrument comprises the following steps: triple Quad ^TM6500 Mass spectrometer, AB SCIEX, USA;

an ion source: electrospray ionization source (Turbo Spray);

the detection mode is as follows: a positive ion;

the scanning mode is as follows: MRM;

source ejection voltage: 5500V;

inlet voltage: 10V;

temperature: 500 ℃;

collision gas: 9 Psi;

air curtain air: 35 Psi;

ion source gas 1: 55 Psi;

ion source gas 2: 55 Psi;

mass to charge ratio (m/z) and MRM parameters: as shown in table 2.

TABLE 2

4. Standard curve preparation and quantitative analysis: taking the ratio (Y) of the areas of the vildagliptin and the internal standard peak as an ordinate, taking the concentration (X) of the vildagliptin in the plasma sample as an abscissa, and adopting a weighted least square method (weight factor l/X)²) And performing linear regression to prepare a standard curve equation of the vildagliptin, and quantifying the quality control sample and the human plasma sample to be detected through the standard curve.

Methodology validation

(1) Selectivity is

In order to demonstrate the selectivity of the method of the invention, the following experimental protocol is set: (a) and (3) respectively taking blank plasma from different individual sources and processing according to the step (2). (b) And (3) adding blank plasma from different individual sources into the internal standard working solution, and processing according to the step (2). (c) And (3) respectively taking blank plasma from different individual sources, adding vildagliptin solution, and processing according to the step (2). (d) And (3) respectively taking blank plasma from different individual sources, adding vildagliptin and an internal standard solution, and processing according to the step (2). (e) Plasma samples were taken 1 hour after the subjects had taken the drug and processed according to step (2).

According to the measuring method of the step (3), the experimental sample is measured, and as shown in fig. 2-fig. 6, in the chromatogram of the blank plasma, no peak is seen at the retention time of the analyte and the internal standard (fig. 2), in the chromatogram of the internal standard control added to the blank normal plasma, 2.29min at the retention time of the internal standard, there is a dedicated peak (fig. 3), in the chromatogram of the vildagliptin control added to the blank normal plasma, 2.26min at the retention time of vildagliptin, there is a dedicated peak (fig. 4), in the chromatogram of the internal standard and the vildagliptin control added to the blank normal plasma, there is a dedicated peak at the retention time of the internal standard and the vildagliptin (fig. 5), in the chromatogram of the internal standard control added to the subject plasma, there is a dedicated peak at the retention time of the internal standard and the vildagliptin (fig. 6), the result shows that the blank plasma matrix does not contain endogenous substances interfering with vildagliptin and the internal standard, and the method has good selectivity.

(2) Linearity and sensitivity

Under the measuring conditions of the step (3), 3 calibration curves are examined within two consecutive days, and the prepared standard curve shows that the vildagliptin concentration is between 1.113ng/ml and 534.0ng/ml, and shows a good linear relation, R²Is 0.9946. The standard curve parameters and the coefficient of variation are shown in Table 3. A representative diagram is shown in fig. 1.

The lower limit of quantitation (LLOQ) of this method was 1.113 ng/ml. The differences between the measured and theoretical values of the lower limit of quantitation and the precision data are shown in Table 4. The quantitative limit signal-to-noise ratio is more than 5.

TABLE 3 summary of Standard Curve parameters

Batch number	Slope of	Intercept of a beam	R²	Fitting equation
					1	0.0383	-0.00771	0.9970	Y＝0.0383+-0.00771
2	0.0354	-8.78E-05	0.9940	Y＝0.0354+-0.00008
					3	0.0355	-0.00135	0.9928	Y＝0.0355+-0.00135
Mean value of	0.0364	-0.00305	0.9946
					SD	0.0016	0.0041	0.0022
％CV	4.52	-133.98	0.22

TABLE 4 difference between measured and theoretical values of the lower limit of quantitation and precision

(3) Precision and accuracy

In order to prove the precision and the accuracy of the method, the invention sets the following experimental scheme: preparing quality control plasma samples with four concentration levels of LLOQ, low, medium and high, preparing and measuring 6 samples for each concentration level, processing according to the method in the step (2), calculating the concentration of each sample by using the random calibration standard curve under the measuring condition in the step (3), and performing 3 batch verification at least for two days according to the method. The precision and accuracy between and within the batches were then calculated. The precision within the batch is indicated by the relative standard deviation (RSD%) of the measurements of 6 samples at each concentration level. The accuracy is calculated according to the following formula: accuracy (%) — Cm/Cn x 100%, where Cm is the average of the measured values of 6 samples at each concentration point and Cn is the theoretical value at each concentration point. The batch-to-batch precision is represented by the relative standard deviation (RSD%) of the measurements of 18 samples of 3 batches at each concentration level. The accuracy is calculated according to the above formula, where Cm is the average of the 18 sample measurements at each concentration point. The results of the precision and accuracy measurements are shown in Table 5.

TABLE 5 Interbatch, Intrabatch precision and accuracy

	LLOQ	LQC ng/ml	MQC	HQC ng/ml
					Mean value in batch	1.044	2.375	28.856	462.685
Internal% CV of batch	3.59	9.83	1.04	2.32
					Accuracy in batch%	93.77	106.56	107.87	108.10
Mean value between batches	1.110	2.319	28.530	462.992
					Inter-batch% CV	8.46	7.48	1.62	2.10
Inter-batch accuracy%	99.77	104.02	106.65	108.18

(4) Recovery rate

In order to examine the recovery rate of the method, the following experimental scheme is set in the invention: quality control plasma samples of low, medium and high concentration levels were prepared, and 6 aliquots were prepared for each concentration in parallel. The peak areas of vildagliptin (a1) and internal standard (a2) in the plasma sample were obtained under the measurement conditions of step (3) by performing the treatment according to the method of step (2).

Taking 100 mu l of blank plasma, adding no vildagliptin and internal standard working solution, adding 1.0ml acetonitrile containing 3.15ng/ml internal standard, automatically shaking for 5min on a shaker, centrifuging (15000r/min) for 10min, sucking 1.0ml supernatant into another clean 2ml EP tube, then respectively adding 5 mul of vildagliptin working solution with low, medium and high concentration levels, automatically shaking for 2min on a shaker, centrifuging at 4 ℃ (15000r/min) for 1min, sucking 50 mul of supernatant into another clean 2ml EP tube, adding 300 μ l of 5mM ammonium formate aqueous solution and acetonitrile mixed solution (1: 1), shaking automatically for 2min on a shaker, centrifuging (15000r/min) for 5min, transferring the supernatant to a sampling bottle, under the measurement conditions of the above step (3), peak areas of vildagliptin (a1 ') and internal standard (a 2') were obtained, respectively.

The extraction recovery rates of vildagliptin and the internal standard were evaluated by comparing the average peak area of the former treatment with the average peak area of the latter treatment at each concentration, and the calculation formulas were a1/a1 'x 100% and a2/a 2' x 100%, respectively. The results of recovery measurements are shown in Table 6.

Table 6 vildagliptin extraction recovery

(5) Stability of

In order to examine the stability of the method, the following experimental scheme is set: preparing quality control plasma samples with low and high concentration levels, wherein 6 parts of each concentration are parallel, respectively placing at room temperature (23.2 ℃) for 1 hour, repeatedly freezing and thawing for 3 times, after freezing and storing for 90 days at minus 80 ℃ in a low-temperature refrigerator, measuring according to the measuring conditions in the step (3) after processing in the step (2) in the detection method, calculating the concentration of each sample by using a random calibration standard curve, and when the RSD of the obtained result is less than 15%, considering that the sample is stable. The results of the stability measurements are shown in Table 7.

TABLE 7 stability test results

The embodiment of the invention detects vildagliptin in blank plasma, and proves that the method has higher sensitivity, accuracy and precision and higher recovery rate.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Claims

1. An LC-MS/MS detection method for quantitatively analyzing human plasma vildagliptin concentration is characterized by comprising the following steps:

(1) sample preparation:

a. preparation of a standard sample: accurately weighing 10.68mg of vildagliptin reference substance into a 10ml volumetric flask, respectively washing the tube wall and the weighed paper residue into the volumetric flask by using 4ml and 3ml of methanol solution, uniformly mixing and dissolving, adding methanol to a constant volume, and reversely and uniformly mixing to prepare vildagliptin standard stock solution A with the concentration of 1.068 mg/ml; using methanol: aqueous solution 50: 50, diluting and preparing a standard curve working solution of vildagliptin at a V/V ratio, wherein the concentration is respectively as follows: 22.25ng/ml, 44.5ng/ml, 89.0ng/ml, 178.0ng/ml, 534.0ng/ml, 1186.67ng/ml, 3560.0ng/ml and 10680.0 ng/ml;

taking human blank plasma as a blank matrix, adding vildagliptin standard working solution, and preparing 8 known vildagliptin standard samples with different concentrations;

b. preparing a quality control sample: accurately weighing 10.70mg of vildagliptin reference substance into a 10ml volumetric flask, respectively washing the tube wall and the weighed paper residue into the volumetric flask by using 4ml and 3ml of methanol solutions, uniformly mixing and dissolving, adding methanol to a constant volume, and reversely and uniformly mixing to prepare vildagliptin standard stock solution B with the concentration of 1.070 mg/ml; using methanol: aqueous solution 50: 50, diluting and preparing a quality control working solution of vildagliptin at a ratio of V/V, wherein the concentration is respectively as follows: 44.583ng/ml, 535.0ng/ml, 8560.0 ng/ml;

taking a plasma sample 100 mu l to a 2ml EP tube, adding 1.0ml of precipitating agent acetonitrile containing 3.15ng/ml internal standard, automatically shaking for 5min on a shaker, centrifuging for 10min at 15000r/min, sucking 50 mu l of supernatant into another clean 2ml EP tube, and adding 300 mu l of a mixture of 5mM ammonium formate aqueous solution and acetonitrile, wherein the volume ratio of the ammonium formate aqueous solution to the acetonitrile mixture is 1: 1, automatically shaking for 2min on a shaker, centrifuging for 5min at 15000r/min, and transferring the supernatant to a sample injection bottle;

(4) standard curve preparation and quantitative analysis: after the step (3) is finished, taking the ratio (Y) of the areas of the vildagliptin and the internal standard peak as a vertical coordinate, taking the concentration X of the vildagliptin in the plasma sample as a horizontal coordinate, and adopting a weighted least square method to obtain a weight factor l/X²And performing linear regression to prepare a standard curve equation of the vildagliptin, and quantifying the quality control sample and the human plasma sample to be detected through the standard curve.

2. The LC-MS/MS detection method for quantitatively analyzing human plasma vildagliptin concentration according to claim 1, wherein in the step (1), the series of vildagliptin standard samples are 1.113ng/ml, 2.225ng/ml, 4.45ng/ml, 8.90ng/ml, 26.70ng/ml, 59.33ng/ml, 178.0ng/ml and 534.0 ng/ml.

3. The LC-MS/MS detection method for quantitatively analyzing the concentration of human plasma vildagliptin according to claim 2, wherein in the step (1), the low, medium and high concentrations of the vildagliptin quality control sample are 2.229ng/ml, 26.75ng/ml and 428.0ng/ml respectively.

4. The LC-MS/MS detection method for quantitatively analyzing human plasma vildagliptin concentration according to claim 1, wherein in the step (2), a stable isotope label is adopted¹³C₅-¹⁵Taking N-vildagliptin as an internal standard; the preparation process comprises the following steps: precise weighing isotope¹³C₅-¹⁵Placing 3.15mg of N-vildagliptin reference substance in a 10ml volumetric flask, respectively washing the tube wall and the weighed paper residue in the volumetric flask with 4ml and 3ml of methanol solution, uniformly mixing and dissolving, adding methanol to a constant volume, reversely mixing and preparing into the product with the concentration of 0.315mg/ml¹³C₅-¹⁵N-vildagliptin standard stock solution C; using methanol: aqueous solution 50: and diluting by 50V/V to prepare an internal standard working solution, and diluting the finally used working solution by using acetonitrile to prepare the internal standard working solution.

5. The LC-MS/MS detection method for quantitatively analyzing human plasma vildagliptin concentration according to claim 1, characterized in that, in the step (3), the analysis is used for determining the vildagliptin concentration in the plasma sample, and gradient elution reverse phase chromatography is adopted, wherein the mobile phase A is 5mM ammonium formate aqueous solution, the mobile phase B is acetonitrile, the chromatographic column is a Hyperity C18 column 150mM x 2.1mM, 5 μm, ThermoHypersil, the flow rate of the mobile phase is 0.5ml/min, and the column temperature is 40 ℃.

6. The LC-MS/MS detection method for quantitatively analyzing the concentration of human plasma vildagliptin according to claim 5, is characterized in that the gradient elution procedure is as follows: 0.0-0.5min, 70% of mobile phase B, 0.5-2.0min, gradually increasing the content of the mobile phase B from 70% to 90%, 2.0-3.5min, 90% of the mobile phase B, 3.5-4.0min, gradually decreasing the content of the mobile phase B from 90% to 70%, 4.0-6.0min, and 70% of the mobile phase B.

7. The LC-MS/MS detection method for quantitatively analyzing the concentration of human plasma vildagliptin according to claim 1, wherein the mass spectrum conditions in the step (3) are as follows: adopting an electrospray ionization source ESI and a positive ion multi-reaction monitoring mode MRM, wherein the mass-to-charge ratios m/z of parent ions/daughter ions of vildagliptin and internal standards are respectively as follows: 304.3/154.2 and 310.3/160.3, declustering voltage 50V and 80V, collision energy 23eV and 24eV, source firing voltage: 5500V, source temperature: at 500 ℃.