CN110927305A - Method for determining glipizide concentration in plasma by liquid chromatography-mass spectrometry - Google Patents
Method for determining glipizide concentration in plasma by liquid chromatography-mass spectrometry Download PDFInfo
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Abstract
The invention discloses a method for determining glipizide concentration in plasma by liquid chromatography-mass spectrometry, which comprises the steps of firstly taking a sample to be determined, adding a certain amount of mixed organic solvent for extraction, pretreating, separating by a chromatographic column, and detecting by a mass spectrometer. The method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for determining the blood concentration of glipizide; the plasma standard curve linear range of the method is 1-500 ng/mL, the precision RSD in batch and between batches is less than +/-15%, and the method is suitable for measuring the concentration of glipizide in plasma.
Description
Technical Field
The invention belongs to the technical field of medicines, particularly relates to a method for determining a medicine, and particularly relates to a method for determining the concentration of glipizide in plasma by liquid chromatography-mass spectrometry.
Background
Due to the improvement of living standard and the aggravation of aging, the number of diabetics presents an increasing trend year by year; according to incomplete statistics, the incidence of diabetes in all countries around the world is on the rise, and the number of type II diabetes patients is more than 90%. In recent years, the rate of rise of diabetes in developed countries has been 45% and up to 200% in developing countries, and more data has shown that diabetes is very common and prevalent in some developing countries in china, india, africa at the present stage. In recent years, the increasing trend of diabetic patients in China is rising all the time, statistics in 2003 show that the number of patients with impaired glucose tolerance in China is about 3320 ten thousand, and by 2025, the number of patients with impaired glucose tolerance in China may be increased to 3320 ten thousand, and among them, the number of patients with diagnosed diabetes is about 4610 ten thousand.
Glipizide is a lipophilic weak acid, the second generation of sulfonylureas oral hypoglycemic agents, and has lipid-lowering and anticoagulant effects, the chemical name is 5-methyl-N- [2- [4- [ [ [ (cyclohexylamino) carbonyl ] amino ] sulfonyl ] phenyl ] ethyl ] -pyrazine carboxamide, the in vivo action mechanism is mainly to promote insulin secretion of islet β cells, especially to promote insulin β cell secretion stimulated by glucose, and also to enhance insulin action, thereby effectively reducing blood glucose concentration and glycosylated hemoglobin, improving hyperlipidemia, reducing triglyceride and cholesterol levels, increasing the ratio of high-density lipoprotein cholesterol in total cholesterol, and also inhibiting platelet aggregation and promoting fibrinolysis, thus having certain prevention and treatment effects on vascular lesions.
At present, the speed, the precision and the sensitivity of the existing glipizide measuring method are all required to be improved.
Disclosure of Invention
The invention aims to provide a method for determining the concentration of glipizide in plasma by liquid chromatography-mass spectrometry, which can improve the speed, the precision and the sensitivity of detection.
In order to achieve the aim, the method for measuring the concentration of glipizide in plasma comprises the following steps of:
(1) pretreatment of a plasma sample:
plasma with K2EDTA as anticoagulant, glipizide-d 11 as internal standard; precisely adding 100 μ L of plasma sample into a 96-deep-well plate, adding 5 μ L of plasma sample at a volume ratio of 1: 1 methanol aqueous solution, mixing well, adding 5 μ L of 1ng/μ L internal standardMixing glipizide-d 11 solution, adding 1000 μ L acetonitrile into 96 deep-well plate, vortex mixing for 1min, centrifuging at 20 deg.C and 3000rpm for 10min, collecting supernatant 150 μ L into 96 deep-well plate filled with 350 μ L mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid is mixed according to a volume ratio of 25: 75: 0.2, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ and 3000rpm for 5min, and taking the mixture as a test sample to be detected;
(2) and (3) sample measurement:
injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of glipizide and internal standard glipizide-d 11 in the sample, and calculating the glipizide concentration in the plasma sample according to the chromatographic peaks;
(3) liquid chromatography determination, conditions are as follows:
a chromatographic column: agilent ZORBAX XDB-C18, 5 μm, column specification 50X 2.1 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume ratio of water to formic acid is 100/0.2; mobile phase B: the volume ratio of acetonitrile/formic acid is 100/0.2; washing liquid: the volume ratio of acetonitrile/water is 50/50; the autosampler temperature was 15 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 3.5 min;
(4) mass spectrometry under the conditions:
the ion source is an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 500 ℃, the spraying air pressure is 20Psi, the auxiliary heating air pressure is 20Psi, the air curtain air pressure is 30Psi, the collision air pressure is 10Psi, and the declustering voltage is glipizide and glipizide-d 11 of 30eV respectively; the voltage of the entrance of the collision chamber is 8eV of glipizide and glipizide-d 11; glipizide and glipizide-d 11 with collision voltages of 20eV respectively; the voltage of the outlet of the collision chamber is glipizide and glipizide-d 11 of 20eV respectively; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z446.2 → m/z321.2, which is glipizide; and m/z457.3 → m/z321.1, which is glipizide-d 11.
Preferably, the gradient elution in step (3) is performed by the following procedure:
preferably, in the step (2), an internal standard method is adopted, and the peak area ratio of glipizide and internal standard glipizide-d 11 is substituted into a standard curve equation to calculate the glipizide concentration in the plasma sample.
Preferably, the establishment of the standard curve equation comprises the following steps:
taking 10 parts of 100 mu L blank plasma, placing the blank plasma in a 96-deep-well plate, adding 5 mu L of glipizide solution with the concentration of 0.02 ng/mu L, 0.04 ng/mu L, 0.1 ng/mu L, 0.2 ng/mu L, 1 ng/mu L, 2 ng/mu L, 4 ng/mu L and 10 ng/mu L to the lowest quantitative lower limit sample, the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6 and the highest quantitative upper limit sample in the form of stock solution, respectively adding 5 mu L of the glipizide solution with the volume ratio of 1: 1 to a blank sample and a zero-concentration sample, respectively adding 5 mu L of 1 ng/mu L of internal standard glipizide-d 11 solution into the lowest quantitative lower limit sample, the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6, the highest quantitative upper limit sample and the zero-concentration sample after uniformly mixing, and adding 5 mu L of internal standard glipizide-d 11 solution into the blank sample according to the volume ratio of 1: 1, adding 1000 mu L of acetonitrile into 10 samples respectively after uniformly mixing, mixing for 1min in a vortex mode, centrifuging for 10min at 20 ℃ at 3000rpm, taking 150 mu L of supernatant liquid into a 96 deep-well plate filled with 350 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid is mixed according to a volume ratio of 25: 75: 0.2, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ at 3000rpm for 5min, and taking the mixture as 10 parts of standard sample to be detected;
and respectively injecting 10 mu L of standard sample into a high performance liquid chromatography-tandem mass spectrometer, detecting the chromatographic peaks of glipizide and internal standard glipizide-d 11 in the sample, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of glipizide in the plasma.
Compared with the prior art, the invention has the following advantages:
(1) the pretreatment method is simple and convenient, two-step organic solution extraction is adopted, and the method is suitable for conventional determination;
(2) the specificity is strong: under the chromatographic conditions adopted in the experiment, the retention time of glipizide is about 1.132min, and the retention time of internal standard glipizide-d 11 is about 1.106 min. The glipizide and the internal standard glipizide-d 11 have good peak shapes, no interference of miscellaneous peaks for measurement and stable base line;
(3) the sensitivity is high: the minimum limit of plasma quantification is 1ng/mL, the concentration of glipizide in plasma can be accurately determined, the sensitivity is high, and the specificity is strong;
(4) the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for determining the blood concentration of glipizide. The linear range of the plasma standard curve of the method is 1-500 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.
Drawings
FIG. 1 is a standard graph of glipizide in human plasma measured by HPLC-MS/MS;
FIG. 2 is a HPLC-MS/MS graph of human blank plasma;
FIG. 3 is a HPLC-MS/MS graph of human blank plasma added with glipizide and glipizide-d 11;
FIG. 4 is a HPLC-MS/MS graph of plasma samples after oral administration of glipizide or a pharmaceutically acceptable salt thereof to healthy subjects followed by the addition of internal standard glipizide-d 11.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example (b): human K2Determination of glipizide concentration in EDTA plasma
First, experimental material and analytical equipment
Glipizide (analyte): TLC Pharmaceutical Standards or equivalent, higher-grade Standards
Glipizide-d 11 (internal standard): TLC Pharmaceutical Standards or equivalent, higher-grade Standards
The reagents used are shown in table 1 below:
TABLE 1 details of reagents
| Name of reagent | Rank of | Manufacturer(s) |
| Acetonitrile | HPLC | J.T.Baker |
| Methanol | HPLC | J.T.Baker |
| Formic acid | ACS | Adamas |
Note: the same or higher level of reagents may also be used
The analytical equipment used is shown in table 2 below:
TABLE 2 details of the devices used
The same LC/MS system may also be used.
Second, liquid condition
1. Conditions of liquid chromatography
A chromatographic column: agilent ZORBAX XDB-C18, 5 μm, column specification 50X 2.1 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume ratio of water to formic acid is 100/0.2; mobile phase B: the volume ratio of acetonitrile/formic acid is 100/0.2; washing liquid: the volume ratio of acetonitrile/water is 50/50; the autosampler temperature was 15 ℃; gradient elution, flow rate of 0.4mL/min, sample size of 10 μ L, analysis time of 3.5 min.
TABLE 3 gradient elution procedure
| Step (ii) of | Total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 1 | 0 | 60 | 40 |
| 2 | 0.8 | 60 | 40 |
| 3 | 0.9 | 30 | 70 |
| 4 | 1.5 | 30 | 70 |
| 5 | 1.6 | 60 | 40 |
| 6 | 3.5 | 60 | 40 |
2. Conditions of Mass Spectrometry
The ion source is an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 500 ℃, the spraying air pressure is 20Psi, the auxiliary heating air pressure is 20Psi, the air curtain air pressure is 30Psi, the collision air pressure is 10Psi, and the declustering voltage is glipizide and glipizide-d 11 of 30eV respectively; the voltage of the entrance of the collision chamber is 8eV of glipizide and glipizide-d 11; glipizide and glipizide-d 11 with collision voltages of 20eV respectively; the voltage of the outlet of the collision chamber is glipizide and glipizide-d 11 of 20eV respectively; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z446.2 → m/z321.2, which is glipizide; and m/z457.3 → m/z321.1, which is glipizide-d 11.
Third, the experimental process
1. Preparation of glipizide standard solution
Preparing a glipizide standard solution: accurately weighing 0.001g of glipizide (analyte), placing the glipizide in a 10mL volumetric flask, adding a methanol solution to dissolve and fix the volume to a scale, shaking up to obtain 100 ng/mu L of glipizide stock solution, and then mixing the glipizide stock solution with the volume ratio of 1: 1, sequentially diluting the methanol aqueous solution to prepare a glipizide standard solution, wherein the specific dilution concentration is shown in the following table 4:
TABLE 4 glipizide Standard solution preparation concentration
| Source solution (ng/. mu.L) | Volume of source solution (μ L) | Final volume (mL) | Final concentration (ng/. mu.L) |
| 100a | 1000 | 10 | 10 |
| 100a | 800 | 10 | 8 |
| 100a | 500 | 10 | 5 |
| 100a | 400 | 10 | 4 |
| 100a | 200 | 10 | 2 |
| 100a | 100 | 10 | 1 |
| 10 | 200 | 10 | 0.2 |
| 10 | 100 | 10 | 0.1 |
| 10 | 60 | 10 | 0.06 |
| 10 | 40 | 10 | 0.04 |
| 10 | 20 | 10 | 0.02 |
a: directly from glipizide (analyte)
The glipizide standard solution is stored in a plastic container and a refrigerator (4 ℃) for storage when not used, and the volume can be increased or reduced according to the proportion as required.
2. Preparation of glipizide-d 11 internal standard solution
Preparing a glipizide-d 11 internal standard solution: accurately weighing 0.001g of glipizide-d 11 (internal standard), placing the glipizide-d 11 (internal standard) into a 10mL volumetric flask, adding a methanol solution for dissolving, fixing the volume to a scale, shaking up to obtain 40 ng/mu L of glipizide-d 11 stock solution, and then mixing the stock solution with the volume ratio of 1: 1, diluting the methanol aqueous solution to prepare 1 ng/mu L glipizide-d 11 internal standard solution, wherein the specific dilution concentration is shown in the following table 5:
TABLE 5 glipizide d11 Standard solution preparation concentration
| Source solution (ng/. mu.L) | Volume of source solution (μ L) | Final volume (mL) | Final concentration (ng/. mu.L) |
| 100a | 1000 | 100 | 1b |
a: directly prepared from glipizide-d 11 (internal standard)
b: for sample preparation procedures
The standard solution of glipizide-d 11 internal standard is stored in plastic container and refrigerator (4 ℃) when not in use, and the volume can be increased or decreased proportionally according to the requirement.
3. Linear test
Putting the blank plasma into a water bath at room temperature for unfreezing; transferring 10 parts of 100 mu L of blank plasma into a 96-deep-well plate (each standard curve sample, blank sample-00 and zero concentration sample-0), respectively and precisely adding 5 mu L of glipizide standard solution or diluted solution with different concentrations according to the list in the following table 6 to prepare each sample, uniformly mixing the samples to prepare the drug-containing plasma with different concentrations, and carrying out the operation according to 'plasma sample pretreatment'. Calculating the ratio Y (Y is As/Ai) of the area As of the glipizide peak and the area Ai of the internal standard glipizide-d 11 peak, and performing regression calculation on the blood concentration X by using the area ratio Y, wherein the result is shown in a figure 1 and a table 7. And performing regression calculation on the blood concentration X by using the average ratio Y to obtain a regression equation Y of 0.0188X +0.00119 and r of 0.9994. The weight coefficient W is 1/X, and the lowest quantitative limit of the glipizide blood concentration measured by the method is as follows: 1 ng/mL.
TABLE 6 glipizide Standard Curve formulation concentration
b: diluted solution of analyte: MeOH/H2O＝50/50
TABLE 7 Standard Curve of glipizide in human plasma by HPLC-MS/MS method (n ═ 10)
4. Accuracy and precision
Putting the blank plasma into a water bath at room temperature for unfreezing; the appropriate volume of blank plasma was transferred to appropriate containers and glipizide standard solution was added to prepare 5 drug-containing plasma quality control samples (LLOQ, QL, QLM, QM, QH) of different concentrations and a running standard curve, following the "pretreatment of plasma samples" procedure, the quality control sample preparation was as shown in table 8 below. Making one batch and one following standard curve every day, continuously making 3 days, making 6 samples for each concentration of the first batch and the second batch, making 15 samples for each concentration of the third batch, calculating the ratio Y of the peak area As of glipizide and the peak area Ai of the peak area of internal standard glipizide-d 11, substituting the ratio Y into the standard curve on the day to obtain the actually measured concentration, calculating the precision between batches and the actually measured concentration, wherein the ratio of the actually measured concentration to the added concentration is the accuracy, and the result is shown in a table 9. The result shows that the precision and accuracy of the glipizide plasma sample in and among batches are less than +/-15 percent and meet the requirements.
TABLE 8 quality control sample preparation concentration
a: final volume is source solution volume + plasma volume
Sufficient volume was dispensed into the labeled sample vials as required for each assay batch and stored at the theoretical temperature-80 ℃. The volume may be scaled up or down as desired.
TABLE 9 Intra-batch, inter-batch precision and accuracy of glipizide assay in plasma by HPLC-MS/MS method
a: out of standard but including calculations; b: the samples were re-injected while in the balance system, thus eliminating the calculation.
5. Interference
Nine different blank plasma samples are respectively from different healthy human bodies, and the nine different blank plasma samples are prepared and analyzed in the same analysis batch according to the sample preparation steps to evaluate the interference of the different blank plasma on the glipizide analyte and the internal standard glipizide-d 11.
After the preparation and analysis of the blank healthy human plasma samples from nine different sources, the interference peak responses at the positions meeting the glipizide retention time are all lower than 20.0% of the glipizide response of the quantitative lower limit sample in the standard curve of the analysis batch, and the results are shown in table 10. The result shows that the analysis method has specificity on analysis of glipizide.
After the analysis of the nine blank healthy human plasma samples from different sources, the interference peak responses at the retention times corresponding to the internal standards were all less than 5.0% of the internal standard response of the quantitative lower limit sample in the standard curve of the analysis lot, see table 11 in the appendix. The results show that the assay is selective for the analysis of internal standards.
TABLE 10 comparison of interference data of blank healthy human plasma from nine different sources on glipizide analytes
a: analyte peak area (selective sample)/analyte peak area (LLOQ of standard curve)
×100.0％≤20.0％
b: the area peak area is considered zero when "no significant peak can be integrated (or no peak)" or "the retention time of the peak area does not match the retention time of the analyte in the sample".
TABLE 11 comparison of interference data of blank healthy human plasma from nine different sources on internal standard glipizide-d 11
a: analyte peak area (selective sample)/internal standard peak area (LLOQ of standard curve) x 100.0% ≦ 5.0% b: the area peak area is considered zero when "no significant peak can be integrated (or no peak)" or "the retention time of the peak area does not match the retention time of the analyte in the sample".
As can be seen from tables 10 and 11, the blank plasma of different human bodies did not interfere with the detection result of glipizide. Therefore, the method can be used for detecting the glipizide concentration in different human plasmas.
6. Human plasma sample detection
(1) Taking human blank plasma without glipizide, precisely adding 100 mu L of blank plasma samples into a 96-deep-well plate, and adding 10 mu L of blank plasma samples in a volume ratio of 1: 1, adding 1000 mu L of acetonitrile into a 96 deep-hole plate after uniformly mixing, carrying out vortex mixing for 1min, centrifuging for 10min at 20 ℃ at 3000rpm, taking 150 mu L of supernatant liquid into the 96 deep-hole plate filled with 350 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid is mixed according to a volume ratio of 25: 75: 0.2 the resulting mixture was mixed, vortexed, centrifuged at 3000rpm for 5min at 20 ℃ and 10. mu.L of the sample was analyzed by LC-MS/MS, the results of which are shown in FIG. 2.
(2) Taking human blank plasma without glipizide, precisely adding 100 mu L of blank plasma sample into a 96 deep-well plate, adding 5 mu L of glipizide standard solution with the concentration of 1 ng/mu L, uniformly mixing, adding 5 mu L of 1 ng/mu L of internal standard glipizide-d 11 solution, uniformly mixing, adding 1000 mu L of acetonitrile into the 96 deep-well plate, carrying out vortex mixing for 1min, centrifuging at 20 ℃ at 3000rpm for 10min, taking 150 mu L of supernatant into the 96 deep-well plate filled with 350 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid is mixed according to a volume ratio of 25: 75: 0.2 the resulting mixture was mixed, vortexed, centrifuged at 3000rpm for 5min at 20 ℃ and 10. mu.L of the sample was analyzed by LC-MS/MS, the results of which are shown in FIG. 3.
(3) Collecting plasma of healthy subjects after oral administration of glipizide or pharmaceutically acceptable salts thereof, precisely adding 100 mu L of collected human plasma samples into a 96-deep-well plate, and adding 5 mu L of plasma samples with a volume ratio of 1: 1, uniformly mixing the methanol aqueous solution, adding 5 mu L of 1 ng/mu L internal standard glipizide-d 11 solution, uniformly mixing, adding 1000 mu L acetonitrile into a 96 deep-well plate, carrying out vortex mixing for 1min, centrifuging at 20 ℃ at 3000rpm for 10min, taking 150 mu L supernatant liquid into the 96 deep-well plate filled with 350 mu L mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid is mixed according to a volume ratio of 25: 75: 0.2 the resulting mixture was mixed, vortexed, centrifuged at 3000rpm for 5min at 20 ℃ and 10. mu.L of the sample was analyzed by LC-MS/MS, the results of which are shown in FIG. 4.
In conclusion, the invention provides a simple and convenient method for determining the concentration of glipizide in plasma by a pretreatment method, adopts a two-step organic solution extraction method, and is suitable for conventional determination; meanwhile, under the chromatographic conditions adopted in the experiment, the retention time of glipizide is about 1.132min, the retention time of internal standard glipizide-d 11 is about 1.106min, the peak shapes of glipizide and internal standard glipizide-d 11 are good, the measurement is free from the interference of miscellaneous peaks, and the base line is stable; the method has high specificity, can accurately determine the concentration of glipizide in plasma, has high sensitivity, and has the minimum limit of plasma quantification of 1 ng/mL; meanwhile, the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for determining the blood concentration of glipizide. The linear range of the plasma standard curve of the method is 1-500 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.
Claims (4)
1. A method for determining glipizide concentration in plasma by liquid chromatography-mass spectrometry is characterized by comprising the following steps: the plasma sample is pretreated and then the concentration of the plasma sample is detected by high performance liquid chromatography-tandem mass spectrometry, and the specific method comprises the following steps:
(1) pretreatment of a plasma sample:
plasma with K2EDTA as anticoagulant, glipizide-d 11 as internal standard; precisely adding 100 μ L of plasma sample into a 96-deep-well plate, adding 5 μ L of plasma sample at a volume ratio of 1: 1, uniformly mixing the mixture with 5 mu L of 1 ng/mu L internal standard glipizide-d 11 solution, uniformly mixing the mixture with 1000 mu L of acetonitrile in a 96 deep-well plate, carrying out vortex mixing for 1min, centrifuging at 20 ℃ at 3000rpm for 10min, taking 150 mu L of supernatant to a 96 deep-well plate filled with 350 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid is mixed according to a volume ratio of 25: 75: 0.2, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ and 3000rpm for 5min, and taking the mixture as a test sample to be detected;
(2) and (3) sample measurement:
injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of glipizide and internal standard glipizide-d 11 in the sample, and calculating the glipizide concentration in the plasma sample according to the chromatographic peaks;
(3) liquid chromatography determination, conditions are as follows:
a chromatographic column: agilent ZORBAX XDB-C18, 5 μm, column specification 50X 2.1 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume ratio of water to formic acid is 100/0.2; mobile phase B: the volume ratio of acetonitrile/formic acid is 100/0.2; washing liquid: the volume ratio of acetonitrile/water is 50/50; the autosampler temperature was 15 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10 μ L, and analysis time of 3.5 min;
(4) mass spectrometry under the conditions:
the ion source is an electrospray ion source, the spraying voltage is 5500V, the atomizing temperature is 500 ℃, the spraying air pressure is 20Psi, the auxiliary heating air pressure is 20Psi, the air curtain air pressure is 30Psi, the collision air pressure is 10Psi, and the declustering voltage is glipizide and glipizide-d 11 of 30eV respectively; the voltage of the entrance of the collision chamber is 8eV of glipizide and glipizide-d 11; glipizide and glipizide-d 11 with collision voltages of 20eV respectively; the voltage of the outlet of the collision chamber is glipizide and glipizide-d 11 of 20eV respectively; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring;
the ion reactions for quantitative analysis were: m/z446.2 → m/z321.2, which is glipizide; and m/z457.3 → m/z321.1, which is glipizide-d 11.
2. The method for determining the concentration of glipizide in plasma by liquid chromatography-mass spectrometry according to claim 1, wherein: the gradient elution procedure in the step (3) is as follows:
3. the method for determining glipizide concentration in plasma by LC-MS according to claim 1 or 2, wherein: and (2) adopting an internal standard method, and substituting the peak area ratio of glipizide and the internal standard glipizide-d 11 into a standard curve equation to calculate the concentration of glipizide in the plasma sample.
4. The method for determining the concentration of glipizide in plasma by liquid chromatography-mass spectrometry according to claim 3, wherein: the establishment of the standard curve equation comprises the following steps:
taking 10 parts of 100 mu L blank plasma, placing the blank plasma in a 96-deep-well plate, adding 5 mu L of glipizide solution with the concentration of 0.02 ng/mu L, 0.04 ng/mu L, 0.1 ng/mu L, 0.2 ng/mu L, 1 ng/mu L, 2 ng/mu L, 4 ng/mu L and 10 ng/mu L to the lowest quantitative lower limit sample, the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6 and the highest quantitative upper limit sample in the form of stock solution, respectively adding 5 mu L of the glipizide solution with the volume ratio of 1: 1 to a blank sample and a zero-concentration sample, respectively adding 5 mu L of 1 ng/mu L of internal standard glipizide-d 11 solution into the lowest quantitative lower limit sample, the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6, the highest quantitative upper limit sample and the zero-concentration sample after uniformly mixing, and adding 5 mu L of internal standard glipizide-d 11 solution into the blank sample according to the volume ratio of 1: 1, adding 1000 mu L of acetonitrile into 10 samples respectively after uniformly mixing, mixing for 1min in a vortex mode, centrifuging for 10min at 20 ℃ at 3000rpm, taking 150 mu L of supernatant liquid into a 96 deep-well plate filled with 350 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: formic acid is mixed according to a volume ratio of 25: 75: 0.2, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ at 3000rpm for 5min, and taking the mixture as 10 parts of standard sample to be detected;
and respectively injecting 10 mu L of standard sample into a high performance liquid chromatography-tandem mass spectrometer, detecting the chromatographic peaks of glipizide and internal standard glipizide-d 11 in the sample, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of glipizide in the plasma.
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