Method for detecting omeprazole in human plasma by HPLC-MS/MS combination
阅读说明：本技术 一种hplc-ms/ms联用检测人血浆中奥美拉唑的方法 (Method for detecting omeprazole in human plasma by HPLC-MS/MS combination ) 是由 高倩倩 夏云燕 李艳静 于 2021-07-26 设计创作，主要内容包括：本发明涉及一种HPLC-MS/MS联用检测人血浆中奥美拉唑的方法,它包括以下步骤：(1)人血浆样品前处理；(2)液相色谱-质谱联用检测,采用流动相A和流动相B为混合流动相进行梯度洗脱,其中：流动相A为甲醇-乙腈混合溶液,在混合溶液中甲醇与乙腈的体积比为3～5:7～5；流动相B为2～20mM甲酸铵水溶液；(3)人血浆中奥美拉唑浓度的测定。本发明以氘代奥美拉唑作为内标物,采用Welch Ultimate XB-C18进行梯度洗脱,氘代内标和待测物具有相同的保留时间、化学性质和基质效应,测定血浆中奥美拉唑浓度的重现性、准确度均较好。本发明方法可用于评价奥美拉唑各剂型的生物等效性。(The invention relates to a method for detecting omeprazole in human plasma by HPLC-MS/MS combination, which comprises the following steps: (1) pretreating a human plasma sample; (2) performing liquid chromatography-mass spectrometry detection, and performing gradient elution by using a mobile phase A and a mobile phase B as a mixed mobile phase, wherein: the mobile phase A is a methanol-acetonitrile mixed solution, and the volume ratio of methanol to acetonitrile in the mixed solution is 3-5: 7-5; the mobile phase B is 2-20 mM ammonium formate aqueous solution; (3) determination of omeprazole concentration in human plasma. The method takes deuterated omeprazole as an internal standard substance, adopts Welch Ultimate XB-C18 to carry out gradient elution, the deuterated internal standard substance and the substance to be detected have the same retention time, chemical property and matrix effect, and the reproducibility and accuracy of the concentration of omeprazole in the plasma are better. The method of the present invention can be used to evaluate the bioequivalence of each dosage form of omeprazole.)
1. A method for detecting omeprazole in human plasma by HPLC-MS/MS combination is characterized by comprising the following steps: (1) pretreating a human plasma sample; (2) performing liquid chromatography-mass spectrometry detection, namely performing gradient elution by using a mobile phase A and a mobile phase B as a mixed mobile phase, wherein the mobile phase A is a methanol-acetonitrile mixed solution, and the volume ratio of methanol to acetonitrile in the mixed solution is 3-5: 7-5; the mobile phase B is 2-20 mM ammonium formate aqueous solution; (3) measuring the concentration of omeprazole in human plasma; the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is 45:55 within 0-0.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 45:55 to 95:5 at a constant speed within 0.5-2.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is 95:5 within 2.0-2.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is uniformly and gradually changed from 95:5 to 45:55 within 2.8-3.2 minutes; the volume ratio of mobile phase A to mobile phase B was 45:55 in 3.2-4.5 minutes.
2. The method for detecting omeprazole in human plasma by HPLC-MS/MS combination according to claim 1, wherein in the gradient elution process, the weak needle washing solution is a methanol-acetonitrile-water mixed solution, preferably, the volume ratio of methanol, acetonitrile and water in the weak needle washing solution is 1-3: 4-2: 5, more preferably, the volume ratio of methanol, acetonitrile and water in the weak needle washing solution is 2:3: 5; the strong needle washing liquid is a methanol-acetonitrile-isopropanol mixed solution, preferably, the volume ratio of methanol to acetonitrile to isopropanol in the strong needle washing liquid is 1-2: 2-3: 1, and more preferably, the volume ratio of methanol to acetonitrile to isopropanol in the strong needle washing liquid is 1:2: 1.
3. The method for detecting omeprazole in human plasma by HPLC-MS/MS combination according to claim 1, wherein the volume ratio of methanol to acetonitrile in the mobile phase A is 4: 6; the mobile phase B is 10mM ammonium formate aqueous solution; preferably, the mobile phase B contains 0.05-0.20% by volume of formic acid based on 100% of the total volume of the ammonium formate aqueous solution; more preferably, in mobile phase B, formic acid is contained in a volume ratio of 0.1% based on 100% of the total volume of the aqueous ammonium formate solution.
4. The method for detecting omeprazole in human plasma by HPLC-MS/MS combination according to claim 1, wherein the liquid chromatography conditions comprise: the chromatographic column is Welch Ultimate XB-C18, and preferably the chromatographic column has a length of 100mm, a diameter of 2.1mm and a filler particle size of 3 μm; the column temperature is 30-45 ℃, and preferably 40 ℃; the flow rate is 0.2-0.6 mL/min, preferably 0.4 mL/min.
5. The method for detecting omeprazole in human plasma by HPLC-MS/MS combination according to claim 1, wherein in the step (1), the pretreatment of the human plasma sample comprises: adding internal standard working solution and a precipitator into a human plasma sample, taking supernate after vortex and centrifugation, and mixing the supernate with a diluent to obtain a sample to be detected; the internal standard in the internal standard working solution is omeprazole-d 3; the precipitant is acetonitrile; the diluent is a mixed solution of methanol and water, and preferably, the volume ratio of the methanol to the water in the diluent is 40-60: 6-40; more preferably, the volume ratio of methanol to water in the diluent is 50: 50.
6. The method for detecting omeprazole in human plasma by HPLC-MS/MS combination according to claim 5, wherein in step (1), the internal standard working solution is prepared as follows: weighing omeprazole-d 3 reference substance, dissolving with methanol to obtain omeprazole-d 3 stock solution with the concentration of 1.00mg/mL, and diluting with mixed solution of methanol and water with the volume ratio of 50:50 to obtain omeprazole-d 3 internal standard working solution with the concentration of 200 ng/mL.
7. The method for detecting omeprazole in human plasma by HPLC-MS/MS combination according to claim 6, wherein in the step (1), the pretreatment of the human plasma sample comprises: taking 20 mu L of human plasma sample, adding 20 mu L of internal standard working solution and 160 mu L of acetonitrile, taking 50 mu L of supernatant after vortex and centrifugation, and mixing with 200 mu L of diluent to obtain a sample to be detected; the volume ratio of methanol to water in the diluent is 50: 50.
8. The method for detecting omeprazole in human plasma by HPLC-MS/MS combination according to claim 6 or 7, wherein the conditions of vortexing and centrifugation are as follows: vortexing for 10min, and centrifuging at 4000rpm/min for 10min at 4 ℃; and (3) placing the sample to be detected in an automatic sample injector, and performing LC-MS/MS analysis, wherein the sample injection volume is 2 mu L, and the temperature of the sample injector is 5 ℃.
9. The method for detecting omeprazole in human plasma by HPLC-MS/MS combination according to claim 1, wherein the mass spectrometry conditions comprise: adopting an electrospray ion source, monitoring and scanning positive ion multiple reaction, wherein the spraying voltage is 5500V, and the ion source temperature is 550 ℃; omeprazole, [ M + H ]]+M/z 346.2 → 198.1, DP value 70V, CE value 30V; omeprazole-d 3, [ M + H]+M/z 349.3 → 198.2, DP 80V, CE 30V.
10. The method for the combined HPLC-MS/MS detection of omeprazole in human plasma according to claim 1, wherein the method can be used for clinical pharmacokinetic sample monitoring.
Technical Field
The invention belongs to the technical field of biological analysis, and particularly relates to a method for detecting omeprazole in human plasma by using HPLC-MS/MS in a combined manner.
Background
Omeprazole belongs to benzimidazole antacid secretion compounds and can specifically inhibit H on cell surface of gastric parietal cell+/K+The ATPase system inhibits gastric acid secretion. Omeprazole is alkalescent and is easily converted into a sulfenamide compound after encountering acid, and the intestinal bioavailability of the sulfenamide compound is very low. The bioavailability of the drug for the first administration is 35%, and the bioavailability can be improved to 60% after continuous administration, which is probably because the gastric acid is inhibited by the drug and the decomposition of the drug in the stomach is reduced. The commercially available compound omeprazole can specifically act on secretory microtubules formed by apical membranes of gastric parietal cells and tubular vesicles in cytoplasm, namely gastric parietal cell proton pump H+-K+The last step of inhibiting the acid secretion of parietal cells at the site of ATPase, so that the parietal cells have H+Can not be transferred into the gastric cavity, so that the gastric acid amount in gastric juice is greatly reduced, and the gastric acid secretion inhibitor has strong inhibiting effect on gastric acid secretion caused by various stimulation factors.
At present, domestic documents report a method for determining the content of omeprazole by adopting liquid chromatography-mass spectrometry. For example, the literature liquid chromatography-tandem mass spectrometry method for simultaneously determining the mass concentration of omeprazole and its metabolite, omeprazole sulfone, in human plasma (morning, shenyang university of pharmacy, 2020) discloses a method for detecting omeprazole in plasma, but does not disclose the use of a needle washing solution during elution, and the detection method has residues, which may cause the difference between the detection result of a sample and the true value. The method for determining the blood concentration of omeprazole in human plasma by using a liquid chromatography-tandem mass spectrometry method and the pharmacokinetics (Rixiua, the department of pharmacy of the same-economic hospital affiliated to the same-economic college of the same-economic medical college of Huazhong university of science and technology) also discloses a method for detecting omeprazole in plasma, but in the detection method, the non-deuterated internal standard lansoprazole is used as an internal standard, the matrix effect of an analyte and the internal standard cannot be controlled to be the same, the difference between the detection result of a sample and the true value can be caused, the plasma dosage is 200 mu L, and the plasma dosage is large, so that the method is not suitable for analyzing the sample in a large batch.
Chinese patent CN 111337615A discloses a method for detecting omeprazole in blood plasma, but isocratic elution is adopted in the detection method, the retention time of omeprazole is 6.63min, the running time is 10min, and the method is not suitable for analyzing large-batch samples.
In order to meet the requirement of clinical large-batch sample analysis and evaluation on the biological equivalence of the drug, a simpler, reliable and high-flux sample pretreatment method and a method for detecting the concentration of omeprazole in human plasma need to be developed.
Disclosure of Invention
The invention aims to provide a method for detecting the concentration of omeprazole in human plasma, which has the advantages of good reproducibility, high sensitivity, high analysis speed, small matrix effect influence and high extraction recovery rate, on the basis of the prior art.
The technical scheme of the invention is as follows:
a method for detecting omeprazole in human plasma by HPLC-MS/MS combination comprises the following steps: (1) pretreating a human plasma sample; (2) performing liquid chromatography-mass spectrometry detection, and performing gradient elution by using a mobile phase A and a mobile phase B as a mixed mobile phase, wherein the mobile phase A is a methanol-acetonitrile mixed solution, and the volume ratio of methanol to acetonitrile in the mixed solution is 3-5: 7-5; the mobile phase B is 2-20 mM ammonium formate aqueous solution; (3) measuring the concentration of omeprazole in human plasma; the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is 45:55 within 0-0.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 45:55 to 95:5 at a constant speed within 0.5-2.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is 95:5 within 2.0-2.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is uniformly and gradually changed from 95:5 to 45:55 within 2.8-3.2 minutes; the volume ratio of mobile phase A to mobile phase B was 45:55 in 3.2-4.5 minutes. The specific gradient elution procedure is shown in table 1:
table 1: omeprazole liquid chromatography gradient
When the HPLC-MS/MS combination is used for detecting omeprazole in human plasma, in a preferred scheme, in the gradient elution process, the weak needle washing liquid is a methanol-acetonitrile-water mixed solution, in order to better implement the method, the volume ratio of methanol, acetonitrile and water in the weak needle washing liquid is 1-3: 4-2: 5, and under the condition that the effect of the method is not influenced, in a further preferred scheme, the volume ratio of methanol, acetonitrile and water in the weak needle washing liquid is 2:3: 5. In the experimental process, it is found that by using the commonly used strong needle washing liquid, such as methanol, acetonitrile, methanol-ethanol, methanol-isopropanol and methanol-acetonitrile-isopropanol, as the strong needle washing liquid, the peak area of the analyte in the residual sample after the high-concentration sample appears in other solvents as the strong needle washing liquid except for methanol-acetonitrile-isopropanol exceeds 20% of the peak area of the analyte in the lower limit sample, which affects the accurate determination of the sample after the high-concentration sample. For the invention, the strong needle washing liquid is a methanol-acetonitrile-isopropanol mixed solution, residues can be effectively removed, and the detection result is accurate and reliable, in a preferable scheme, the volume ratio of methanol to acetonitrile to isopropanol in the strong needle washing liquid is 1-2: 2-3: 1, and in a further preferable scheme, the volume ratio of methanol to acetonitrile to isopropanol in the strong needle washing liquid is 1:2:1 under the condition of not influencing the effect of the invention.
When the invention adopts HPLC-MS/MS combination to detect omeprazole in human plasma, in a preferred scheme, the volume ratio of methanol to acetonitrile in the mobile phase A is 4: 6. Further, mobile phase B was 10mM aqueous ammonium formate. In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. In the mobile phase B, the total volume of the ammonium formate aqueous solution is 100 percent as a reference, and the mobile phase B contains 0.05 to 0.20 percent of formic acid in volume ratio; in a preferable embodiment, the mobile phase B contains 0.1% by volume of formic acid based on 100% by volume of the total ammonium formate solution, i.e., the mobile phase B is 0.1% formic acid to 10mM ammonium formate solution.
When the HPLC-MS/MS combination is used for detecting omeprazole in human plasma, Welch Ultimate XB-C18 is used as a chromatographic column, and further, the length of the chromatographic column is 100mm, the diameter of the chromatographic column is 2.1mm, and the particle size of a filler is 3 mu m, namely Welch Ultimate XB-C18(2.1 multiplied by 100mm, 3 mu m).
In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. The invention adopts the mobile phase mentioned above to carry out gradient elution, takes Welch Ultimate XB-C18 as a chromatographic column, and endogenous substances do not interfere the determination of the sample under the coordination of other conditions, and the invention has good reproducibility, high sensitivity and fast analysis speed and has little influence on matrix effect.
When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. When the HPLC-MS/MS combined detection method is used for detecting omeprazole in human plasma, the omeprazole-d 3 is used as an internal standard, deuterated omeprazole is used as an internal standard substance, the deuterated internal standard and a substance to be detected have the same retention time, chemical properties and matrix effect, and the reproducibility and accuracy of the concentration of omeprazole in the plasma are better.
In the step (1), the human plasma sample is pretreated by adopting a protein precipitation method, and acetonitrile is used as a precipitator. The method for pretreating the human plasma sample by adopting the protein precipitation method can avoid the complicated and time-consuming liquid-liquid extraction process and simultaneously obtain the surprisingly high recovery rate. The invention adopts a protein precipitation method to pretreat a human blood plasma sample, uses Ultimate XB-C18 as a chromatographic column, and under the coordination of other conditions, the total extraction recovery rate of omeprazole is 99.99%.
In one embodiment, in step (1), the pretreatment of the human plasma sample comprises: adding internal standard working solution and a precipitator into a human plasma sample, taking supernate after vortex and centrifugation, and mixing the supernate with a diluent to obtain a sample to be detected; wherein the internal standard in the internal standard working solution is omeprazole-d 3; the precipitant is acetonitrile. The diluent is a mixed solution of methanol and water, and preferably, the volume ratio of the methanol to the water in the diluent is 40-60: 6-40; more preferably, the volume ratio of methanol to water in the diluent is 50: 50.
The detection method of the invention also comprises the steps of preparing an internal standard working solution: weighing omeprazole-d 3 reference substance, dissolving with methanol to obtain omeprazole-d 3 stock solution with the concentration of 1.00mg/mL, and diluting with mixed solution of methanol and water with the volume ratio of 50:50 to obtain omeprazole-d 3 internal standard working solution with the concentration of 200 ng/mL.
In a more preferred embodiment, in step (1), the pretreatment of the human plasma sample comprises: taking 20 mu L of human plasma sample, adding 20 mu L of internal standard working solution and 160 mu L of acetonitrile, taking 50 mu L of supernatant after vortex and centrifugation, and mixing with 200 mu L of diluent to obtain a sample to be detected; the volume ratio of methanol to water in the diluent is 50: 50.
The invention adopts a protein precipitation method for pretreatment of a human plasma sample, wherein the conditions of vortex and centrifugation are as follows: vortex for 10min and centrifuge at 4000rpm/min for 10min at 4 ℃.
In the invention, during the chromatographic detection, a sample to be detected is placed in an automatic sample injector for LC-MS/MS analysis, the sample injection volume is 2 mu L, and the temperature of the sample injector is 5 ℃.
The detection method of the invention, the step (2) of adopting the liquid chromatography-mass spectrometry combined detection comprises the following steps: the detailed chromatographic conditions of the invention are as follows: using Welch Ultimate XB-C18(2.1 multiplied by 100mm, 3 mu m) as a chromatographic column, and carrying out gradient elution according to the elution process, wherein the column temperature is 30-45 ℃, and preferably 40 ℃; the flow rate is 0.2-0.6 mL/min, preferably 0.4 mL/min.
Hair brushThe bright mass spectral conditions include: adopting an electrospray ion source, monitoring and scanning positive ion multiple reaction, wherein the spraying voltage is 5500V, and the ion source temperature is 550 ℃; omeprazole, [ M + H ]]+M/z 346.2 → 198.1, DP value 70V, CE value 30V; omeprazole-d 3, [ M + H]+M/z 349.3 → 198.2, DP 80V, CE 30V.
In the detection method, the step (3) of measuring the concentration of omeprazole in human plasma comprises the following steps: preparing the plasma to be detected according to the sample pretreatment method in the step (1), detecting according to the liquid chromatography-mass spectrometry in the step (2), recording the peak area corresponding to omeprazole, and determining the peak area ratio of omeprazole and the internal standard according to the weight coefficient w which is 1/x2And (4) performing linear regression, wherein the formula is expressed as y ═ ax + b, and calculating to obtain the concentration of omeprazole in the blood plasma to be detected.
The detection method of the invention can be used for clinical pharmacokinetic sample monitoring. The step of calculating the clinical pharmacokinetic parameters comprises: pharmacokinetic parameters were calculated using WinNonlin 8.0, including: cmax、Tmax、t1/2、AUC0-tAnd simultaneously calculating the average number and the standard deviation of each parameter.
By adopting the technical scheme of the invention, the advantages are as follows:
(1) the method takes the deuterated omeprazole as the internal standard substance, the deuterated internal standard substance and the substance to be measured have the same retention time, chemical properties and matrix effect, and the reproducibility and accuracy of the concentration of the omeprazole in the plasma are better.
(2) The detection method of the invention adopts the methanol-acetonitrile-isopropanol mixed solution as the needle washing liquid (for example, the volume ratio is 1:2:1) in the gradient elution process, and can effectively remove residues, so that the detection result is accurate and reliable.
(3) The method uses only 20 mu L of sample, uses less sample and is suitable for detecting large-batch plasma samples. The linear range of the detection method is 3.00-3000 ng/mL, the linear range is wide, the quantitative limit is low, the method is suitable for analyzing plasma samples with different specifications after administration, and the application range is wide.
(4) The detection method selects a specific mobile phase, optimizes the elution time and the proportion of the mobile phase in the gradient elution process, has the advantages of good reproducibility, high sensitivity, high analysis speed, small matrix effect influence, high recovery rate and the like, is verified by all methods including specificity, accuracy, precision, matrix effect, extraction recovery rate and stability, and can be reliably used for evaluating the bioequivalence of each formulation of omeprazole in a human body.
Drawings
Figure 1 is an omeprazole ion scan;
FIG. 2 is an omeprazole-d 3 daughter ion scan;
FIG. 3 is a specific chromatogram for measuring omeprazole in plasma by LC-MS/MS method;
(3-1-3-6) 6 batches of blank plasma chromatograms of different individuals;
in each of the diagrams of fig. 3-1 to 3-6, the left chromatogram is omeprazole, and the right chromatogram is omeprazole-d 3;
FIG. 4 is a mixed blank plasma chromatogram;
wherein the left chromatogram is omeprazole, and the right chromatogram is omeprazole-d 3;
FIG. 5 is a quantitative lower limit sample chromatogram;
wherein the left chromatogram is omeprazole, and the right chromatogram is omeprazole-d 3;
FIG. 6 is a chromatogram of omeprazole in comparative example 2;
wherein, the left chromatogram is the chromatogram of omeprazole in a quantitative upper limit sample, and the right chromatogram is the chromatogram of omeprazole in a residual sample;
FIG. 7 is a chromatogram of omeprazole in comparative example 3;
wherein, the left chromatogram is the chromatogram of omeprazole in the residual sample with the strong needle washing liquid being acetonitrile, and the right chromatogram is the chromatogram of omeprazole in the residual sample with the strong needle washing liquid being methanol to acetonitrile to isopropanol (1:2:1, v/v/v).
Detailed Description
The detection method of the present invention is further illustrated by the following examples in conjunction with the drawings, but the present invention is not limited to these examples.
Materials and methods
1. Instruments and reagents
High phase liquid chromatography (Shimadzu LC-30AD series); mass Spectrometry (API 4000, Applied Biosystems/Sciex); water purifiers (MilliDirectQ, Millipore); microbalance (XP6, METTLER TOLEDO); centrifuge (heraeus muitifiduge X1R, ThermoFisher); an oscillator (LPD2500, LE PARD).
Methanol (Merck, HPLC grade), acetonitrile (Merck, HPLC grade), water (ultrapure water, self-made in the laboratory), formic acid (aladin, HPLC grade), ammonium formate (aladin, HPLC grade), isopropanol (national pharmaceutical group chemicals limited, HPLC grade). Blank plasma was derived from healthy subjects. Omeprazole (China institute for testing food and drug, batch No. 100367-201706), omeprazole-d 3(TLC, batch No. 1194-005A3)
2. Liquid condition
Liquid phase conditions: a chromatographic column: welch Ultimate XB-C18 (2.1X 100mm, 3 μm); column temperature: 40 ℃; sample injector temperature: 5 ℃; the mobile phase A is methanol-acetonitrile (4:6, v/v); mobile phase B was 0.1% formic acid-10 mM aqueous ammonium formate solution (containing 0.1% formic acid by volume based on 100% total volume of 10mM aqueous ammonium formate solution); the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is 45:55 within 0-0.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is 45:55 within 0.5-2.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 45:55 to 95:5 at a constant speed within 0.5-2.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is 95:5 within 2.0-2.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is uniformly and gradually changed from 95:5 to 45:55 within 2.8-3.2 minutes; the volume ratio of mobile phase A to mobile phase B was 45:55 in 3.2-4.5 minutes. The flow rate is 0.4 mL/min; the needle washing mode comprises the following steps: rinse Port Only; weak needle washing liquid: methanol acetonitrile water (2:3:5, v/v/v); washing volume: 500 mu L of the solution; strong needle washing liquid: methanol acetonitrile isopropanol (1:2:1, v/v/v).
Mass spectrum conditions: ion detection mode: multiple Reaction Monitoring (MRM); an ionization mode: pneumatically assisted electrospray ionizationChemolysis (ESI); ion polarity: positive ions (Positive); detecting an object: omeprazole, [ M + H ]]+M/z 346.2 → 198.1, DP value 70V, CE value 30V; omeprazole-d 3, [ M + H]+M/z 349.3 → 198.2, DP 80V, CE 30V. Mass spectrum parameters: IonSpray Voltage: 5500V; TEM: at 550 ℃. The ion scanning patterns of the omeprazole and omeprazole-d 3 are shown in figure 1 and figure 2.
3. Preparation of standard solution
Preparing an omeprazole working solution: precisely weighing two parts of omeprazole reference substance, correcting by a mass correction coefficient, dissolving by methanol to obtain two parts of omeprazole stock solution with the concentration of 1.00mg/mL, and storing the stock solution in a refrigerator at the temperature of 20 ℃ below zero. After the stock solution is qualified, precisely measuring one part of omeprazole stock solution, diluting the omeprazole stock solution with methanol to water (50:50, v/v), preparing a series of omeprazole standard curve sample working solutions with the concentrations of 60,000, 54,000, 30,000, 12,000, 3,000, 600, 120 and 60.0ng/mL, precisely measuring the other part of omeprazole stock solution, diluting the omeprazole stock solution with methanol to water (50:50, v/v), and preparing QC working solutions with the concentrations of 48,000, 2,400 and 180ng/mL respectively.
Preparation of omeprazole-d 3 working solution: precisely weighing omeprazole-d 3 reference substance, correcting by mass correction coefficient, dissolving with methanol to obtain omeprazole-d 3 stock solution with concentration of 1.00mg/mL, and storing the stock solution in a refrigerator at-20 deg.C. A certain amount of omeprazole-d 3 stock solution is precisely measured and diluted by methanol and water (50:50, v/v) to prepare internal standard working solution with the concentration of 200 ng/mL.
4. Preparation of standard curve sample and quality control sample
For the standard curve samples and quality control samples at each concentration level, the formulation procedure is exemplified as follows: adding 10 μ L of corresponding working solution into 190 μ L of blank plasma, mixing uniformly, adjusting the preparation volume according to the actual situation, and sequentially preparing standard curve samples with omeprazole concentration of 3.00, 6.00, 30.0, 150, 600, 1500, 2700, 3000ng/mL and quality control samples with omeprazole concentration of 3.00ng/mL (LLOQ QC), 9.0ng/mL (LQC), 120ng/ML (MQC), 2400ng/mL (HQC).
5. Sample pretreatment
Adding 20 mu L of samples (biological samples to be detected, standard curve samples and quality control samples) into a 1.0mL 96-well plate; for both the double blank samples and the blank samples, 20. mu.L of blank matrix was added. Add 50. mu.L of solvent methanol water (1:1, v/v) to the double blank samples, add 20. mu.L of internal standard working solution to all wells except the double blank samples, add 160. mu.L of precipitant acetonitrile, vortex the 96-well plate at 1600rpm/min for 10min, continue to centrifuge the 96-well plate at 4 ℃ for 10min at 4000 rpm/min. Adding 50 mu L of supernatant into a clean 96-well plate, adding 200 mu L of diluent methanol and water (1:1, v/v), uniformly mixing, and placing in a sample chamber or a refrigerator at the same temperature for testing.
6. Methodology review content
And (3) carrying out methodology verification on the detection method according to a verification guiding principle of the quantitative analysis method of the biological sample in Chinese pharmacopoeia of 2015 edition to ensure the accuracy, repeatability and stability of detection. The verification comprises the following steps: specificity, standard curve, precision and accuracy, matrix effect, extraction recovery rate, stability.
Results and discussion
1. Specificity
Under the chromatographic conditions adopted in the test, the retention time of omeprazole is about 1.82min, as shown in figure 5; the retention time of the internal standard (omeprazole-d 3) is about 1.81min, as shown in figure 4; respectively taking 20 μ L of blank plasma from 6 different sources, and performing sample pretreatment operation except for no internal standard to obtain blank plasma sample chromatograms as shown in FIGS. 3-1-3-6; the chromatogram of the lower limit sample is shown in FIG. 5; the results show that endogenous substances in the plasma do not influence the detection of omeprazole, and meanwhile, the internal standard does not influence the detection of omeprazole.
2. Accuracy and precision test
Preparing quality control samples containing omeprazole with the concentration of 3.00ng/mL, 9.00ng/mL, 120ng/mL and 2400ng/mL respectively, preparing 6 samples for each concentration, preparing two standard curves (obtained by regression of two sets of standard curve samples), calculating the ratio f of the peak area As of omeprazole to the peak area Ai of an internal standard, marking the ratio f As f, substituting f into the standard curve on the day to obtain the average value and the accuracy of the actually measured concentration and the actually measured concentration, and calculating the precision and the accuracy in batches, wherein the results are shown in Table 2. The results show that: except for the lowest limit of quantitation (LLOQ), the precision RSD of the omeprazole quality control sample in batch is less than 15%, the accuracy RE in batch is at least 67% and not more than +/-15%, and the deviation of the quality control sample with the quantity of at least 50% of each concentration level from the theoretical value is not more than +/-15%. The precision RSD of the quality control samples in batches of the omeprazole samples with the lowest quantitative limit is less than 20%, the accuracy RE in batches is at least 67% and is not more than +/-20%, and the deviation between the quality control samples with the quantity of at least 50% of each concentration level and the theoretical value of the quality control samples is not more than +/-20%.
TABLE 2 precision and accuracy of in-batch and inter-batch sample testing
3. Matrix effect investigation
Preparing a matrix sample: using 6 batches of blank plasma from different donors, preparing nine repeated double blank samples from each batch of blank plasma, obtaining a blank matrix extracting solution according to the sample pretreatment operation, and adding a certain amount of analyte and internal standard after extraction so as to lead the final concentration of the blank matrix extracting solution to be consistent with the sample injection concentration of low, medium and high concentration quality control samples respectively (each concentration level, three repetitions).
Preparing a solution sample: and (3) replacing blank plasma with pure water to carry out a pretreatment step, and then diluting the working solution to prepare a corresponding concentration so that the concentration of the added blank matrix extracting solution of 50 mu L or the pure water extracting solution of 50 mu L is consistent with the concentration of the pre-treated low, medium and high concentration quality control samples. And 3 replicate samples per concentration.
The result shows that the total matrix effect factor of omeprazole matrix effect (calculated by peak area ratio) is 0.99-1.03, the precision is less than 1.7%, and the accurate quantification of omeprazole is not influenced by the plasma matrix. The results of the matrix effect data are shown in table 3.
TABLE 3 matrix Effect
4. Investigation of extraction recovery
Preparing a matrix sample: using plasma formed by mixing 6 batches of blank plasma of different donors to prepare 18 repeated double blank samples, obtaining blank plasma extract according to sample pretreatment operation, adding a certain amount of analyte and internal standard after extraction so as to lead the final concentration to be consistent with the sample injection concentration of low, medium and high concentration quality control samples respectively (6 repetitions at each concentration level).
Preparing a quality control sample: and (3) taking quality control samples with low, medium and high concentrations to process according to a sample processing method, and preparing 6 parts of each concentration level.
And evaluating the recovery rate by comparing the response value of the analyte or the internal standard in a single quality control sample with the average value of the response values of the double blank samples added with the analyte and the internal standard after extraction.
The accepted criteria for recovery are: the precision of recovery should be within 15.0% for each concentration level and all concentration levels. The extraction recovery rate (calculated by peak area ratio) of omeprazole is 99.99%, wherein the extraction recovery rates of the low, medium and high concentrations are 100.69%, 99.75% and 99.54% respectively. The results are shown in Table 4.
TABLE 4 recovery rate of extraction
5. Stability survey
Sample stability after treatment: after the analysis of precision and accuracy is examined, the sample is placed in an automatic sample injector (5 ℃) for 96 hours, a newly prepared standard curve sample and an analyzed sample are subjected to sample injection analysis, and a chromatogram is recorded, wherein the result is shown in table 5, and the sample injection solution after the omeprazole plasma sample is treated is placed in the sample injector for 96 hours, so that the stability is good, and the requirement of biological sample analysis is met.
Stability at room temperature: the prepared quality control samples with low and high concentration levels, wherein the omeprazole concentration is respectively 9.00ng/mL and 2400ng/mL, after the samples with each concentration level are uniformly mixed, the samples are placed at room temperature for 23h, LC-MS/MS analysis is carried out, a chromatogram is recorded, and the results are shown in Table 6, and the stability of the plasma samples is good after the plasma samples are placed at room temperature for 23 h.
Freeze-thaw stability: samples containing omeprazole with the concentration of 9.00ng/mL and 2400ng/mL are prepared in a fresh way and are respectively put into a refrigerator with the temperature of 80 ℃ below zero for 4 times of freeze thawing circulation. The acceptance criteria were: the average measured value of the stability sample and the% RE of the theoretical value of the stability sample should not exceed +/-l 5.0%, the% RSD of the measured value of the stability sample at each concentration level should be less than or equal to 15.0%, the result is shown in Table 7, and the stability of the sample is good after 4 freeze-thaw cycles at-80 ℃.
Long-term stability: samples containing omeprazole with the concentration of 9.00ng/mL and 2400ng/mL are prepared freshly and are respectively put into a refrigerator with the temperature of minus 80 ℃ for frozen storage for 78 days and then are detected. The acceptance criteria were: the average measured value of the stability sample and the% RE of the theoretical value of the stability sample should not exceed +/-l 5.0%, the% RSD of the measured value of the stability sample at each concentration level should be less than or equal to 15.0%, the result is shown in Table 8, and the stability is good when the sample is frozen and stored for 78 days at the temperature of minus 80 ℃.
TABLE 5 post-preparation sample stability
TABLE 6 stability during pretreatment of biological samples (stability at room temperature)
TABLE 7 Freeze thaw stability
TABLE 8 Long term stability
The method establishes an HPLC-MS/MS determination method for omeprazole in blood plasma, has good specificity, endogenous substances in the blood plasma do not interfere with the determination of a sample, the linear range of an omeprazole standard curve is 3.00-3000 ng/mL, and the linear relation is good: the precision of the detection results of the quality control samples with high concentration (2400ng/mL), medium concentration (120ng/mL) and low concentration (9.00ng/mL) in batches and between batches is less than 15.0 percent; the precision of the detection result of the quality control sample (3.00ng/mL) with the lower limit of quantification is less than 20.0 percent in batch and between batches. The omeprazole total matrix effect factor is 0.99-1.03, the precision is less than 1.7%, and the accurate quantification of omeprazole is not influenced by a plasma matrix. The extraction recovery rate of omeprazole is 99.99%. The omeprazole plasma has good stability after being placed at room temperature for 23 hours; the stability is good after 4 cycles of freezing/unfreezing; after the plasma sample is treated, the plasma sample is placed in an automatic sample injector at 5 ℃ for 96 hours, and the stability is good; the omeprazole plasma sample has good stability after being placed for 78 days at the temperature of-80 ℃, and meets the analysis requirement of a biological sample.
In conclusion, the method for determining the concentration of omeprazole in human plasma by the HPLC-MS/MS method established by the method meets the relevant requirements in the 'biological sample quantitative analysis method verification guiding principle' in the 2020 pharmacopoeia, and can be used for analyzing and detecting plasma samples in clinical tests.
Comparative example 1
Liquid phase conditions: a chromatographic column: welch Ultimate XB-C18 (2.1X 100mm, 3 μm); column temperature: 40 ℃; sample injector temperature: 5 ℃; the mobile phase A is acetonitrile; mobile phase B was 0.1% formic acid-10 mM aqueous ammonium formate solution (containing 0.1% formic acid by volume based on 100% total volume of 10mM aqueous ammonium formate solution); the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is 45:55 within 0-0.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 45:55 to 95:5 at a constant speed within 0.5-2.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is 95:5 within 2.0-2.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is uniformly and gradually changed from 95:5 to 45:55 within 2.8-3.2 minutes; the volume ratio of mobile phase A to mobile phase B was 45:55 in 3.2-4.5 minutes. The flow rate is 0.4 mL/min; the needle washing mode comprises the following steps: rinse Port Only; weak needle washing liquid: methanol acetonitrile water (2:3:5, v/v/v); washing volume: 500 mu L of the solution; strong needle washing liquid: methanol acetonitrile isopropanol (1:2:1, v/v/v).
Mass spectrum conditions: ion detection mode: multiple Reaction Monitoring (MRM); an ionization mode: pneumatically assisted electrospray ionization (ESI); ion polarity: positive ions (Positive); detecting an object: omeprazole, [ M + H ]]+M/z 346.2 → 198.1, DP value 70V, CE value 30V; omeprazole-d 3, [ M + H]+M/z 349.3 → 198.2, DP 80V, CE 30V. Mass spectrum parameters: IonSpray Voltage: 5500V; TEM: at 550 ℃.
As a result, it was found that: the mobile phase A is adjusted to be acetonitrile, a tailing phenomenon appears on a chromatographic peak, the peak shape is asymmetric, the lower limit of quantification is 6.00ng/mL, and the linear range is narrowed.
Comparative example 2
Liquid phase conditions: a chromatographic column: welch Ultimate XB-C18 (2.1X 100mm, 3 μm); column temperature: 40 ℃; sample injector temperature: 5 ℃; the mobile phase A is methanol-acetonitrile (4:6, v/v); mobile phase B was 0.1% formic acid-10 mM aqueous ammonium formate solution (containing 0.1% formic acid by volume based on 100% total volume of 10mM aqueous ammonium formate solution); the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is 30:70 within 0-0.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 30:70 to 80:20 at a constant speed within 0.5-0.7 min; the volume ratio of the mobile phase A to the mobile phase B is 80:20 within 0.7-1.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 80:20 to 95:5 at a constant speed within 1.8-2.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is 95:5 within 2.0-2.8 minutes; in 2.8-2.9 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly and gradually changed from 95:5 to 30: 70; the volume ratio of mobile phase A to mobile phase B is 30:70 within 2.9-4.0 minutes. The flow rate is 0.4 mL/min; the needle washing mode comprises the following steps: rinse Port Only; weak needle washing liquid: methanol acetonitrile water (2:3:5, v/v/v); washing volume: 500 mu L of the solution; strong needle washing liquid: methanol acetonitrile isopropanol (1:2:1, v/v/v).
Mass spectrum conditions: ion detection mode: multiple Reaction Monitoring (MRM); an ionization mode: pneumatically assisted electrospray ionization (ESI); ion polarity: positive ions (Positive); detecting an object: omeprazole, [ M + H ]]+M/z 346.2 → 198.1, DP value 70V, CE value 30V; omeprazole-d 3, [ M + H]+M/z 349.3 → 198.2, DP 80V, CE 30V. Mass spectrum parameters: IonSpray Voltage: 5500V; TEM: at 550 ℃.
As a result, it was found that: as shown in fig. 6, when the gradient elution process is adjusted, the analyte in the residual sample after the upper limit sample is quantified is likely to remain, which affects the accuracy of the sample measurement after the high concentration sample, and the possible reason for the occurrence of the residue is that the initial water phase ratio is relatively high, which causes the residue to accumulate and be eluted at the peak position.
Comparative example 3
Liquid phase conditions: a chromatographic column: welch Ultimate XB-C18 (2.1X 100mm, 3 μm); column temperature: 40 ℃; sample injector temperature: 5 ℃; the mobile phase A is methanol-acetonitrile (4:6, v/v); mobile phase B was 0.1% formic acid-10 mM aqueous ammonium formate solution (containing 0.1% formic acid by volume based on 100% total volume of 10mM aqueous ammonium formate solution); the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is 45:55 within 0-0.5 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 45:55 to 95:5 at a constant speed within 0.5-2.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is 95:5 within 2.0-2.8 minutes; the volume ratio of the mobile phase A to the mobile phase B is uniformly and gradually changed from 95:5 to 45:55 within 2.8-3.2 minutes; the volume ratio of mobile phase A to mobile phase B was 45:55 in 3.2-4.5 minutes. The flow rate is 0.4 mL/min; the needle washing mode comprises the following steps: rinse Port Only; weak needle washing liquid: methanol acetonitrile water (2:3:5, v/v/v); washing volume: 500 mu L of the solution; strong needle washing liquid: and (3) acetonitrile.
Mass spectrum conditions: ion detection mode: multiple Reaction Monitoring (MRM);an ionization mode: pneumatically assisted electrospray ionization (ESI); ion polarity: positive ions (Positive); detecting an object: omeprazole, [ M + H ]]+M/z 346.2 → 198.1, DP value 70V, CE value 30V; omeprazole-d 3, [ M + H]+M/z 349.3 → 198.2, DP 80V, CE 30V. Mass spectrum parameters: IonSpray Voltage: 5500V; TEM: at 550 ℃.
As a result, it was found that: as shown in fig. 7, when the strong needle wash is adjusted, the analyte is likely to remain in the sample remaining after the upper limit sample is quantified, and the accuracy of the sample measurement after the high concentration sample is affected.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.