CN112782323A - Method for determining concentration of omeprazole in blood plasma by liquid chromatography-mass spectrometry - Google Patents
Method for determining concentration of omeprazole in blood plasma by liquid chromatography-mass spectrometry Download PDFInfo
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Abstract
The invention discloses a method for determining omeprazole concentration in blood 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 measuring the blood concentration of omeprazole; the plasma standard curve linear range of the method is 2-1500 ng/mL, the precision RSD in batch and between batches is less than +/-15%, and the method is suitable for measuring the concentration of omeprazole 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 omeprazole in blood plasma by liquid chromatography-mass spectrometry.
Background
Omeprazole is a substituted benzimidazole compound, and is a racemate of a pair of active optical antipodes. Omeprazole acts on the proton pump in parietal cells by a special mechanism to reduce gastric acid secretion, which is reversible. Omeprazole, a weak base, is concentrated in the acidic environment of parietal cells and converted to the active form, inhibiting the last link in the production of hydrochloric acid in gastric juice: h + -K + -ATPase, which is dose-dependent, has an effect on both basal and post-stimulated gastric acid secretion, regardless of the type of stimulus. Omeprazole has no effect on cholinergic and histamine receptors. Similar to the H2 receptor blockers, omeprazole decreases the acidity in the stomach, thereby causing an increase in gastrin, which is reversible, in proportion to the decrease in acidity. It has been reported that the incidence of gastric gland cysts increases in long-term treatment. These changes are both physiological consequences of inhibited gastric acid secretion, and are benign and reversible. The reduction of gastric acid by proton pump inhibitors or other acid inhibitors increases the number of normal bacteria in the gastrointestinal tract, and thus treatment may result in a slight increase in the risk of gastrointestinal infections (e.g., salmonella and campylobacter). At present, the speed, the precision, the sensitivity and the selectivity of the existing omeprazole determination method need to be improved.
Disclosure of Invention
The invention aims to provide a method for determining the concentration of omeprazole in blood plasma by liquid chromatography-mass spectrometry, which can improve the sensitivity, precision, selectivity and speed of detection.
In order to achieve the aim, the invention provides a method for determining the concentration of omeprazole in blood plasma by liquid chromatography-mass spectrometry, wherein a blood plasma sample is pretreated and then the concentration of omeprazole is detected by high performance liquid chromatography-tandem mass spectrometry, and the specific method comprises the following steps:
(1) plasma sample pretreatment:
plasma with K2EDTA as anticoagulant, omeprazole-d 3 as internal standard; precisely adding 100 mu L of plasma sample into a 96 deep-well plate, adding 5 mu L of mixed organic solvent I, uniformly mixing, adding 5 mu L of 1 ng/mu L of internal standard omeprazole-d 3 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 20 mu L of supernatant into the 96 deep-well plate filled with 1000 mu L of mixed organic solvent II, carrying out vortex mixing, centrifuging at 20 ℃ at 3000rpm for 5min, and taking the supernatant as a test sample to be detected; wherein, the mixed organic solvent I is methanol: water: ammonium hydroxide is added according to a volume ratio of 50: 50: 0.2 mixing the obtained mixture, wherein the mixed organic solvent II is water: methanol: 1M ammonium acetate: ammonium hydroxide is mixed according to a volume ratio of 55: 45: 0.5: 0.2 mixing the obtained mixture;
(2) and (3) sample measurement:
and (3) injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of omeprazole and internal standard omeprazole-d 3 in the sample, and calculating the concentration of omeprazole in the plasma sample according to the chromatographic peaks.
The liquid chromatography determination conditions were: the chromatographic column is SynergiTM Polar-RP
4 μm, column size 50 × 2 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: methanol: 1M ammonium acetate in a volume ratio of 90: 10: 0.5 mixing the obtained mixture; the mobile phase B is methanol: water: 1M ammonium acetate in a volume ratio of 90: 10: 0.5 mixing the obtained mixture; the washing liquid is methanol: water: 1M ammonium acetate: ammonium hydroxide is added according to a volume ratio of 25: 75: 0.5: 1 mixing the obtained mixture; 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 min;
the mass spectrometry conditions are as follows: the ion source is an electrospray ion source, the spraying voltage is 5000V, the atomizing temperature is 520 ℃, the spraying air pressure is 30Psi, the auxiliary heating air pressure is 30Psi, the air curtain air pressure is 25Psi, the collision air pressure is Low Psi, and the declustering voltage of omeprazole and internal standard omeprazole-d 3 is 20 eV; the entrance voltage of the collision chamber of omeprazole and the internal standard omeprazole-d 3 is 11 eV; the collision voltage of omeprazole and internal standard omeprazole-d 3 is 15 eV; the outlet voltage of the collision chamber of omeprazole and the internal standard omeprazole-d 3 is 15 eV; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z346.2 → m/z198.1, which is omeprazole; and m/z349.1 → m/z198.1, which is omeprazole-d 3.
Preferably, the gradient elution in step (2) is performed by the following procedure:
preferably, in the step (2), the concentration of omeprazole in the plasma sample is calculated by taking the peak area ratio of omeprazole and internal standard omeprazole-d 3 into a standard curve equation by using an internal standard method.
Preferably, the establishment of the standard curve equation comprises the following steps:
placing ten 100-microliter blank blood plasmas into a 96-deep-hole plate, and sequentially naming the blank blood plasmas as a lowest quantitative lower limit sample, a standard sample 1, a standard sample 2, a standard sample 3, a standard sample 4, a standard sample 5, a standard sample 6, a highest quantitative upper limit sample, a zero-concentration sample and a blank sample to total ten samples, wherein the zero-concentration sample contains an internal standard omeprazole-d 3 solution and does not contain an omeprazole solution and is used for eliminating the interference of the internal standard omeprazole-d 3 solution on a detection result; the blank sample does not contain an omeprazole solution and an internal standard omeprazole-d 3 solution, and is used for eliminating the interference of the blank plasma on the detection result;
adding 5 mul of omeprazole solution with the concentration of 0.04 ng/mul, 0.08 ng/mul, 0.2 ng/mul, 1 ng/mul, 2 ng/mul, 10 ng/mul, 20 ng/mul and 30 ng/mul into a sample with the lowest limit of quantitation, a sample with the value of 1-6 and a sample with the highest limit of quantitation, respectively adding 5 mul of mixed organic solvent I into a sample with the concentration of zero and a sample with the concentration of blank, respectively mixing the ten samples, respectively adding 5 mul of internal standard omeprazole-d 3 solution with the concentration of 1 ng/mul into nine samples except the sample with the sample of blank, respectively adding 5 mul of mixed organic solvent I into the sample with the concentration of 5 mul, respectively adding the ten samples into the ten samples, respectively adding 1000 mul of acetonitrile into a 96 deep-well plate, mixing for 1min by vortex, centrifuging at 20 deg.C at 3000rpm for 10min, collecting supernatant 20 μ L into 96 deep-well plate containing 1000 μ L mixed organic solvent II, mixing by vortex, centrifuging at 20 deg.C at 3000rpm for 5min, and detecting as 10 standard samples; wherein, the mixed organic solvent I is methanol: water: ammonium hydroxide is added according to a volume ratio of 50: 50: 0.2 mixing the obtained mixture, wherein the mixed organic solvent II is water: methanol: 1M ammonium acetate: ammonium hydroxide is mixed according to a volume ratio of 55: 45: 0.5: 0.2 mixing the obtained mixture;
and respectively injecting 10 mu L of standard sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of omeprazole and internal standard omeprazole-d 3 in the sample, and obtaining a standard curve according to the chromatographic peaks for calculating the concentration of omeprazole in the plasma.
Further, the liquid chromatography determination conditions in the step (3) further include: the volume of the syringe body of the automatic sample injector is 1000 mu L; the depth of a sample injection needle of the automatic sample injector is 30 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 5 mu L/s; the soaking time is 60s when the sample injection needle of the automatic sample injector is cleaned; the automatic sample injector cleaning mode is before sample injection and after sample injection.
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 omeprazole is about 1.432min, the retention time of internal standard omeprazole-d 3 is about 1.420min, the peak shapes of omeprazole and internal standard omeprazole-d 3 are good, the measurement is not interfered by a miscellaneous peak, and the base line is stable;
(3) the sensitivity is high: the minimum limit of quantitation of the plasma is 2ng/mL, the concentration of omeprazole in the 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 measuring the blood concentration of omeprazole. The linear range of the plasma standard curve of the method is 2-1500 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.
Drawings
FIG. 1 is a standard graph of omeprazole 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 omeprazole-d 3;
FIG. 4 is a HPLC-MS/MS graph of human blank plasma supplemented with omeprazole and omeprazole-d 3;
FIG. 5 is a HPLC-MS/MS graph of plasma samples with omeprazole-d 3 added as an internal standard after oral administration of omeprazole drug to healthy subjects.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example (b): human K2Determination of omeprazole concentration in EDTA plasma
First, experimental material and analytical equipment
Omeprazole (analyte) chinese food and drug assay institute or same, higher grade standard omeprazole-d 3 (internal standard) TLC Pharmaceutical Standards or same, higher grade standard reagents used are given in table 1 below:
TABLE 1 details of reagents
| Name of reagent | Rank of | Manufacturer(s) |
| Acetonitrile (ACN) | HPLC | J.T.Baker |
| Methanol (MeOH) | HPLC | J.T.Baker |
| Ammonium acetate (CH)3COONH4) | HPLC | J.T.Baker |
| Ammonium hydroxide (NH)4OHa) | ACS | J.T.Baker |
a：NH4OH is expressed as A.C.S. grade ammonia water, and the ammonia content is 28-30%.
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
The chromatographic column is SynergiTM Polar-RP
4 μm, column size 50 × 2 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: methanol: 1M ammonium acetate in a volume ratio of 90: 10: 0.5 mixing the obtained mixture; the mobile phase B is methanol: water: 1M ammonium acetate in a volume ratio of 90: 10: 0.5 mixing the obtained mixture; the washing liquid is methanol: water: 1M ammonium acetate: ammonium hydroxide is added according to a volume ratio of 25: 75: 0.5: 1 mixing the obtained mixture; the autosampler temperature was 15 ℃; gradient elution, flow rate of 0.4mL/min, sample size of 10 μ L, analysis time of 3 min.
The volume of the syringe body of the automatic sample injector is 1000 mu L; the depth of a sample injection needle of the automatic sample injector is 30 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 5 mu L/s; the soaking time is 60s when the sample injection needle of the automatic sample injector is cleaned; the automatic sample injector cleaning mode is before sample injection and after sample injection.
TABLE 3 gradient elution procedure
2. Conditions of Mass Spectrometry
The ion source is an electrospray ion source, the spraying voltage is 5000V, the atomizing temperature is 520 ℃, the spraying air pressure is 30Psi, the auxiliary heating air pressure is 30Psi, the air curtain air pressure is 25Psi, the collision air pressure is Low Psi, and the declustering voltage of omeprazole and internal standard omeprazole-d 3 is 20 eV; the entrance voltage of the collision chamber of omeprazole and the internal standard omeprazole-d 3 is 11 eV; the collision voltage of omeprazole and internal standard omeprazole-d 3 is 15 eV; the outlet voltage of the collision chamber of omeprazole and the internal standard omeprazole-d 3 is 15 eV; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z346.2 → m/z198.1, which is omeprazole; and m/z349.1 → m/z198.1, which is omeprazole-d 3.
Third, the experimental process
1. Preparation of omeprazole standard solution
The standard solution (containing stock solution and working solution) for the omeprazole standard curve is weighed and prepared by the following steps:
| weighing weight (mg) | Dissolved volume (μ l) | Final concentration (ng/. mu.l) |
| 2.026 | 10130 | 200 |
Preparing 200 ng/mu L omeprazole stock solution according to the preparation process, and then mixing the omeprazole stock solution with the mixture according to the volume ratio of 50: 50: 0.2 methanol: water: the mixed solution of ammonium hydroxide is diluted in sequence to prepare an omeprazole standard solution, and the specific dilution concentration is shown in the following table 4:
table 4 omeprazole standard solution preparation concentration
a: directly from omeprazole (analyte)
The omeprazole standard solution is stored in a plastic container and a refrigerator (4 ℃) when not used, and the volume can be increased or reduced according to the proportion as required.
2. Preparation of omeprazole-d 3 internal standard solution
The weighing and preparation process of the omeprazole-d 3 internal standard solution is as follows:
| weighing weight (mg) | Dissolved volume (μ l) | Final concentration (ng/. mu.l) |
| 1.058 | 10465 | 100 |
Preparing 100 ng/mu L of omeprazole-d 3 stock solution according to the preparation process, and mixing the omeprazole-d 3 stock solution with the volume ratio of 50: 50: 0.2 methanol: water: ammonium hydroxide solution is diluted to prepare omeprazole-d 3 internal standard solution with the concentration of 1 ng/. mu.L, and the specific dilution concentration is shown in the following table 5:
TABLE 5 omeprazole-d 3 Standard solution preparation concentration
a: directly prepared from omeprazole-d 3 (internal standard)
b: for sample preparation procedures
The standard solution of omeprazole-d 3 internal standard is stored in a plastic container and a refrigerator (4 ℃) when not used, and the volume can be increased or reduced according to the proportion.
3. Linear test
Putting the blank plasma into a water bath at room temperature for unfreezing; transferring 10 parts of 100 mu L 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 omeprazole standard solution or diluted solution with different concentrations according to the list in the following table 6 to prepare each sample, mixing uniformly to prepare drug-containing plasma with different concentrations, and carrying out the operation according to 'plasma sample pretreatment'. The ratio Y (Y As/Ai) of the omeprazole peak area As and the omeprazole-d 3 peak area Ai was calculated, and the peak area ratio Y was used to perform regression calculation on the blood concentration X, and the results are shown in fig. 1 and table 7. The average ratio Y is used for carrying out regression calculation on the blood concentration X to obtain a regression equation Y which is 0.0192X +0.00151, r which is 0.9994, and a weight coefficient W which is 1/X2The lowest quantitative limit of the blood concentration of omeprazole measured by the method is as follows: 2 ng/mL.
TABLE 6 omeprazole Standard Curve formulation concentration
b: diluted solution of analyte: MeOH/H2O/NH4OH＝50/50/0.2
Table 7 standard curve of omeprazole in human plasma by HPLC-MS/MS method (n ═ 14)
4. Accuracy and precision
Putting the blank plasma into a water bath at room temperature for unfreezing; transfer appropriate volume of blank plasma to appropriate container and add omeprazole standard solution to prepare 5 drug-containing plasma quality control samples (LLOQ, QL, QLM, QM, QH) of different concentrations and a follow-up standard curve, run as "plasma sample pretreatment" and quality control sample preparation 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 16 samples for each concentration of the third batch, calculating the ratio Y of the omeprazole peak area As and the internal standard omeprazole-d 3 peak area Ai, substituting the ratio Y into the standard curve on the day to obtain the actually measured concentration, calculating the precision between batches according to the actually measured concentration, and determining the ratio of the actually measured concentration to the added concentration As the accuracy, wherein the result is shown in Table 9. The result shows that the precision and the accuracy of the omeprazole plasma sample in batches 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 HPLC-MS/MS method for determining the accuracy and precision of omeprazole in plasma
Note: the data of the evaluation results are from the data of 3 batches of 26 groups of quality control samples in table 9.
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 omeprazole analyte and the internal standard omeprazole-d 3.
After nine blank healthy human plasma samples from different sources were prepared and analyzed, the interference peak responses at the retention time of omeprazole were all less than 20.0% of the omeprazole 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 for the analysis of omeprazole.
After nine blank healthy human plasma samples from different sources were prepared and analyzed, the interference peak responses at retention times consistent with internal standard omeprazole-d 3 were all less than 5.0% of the response of internal standard omeprazole-d 3 from the lower limit quantitation sample in the standard curve of the assay lot, see table 11 in the appendix. The results show that the analysis method has selectivity to the analysis of the internal standard omeprazole-d 3.
TABLE 10 comparison of interference data on omeprazole analytes from blank healthy human plasma from nine different sources
a: analyte peak area (selective sample)/analyte peak area (LLOQ of standard curve). times.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 against internal standard omeprazole-d 3
a: analyte peak area (selective sample)/internal standard peak area (LLOQ of standard curve). times.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, blank plasma from different human bodies did not interfere with the test results of omeprazole. Therefore, the method can be used for detecting the concentration of omeprazole in plasma of different human bodies.
6. Detection of human plasma samples
(1) Accurately adding 100 mu L of plasma sample into a 96 deep-well plate without omeprazole, adding 10 mu L of mixed organic solvent I, 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 20 mu L of supernatant into the 96 deep-well plate filled with 1000 mu L of mixed organic solvent II, carrying out vortex mixing, centrifuging at 20 ℃ at 3000rpm for 5min, taking 10 mu L of sample, and carrying out LC-MS/MS analysis, wherein the result of a representative map is shown in figure 2; wherein, the mixed organic solvent I is methanol: water: ammonium hydroxide is added according to a volume ratio of 50: 50: 0.2 mixing the obtained mixture, wherein the mixed organic solvent II is water: methanol: 1M ammonium acetate: ammonium hydroxide is mixed according to a volume ratio of 55: 45: 0.5: 0.2 mixing the resulting mixture.
(2) Accurately adding 100 mu L of plasma sample into a 96 deep-well plate without omeprazole, adding 5 mu L of mixed organic solvent I, uniformly mixing, adding 5 mu L of 1 ng/mu L of internal standard omeprazole-d 3 solution, uniformly mixing, adding 1000 mu L of acetonitrile into the 96 deep-well plate, carrying out vortex mixing for 1min, centrifuging at 20 ℃ for 10min at 3000rpm, taking 20 mu L of supernatant into the 96 deep-well plate containing 1000 mu L of mixed organic solvent II, carrying out vortex mixing, centrifuging at 20 ℃ for 5min at 3000rpm, taking 10 mu L of sample, carrying out LC-MS/MS analysis, wherein the representative map result is shown in figure 3; wherein, the mixed organic solvent I is methanol: water: ammonium hydroxide is added according to a volume ratio of 50: 50: 0.2 mixing the obtained mixture, wherein the mixed organic solvent II is water: methanol: 1M ammonium acetate: ammonium hydroxide is mixed according to a volume ratio of 55: 45: 0.5: 0.2 mixing the resulting mixture.
(3) Taking human blank plasma without omeprazole, precisely adding 100 mu L blank plasma sample into a 96 deep-well plate, adding 5 mu L omeprazole standard solution, uniformly mixing, adding 5 mu L1 ng/mu L internal standard omeprazole-d 3 solution, uniformly mixing, adding 1000 mu L acetonitrile into the 96 deep-well plate, vortex mixing for 1min, centrifuging at 20 ℃ for 10min at 3000rpm, taking 20 mu L supernatant into the 96 deep-well plate filled with 1000 mu L mixed organic solvent II, wherein the mixed organic solvent II is water: methanol: 1M ammonium acetate: ammonium hydroxide is mixed according to a volume ratio of 55: 45: 0.5: 0.2, vortex the resulting mixture, mix well, centrifuge at 3000rpm for 5min at 20 ℃ and then take 10 μ L sample for LC-MS/MS analysis, representative profile results are shown in figure 4.
(4) Collecting blood plasma of healthy subjects after oral administration of omeprazole or pharmaceutical salt thereof, precisely adding 100 mu L of collected human plasma samples into a 96 deep-well plate, adding 5 mu L of mixed organic solvent I, uniformly mixing, adding 5 mu L of 1 ng/mu L of internal standard omeprazole-d 3 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 20 mu L of supernatant into the 96 deep-well plate filled with 1000 mu L of mixed organic solvent II, carrying out vortex mixing, centrifuging at 20 ℃ at 3000rpm for 5min, taking 10 mu L of samples, and carrying out LC-MS/MS analysis, wherein the representative map result is shown in figure 5; wherein, the mixed organic solvent I is methanol: water: ammonium hydroxide is added according to a volume ratio of 50: 50: 0.2 mixing the obtained mixture, wherein the mixed organic solvent II is water: methanol: 1M ammonium acetate: ammonium hydroxide is mixed according to a volume ratio of 55: 45: 0.5: 0.2 mixing the resulting mixture.
In conclusion, the invention provides a method for determining the concentration of omeprazole in blood plasma, which is simple and convenient in 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 omeprazole is about 1.432min, the retention time of internal standard omeprazole-d 3 is about 1.420min, the omeprazole and internal standard omeprazole-d 3 have good peak shapes, no interference of miscellaneous peaks is caused in measurement, and the base line is stable; the method has high specificity, can accurately determine the concentration of omeprazole in blood plasma, and has high sensitivity, and the minimum limit of quantitation of the blood plasma is 2 ng/mL; meanwhile, the method is rapid, accurate, high in sensitivity and simple and convenient to operate, and provides a basis for measuring the blood concentration of omeprazole. The linear range of the plasma standard curve of the method is 2-1500 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.
Claims (5)
1. A method for determining omeprazole concentration in blood plasma by liquid chromatography-mass spectrometry is characterized in that: 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) plasma sample pretreatment:
plasma with K2EDTA as anticoagulant, omeprazole-d 3 as internal standard; precisely adding 100 mu L of plasma sample into a 96 deep-well plate, adding 5 mu L of mixed organic solvent I, uniformly mixing, adding 5 mu L of 1 ng/mu L of internal standard omeprazole-d 3 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 20 mu L of supernatant into the 96 deep-well plate filled with 1000 mu L of mixed organic solvent II, carrying out vortex mixing, centrifuging at 20 ℃ at 3000rpm for 5min, and taking the supernatant as a test sample to be detected; wherein, the mixed organic solvent I is methanol: water: ammonium hydroxide is added according to a volume ratio of 50: 50: 0.2 mixing the obtained mixture, wherein the mixed organic solvent II is water: methanol: 1M ammonium acetate: ammonium hydroxide is mixed according to a volume ratio of 55: 45: 0.5: 0.2 mixing the obtained mixture;
(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 omeprazole and internal standard omeprazole-d 3 in the sample, and calculating the concentration of omeprazole in the plasma sample according to the chromatographic peaks;
the liquid chromatography determination conditions were: the chromatographic column is SynergiTMPolar-
4 μm, column size 50 × 2 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: methanol: 1M ammonium acetate in a volume ratio of 90: 10: 0.5 mixing the obtained mixture; the mobile phase B is methanol: water: 1M ammonium acetate in a volume ratio of 90: 10: 0.5 mixing the obtained mixture; the washing liquid is methanol: water: 1M ammonium acetate: ammonium hydroxide is added according to a volume ratio of 25: 75: 0.5: 1 mixing the obtained mixture; 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 min;
the mass spectrometry conditions are as follows: the ion source is an electrospray ion source, the spraying voltage is 5000V, the atomizing temperature is 520 ℃, the spraying air pressure is 30Psi, the auxiliary heating air pressure is 30Psi, the air curtain air pressure is 25Psi, the collision air pressure is Lowpsi, and the declustering voltage of omeprazole and internal standard omeprazole-d 3 is 20 eV; the entrance voltage of the collision chamber of omeprazole and the internal standard omeprazole-d 3 is 11 eV; the collision voltage of omeprazole and internal standard omeprazole-d 3 is 15 eV; the outlet voltage of the collision chamber of omeprazole and the internal standard omeprazole-d 3 is 15 eV; detecting in a positive ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z346.2 → m/z198.1, which is omeprazole; and m/z349.1 → m/z198.1, which is omeprazole-d 3.
2. The method for determining the concentration of omeprazole in blood plasma by LC-MS according to claim 1, wherein: the gradient elution procedure in the step (2) is as follows:
3. the method for determining the concentration of omeprazole in blood plasma according to claim 1 or 2, wherein: in the step (2), an internal standard method is adopted, and the concentration of omeprazole in the plasma sample is calculated by substituting the peak area ratio of omeprazole and internal standard omeprazole-d 3 into a standard curve equation.
4. The method for determining the concentration of omeprazole in blood plasma by LC-MS according to claim 3, wherein: the establishment of the standard curve equation comprises the following steps:
placing ten 100-microliter blank blood plasmas into a 96-deep-hole plate, and sequentially naming the blank blood plasmas as a lowest quantitative lower limit sample, a standard sample 1, a standard sample 2, a standard sample 3, a standard sample 4, a standard sample 5, a standard sample 6, a highest quantitative upper limit sample, a zero-concentration sample and a blank sample to total ten samples, wherein the zero-concentration sample contains an internal standard omeprazole-d 3 solution and does not contain an omeprazole solution and is used for eliminating the interference of the internal standard omeprazole-d 3 solution on a detection result; the blank sample does not contain an omeprazole solution and an internal standard omeprazole-d 3 solution, and is used for eliminating the interference of the blank plasma on the detection result;
adding 5 mul of omeprazole solution with the concentration of 0.04 ng/mul, 0.08 ng/mul, 0.2 ng/mul, 1 ng/mul, 2 ng/mul, 10 ng/mul, 20 ng/mul and 30 ng/mul into a sample with the lowest limit of quantitation, a sample with the value of 1-6 and a sample with the highest limit of quantitation, respectively adding 5 mul of mixed organic solvent I into a sample with the concentration of zero and a sample with the concentration of blank, respectively mixing the ten samples, respectively adding 5 mul of internal standard omeprazole-d 3 solution with the concentration of 10 ng/mul into nine samples except the sample with the sample of blank, respectively adding 5 mul of mixed organic solvent I into the sample with the concentration of 5 mul, respectively adding the ten samples, respectively adding 1000 mul of acetonitrile into the ten samples into a 96 deep-well plate, mixing for 1min by vortex, centrifuging at 20 deg.C at 3000rpm for 10min, collecting supernatant 20 μ L into 96 deep-well plate containing 1000 μ L mixed organic solvent II, mixing by vortex, centrifuging at 20 deg.C at 3000rpm for 5min, and detecting as 10 standard samples; wherein, the mixed organic solvent I is methanol: water: ammonium hydroxide is added according to a volume ratio of 50: 50: 0.2 mixing the obtained mixture, wherein the mixed organic solvent II is water: methanol: 1M ammonium acetate: ammonium hydroxide is mixed according to a volume ratio of 55: 45: 0.5: 0.2 mixing the obtained mixture;
and respectively injecting 10 mu L of standard sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of omeprazole and internal standard omeprazole-d 3 in the sample, and obtaining a standard curve according to the chromatographic peaks for calculating the concentration of omeprazole in the plasma.
5. The method for determining the concentration of omeprazole in blood plasma according to claim 1 or 2, wherein: the conditions for the liquid chromatography determination in the step (2) further include: the volume of the syringe body of the automatic sample injector is 1000 mu L; the depth of a sample injection needle of the automatic sample injector is 30 mm; the cleaning speed of the automatic sample injector is 35 mu L/s; the sample injection speed of the automatic sample injector is 5 mu L/s; the soaking time is 60s when the sample injection needle of the automatic sample injector is cleaned; the automatic sample injector cleaning mode is before sample injection and after sample injection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113533597A (en) * | 2021-07-29 | 2021-10-22 | 南京西默思博检测技术有限公司 | Method for determining esomeprazole in human plasma |
| CN113720930A (en) * | 2021-07-26 | 2021-11-30 | 南京一诺医药科技有限公司 | Method for detecting omeprazole in human plasma by HPLC-MS/MS combination |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113720930A (en) * | 2021-07-26 | 2021-11-30 | 南京一诺医药科技有限公司 | Method for detecting omeprazole in human plasma by HPLC-MS/MS combination |
| CN113533597A (en) * | 2021-07-29 | 2021-10-22 | 南京西默思博检测技术有限公司 | Method for determining esomeprazole in human plasma |
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