CN112834660A - Method for determining concentration of diclofenac in blood plasma by liquid chromatography-mass spectrometry - Google Patents
Method for determining concentration of diclofenac in blood plasma by liquid chromatography-mass spectrometry Download PDFInfo
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Abstract
The invention discloses a method for determining the concentration of diclofenac in blood plasma by liquid chromatography-mass spectrometry, which adopts a liquid chromatography-mass spectrometry system to determine, firstly takes a sample to be determined, adds a certain amount of mixed organic solvent to extract, pretreats, separates by a chromatographic column, and detects 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 diclofenac; the linear range of the plasma standard curve of the method is 2-3000 ng/mL, the precision RSD in batch and between batches is less than +/-15%, and the method is suitable for measuring the concentration of diclofenac 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 diclofenac in blood plasma by liquid chromatography-mass spectrometry.
Background
Diclofenac is a nonsteroidal anti-inflammatory analgesic derived from phenylacetic acid, and has the action mechanism of inhibiting cyclooxygenase activity so as to block arachidonic acid from converting prostaglandin, which is a main factor causing phenomena such as pain, fever, inflammation and the like. Meanwhile, it can also promote the combination of arachidonic acid and triglyceride (triacylglycerol), reduce the concentration of free arachidonic acid in cells, and indirectly inhibit the synthesis of leukotriene. Diclofenac is one of the nonsteroidal anti-inflammatory drugs with stronger effect, and has stronger inhibition effect on prostaglandin synthesis than aspirin and indometacin. At present, the speed, precision, sensitivity and selectivity of the existing diclofenac determination methods are all to be improved.
Disclosure of Invention
The invention aims to provide a method for determining the concentration of diclofenac in 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 diclofenac in plasma by liquid chromatography-mass spectrometry, wherein a plasma sample is pretreated and then the concentration of the sample is detected by high performance liquid chromatography-tandem mass spectrometry, and the method comprises the following steps:
(1) plasma sample pretreatment:
plasma with K2EDTA as anticoagulant, diclofenac-d 4 as internal standard; precisely adding 100 μ L of plasma sample into a 96-deep-well plate, adding 5 μ L of a volume ratio of 1: 1, uniformly mixing the methanol aqueous solution, adding 5 mu L of 8 ng/mu L internal standard diclofenac-d 4 solution, uniformly mixing the solution, adding 500 mu L acetonitrile, carrying out vortex mixing for 1min, centrifuging the mixture at 20 ℃ for 10min at 3000rpm, taking 150 mu L supernatant of the supernatant into another 96 deep-well plate filled with 350 mu L mixed organic solvent, carrying out vortex mixing for 10min, centrifuging the mixture at 20 ℃ for 5min at 3000rpm, and taking the supernatant as a test sample to be detected; wherein: the mixed organic solvent is acetonitrile: water: 1M ammonium acetate in a volume ratio of 5: 95: 0.1 mixing the obtained mixed solution;
(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 diclofenac acid and internal standard diclofenac-d 4 in the sample, and calculating the concentration of diclofenac acid in the plasma sample according to the chromatographic peaks;
the liquid chromatography determination conditions were: the chromatographic column is Agilent ZORBAX XDB-C18, and the specification of the column is 2.1 × 50 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: 1M ammonium acetate is 100: 1 mixing the obtained mixture; the mobile phase B is acetonitrile: water: 1M ammonium acetate is 70: 30: 0.1 mixing the obtained mixture; the washing liquid is acetonitrile: water is mixed according to the volume ratio of 50: 50 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-4500V, the atomizing temperature is 450 ℃, 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 Medium, and the declustering voltages of diclofenac and internal standard diclofenac-d 4 are both-20 eV; the entrance voltages of the collision chambers of the diclofenac and the internal standard diclofenac-d 4 are both-8 eV; the collision voltage of the diclofenac and the internal standard diclofenac-d 4 is-20 eV; the outlet voltages of the collision chambers of the diclofenac and the internal standard diclofenac-d 4 are both-20 eV; detecting in a negative ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z 293.8 → m/z 249.9, which is diclofenac; and m/z 299.8 → m/z 256.0, which is diclofenac-d 4.
Preferably, the gradient elution in step (2) is performed by the following procedure:
| total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.00 | 60.0 | 40.0 |
| 0.80 | 60.0 | 40.0 |
| 0.90 | 10.0 | 90.0 |
| 1.30 | 10.0 | 90.0 |
| 1.40 | 60.0 | 40.0 |
| 3.00 | 60.0 | 40.0 |
Preferably, in the step (2), the concentration of diclofenac in the plasma sample is calculated by adopting an internal standard method and substituting the peak area ratio of diclofenac to the internal standard diclofenac-d 4 into a standard curve equation.
Preferably, the establishment of the standard curve equation comprises the following steps:
placing ten blank plasma of 50 mu L in a 96-deep-well plate, and sequentially naming the blank plasma 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 diclofenac-d 4 solution and does not contain a diclofenac solution and is used for eliminating the interference of the internal standard diclofenac-d 4 solution on the detection result; the blank sample does not contain a diclofenac solution and an internal standard diclofenac-d 4 solution, and is used for eliminating the interference of used blank plasma on the detection result;
adding 5 mu L of diclofenac solution with the concentration of 0.04 ng/mu L, 0.2 ng/mu L, 1 ng/mu L, 2 ng/mu L, 10 ng/mu L, 20 ng/mu L, 40 ng/mu L and 60 ng/mu L to the lowest quantitative limit sample, the standard samples of 1-6 and the highest quantitative limit sample in the form of stock solution, and adding 5 mu L of diclofenac solution with the volume ratio of 1: 1, respectively mixing the ten samples, respectively adding 5 mu L of 8 ng/mu L internal standard diclofenac-d 4 solution into nine samples except a blank sample, and adding 5 mu L of 1: 1, uniformly mixing the ten samples, adding 500 mu L of acetonitrile into the ten samples, mixing for 1min in a vortex manner, centrifuging for 10min at 20 ℃ at 3000rpm, taking 150 mu L of supernatant to another 96 deep-hole plate filled with 350 mu L of mixed organic solvent, mixing for 10min in a vortex manner, centrifuging for 5min at 20 ℃ at 3000rpm, and taking the supernatant as ten standard samples to be detected; wherein: the mixed organic solvent is acetonitrile: water: 1M ammonium acetate in a volume ratio of 5: 95: 0.1 mixing the obtained mixed solution;
and respectively taking 10 mu L of standard samples, injecting the standard samples into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of the diclofenac acid and the internal standard diclofenac-d 4 in the samples, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of the diclofenac acid 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 45 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 5s 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 the diclofenac is about 0.955min, the retention time of the internal standard diclofenac-d 4 is about 0.937min, the peak shapes of the diclofenac and the internal standard diclofenac-d 4 are good, the measurement is free of the interference of the miscellaneous peaks, and the baseline is stable;
(3) the sensitivity is high: the minimum limit of plasma quantification is 2ng/mL, the concentration of diclofenac 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 measuring the blood concentration of the diclofenac. The linear range of the plasma standard curve of the method is 2-3000 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.
Drawings
FIG. 1 is a standard graph of diclofenac in human plasma 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 diclofenac-d 4 added to human blank plasma;
FIG. 4 is a HPLC-MS/MS graph of diclofenac and diclofenac-d 4 added to human blank plasma;
FIG. 5 is a HPLC-MS/MS chart of plasma samples with the addition of the internal standard diclofenac-d 4 after oral administration of diclofenac drug to healthy subjects.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example (b): human K2Determination of diclofenac concentration in EDTA plasma
First, the experimental materials and analytical equipment diclofenac (analyte) Toronto Research Chemicals or the same, higher-grade standard diclofenac-d 4 (internal standard) Toronto Research Chemicals or the same, higher-grade standard use reagents as 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 |
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 Agilent ZORBAX XDB-C18, and the specification of the column is 2.1 × 50 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: 1M ammonium acetate is 100: 1 mixing the obtained mixture; the mobile phase B is acetonitrile: water: 1M ammonium acetate is 70: 30: 0.1 mixing the obtained mixture; the washing liquid is acetonitrile: water is mixed according to the volume ratio of 50: 50 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 45 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 5s 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
| Total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.00 | 60.0 | 40.0 |
| 0.80 | 60.0 | 40.0 |
| 0.90 | 10.0 | 90.0 |
| 1.30 | 10.0 | 90.0 |
| 1.40 | 60.0 | 40.0 |
| 3.00 | 60.0 | 40.0 |
2. Conditions of Mass Spectrometry
The ion source is an electrospray ion source, the spraying voltage is-4500V, the atomizing temperature is 450 ℃, 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 Medium, and the declustering voltages of diclofenac and internal standard diclofenac-d 4 are both-20 eV; the entrance voltages of the collision chambers of the diclofenac and the internal standard diclofenac-d 4 are both-8 eV; the collision voltage of the diclofenac and the internal standard diclofenac-d 4 is-20 eV; the outlet voltages of the collision chambers of the diclofenac and the internal standard diclofenac-d 4 are both-20 eV; detecting in a negative ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z 293.8 → m/z 249.9, which is diclofenac; and m/z 299.8 → m/z 256.0, which is diclofenac-d 4.
Third, the experimental process
1. Preparation of diclofenac standard solution
The weighing and preparation process of the standard solution (containing stock solution and working solution) for the standard curve of diclofenac is as follows:
| weighing weight (mg) | Dissolved volume (μ l) | Final concentration (ng/. mu.l) |
| 5.145 | 10084 | 500 |
According to the preparation process, 500 ng/mu L of diclofenac sodium stock solution is obtained by methanol, and then the volume ratio of the diclofenac sodium stock solution to the diclofenac sodium stock solution is 1: 1, sequentially diluting the methanol aqueous solution to prepare a diclofenac standard solution, wherein the specific dilution concentration is shown in the following table 4:
TABLE 4 diclofenac standard solution formulation concentration
a: prepared directly from diclofenac (analyte)
The standard diclofenac solution is stored in plastic container and refrigerator (4 deg.C) when not in use, and the volume can be increased or decreased proportionally according to the requirement.
2. Preparation of standard solution of diclofenac-d 4 internal standard
The weighing and preparation process of the standard solution of diclofenac-d 4 internal standard is as follows:
| weighing weight (mg) | Dissolved volume (μ l) | Final concentration (ng/. mu.l) |
| 1.095 | 10092 | 100 |
Preparing 100 ng/mu L of diclofenac-d 4 stock solution according to the preparation process, and mixing the diclofenac-d 4 stock solution according to the volume ratio of 1: 1, the methanol aqueous solution is diluted to prepare an diclofenac-d 4 internal standard solution with the concentration of 8 ng/. mu.L, and the specific dilution concentration is shown in the following table 5:
TABLE 5 diclofenac-d 4 Standard solution preparation concentration
a: prepared directly from diclofenac-d 4 (internal standard)
b: for sample preparation procedures
The standard solution of diclofenac-d 4 internal standard is stored in plastic container and refrigerator (4 deg.C) 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 diclofenac 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 is As/Ai) of the peak area As of diclofenac acid and the peak area Ai of diclofenac-d 4 is calculated, and the peak area ratio Y is used for carrying out regression calculation on the blood concentration X, and the result is shown in a figure 1 and a 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 0.00315X +0.000336, r is 0.9995, and a weight coefficient W is 1/X2The lowest quantitative limit of the blood concentration of diclofenac measured by the method is as follows: 2 ng/mL.
TABLE 6 standard curve for diclofenac acid
b: diluted solution of analyte: MeOH/H2O＝50/50
Table 7 standard curve of diclofenac in human plasma by HPLC-MS/MS method (n-12)
4. Accuracy and precision
Putting the blank plasma into a water bath at room temperature for unfreezing; appropriate volumes of blank plasma were transferred to appropriate containers and diclofenac standard solution was added to prepare 5 drug-containing plasma quality control samples (LLOQ, QL, QLM, QM, QH) of different concentrations and a follow-up standard curve, which were prepared as shown in table 8 below, according to the "plasma sample pretreatment" procedure. 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 third batch, making 16 samples for each concentration of the second batch, calculating the ratio Y of the peak area As of the diclofenac acid and the peak area Ai of the internal standard diclofenac-d 4, 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 diclofenac 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 analysis batch.
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 the HPLC-MS/MS method for determining diclofenac acid in plasma
Note: the data of the evaluation results are from the data of 28 groups of quality control samples in 3 batches of 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 diclofenac analytes and the internal standard diclofenac-d 4.
After the plasma samples of blank healthy human from nine different sources were prepared and analyzed, the interference peak responses at the time of meeting the retention time of diclofenac were all less than 20.0% of the diclofenac response of the quantitative lower limit sample in the standard curve of the analysis batch, and the results are shown in table 10. The results show that the analysis method has specificity for analyzing diclofenac.
After the plasma samples of blank healthy persons from nine different sources were prepared and analyzed, the interference peak responses at retention times consistent with the internal standard diclofenac-d 4 were all less than 5.0% of the response of the internal standard diclofenac-d 4 of the lower limit quantification sample in the standard curve of the assay lot, see table 11 in the appendix. The results show that the analysis method has selectivity on the analysis of the internal standard diclofenac-d 4.
TABLE 10 comparison of interference data of blank healthy human plasma from nine different sources on diclofenac analytes
a: analyte peak area (selective sample)/analyte peak area (LLOQ of standard curve). times.100.0% to 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 diclofenac-d 4
a: the area of the peak of the analyte (selective sample)/the area of the peak of the internal standard (LLOQ of the standard curve) multiplied by 100.0 percent is less than or equal to 5.0 percent
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 diclofenac. Therefore, the method can be used for detecting the concentration of diclofenac in the plasma of different human bodies.
6. Detection of human plasma samples
(1) A blank plasma of human, to which diclofenac had not been administered, was taken, 100. mu.L of plasma samples were precisely added to a 96-deep well plate, and 10. mu.L of a mixture of 1: 1, adding 500 mu L of acetonitrile after uniformly mixing, carrying out vortex mixing for 1min, centrifuging for 10min at 20 ℃ at 3000rpm, taking 150 mu L of supernatant to another 96 deep-hole plate filled with 350 mu L of mixed organic solvent, carrying out vortex mixing for 10min, centrifuging for 5min at 20 ℃ at 3000rpm, 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 is acetonitrile: water: 1M ammonium acetate in a volume ratio of 5: 95: 0.1 mixing the resulting mixed solution.
(2) A blank plasma of human, to which diclofenac had not been administered, was taken, 100. mu.L of plasma samples were added precisely to a 96-deep well plate, and 5. mu.L of a mixture of plasma samples in a volume ratio of 1: 1, adding 5 mu L of 8 ng/mu L internal standard diclofenac-d 4 solution after uniformly mixing, adding 500 mu L acetonitrile after uniformly mixing, carrying out vortex mixing for 1min, centrifuging at 20 ℃ for 10min at 3000rpm, taking 150 mu L supernatant of the supernatant to another 96 deep-well plate filled with 350 mu L mixed organic solvent, carrying out vortex mixing for 10min, centrifuging at 20 ℃ for 5min at 3000rpm, taking 10 mu L sample, and carrying out LC-MS/MS analysis, wherein the representative map result is shown in figure 3; wherein: the mixed organic solvent is acetonitrile: water: 1M ammonium acetate in a volume ratio of 5: 95: 0.1 mixing the resulting mixed solution.
(3) Accurately adding 100 mu L of blank plasma sample into a 96 deep-well plate without diclofenac, adding 5 mu L of diclofenac standard solution, uniformly mixing, adding 5 mu L of 8 ng/mu L of internal standard diclofenac-d 4 solution, uniformly mixing, adding 500 mu L of acetonitrile, carrying out vortex mixing for 1min, centrifuging at 20 ℃ at 3000rpm for 10min, taking 150 mu L of supernatant into another 96 deep-well plate filled with 350 mu L of mixed organic solvent, carrying out vortex mixing for 10min, centrifuging at 20 ℃ at 3000rpm for 5min, taking 10 mu L of sample, and carrying out LC-MS/MS analysis, wherein the representative map result is shown in figure 4; wherein: the mixed organic solvent is acetonitrile: water: 1M ammonium acetate in a volume ratio of 5: 95: 0.1 mixing the resulting mixed solution.
(4) Plasma of healthy subjects after oral administration of diclofenac or pharmaceutically acceptable salts thereof is collected, 100 μ L of collected human plasma samples are precisely added to a 96-deep-well plate, and 5 μ L of a sample solution with a volume ratio of 1: 1, adding 5 mu L of 8 ng/mu L internal standard diclofenac-d 4 solution after uniformly mixing, adding 500 mu L acetonitrile after uniformly mixing, carrying out vortex mixing for 1min, centrifuging at 20 ℃ for 10min at 3000rpm, taking 150 mu L supernatant of the supernatant to another 96 deep-well plate filled with 350 mu L mixed organic solvent, carrying out vortex mixing for 10min, centrifuging at 20 ℃ for 5min at 3000rpm, taking 10 mu L sample, and carrying out LC-MS/MS analysis, wherein the representative map result is shown in figure 5; wherein: the mixed organic solvent is acetonitrile: water: 1M ammonium acetate in a volume ratio of 5: 95: 0.1 mixing the resulting mixed solution.
In conclusion, the invention provides a simple and convenient method for measuring the concentration of diclofenac in blood plasma by a pretreatment method, adopts a protein precipitation method, and is suitable for conventional measurement; meanwhile, under the chromatographic conditions adopted in the experiment, the retention time of the diclofenac is about 0.937min, the retention time of the internal standard diclofenac-d 4 is about 0.955min, the peak shapes of the diclofenac and the internal standard diclofenac-d 4 are good, the measurement is free of the interference of miscellaneous peaks, and the baseline is stable; the method has higher specificity, can accurately measure the concentration of the diclofenac in the plasma, has higher sensitivity, and has the minimum limit of plasma quantification of 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 diclofenac. The linear range of the plasma standard curve of the method is 2-3000 ng/mL, and the precision RSD in batch and between batches is less than +/-15%.
Claims (5)
1. A method for determining the concentration of diclofenac in 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, diclofenac-d 4 as internal standard; precisely adding 100 μ L of plasma sample into a 96-deep-well plate, adding 5 μ L of a volume ratio of 1: 1, uniformly mixing the methanol aqueous solution, adding 5 mu L of 8 ng/mu L internal standard diclofenac-d 4 solution, uniformly mixing the solution, adding 500 mu L acetonitrile, carrying out vortex mixing for 1min, centrifuging the mixture at 20 ℃ for 10min at 3000rpm, taking 150 mu L supernatant of the supernatant into another 96 deep-well plate filled with 350 mu L mixed organic solvent, carrying out vortex mixing for 10min, centrifuging the mixture at 20 ℃ for 5min at 3000rpm, and taking the supernatant as a test sample to be detected; wherein: the mixed organic solvent is acetonitrile: water: 1M ammonium acetate in a volume ratio of 5: 95: 0.1 mixing the obtained mixed solution;
(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 diclofenac acid and internal standard diclofenac-d 4 in the sample, and calculating the concentration of diclofenac acid in the plasma sample according to the chromatographic peaks;
the liquid chromatography determination conditions were: the chromatographic column is Agilent ZORBAX XDB-C18, and the specification of the column is 2.1 × 50 mm; the temperature of the chromatographic column is 40 ℃; the mobile phase A is water: 1M ammonium acetate is 100: 1 mixing the obtained mixture; the mobile phase B is acetonitrile: water: 1M ammonium acetate is 70: 30: 0.1 mixing the obtained mixture; the washing liquid is acetonitrile: water is mixed according to the volume ratio of 50: 50 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-4500V, the atomizing temperature is 450 ℃, 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 Medium, and the declustering voltages of diclofenac and internal standard diclofenac-d 4 are both-20 eV; the entrance voltages of the collision chambers of the diclofenac and the internal standard diclofenac-d 4 are both-8 eV; the collision voltage of the diclofenac and the internal standard diclofenac-d 4 is-20 eV; the outlet voltages of the collision chambers of the diclofenac and the internal standard diclofenac-d 4 are both-20 eV; detecting in a negative ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z 293.8 → m/z 249.9, which is diclofenac; and m/z 299.8 → m/z 256.0, which is diclofenac-d 4.
2. The method for determining diclofenac concentration in plasma by LC-MS as claimed in claim 1, wherein: the gradient elution procedure in the step (2) is as follows:
3. the method for determining diclofenac concentrations in plasma by LC-MS according to claim 1 or 2, characterized in that: in the step (2), an internal standard method is adopted, and the peak area ratio of the diclofenac to the internal standard diclofenac-d 4 is substituted into a standard curve equation to calculate the concentration of the diclofenac in the plasma sample.
4. The method for determining diclofenac concentration in plasma by LC-MS as claimed in 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-well 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 diclofenac-d 4 solution and does not contain a diclofenac solution and is used for eliminating the interference of the internal standard diclofenac-d 4 solution on a detection result; the blank sample does not contain a diclofenac solution and an internal standard diclofenac-d 4 solution, and is used for eliminating the interference of used blank plasma on the detection result;
adding 5 mu L of diclofenac solution with the concentration of 0.04 ng/mu L, 0.2 ng/mu L, 1 ng/mu L, 2 ng/mu L, 10 ng/mu L, 20 ng/mu L, 40 ng/mu L and 60 ng/mu L to the lowest quantitative limit sample, the standard samples of 1-6 and the highest quantitative limit sample in the form of stock solution, and adding 5 mu L of diclofenac solution with the volume ratio of 1: 1, respectively mixing the ten samples, respectively adding 5 mu L of 8 ng/mu L internal standard diclofenac-d 4 solution into nine samples except a blank sample, and adding 5 mu L of 1: 1, uniformly mixing the ten samples, adding 500 mu L of acetonitrile into the ten samples, mixing for 1min in a vortex manner, centrifuging for 10min at 20 ℃ at 3000rpm, taking 150 mu L of supernatant to another 96 deep-hole plate filled with 350 mu L of mixed organic solvent, mixing for 10min in a vortex manner, centrifuging for 5min at 20 ℃ at 3000rpm, and taking the supernatant as ten standard samples to be detected; wherein: the mixed organic solvent is acetonitrile: water: 1M ammonium acetate in a volume ratio of 5: 95: 0.1 mixing the obtained mixed solution;
and respectively taking 10 mu L of standard samples, injecting the standard samples into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of the diclofenac acid and the internal standard diclofenac-d 4 in the samples, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of the diclofenac acid in the plasma.
5. The method for determining diclofenac concentrations in plasma by LC-MS according to claim 1 or 2, characterized in that: 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 45 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 5s 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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