CN110927306A - Method for determining concentration of ibuprofen in blood plasma by liquid chromatography-mass spectrometry - Google Patents
Method for determining concentration of ibuprofen in blood plasma by liquid chromatography-mass spectrometry Download PDFInfo
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Abstract
The invention discloses a method for determining ibuprofen concentration in blood plasma by liquid chromatography-mass spectrometry, which adopts a liquid chromatography-mass spectrometry system to determine, and 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 ibuprofen; the plasma standard curve linear range of the method is 0.1-40 mu g/mL, the precision RSD in batch and between batches is less than +/-15%, and the method is suitable for measuring the concentration of ibuprofen 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 ibuprofen in blood plasma by liquid chromatography-mass spectrometry.
Background
Ibuprofen (Ibuprofen) is the only commonly recommended antipyretic for children by the world health organization and the FDA in the United states, is a well-known first-choice nonsteroidal anti-inflammatory drug for children, and has the effects of resisting inflammation, relieving pain and relieving fever. The chemical name is 2-methyl-4- (2-methylpropyl) phenylacetic acid, and the molecular formula is as follows: c13H18O2Molecular weight: 206.29, having the chemical formula:
non-steroidal anti-inflammatory drugs (NSAIDs) are anti-inflammatory drugs that do not contain a steroidal structure, and the drugs exert their antipyretic, analgesic, and anti-inflammatory effects by inhibiting the synthesis of prostaglandins. Currently, the non-steroidal anti-inflammatory drug is one of the most used drugs in the world, is the first choice drug for treating rheumatoid arthritis, and is used by about 3000 million people every day in the world. Ibuprofen has been used clinically as an anti-inflammatory analgesic for over 30 years and is considered to be the safest and most effective of the non-steroidal anti-inflammatory drugs. Ibuprofen can stimulate the cellular immune function of human body and experimental animals, increase the activity of natural killer cells and improve the anti-infection capability.
At present, the speed, the precision, the sensitivity and the selectivity of the existing ibuprofen determination method are all to be improved.
Disclosure of Invention
The invention aims to provide a method for determining the concentration of ibuprofen in blood plasma by liquid chromatography-mass spectrometry, which can improve the detection speed, accuracy and sensitivity.
In order to realize the aim, the method for determining the concentration of ibuprofen in plasma by liquid chromatography-mass spectrometry comprises the following steps of:
(1) pretreatment of a plasma sample:
plasma with K2EDTA as anticoagulant, ibuprofen-d 3 as internal standard; precisely adding 100 μ L of plasma sample into a 96-deep-well plate, adding 5 μ L of plasma sample at a volume ratio of 1: 1, uniformly mixing, adding 5 mu L of 0.02 mu g/mu L internal standard ibuprofen-d 3 solution, uniformly mixing, adding 500 mu L of acetonitrile into a 96 deep-well plate, carrying out vortex mixing for 1min, centrifuging at 20 ℃ at 3000rpm for 10min, taking 70 mu L of supernatant into the 96 deep-well plate filled with 500 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: 1mol/L of ammonium acetate according to the volume ratio of 20: 80: 0.1, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ and 3000rpm for 5min, and taking the mixture as a test sample to be detected;
(2) and (3) sample measurement:
injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of ibuprofen and internal standard ibuprofen-d 3 in the sample, and calculating the concentration of ibuprofen in the plasma sample according to the chromatographic peaks;
(3) liquid chromatography determination, conditions are as follows:
a chromatographic column: agilent ZORBAX XDB-C18, 5 μm, column specification 50X 2.1 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume ratio of water to 1mol/L ammonium acetate is 100/0.1; mobile phase B: the volume ratio of acetonitrile/water/1 mol/L ammonium acetate is 70/30/0.1; washing liquid: the volume ratio of acetonitrile/water is 50/50; the autosampler temperature was 15 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10uL and analysis time of 3.5 min;
(4) mass spectrometry under the conditions:
the ion source is an electrospray ion source, the spraying voltage is-4500V, the atomizing temperature is 450 ℃, the spraying air pressure is 10Psi, the auxiliary heating air pressure is 10Psi, the air curtain air pressure is 20Psi, the collision air pressure is 8Psi, and the cluster removing voltage is ibuprofen of-25 eV and ibuprofen-d 3 respectively; the inlet voltage of the collision chamber is ibuprofen with the voltage of-10 eV and ibuprofen-d 3; ibuprofen and ibuprofen-d 3 with collision voltages of-10 eV respectively; the outlet voltage of the collision chamber is ibuprofen with the voltage of-10 eV and ibuprofen-d 3; detecting in a negative ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z205.1 → m/z160.9, which is ibuprofen; and m/z208.1 → m/z164.0, which is ibuprofen-d 3.
Preferably, the gradient elution in step (3) is performed by the following procedure:
preferably, in the step (2), the concentration of ibuprofen in the plasma sample is calculated by adopting an internal standard method and substituting the peak area ratio of ibuprofen and internal standard ibuprofen-d 3 into a standard curve equation.
Preferably, the establishment of the standard curve equation comprises the following steps:
taking 10 parts of 100 mu L blank plasma, placing the blank plasma in a 96-deep-well plate, adding 5 mu L of ibuprofen solution with the concentration of 0.002 mu g/mu L, 0.004 mu g/mu L, 0.01 mu g/mu L, 0.02 mu g/mu L, 0.1 mu g/mu L, 0.2 mu g/mu L, 0.5 mu g/mu L and 0.8 mu g/mu L to the lowest quantitative lower limit sample, the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6 and the highest quantitative upper limit sample in the form of stock solution, respectively adding 5 mu L of the ibuprofen solution with the volume ratio of 1: 1 to a blank sample and a zero-concentration sample, respectively adding 5 mu L of 0.02 mu g/mu L internal standard ibuprofen-d 3 solution into the lowest quantitative lower limit sample, the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6, the highest quantitative upper limit sample and the zero-concentration sample after uniformly mixing, and adding 5 mu L of the internal standard ibuprofen-d 3 solution into the blank sample according to the volume ratio of 1: 1, after uniformly mixing, respectively adding 500 mu L of acetonitrile into 10 samples, mixing in a 96 deep-well plate in a vortex mode for 1min, centrifuging at the temperature of 20 ℃ at 3000rpm for 10min, respectively taking 70 mu L of supernatant liquid, and putting the supernatant liquid into the 96 deep-well plate filled with 500 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: 1mol/L of ammonium acetate according to the volume ratio of 20: 80: 0.1, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ at 3000rpm for 5min, and taking the mixture as 10 parts of standard sample to be detected;
and respectively injecting 10 mu L of standard sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of ibuprofen and internal standard ibuprofen-d 3 in the sample, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of ibuprofen in the plasma.
Compared with the prior art, the invention has the following advantages:
(1) the pretreatment method is simple and convenient, two-step organic solution extraction is adopted, and the method is suitable for conventional determination;
(2) the specificity is strong: under the chromatographic conditions adopted in the experiment, the retention time of ibuprofen is about 1.368min, and the retention time of internal standard ibuprofen-d 3 is about 1.363 min. The peak shapes of the ibuprofen and the internal standard ibuprofen-d 3 are good, the measurement is free from the interference of miscellaneous peaks, and the base line is stable;
(3) the sensitivity is high: the minimum limit of quantitation of the plasma is 0.1 mug/mL, the concentration of ibuprofen 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 ibuprofen. The linear range of the plasma standard curve of the method is 0.1-40 mu g/mL, and the precision RSD in batch and between batches is less than +/-15%.
Drawings
FIG. 1 is a standard graph of ibuprofen 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 plot of ibuprofen and ibuprofen-d 3 added to human blank plasma;
figure 4 is a HPLC-MS/MS plot of plasma samples after oral administration of ibuprofen or a pharmaceutically acceptable salt thereof to healthy subjects followed by the addition of the internal standard ibuprofen-d 3.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example (b): human K2Determination of ibuprofen concentration in EDTA plasma
First, experimental material and analytical equipment
Ibuprofen (analyte): USP or equivalent, higher grade standards
Ibuprofen-d 3 (internal standard): TLC Pharmaceutical Standards or equivalent, higher-grade Standards
The reagents used are shown in table 1 below:
TABLE 1 details of reagents
| Name of reagent | Rank of | Manufacturer(s) |
| Acetonitrile | HPLC | J.T.Baker |
| Methanol | HPLC | J.T.Baker |
| Ammonium acetate | 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
A chromatographic column: agilent ZORBAX XDB-C18, 5 μm, column specification 50X 2.1 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume ratio of water to 1mol/L ammonium acetate is 100/0.1; mobile phase B: the volume ratio of acetonitrile/water/1 mol/L ammonium acetate is 70/30/0.1; washing liquid: the volume ratio of acetonitrile/water is 50/50; the autosampler temperature was 15 ℃; gradient elution, flow rate of 0.4mL/min, sample size of 10uL, analysis time of 3.5 min.
TABLE 3 gradient elution procedure
| Step (ii) of | Total time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 1 | 0 | 60 | 40 |
| 2 | 0.9 | 60 | 40 |
| 3 | 0.91 | 0 | 100 |
| 4 | 1.50 | 0 | 100 |
| 5 | 1.51 | 60 | 40 |
| 6 | 3.50 | 60 | 40 |
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 10Psi, the auxiliary heating air pressure is 10Psi, the air curtain air pressure is 20Psi, the collision air pressure is 8Psi, and the cluster removing voltage is ibuprofen of-25 eV and ibuprofen-d 3 respectively; the inlet voltage of the collision chamber is ibuprofen with the voltage of-10 eV and ibuprofen-d 3; ibuprofen and ibuprofen-d 3 with collision voltages of-10 eV respectively; the outlet voltage of the collision chamber is ibuprofen with the voltage of-10 eV and ibuprofen-d 3; detecting in a negative ion mode; the scanning mode is multiple reaction monitoring; the ion reactions for quantitative analysis were: m/z205.1 → m/z160.9, which is ibuprofen; and m/z208.1 → m/z164.0, which is ibuprofen-d 3.
Third, the experimental process
1. Preparation of ibuprofen standard solution
Preparing an ibuprofen standard solution: accurately weighing 0.05g of ibuprofen (analyte), placing the ibuprofen into a 10mL volumetric flask, adding methanol to dissolve the ibuprofen, fixing the volume to a scale, shaking up the solution to obtain 5 mu g/mu L of ibuprofen stock solution, and then mixing the ibuprofen stock solution with the volume ratio of 1: 1, sequentially diluting the methanol aqueous solution to prepare an ibuprofen standard solution, wherein the specific dilution concentration is shown in the following table 4:
table 4 ibuprofen standard solution preparation concentration
| Source solution (μ g/. mu.L) | Volume of source solution (μ L) | Final volume (mL) | Final concentration (μ g/. mu.L) |
| 5a | 1600 | 10 | 0.8 |
| 5a | 1280 | 10 | 0.64 |
| 5a | 1000 | 10 | 0.5 |
| 5a | 800 | 10 | 0.4 |
| 5a | 400 | 10 | 0.2 |
| 5a | 200 | 10 | 0.1 |
| 5a | 40 | 10 | 0.02 |
| 0.5 | 200 | 10 | 0.01 |
| 0.5 | 120 | 10 | 0.006 |
| 0.5 | 80 | 10 | 0.004 |
| 0.5 | 40 | 10 | 0.002 |
a: directly from ibuprofen (analyte)
The ibuprofen 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 ibuprofen-d 3 internal standard solution
Preparation of ibuprofen-d 3 internal standard solution: accurately weighing 0.01g of ibuprofen-d 3 (internal standard), placing the weighed solution in a 10mL volumetric flask, adding methanol to dissolve the solution and fixing the volume to scale, shaking up the solution to obtain 1 mu g/mu L of ibuprofen-d 3 stock solution, and then mixing the solution in a volume ratio of 1: 1, diluting the methanol aqueous solution to prepare an internal standard solution with the concentration of 2ng/uL ibuprofen-d 3, wherein the specific dilution concentration is shown in the following table 5:
TABLE 5 ibuprofen-d 3 Standard solution preparation concentration
| Source solution (μ g/. mu.L) | Volume of source solution (μ L) | Final volume (mL) | Final concentration (μ g/. mu.L) |
| 1a | 1000 | 50 | 0.02b |
a: prepared directly from ibuprofen-d 3 (internal standard)
b: for sample preparation procedures
The standard solution of ibuprofen-d 3 internal standard was stored in plastic containers and in a refrigerator (4 ℃) when not in use, and the volume was scaled up or down as required.
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 ibuprofen standard solution or diluted solution with different concentrations according to the list in the following table 6 to prepare each sample, uniformly mixing the samples to prepare drug-containing plasma with different concentrations, and carrying out the operation according to 'plasma sample pretreatment'. The ratio Y (Y As/Ai) of the peak area As of ibuprofen and the peak area Ai of ibuprofen-d 3 in the internal standard 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. And performing regression calculation on the blood concentration X by using the average ratio Y to obtain a regression equation Y which is 0.756X-0.00527 and r which is 0.9988. The weight coefficient W is 1/X, and the lowest quantitative limit of the blood concentration of the ibuprofen measured by the method is as follows: 0.1. mu.g/mL.
Table 6 ibuprofen Standard Curve formulation concentrations
b: diluted solution of analyte: MeOH/H2O＝50/50
TABLE 7 Standard Curve of ibuprofen in human plasma by HPLC-MS/MS (n ═ 13)
4. Accuracy and precision
Putting the blank plasma into a water bath at room temperature for unfreezing; the appropriate volume of blank plasma was transferred to the appropriate container and ibuprofen 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 second batch, making 14 samples for each concentration of the third batch, calculating the ratio Y of the peak area As of ibuprofen and the peak area Ai of the internal standard ibuprofen-d 3, 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 accuracy and precision of the ibuprofen plasma sample in batch and between batches are less than +/-15 percent and meet the requirements.
TABLE 8 quality control sample preparation concentration
a: final volume is source solution volume + plasma volume
Sufficient volume was dispensed into the labeled sample vials as required for each assay batch and stored at the theoretical temperature-80 ℃. The volume may be scaled up or down as desired.
TABLE 9 Intra-batch, inter-batch precision and accuracy of HPLC-MS/MS method for determination of ibuprofen in plasma
a-out of standard but in calculations.
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 ibuprofen analyte and the internal standard ibuprofen-d 3.
After the analysis of the nine different sources of blank healthy human plasma samples, the interference peak responses at the retention times corresponding to ibuprofen were all less than 20.0% of the ibuprofen response of the lower limit quantitation 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 ibuprofen analysis.
After the analysis of the nine blank healthy human plasma samples from different sources, the interference peak responses at the retention times corresponding to the internal standards were all less than 5.0% of the internal standard response of the quantitative lower limit sample in the standard curve of the analysis lot, see table 11 in the appendix. The results show that the assay is selective for the analysis of internal standards.
TABLE 10 comparison of interference data on ibuprofen analyte from nine different sources of blank healthy human plasma
a: analyte peak area (selective sample)/analyte peak area (LLOQ of standard curve)
×100.0％≤20.0％
b: the area peak area is considered zero when "no significant peak can be integrated (or no peak)" or "the retention time of the peak area does not match the retention time of the analyte in the sample".
TABLE 11 comparison of interference data of blank healthy human plasma from nine different sources against internal standard ibuprofen-d 3
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 ibuprofen. Therefore, the method can be used for detecting the ibuprofen concentration in different human plasmas.
6. Human plasma sample detection
(1) Human blank plasma without ibuprofen administration was taken and 100 μ L of blank plasma samples were added to a 96-well plate with precision, 10 μ L of the blank plasma samples being added in a volume ratio of 1: 1, adding 500 mu L of acetonitrile into a 96 deep-hole plate after uniformly mixing, carrying out vortex mixing for 1min, centrifuging for 10min at 20 ℃ at 3000rpm, taking 70 mu L of supernatant liquid into the 96 deep-hole plate filled with 500 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: 1mol/L of ammonium acetate according to the volume ratio of 20: 80: 0.1, vortex the resulting mixture, centrifuge at 3000rpm for 5min at 20 ℃ and then take 10uL of sample for LC-MS/MS analysis, the results are shown in FIG. 2.
(2) Taking human blank plasma without ibuprofen, precisely adding 100 mu L of blank plasma sample into a 96-deep-well plate, adding 5uL of ibuprofen standard solution with the concentration of 0.02 mu g/mu L, uniformly mixing, adding 5 mu L of 0.02 mu g/mu L of internal standard ibuprofen-d 3 solution, uniformly mixing, adding 500 mu L of acetonitrile into the 96-deep-well plate, vortex mixing for 1min, centrifuging at 20 ℃ at 3000rpm for 10min, taking 70 mu L of supernatant into the 96-deep-well plate filled with 500 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: 1mol/L of ammonium acetate according to the volume ratio of 20: 80: 0.1, vortex the resulting mixture, centrifuge at 3000rpm for 5min at 20 ℃ and then take 10uL of sample for LC-MS/MS analysis, the results are shown in FIG. 3.
(3) Plasma of healthy subjects after oral administration of ibuprofen or pharmaceutically acceptable salts thereof is collected, 100 mul of collected human plasma samples are precisely added into a 96-deep-well plate, and 5 mul of plasma samples are added, wherein the volume ratio of the samples is 1: 1, uniformly mixing, adding 5 mu L of 0.02 mu g/mu L internal standard ibuprofen-d 3 solution, uniformly mixing, adding 500 mu L of acetonitrile into a 96 deep-well plate, carrying out vortex mixing for 1min, centrifuging at 20 ℃ at 3000rpm for 10min, taking 70 mu L of supernatant into the 96 deep-well plate filled with 500 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: 1mol/L of ammonium acetate according to the volume ratio of 20: 80: 0.1, vortex the resulting mixture, centrifuge at 3000rpm for 5min at 20 ℃ and then take 10uL of sample for LC-MS/MS analysis, the results are shown in FIG. 4.
In conclusion, the invention provides a simple and convenient method for determining the concentration of ibuprofen in blood plasma by a pretreatment method, adopts a two-step organic solution extraction method, and is suitable for conventional determination; meanwhile, under the chromatographic conditions adopted in the experiment, the retention time of ibuprofen is about 1.368min, the retention time of internal standard ibuprofen-d 3 is about 1.363min, the peak shapes of ibuprofen and internal standard ibuprofen-d 3 are good, the measurement is free of the interference of miscellaneous peaks, and the base line is stable; the method has high specificity, can accurately measure the concentration of ibuprofen in blood plasma, and has high sensitivity, and the minimum quantitative limit of the blood plasma is 0.1 mu g/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 ibuprofen. The linear range of the plasma standard curve of the method is 0.1-40 mu g/mL, and the precision RSD in batch and between batches is less than +/-15%.
Claims (4)
1. A method for determining ibuprofen 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) pretreatment of a plasma sample:
plasma with K2EDTA as anticoagulant, ibuprofen-d 3 as internal standard; precision machining in 96 deep-hole plateTo 100 μ L of plasma sample, 5 μ L of a volume ratio of 1: 1, uniformly mixing, adding 5 mu L of 0.02 mu g/mu L internal standard ibuprofen-d 3 solution, uniformly mixing, adding 500 mu L of acetonitrile into a 96 deep-well plate, carrying out vortex mixing for 1min, centrifuging at 20 ℃ at 3000rpm for 10min, taking 70 mu L of supernatant into the 96 deep-well plate filled with 500 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: 1mol/L of ammonium acetate according to the volume ratio of 20: 80: 0.1, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ and 3000rpm for 5min, and taking the mixture as a test sample to be detected;
(2) and (3) sample measurement:
injecting 10 mu L of test sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of ibuprofen and internal standard ibuprofen-d 3 in the sample, and calculating the concentration of ibuprofen in the plasma sample according to the chromatographic peaks;
(3) liquid chromatography determination, conditions are as follows:
a chromatographic column: agilent ZORBAX XDB-C18, 5 μm, column specification 50X 2.1 mm; temperature of the chromatographic column: 40 ℃; mobile phase A: the volume ratio of water to 1mol/L ammonium acetate is 100/0.1; mobile phase B: the volume ratio of acetonitrile/water/1 mol/L ammonium acetate is 70/30/0.1; washing liquid: the volume ratio of acetonitrile/water is 50/50; the autosampler temperature was 15 ℃; gradient elution with flow rate of 0.4mL/min, sample size of 10uL and analysis time of 3.5 min;
(4) mass spectrometry under the conditions:
the ion source is an electrospray ion source, the spraying voltage is-4500V, the atomizing temperature is 450 ℃, the spraying air pressure is 10Psi, the auxiliary heating air pressure is 10Psi, the air curtain air pressure is 20Psi, the collision air pressure is 8Psi, and the cluster removing voltage is ibuprofen of-25 eV and ibuprofen-d 3 respectively; the inlet voltage of the collision chamber is ibuprofen with the voltage of-10 eV and ibuprofen-d 3; ibuprofen and ibuprofen-d 3 with collision voltages of-10 eV respectively; the outlet voltage of the collision chamber is ibuprofen with the voltage of-10 eV and ibuprofen-d 3; detecting in a negative ion mode; the scanning mode is multiple reaction monitoring;
the ion reactions for quantitative analysis were: m/z205.1 → m/z160.9, which is ibuprofen; and m/z208.1 → m/z164.0, which is ibuprofen-d 3.
2. The method for determining ibuprofen concentration in plasma according to claim 1, which comprises the following steps: the gradient elution procedure in the step (3) is as follows:
3. the method for the LC-MS measurement of ibuprofen concentration in plasma according to claim 1 or 2, wherein: in the step (2), an internal standard method is adopted, and the concentration of ibuprofen in the plasma sample is calculated by taking the peak area ratio of ibuprofen and internal standard ibuprofen-d 3 into a standard curve equation.
4. The method for determining ibuprofen concentration in plasma according to claim 3, wherein: the establishment of the standard curve equation comprises the following steps:
taking 10 parts of 100 mu L blank plasma, placing the blank plasma in a 96-deep-well plate, adding 5 mu L of ibuprofen solution with the concentration of 0.002 mu g/mu L, 0.004 mu g/mu L, 0.01 mu g/mu L, 0.02 mu g/mu L, 0.1 mu g/mu L, 0.2 mu g/mu L, 0.5 mu g/mu L and 0.8 mu g/mu L to the lowest quantitative lower limit sample, the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6 and the highest quantitative upper limit sample in the form of stock solution, respectively adding 5 mu L of the ibuprofen solution with the volume ratio of 1: 1 to a blank sample and a zero-concentration sample, respectively adding 5 mu L of 0.02 mu g/mu L internal standard ibuprofen-d 3 solution into the lowest quantitative lower limit sample, the standard sample 1, the standard sample 2, the standard sample 3, the standard sample 4, the standard sample 5, the standard sample 6, the highest quantitative upper limit sample and the zero-concentration sample after uniformly mixing, and adding 5 mu L of the internal standard ibuprofen-d 3 solution into the blank sample according to the volume ratio of 1: 1, after uniformly mixing, respectively adding 500 mu L of acetonitrile into 10 samples, mixing in a 96 deep-well plate in a vortex mode for 1min, centrifuging at the temperature of 20 ℃ at 3000rpm for 10min, respectively taking 70 mu L of supernatant liquid, and putting the supernatant liquid into the 96 deep-well plate filled with 500 mu L of mixed organic solvent, wherein the mixed organic solvent is acetonitrile: water: 1mol/L of ammonium acetate according to the volume ratio of 20: 80: 0.1, mixing the obtained mixture, uniformly mixing the mixture by vortex, centrifuging the mixture at the temperature of 20 ℃ at 3000rpm for 5min, and taking the mixture as 10 parts of standard sample to be detected;
and respectively injecting 10 mu L of standard sample into a high performance liquid chromatography-tandem mass spectrometer, detecting chromatographic peaks of ibuprofen and internal standard ibuprofen-d 3 in the sample, and obtaining a standard curve according to the chromatographic peaks so as to calculate the concentration of ibuprofen in the plasma.
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