Method for detecting ketoprofen in human plasma through HPLC-MS / MS combination

Abstract

The invention provides a method for detecting ketoprofen in human plasma by HPLC-MS / MS (High Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry). The method comprises the following steps: (1) pretreating a human plasma sample; (2) carrying out liquid chromatography-mass spectrometry detection; a mobile phase A and a mobile phase B are adopted as a mixed mobile phase for gradient elution, and the mobile phase A is acetonitrile; the mobile phase B is a formic acid aqueous solution with the concentration of 0.05-0.20%; and (3) determining ketoprofen in the human plasma. According to the method, Agilent and ZORBAX Eclipse XDB-Phenyl are adopted as chromatographic columns, a specific mobile phase is screened out, the proportion and time of the mobile phase in the gradient elution process are optimized, and the method has the advantages of being good in reproducibility, high in sensitivity, high in analysis speed, small in matrix effect influence, high in recovery rate and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioanalysis, and particularly relates to a method for detecting ketoprofen in human plasma by HPLC-MS / MS coupling. Background Art

[0002] Pain is the most prominent symptom of musculoskeletal diseases and usually requires treatment with analgesics and non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs mainly play an analgesic and anti-inflammatory role by inhibiting cyclooxygenase (COX) required for the conversion of arachidonic acid (AA) into prostaglandin (PG) and thromboxane A2 (TAX2), thereby reducing the generation of inflammatory mediators.

[0003] Systemic (oral or intravenous administration) application of NSAIDs has good efficacy for musculoskeletal pain, but its safety and tolerance issues have been widely concerned. Systemic application of NSAIDs has many adverse reactions, the most common being gastrointestinal reactions, and other adverse reactions including: cardiovascular adverse reactions, effects on platelet aggregation function, effects on renal function (water and sodium retention, renal insufficiency), effects on liver function, etc. Topical NSAIDs, compared with oral or intravenous administration routes, can be directly applied to the skin of the lesion site, penetrate through the skin to reach the painful tissue and play an analgesic role, with advantages such as fast onset, high local concentration, low systemic exposure, and few systemic adverse reactions, and are more suitable for the treatment of acute and chronic pain of the musculoskeletal system.

[0004] Ketoprofen is a phenylpropionic acid non-steroidal anti-inflammatory drug, whose analgesic effect is better than that of similar drugs and the duration of action is long, the anti-inflammatory effect is stronger than that of ibuprofen, the adverse reactions are small, the toxicity is low, and the safety and tolerance are good. Ketoprofen is easily absorbed from the gastrointestinal tract orally, and its absorption process is affected by food, with low oral bioavailability and large individual differences in blood drug concentration, a half-life of 1.6 - 1.9 h, a short duration of maintaining the effective blood drug concentration, and continuous medication having a stimulating effect on the gastrointestinal tract. Long-term use will cause symptoms such as digestive tract ulcers and bleeding. At present, the marketed dosage forms at home and abroad mainly include tablets, capsules, injections, suppositories, topical lotions and other dosage forms. In order to increase the solubility of the drug, reduce adverse reactions and improve bioavailability, in recent years, domestic and foreign scholars have conducted in-depth research on the new dosage forms of ketoprofen and their preparation processes. At present, it has been confirmed that the sustained-release dosage form and transdermal topical dosage form of ketoprofen can effectively avoid adverse stimulation to the gastrointestinal tract and have better therapeutic effects. Existing literature shows that ketoprofen gel patch (cataplasm), this new type of transdermal preparation not only has the advantages of avoiding the first-pass effect of the liver, reducing gastrointestinal irritation, convenient administration, and being able to stop medication at any time of the transdermal drug delivery system; but also can overcome the disadvantages of traditional patches such as allergy, sticking to hair, and soiling clothes; in addition, it also has the characteristics of small irritation, reducing drug adverse reactions, good air permeability, large drug loading capacity, mild viscosity, soft texture, and being easy to stick repeatedly, greatly improving the medication compliance of patients and being worthy of popularization and application.

[0005] At present, domestic literature has reported a method for determining the content of dexketoprofen in human plasma by liquid chromatography-mass spectrometry. For example, the document Detection of Dexketoprofen Concentration in Human Plasma by LC-MS (Song Ying, Jia Yanyan, Ge Jie (Department of Pharmacy, First Affiliated Hospital of Fourth Military Medical University, Xi'an 710032) discloses a method for detecting dexketoprofen in plasma. However, the non-deuterated internal standard febuxostat is used as the internal standard in the detection method, and the matrix effect of the analyte and the internal standard cannot be controlled to be the same, which may cause the sample test result to be different from the true value; the plasma dosage in the detection method of the document is 200 μL, and the detection method of the present invention is 100 μL. The amount of plasma used is only 50 μL, and the amount of plasma used in the present invention is less, which can reduce the amount of blood collected during clinical blood collection; the minimum quantitative concentration of dexketoprofen in the detection method of the document is 10 ng / mL, while the minimum quantitative concentration of ketoprofen in the invention is 0.1 ng / mL; in the detection method of the document, the supernatant is taken after protein precipitation and directly sampled and analyzed, while in the invention, the supernatant is taken after protein precipitation and then mixed with 100 μL of diluent (the volume ratio of acetonitrile and water is 50:50) before sampled and analyzed.

[0006] Chinese patent CN 108828094 discloses a method and application of detecting ketoprofen in plasma using high performance liquid chromatography-tandem mass spectrometry. In the detection method, the lower limit of quantification of ketoprofen is 5ng / mL, and the sensitivity is low, which cannot meet the needs of biological sample analysis of new drugs with a lower pharmacokinetic range and bioequivalence tests. Secondly, the amount of plasma used in the pretreatment process is 500μL, and the blood collection volume required in the clinical blood collection process is relatively large. In addition, the analysis time in the detection method is 12min, and the cycle of large-scale sample testing in clinical trials is relatively long.

[0007] In order to meet the needs of large-scale clinical sample analysis and evaluation of drug bioequivalence, it is necessary to develop simpler, more reliable, and high-throughput sample pretreatment methods and methods for detecting ketoprofen concentrations in human plasma. Summary of the invention

[0008] The purpose of the present invention is to provide a method for detecting ketoprofen in human plasma by using HPLC-MS / MS, based on the prior art, which has the advantages of small amount of plasma required for pretreatment, simple operation, good reproducibility, high sensitivity, fast analysis speed, small influence of matrix effect, high extraction recovery rate, etc.

[0009] The technical solution of the present invention is as follows:

[0010] A method for detecting ketoprofen in human plasma by HPLC-MS / MS, comprising the following steps: (1) pretreatment of human plasma samples; (2) liquid chromatography-mass spectrometry detection; gradient elution using mobile phase A and mobile phase B as mixed mobile phases, wherein mobile phase A is acetonitrile; mobile phase B is 0.05-0.20% formic acid aqueous solution; (3) determination of ketoprofen in human plasma; the gradient elution process is as follows: within 0-0.8 minutes, mobile phase A and mobile phase B are mixed mobile phases to perform gradient elution. The volume ratio of mobile phase A and mobile phase B is 40:60; within 0.8-1.0 minutes, the volume ratio of mobile phase A and mobile phase B changes from 40:60 to 55:45 at a constant speed; within 1.0-3.0 minutes, the volume ratio of mobile phase A and mobile phase B is 55:45; within 3.0-3.3 minutes, the volume ratio of mobile phase A and mobile phase B changes from 55:45 to 40:60 at a constant speed; within 3.3-4.0 minutes, the volume ratio of mobile phase A and mobile phase B is 40:60. The specific gradient elution process is shown in Table 1:

[0011] Table 1 Ketoprofen HPLC gradient

[0012]

[0013] The present invention adopts the above-mentioned gradient elution method for analysis when determining ketoprofen in human plasma, and the analysis time is only 4.0 minutes. The analysis time is short, which greatly shortens the analysis time and can achieve a quantitative lower limit of 0.1 ng / mL, making the analysis of large batches of biological samples more convenient and feasible, and can effectively improve the sensitivity and extraction recovery rate.

[0014] When the present invention adopts HPLC-MS / MS combination to detect ketoprofen in human plasma, the strong needle wash solution is methanol; the weak needle wash solution is a methanol-water mixed solution; preferably, the volume ratio of methanol to water in the weak needle wash solution is 1:1.

[0015] When the present invention adopts HPLC-MS / MS combination to detect ketoprofen in human plasma, in a preferred embodiment, mobile phase A is acetonitrile and mobile phase B is 0.05-0.20% formic acid aqueous solution. In order to improve the chromatographic separation selectivity in the present invention, the polarity of the mobile phase is adjusted by adding formic acid. In mobile phase B, the total volume of the aqueous solution is 100% as a reference, and the volume ratio of formic acid is 0.05-0.20%; without affecting the effect of the present invention, in a preferred embodiment, in mobile phase B, the total volume of the aqueous solution is 100% as a reference, and the volume ratio of formic acid is 0.1%, that is, mobile phase B is 0.1% formic acid aqueous solution.

[0016] In chromatography, the selection of the chromatographic column is very important. The requirements for the chromatographic column are: high column efficiency, good selectivity, fast analysis speed, etc. When the present invention uses HPLC-MS / MS to detect ketoprofen in human plasma, Agilent, ZORBAX Eclipse XDB-Phenyl is used as the chromatographic column. Further, the length of the chromatographic column is 75 mm, the diameter is 4.6 mm, and the filler particle size is 3.5 μm, that is, the chromatographic column is Agilent, ZORBAX Eclipse XDB-Phenyl (4.6×75 mm, 3.5 μm). The present invention uses the above-mentioned mobile phase A and mobile phase B as the mixed mobile phase for gradient elution. Using Agilent, ZORBAX Eclipse XDB-Phenyl as the chromatographic column, with the cooperation of other conditions, endogenous substances do not interfere with the determination of the sample, and it has good reproducibility, high sensitivity, short analysis time, and little matrix effect.

[0017] When using the internal standard method, the selection of the internal standard is a very important task. An ideal internal standard should be added to the sample in an accurate and known amount, and have basically the same or as consistent as possible physical and chemical properties, chromatographic behavior, and response characteristics as the sample to be analyzed; under the chromatographic analysis conditions, the internal standard must be fully separated from each component in the sample. When the present invention uses HPLC-MS / MS to detect ketoprofen in human plasma, ketoprofen-d3 is used as the internal standard, and deuterated is used as the internal standard. The deuterated internal standard and the analyte have the same retention time, chemical properties, and matrix effect, and the reproducibility and accuracy of measuring the concentration of ketoprofen in plasma are both good.

[0018] In step (1), the present invention pretreats the human plasma sample by the protein precipitation method, and uses acetonitrile as the precipitant. Pretreating the human plasma sample by the protein precipitation method can avoid the cumbersome and time-consuming liquid-liquid extraction process, and at the same time obtain a surprisingly high recovery rate. The present invention pretreats the human plasma sample by the protein precipitation method. Using Agilent, ZORBAX Eclipse XDB-Phenyl as the chromatographic column, with the cooperation of other conditions, the overall extraction recovery rate of ketoprofen is 92.16%.

[0019] In a preferred embodiment, in step (1), the pretreatment of the human plasma sample includes: adding an internal standard working solution and a precipitant to the human plasma sample, vortexing and centrifuging, and then taking the supernatant and mixing it with a diluent to obtain the sample to be tested; wherein, the internal standard in the internal standard working solution is ketoprofen-d3; the precipitant is acetonitrile. The diluent is a mixed solution of acetonitrile and water. Preferably, the volume ratio of acetonitrile to water in the diluent is 40-60:60-40; more preferably, the volume ratio of acetonitrile to water in the diluent is 50:50.

[0020] The detection method of the present invention further includes preparing an internal standard working solution, and the preparation of the internal standard working solution is as follows: Weigh ketoprofen-d3 reference substance, dissolve it with methanol to obtain an internal standard stock solution with a concentration of 1.00 mg / ml, and then dilute it with a mixed solution of acetonitrile and water with a volume ratio of 50:50 to obtain an internal standard working solution with a ketoprofen-d3 concentration of 10.0 ng / mL.

[0021] In a more preferred embodiment, in step (1), the pretreatment of human plasma sample includes: taking 50.0 μL of human plasma sample, adding 50.0 μL of internal standard working solution and 400 μL of acetonitrile, vortexing and centrifuging, taking 200 μL of supernatant, and then mixing it with 100 μL of diluent to obtain the sample to be tested; the volume ratio of acetonitrile to water in the diluent is 50:50.

[0022] The present invention pretreats the human plasma sample by protein precipitation method. Among them, the conditions of vortexing and centrifuging are: vortexing at 2000 rpm / min for 5 min, and centrifuging at 4000 rpm / min for 5 min under the condition of 4 °C.

[0023] When performing chromatographic detection in the present invention, the sample to be tested is placed in an autosampler for LC-MS / MS analysis. The injection volume is 10 μL, and the temperature of the autosampler is 4 °C.

[0024] In the detection method of the present invention, in step (2), liquid chromatography-mass spectrometry is used for detection. The detailed chromatographic conditions are: using Agilent, ZORBAX Eclipse XDB-Phenyl (4.6×75 mm, 3.5 μm) as the chromatographic column, performing gradient elution according to the above-mentioned elution process, the column temperature is 30-45 °C, preferably 40 °C; the flow rate is 0.8-1.2 mL / min, preferably 1.0 mL / min.

[0025] The mass spectrometry conditions of the present invention include: using an electrospray ion source, positive ion multiple reaction monitoring scan, the spray voltage is 5500 V, and the ion source temperature is 550 °C; for ketoprofen, [M+H] + , m / z 255.1→105.1, DP value 130 V, CE value 20 V; for ketoprofen-d3, [M+H] + , m / z 258.3→105.1, DP value 130 V, CE value 20 V.

[0026] In the detection method of the present invention, for the determination of the concentration of ketoprofen in human plasma in step (3): Prepare the plasma to be tested according to the sample pretreatment method in step (1), perform liquid chromatography-mass spectrometry detection according to step (2), record the peak area of ketoprofen, and use the weight coefficient w = 1 / x for the peak area ratio of ketoprofen and its internal standard 2Perform linear regression, with the equation expression y = ax + b, and calculate the concentration of ketoprofen in the plasma to be tested respectively.

[0027] The detection method of the present invention can be used for the monitoring of clinical pharmacokinetic plasma samples. The steps for calculating clinical pharmacokinetic parameters include: using WinNonlin 8.0 to calculate pharmacokinetic parameters, including: C max 、T max 、t 1 / 2 、AUC 0-t , and at the same time calculate the mean and standard deviation of each parameter.

[0028] Adopting the technical solution of the present invention, the advantages are as follows:

[0029] (1) The detection method of the present invention uses deuterium as an internal standard. The deuterated internal standard and the analyte have the same retention time, chemical properties and matrix effects, and the reproducibility and accuracy of measuring the concentration of ketoprofen in plasma are both good.

[0030] (2) In the detection method of the present invention, the plasma sample volume is only 50 μL, and the sample volume used is small, which is suitable for the detection of a large number of plasma samples.

[0031] (3) In the detection method of the present invention, the linear range of ketoprofen is 0.1 ng / mL to 50.0 ng / mL, the linear range is wide, the limit of quantification is low, and it is suitable for analyzing plasma samples after administration of different specifications and dosage forms, and has a wide application range.

[0032] (4) In the detection method of the present invention, the extraction recovery rate of ketoprofen is high, and the recovery rate is close to 100%.

[0033] (5) In the detection method of the present invention, the retention time of ketoprofen is appropriate and the peak shape is good.

[0034] (6) The detection method of the present invention selects a specific mobile phase, and during gradient elution, optimizes the elution time and the proportion of the mobile phase, and has the advantages of good reproducibility, high sensitivity, fast analysis speed, small influence of matrix effect and high recovery rate. And all method validations including specificity, accuracy, precision, matrix effect, extraction recovery rate, and stability have been carried out, and it can be reliably used to evaluate the bioequivalence of ketoprofen in the human body. Description of the Drawings

[0035] Figure 1 is the daughter ion scan of ketoprofen;

[0036] Figure 2 is the daughter ion scan of ketoprofen-d3;

[0037] Figure 3 is the specific chromatogram of determining ketoprofen in plasma by LC-MS / MS method; among them, Figures 3-1 to 3-6Chromatograms of 6 batches of blank plasma from different individuals respectively; in each figure, the chromatogram on the left is ketoprofen and the chromatogram on the right is ketoprofen-d3.

[0038] Figure 4 is the chromatogram of mixed blank plasma, the chromatogram on the left is ketoprofen and the chromatogram on the right is ketoprofen-d3;

[0039] Figure 5 is the chromatogram of the lower limit of quantitation sample, the chromatogram on the left is ketoprofen and the chromatogram on the right is ketoprofen-d3. Detailed implementation mode

[0040] According to the following examples, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the examples is only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0041] I. Materials and methods

[0042] Example 1

[0043] 1. Instruments and reagents

[0044] High performance liquid chromatography (Shimadzu LC-30AD series); mass spectrometry (API 5500, Applied Biosystems / Sciex); pure water instrument (Milli-Q, Merck Millipore); microbalance (XP6, METTLER TOLEDO); centrifuge (HeraeusMuitifuge X1R, ThermoFisher).

[0045] Methanol (Merck, HPLC grade), acetonitrile (Merck, HPLC grade), water (ultrapure water, self-made in the laboratory), formic acid (Aladdin, HPLC grade). Blank plasma was obtained from healthy subjects. Ketoprofen (TLC, batch number: 1771-082A1), ketoprofen-d3 (TLC, batch number: 1038-047A1).

[0046] 2. LC-MS conditions

[0047] Liquid phase conditions: Chromatographic column: Agilent, ZORBAX Eclipse XDB-Phenyl (4.6×75mm, 3.5μm); column temperature is 40°C; injector temperature: 4°C; flow rate is 1.0 mL / min; mobile phase A is acetonitrile; mobile phase B is 0.1% formic acid aqueous solution; gradient elution process is as follows: within 0 - 0.8 minutes, the volume ratio of mobile phase A to mobile phase B is 40:60; within 0.8 - 1.0 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 40:60 to 55:45; within 1.0 - 3.0 minutes, the volume ratio of mobile phase A to mobile phase B is 55:45; within 3.0 - 3.3 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 55:45 to 40:60; within 3.3 - 4.0 minutes, the volume ratio of mobile phase A to mobile phase B is 40:60. Weak needle washing solution: methanol: water (volume ratio 1:1, v / v); washing volume is 200 μL; strong needle washing solution is methanol.

[0048] Mass spectrometry conditions: Ion detection mode: positive ion multiple reaction monitoring (MRM); ionization mode: pneumatically assisted electrospray ionization (ESI); ion polarity: positive ion (Positive); detection target: ketoprofen, [M+H] + , m / z 255.1→105.1, DP value 130V, CE value 20V; ketoprofen-d3, [M+H] + , m / z 258.3→105.1, DP value 130V, CE value 20V. Mass spectrometry parameters: Spray voltage is 5500V; ion source temperature is 550°C. Specific ion scan diagrams of ketoprofen and ketoprofen-d3 are as Figure 1 and Figure 2 shown.

[0049] 3. Preparation of standard solution

[0050] Preparation of analyte working solution: Weigh two portions of ketoprofen reference substance precisely. After correction with the mass correction factor, dissolve it with methanol to obtain two analyte stock solutions with a concentration of 0.100 mg / mL each. The stock solutions are stored in a -20 °C refrigerator. After passing the inspection, precisely measure a certain volume of the ketoprofen stock solution and dilute it with acetonitrile: water (volume ratio 50:50, v / v) to prepare a series of standard curve sample working solutions with ketoprofen concentrations of 2.00 ng / mL (LLOQ), 4.00 ng / mL, 20.0 ng / mL, 50.0 ng / mL, 100 ng / mL, 500 ng / mL, 800 ng / mL, and 1,000 ng / mL (ULOQ). Precisely measure another portion of the ketoprofen stock solution and dilute it with acetonitrile: water (volume ratio 50:50, v / v) to prepare quality control working solutions with ketoprofen concentrations of 2.00 ng / mL (LLOQ QC), 6.00 ng / mL (LQC), 45.0 ng / mL (GMQC), 400 ng / mL (MQC), and 750 ng / mL (HQC).

[0051] Preparation of internal standard working solution: Weigh ketoprofen-d3 reference substance precisely. After correction with the mass correction factor, dissolve it with methanol to obtain an internal standard stock solution with a concentration of 1.00 mg / mL. The stock solution is stored in a -20 °C refrigerator. Precisely measure a certain volume of the internal standard stock solution and dilute it with acetonitrile: water (volume ratio 50:50, v / v) to prepare an internal standard working solution with a ketoprofen-d3 concentration of 10.0 ng / mL.

[0052] 4. Preparation of standard curve samples and quality control samples

[0053] For standard curve samples and quality control samples at each concentration level, the preparation process is exemplified as follows: Add 20.0 μL of the corresponding working solution to 380 μL of blank plasma, mix well. The preparation volume can be adjusted appropriately according to the actual situation. Sequentially prepare standard curve samples with ketoprofen concentrations of 0.100 ng / mL (LLOQ), 0.200 ng / mL, 1.00 ng / mL, 2.50 ng / mL, 5.00 ng / mL, 25.0 ng / mL, 40.0 ng / mL, and 50.0 ng / mL (ULOQ), and quality control samples with concentrations of 0.100 ng / mL (LLOQ QC), 0.300 ng / mL (LQC), 2.25 ng / mL (GMQC), 20.0 ng / mL (MQC), and 37.5 ng / mL (HQC).

[0054] 5. Sample pretreatment

[0055] Add 50.0 μL of the sample (biological sample to be tested, standard curve sample, quality control sample) to a 96-well plate; for double blank samples and blank samples, add 50.0 μL of blank matrix. Add 50.0 μL of the solvent acetonitrile: water (volume ratio 1:1, v / v) to the double blank samples. Except for the double blank samples, add 50.0 μL of the internal standard working solution (10.0 ng / mL) to all wells, then add 400 μL of the precipitant acetonitrile. Then vortex the 96-well plate at 2000 rpm / min for 5 min, and continue to centrifuge the 96-well plate at 4000 rpm / min for 5 min at 4 °C. Take 200 μL of the supernatant and add it to a clean 96-well plate, then add 100 μL of the diluent acetonitrile: water (volume ratio 1:1, v / v), mix well, and place it in the injection chamber or a refrigerator at the same temperature for testing.

[0056] 6. Content of methodological investigation

[0057] According to the "Guidelines for the Validation of Quantitative Analysis Methods for Biological Samples" in the 2020 Edition of the Chinese Pharmacopoeia, the detection method was validated methodologically to ensure the accuracy, repeatability, and stability of the detection. The validation includes the following contents: specificity, standard curve, precision and accuracy, matrix effect, extraction recovery rate, and stability.

[0058] II. Results and Discussion

[0059] 1. Specificity

[0060] Under the chromatographic conditions used in this experiment, the retention time of ketoprofen is about 2.26 min, as Figure 5 ; the retention time of the internal standard ketoprofen-d3 is about 2.25 min, as Figure 4 ; Take 50.0 μL of blank plasma from 6 different sources respectively. Except for not adding the internal standard, perform sample pretreatment operations to obtain the chromatograms of blank plasma samples, as Figures 3-1 to 3-6 ; The chromatogram of the lower limit of quantification sample is shown in Figure 5 ; The results show that endogenous substances in plasma do not affect the detection of ketoprofen, and at the same time, the internal standard does not affect the detection of ketoprofen.

[0061] 2. Accuracy and precision tests

[0062] Prepare quality control samples containing ketoprofen at concentrations of 0.100 ng / mL (LLOQ QC), 0.300 ng / mL (LQC), 2.25 ng / mL (GMQC), 20.0 ng / mL (MQC), and 37.5 ng / mL (HQC). Prepare 6 samples for each concentration, and prepare two standard curves (regressed from two sets of standard curve samples). Calculate the ratio f of the peak area As of ketoprofen to the corresponding internal standard peak area Ai, denoted as f. Substitute f into the standard curve of the day to obtain the measured concentration, the average measured concentration, and the accuracy. Calculate the within-batch precision and accuracy. The results are shown in Table 2. The results show that: Except for the lowest quantitation limit (LLOQ), the relative standard deviation (RSD) of the within-batch quality control samples of ketoprofen is less than 15%, and at least 67% of the relative error (RE) of the within-batch accuracy does not exceed ±15%, and the deviation of at least 50% of the quality control samples at each concentration level from their theoretical values does not exceed ±15%. The RSD of the within-batch quality control samples of the ketoprofen sample at the lowest quantitation limit is less than 20%, and at least 67% of the RE of the within-batch accuracy does not exceed ±20%, and the deviation of at least 50% of the quality control samples at each concentration level from their theoretical values does not exceed ±20%. In summary, both the precision and accuracy meet the requirements.

[0063] Table 2 Precision and accuracy of within-batch and between-batch sample detections

[0064]

[0065]

[0066]

[0067] 3. Investigation of matrix effect

[0068] Preparation of matrix samples: Use 6 batches of blank plasma from different donors. Prepare six replicate double-blank samples for each batch of blank plasma. According to the sample pretreatment operation, obtain the blank matrix extract. After extraction, add a certain amount of analyte and internal standard to make their final concentrations consistent with the injection concentrations of the low- and high-concentration quality control samples (three replicates for each concentration level).

[0069] Preparation of solution samples: Replace blank plasma with pure water for the pretreatment steps, and then dilute the working solution to the corresponding concentrations so that the concentrations after adding 50.0 μL of blank matrix extract or 50.0 μL of pure water extract are consistent with the injection concentrations of the low-, medium-, and high-concentration quality control samples after pretreatment. And there are 3 replicate samples for each concentration.

[0070] The results show that the total matrix effect factor of the matrix effect of ketoprofen (calculated by the peak area ratio) is 0.95, and the precision is 0.0%. The plasma matrix does not affect the accurate quantification of ketoprofen. The matrix effect data results are shown in Table 3.

[0071] Table 3 Matrix effect

[0072]

[0073] 4. Investigation of extraction recovery

[0074] Preparation of matrix samples: Plasma mixed from blank plasma of 6 different donors was used to prepare 9 replicated double - blank samples. According to the sample pretreatment operation, blank plasma extract was obtained. After extraction, a certain amount of analyte and internal standard were added to make their final concentrations consistent with the injection concentrations of low, medium, and high - concentration quality control samples respectively (3 replicates for each concentration level).

[0075] Preparation of quality control samples: Quality control samples at low, medium, and high concentrations were processed according to the sample treatment method, and 6 portions were prepared for each concentration level.

[0076] The recovery was evaluated by comparing the response value of the analyte or internal standard in a single quality control sample with the mean response value of the double - blank sample after adding the analyte and internal standard after extraction.

[0077] The acceptance criteria for recovery were as follows: The precision of the recovery at each concentration level and for all concentration levels should be within 15.0%. The extraction recovery of ketoprofen (calculated as the ratio of peak areas) was 92.16%, and the extraction recoveries at low, medium, and high concentrations were 90.05%, 92.08%, and 94.35% respectively. The results are shown in Table 4.

[0078] Table 4 Extraction recovery

[0079]

[0080] 5. Stability investigation

[0081] Stability of processed samples: After the first injection analysis of the analytical batch for investigating precision and accuracy, the freshly prepared standard curve samples and the analyzed samples were placed in the autosampler (4°C) for 170 h, then injected for analysis, and the chromatograms were recorded. The results are shown in Table 5. The injection solution of the ketoprofen plasma sample had good stability after being placed in the autosampler for 170 h, meeting the requirements of bio - sample analysis.

[0082] Stability at room temperature: The prepared quality control samples at low and high concentration levels, with ketoprofen concentrations of 0.300 ng / mL and 37.5 ng / mL, were mixed evenly for each concentration level, then placed at room temperature for 27 h, and then analyzed by LC - MS / MS, and the chromatograms were recorded. The results are shown in Table 6. The plasma samples had good stability after being placed at room temperature for 27 h.

[0083] Freeze-thaw stability: Freshly prepared samples containing ketoprofen at concentrations of 0.300 ng / mL and 37.5 ng / mL were placed in a -80 °C refrigerator for 5 freeze-thaw cycles. The acceptance criteria were as follows: the %RE of the average measured value of the stability samples compared to their theoretical values should not exceed ±15.0%, and the %RSD of the measured values of the stability samples at each concentration level should be ≤15.0%. The results are shown in Table 7. After 5 freeze-thaw cycles at -80 °C, the samples had good stability.

[0084] Long-term stability: Freshly prepared samples containing ketoprofen at concentrations of 0.300 ng / mL and 37.5 ng / mL were stored in a -80 °C refrigerator for 48 days and then tested. The acceptance criteria were as follows: the %RE of the average measured value of the stability samples compared to their theoretical values should not exceed ±15.0%, and the %RSD of the measured values of the stability samples at each concentration level should be ≤15.0%. The results are shown in Table 8. After storing the samples at -80 °C for 48 days, the samples had good stability.

[0085] Table 5 Stability of the treated samples

[0086]

[0087] Table 6 Stability during the pretreatment of biological samples (room temperature stability)

[0088]

[0089]

[0090] Table 7 Freeze-thaw stability

[0091]

[0092] Table 8 Long-term stability

[0093]

[0094]

[0095] The HPLC-MS / MS method for the determination of ketoprofen in plasma provided by the present invention has good specificity. Endogenous substances in plasma do not interfere with the determination of samples. The linear range of the ketoprofen standard curve is 0.100 ng / mL to 50.0 ng / mL, and the linear relationship is good. The within-batch and between-batch precisions of the test results of quality control samples at four concentration levels of high concentration (37.5 ng / mL), medium concentration (20.0 ng / mL), sub-medium concentration (2.25 ng / mL), and low concentration (0.300 ng / mL) of ketoprofen are both less than 15.0%. The within-batch and between-batch precisions of the test results of the quality control sample at the lower limit of quantification (0.100 ng / mL) are both less than 20.0%.

[0096] The total matrix effect factor of ketoprofen was 0.95, and the precision was 0.0%. Plasma matrix did not affect the accurate quantification of ketoprofen. The extraction recovery rate of ketoprofen was 92.16%. The stability of ketoprofen plasma samples was good after being placed at room temperature for 27 h; the stability was good after 5 freeze / thaw cycles; the stability was good after the plasma samples were processed and placed in the autosampler at 4 °C for 170 h; the stability of ketoprofen plasma samples was good after being placed at -80 °C for 48 d, meeting the requirements for biological sample analysis.

[0097] In summary, the method for determining the concentration of ketoprofen in human plasma established in the present invention meets the relevant requirements in the "Guidelines for the Validation of Quantitative Analysis Methods for Biological Samples" of the 2020 edition of the Pharmacopoeia and can be used for the analysis and detection of plasma samples in clinical trials.

[0098] Comparative Example 1

[0099] The mass spectrometry conditions and the pretreatment of plasma samples were the same as those in Example 1, except for the liquid phase conditions. The chromatographic column Agilent, ZORBAX Eclipse XDB-Phenyl (4.6 mm * 75 mm, 3.5 μm) was replaced with an Agilent RS-C18 chromatographic column (2.0 * 150 mm, 5 μm).

[0100] It was found that when the chromatographic column changed, the signal of ketoprofen in the sample to be measured decreased, and the lower limit of quantitation of ketoprofen increased from 0.1 ng / mL to 5 ng / mL, with a significant decrease in sensitivity.

[0101] Comparative Example 2

[0102] The mass spectrometry conditions and the pretreatment of plasma samples were the same as those in Example 1, except for the liquid phase conditions. The gradient elution process was adjusted as follows: within 0.0 - 0.8 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55; within 0.8 - 1.2 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed from 45:55 to 85:15 at a uniform speed; within 1.2 - 2.8 minutes, the volume ratio of mobile phase A to mobile phase B was 85:15; within 2.8 - 3.3 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed from 85:15 to 45:55 at a uniform speed; within 3.3 - 4.0 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55.

[0103] It was found that when the gradient elution process changed, the signal of ketoprofen in the sample to be measured decreased, and the lower limit of quantitation of ketoprofen increased from 0.1 ng / mL to 8 ng / mL, with a significant decrease in sensitivity.

[0104] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for detecting ketoprofen in human plasma by HPLC-MS / MS, characterized in that, It includes the following steps: (1) Pretreatment of human plasma samples; (2) Detection by liquid chromatography-mass spectrometry; Gradient elution is carried out using mobile phase A and mobile phase B as the mixed mobile phase. The mobile phase A is acetonitrile; the mobile phase B is an aqueous solution of 0.05 - 0.20% formic acid; (3) Determination of ketoprofen in human plasma; The gradient elution process is as follows: within 0 - 0.8 minutes, the volume ratio of mobile phase A to mobile phase B is 40:60; within 0.8 - 1.0 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 40:60 to 55:45; within 1.0 - 3.0 minutes, the volume ratio of mobile phase A to mobile phase B is 55:45; within 3.0 - 3.3 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes uniformly from 55:45 to 40:60; within 3.3 - 4.0 minutes, the volume ratio of mobile phase A to mobile phase B is 40:

60.

2. The method for detecting ketoprofen in human plasma by HPLC-MS / MS according to claim 1, characterized in that, The liquid chromatography conditions include: the chromatographic column is Agilent, ZORBAX Eclipse XDB - Phenyl; preferably, the length of the chromatographic column is 75 mm, the diameter is 4.6 mm, and the filler particle size is 3.5 μm.

3. The method for detecting ketoprofen in human plasma by HPLC-MS / MS according to claim 2, wherein The mobile phase B is an aqueous solution of 0.08 - 0.12% formic acid; preferably, the mobile phase B is an aqueous solution of 0.1% formic acid; during the gradient elution process, the strong needle washing solution is methanol; the weak needle washing solution is a methanol - water mixed solution; Preferably, the volume ratio of methanol to water in the weak needle washing solution is 1:

1.

4. The method for detecting ketoprofen in human plasma by HPLC-MS / MS combination according to claim 3, wherein, The liquid chromatography conditions include: the column temperature is 30 - 45 °C, preferably 40 °C; the flow rate is 0.8 - 1.2 mL / min, preferably 1.0 mL / min.

5. The method for detecting ketoprofen in human plasma by HPLC-MS / MS combination according to claim 4, characterized in that, In step (1), the pretreatment of human plasma samples includes: adding an internal standard working solution and a precipitant to the human plasma sample, vortexing and centrifuging, and then taking the supernatant, which is mixed with a diluent to obtain the sample to be tested; the internal standard in the internal standard working solution is ketoprofen - d3; the precipitant is acetonitrile; the diluent is a mixed solution of acetonitrile and water. Preferably, the volume ratio of acetonitrile to water in the diluent is 40 - 60:60 - 40; more preferably, the volume ratio of acetonitrile to water in the diluent is 50:

50.

6. The method for detecting ketoprofen in human plasma by HPLC-MS / MS according to claim 5, characterized in that, In step (1), the preparation of the internal standard working solution is as follows: Weigh the ketoprofen - d3 reference substance, dissolve it with methanol to obtain an internal standard stock solution with a concentration of 1.00 mg / ml, and then dilute it with a mixed solution of acetonitrile and water with a volume ratio of 50:50 to obtain an internal standard working solution with a ketoprofen - d3 concentration of 10.0 ng / mL.

7. The method for detecting ketoprofen in human plasma by HPLC-MS / MS according to claim 6, wherein, In step (1), the pretreatment of human plasma samples includes: taking 50.0 μL of human plasma sample, adding 50.0 μL of internal standard working solution and 400 μL of acetonitrile, vortexing and centrifuging, and then taking 200 μL of the supernatant, which is mixed with 100 μL of diluent to obtain the sample to be tested; the volume ratio of acetonitrile to water in the diluent is 50:

50.

8. The method for detecting ketoprofen in human plasma by HPLC-MS / MS according to claim 7, wherein The vortexing and centrifugation conditions are as follows: vortex at 2000 rpm / min for 5 min, and centrifuge at 4000 rpm / min for 5 min under the condition of 4°C; place the sample to be tested in an auto-sampler for LC-MS / MS analysis, with an injection volume of 10 μL and the auto-sampler temperature at 4°C.

9. The method for detecting ketoprofen in human plasma by HPLC-MS / MS according to claim 1, wherein, The mass spectrometry conditions include: using an electrospray ionization source, positive ion multiple reaction monitoring scan, spray voltage of 5500 V, and ion source temperature of 550 °C; ketoprofen, [M+H] + , m / z 255.1→105.1, DP value of 130 V, CE value of 20 V; ketoprofen-d3, [M+H] + , m / z 258.3→105.1, DP value of 130 V, CE value of 20 V.

10. The method for detecting ketoprofen in human plasma by HPLC-MS / MS coupling according to claim 1, wherein This method can be used for the monitoring of clinical pharmacokinetic plasma samples.

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