Method for detecting concentration of flurbiprofen in pig tissue

Abstract

The invention discloses a method for detecting the concentration of flurbiprofen in pig tissue, and belongs to the technical field of biological medicine detection. The method provided by the invention comprises the following steps: cutting a pig tissue sample treated by flurbiprofen plaster into pieces, adding a PBS (Phosphate Buffer Solution), and grinding by using a biological homogenizer to obtain homogenate; performing vortex treatment on the homogenate to obtain vortex homogenate, then mixing the vortex homogenate with an internal standard working solution, performing vortex treatment and centrifugation to obtain a supernatant, then adding a formic acid aqueous solution, performing vortex uniform mixing, and then performing analysis by using liquid chromatography-tandem mass spectrometry. The method provided by the invention has the advantages of good repeatability, wide linear range, high sensitivity, high recovery rate, simple operation and efficient analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine detection, and in particular to a method for detecting the concentration of flurbiprofen in pig tissue. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Flurbiprofen is a nonsteroidal anti-inflammatory drug widely used in rheumatoid arthritis, osteoarthritis, and postoperative pain. The development of flurbiprofen patches is currently attracting considerable attention, and their nonclinical evaluation focuses on the drug's penetration behavior in skin tissue. After patch administration, the drug must sequentially penetrate the epidermis, dermis, and muscle layers to be effective. Therefore, accurately measuring the drug concentration in each tissue layer is crucial for evaluating the efficacy and safety of the patch.

[0004] The skin tissue structure of pigs is highly similar to that of humans, making it a key model for evaluating transdermal drug delivery. However, their skin samples are thick and tough, making it difficult to efficiently break them into a homogenous slurry using traditional methods. Inhomogeneous homogenates not only lead to poor sampling representativeness and high relative standard deviations (RSDs), but may also interfere with the accuracy of measurement results due to matrix effects. Existing pretreatment methods for biological tissue samples rely on strong acids, strong bases, or long periods of high temperature (such as baking for 24 hours after adding alkali). These operations are cumbersome and time-consuming, and can destroy the structures of compounds that are unstable to heat or alkali. In addition, conventional pretreatment techniques such as protein precipitation lack sensitivity and are unable to meet the quantitative requirements for trace drugs in tissues such as the epidermis and dermis. Highly sensitive liquid-liquid extraction or solid-phase extraction methods have complex processes and require a uniform homogenate, otherwise subsequent process steps cannot be performed.

[0005] In the detection process, liquid chromatography coupled with mass spectrometry (LC-MS) is currently the first choice for analyzing trace substances in biological tissue samples. However, it still has problems such as insufficient sensitivity, cumbersome sample pretreatment, retention time drift, long analysis time, and difficulty in completely eluting interfering substances in the biological matrix, which affect the sample detection results. In addition, in mass spectrometry detection, it has been reported that the ionization mode (such as [MH] - ) has insufficient response to flurbiprofen, making it difficult to achieve both high sensitivity and resistance to matrix interference.

[0006] Therefore, there is an urgent need to establish a method with ideal homogenization effect, simple operation, high sensitivity and good repeatability for the determination of flurbiprofen concentration in porcine tissues. Summary of the Invention

[0007] In view of this, the present invention provides a method for detecting the concentration of flurbiprofen in pig tissue. The method provided by the present invention is simple and rapid to operate, has good homogenization effect uniformity, and has high sensitivity and good repeatability in the detection results.

[0008] The present invention provides a method for detecting the concentration of flurbiprofen in pig tissue, comprising the following steps: The pig tissue sample treated with flurbiprofen patch was minced and added with PBS buffer, and then ground using a biological homogenizer to obtain a homogenate. The homogenate is vortexed to obtain a vortexed homogenate, and the vortexed homogenate is then mixed with an internal standard working solution, vortexed, and centrifuged to obtain a supernatant, and then an aqueous formic acid solution is added, vortexed, and analyzed by liquid chromatography-tandem mass spectrometry after mixing; the internal standard working solution is a formic acid-acetonitrile solution containing tolbutamide; In the liquid chromatography-tandem mass spectrometry method, the mass spectrometry system adopts the multiple reaction monitoring mode, and the parent ion of flurbiprofen is the formate adduct ion [M+HCOO] - .

[0009] Preferably, the porcine tissue samples include porcine epidermal tissue samples, porcine dermal tissue samples, and porcine muscle tissue samples. In the present invention, the sampling thickness of the porcine epidermal tissue samples is 0.1-0.2 mm, and the sampling thickness of the porcine dermal tissue samples is 1-2 mm, and the above sampling thicknesses all refer to the distance from the surface of the porcine skin.

[0010] Furthermore, the mass-to-volume ratio of the porcine epidermal tissue sample to the PBS buffer is 1 g: (15-25) mL, more preferably 1 g: (18-22) mL; the mass-to-volume ratio of the porcine dermal tissue sample to the PBS buffer is 1 g: (5-15) mL, more preferably 1 g: (8-12) mL; and the mass-to-volume ratio of the porcine muscle tissue sample to the PBS buffer is 1 g: (2-5) mL, more preferably 1 g: (2-4) mL. Since the concentration of flurbiprofen gradually decreases from the epidermis to the dermis and then to the muscle, the homogenate ratio gradually decreases during homogenization, avoiding excessive dilution of low-concentration samples, resulting in too low a drug concentration and difficulty in detection; at the same time, ensuring that the test results have good repeatability. PBS buffer, as a solvent for the homogenate, has a better homogenization effect than a PBS solution containing acetonitrile, and its detection repeatability is higher.

[0011] Preferably, the specific steps of the grinding process using a bio-homogenizer are as follows: grinding 2 to 4 cycles at a speed of 3 to 7 m / s, with each cycle lasting 20 to 30 seconds, and each cycle interval being 10 to 30 seconds. The grinding process of the bio-homogenizer is performed, and toughness tissue is broken under mild conditions to obtain a homogenized homogenate, so that the flurbiprofen in the porcine tissue sample is evenly dispersed into PBS buffer from tissue. This treatment method of the present invention avoids the treatment steps such as strong acid, strong base, high temperature, and the treatment conditions are mild, and flurbiprofen structural stability, homogenate homogeneity, and detection result repeatability are good.

[0012] Preferably, in the step of vortexing the homogenate to obtain a vortexed homogenate, the vortexing speed is 1000-2000 rpm, and the vortexing time is 8-20 min. The vortexing speed in this step is relatively high, and the purpose is to fully disperse the flurbiprofen drug into the homogenate to ensure the uniformity of the drug concentration in the homogenate.

[0013] Preferably, in the step of mixing the vortexed homogenate with the internal standard working solution, the volume ratio of the homogenate to the internal standard working solution is 1:(3-5). The addition of the internal standard working solution can correct for pre-treatment losses, matrix effects, and instrument fluctuations, thereby achieving high-precision quantification. The present invention does not impose any particular restrictions on the homogenate sampling site, and the site may be an upper layer, an intermediate layer, or a lower layer, or each site may be sampled separately, and the average result of the three samples is finally calculated to further reduce error.

[0014] Preferably, the concentration of tolbutamide in the internal standard working solution is 8-12 ng / mL, and the volume fraction of formic acid in the formic acid-acetonitrile solution is 0.08-0.12%. The sulfonylurea structure of tolbutamide generates a stable [MH] - The ion (m / z 268.9→170.1) exhibits no cross-interference with the flurbiprofen monitoring channel (m / z 289.1→199.1), and the absence of endogenous interfering compounds in porcine tissue ensures specificity and reliability for trace-level detection. Furthermore, the formic acid-acetonitrile solution simultaneously precipitates proteins, effectively removing matrix interferences.

[0015] Preferably, in the step of vortexing and centrifuging to obtain a supernatant, the vortex speed is 300-700 rpm and the vortex time is 20-40 min. In this step, the vortexing homogenate is thoroughly mixed with the internal standard working solution by vortexing at a relatively low speed to ensure the stability and repeatability of the test results. The centrifugation removes unbroken tissue fragments, insoluble particles, and macromolecular interferences such as proteins and lipids in the homogenate, facilitating sample analysis and reducing matrix effects.

[0016] Preferably, the volume ratio of the supernatant to the formic acid aqueous solution is 1:(0.8-1.2), and the volume fraction of formic acid in the formic acid aqueous solution is 0.08-0.12%. Flurbiprofen contains a carboxyl group (-COOH), which easily forms deprotonated ions [MH] in negative ion mode. - (m / z 243.1), but its response is weak. After adding formic acid aqueous solution, formic acid (HCOOH) dissociates into formate (HCOO - ), flurbiprofen forms a stable formate adduct ion [M+HCOO] with formate - (m / z 289.1), significantly improving detection sensitivity. Furthermore, the addition of aqueous formic acid allows the injected sample solution to be aligned with the flow path, making the analytical method more robust.

[0017] Preferably, in the liquid chromatography-tandem mass spectrometry method, the liquid chromatography conditions are as follows: The mobile phase includes mobile phase A and mobile phase B, mobile phase A is 0.08~0.12% formic acid aqueous solution, and mobile phase B is 0.08~0.12% formic acid-acetonitrile solution; Gradient elution was used, and the program of gradient elution was as follows: 0-0.3 min, 10% mobile phase B; 0.3-0.8 min, 10%→45% mobile phase B; 0.8-1.5 min, 45%→90% mobile phase B; 1.5-2.1 min, 90% mobile phase B; 2.2-3.0 min, 10% mobile phase B; The flow rate is 0.4~0.6mL / min; the column temperature is 30~45℃.

[0018] Preferably, in the liquid chromatography-tandem mass spectrometry method, the mass spectrometry conditions are as follows: The negative ion mode of the electrospray ion source was used, with curtain gas pressure of 30-40 psi, collision gas of 6-10 psi, ionization voltage of 4000-5000 psi, ion source temperature of 340-360°C, nebulizer gas of 45-55 psi, and auxiliary heating gas of 50-60 psi.

[0019] More preferably, the mass-to-charge ratio of the parent ion of flurbiprofen is 289.1, and the mass-to-charge ratio of the daughter ion of flurbiprofen is 199.1; the mass-to-charge ratio of the parent ion of tolbutamide is 268.9, and the mass-to-charge ratio of the daughter ion of tolbutamide is 170.1.

[0020] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) The PBS buffer of the present invention is combined with the grinding of the biohomogenizer to achieve tough tissue fragmentation under mild conditions, which not only avoids the risk of degradation of flurbiprofen caused by strong acid, strong base or high temperature, but also ensures the full dispersion of flurbiprofen, good homogenate uniformity and good repeatability of test results.

[0021] (2) The present invention promotes the formation of a highly responsive adduct ion [M+HCOO] of flurbiprofen by using a formic acid aqueous solution. - , and [M+HCOO] was used for mass spectrometry - The trace amount of flurbiprofen in porcine tissues was detected by mass spectrometry using tolbutamide as the parent ion and formic acid-acetonitrile as the internal standard working solution. The method had high sensitivity, wide linear range, high recovery rate, good repeatability, simple and efficient operation, and was of great significance for the tissue distribution study of flurbiprofen and the subsequent trace amount quantification of flurbiprofen in tissue samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute undue limitations thereon. It is obvious that one of ordinary skill in the art could derive other drawings based on these drawings without inventive effort.

[0023] Figure 1 This is a schematic diagram of the drug administration area of ​​the Bama miniature pig in an embodiment of the present invention; Figure 2 is a blank matrix chromatogram of the flurbiprofen monitoring channel in Example 1 of the present invention; Figure 3 is a blank matrix chromatogram of the internal standard tolbutamide monitoring channel in Example 1 of the present invention; Figure 4 is a chromatogram containing an internal standard blank matrix of the flurbiprofen monitoring channel in Example 1 of the present invention; Figure 5 is a chromatogram containing an internal standard blank matrix of the internal standard tolbutamide monitoring channel in Example 1 of the present invention; Figure 6 is a chromatogram of a flurbiprofen-containing blank matrix of the flurbiprofen monitoring channel in Example 1 of the present invention; Figure 7 This is a chromatogram of a blank matrix containing flurbiprofen in the monitoring channel of the internal standard tolbutamide in Example 1 of the present invention; Figure 8 1 is a chromatogram of the flurbiprofen monitoring channel in Example 1 of the present invention containing an internal standard and a flurbiprofen blank matrix; Figure 9is a chromatogram of a blank matrix containing internal standard and flurbiprofen for the internal standard tolbutamide monitoring channel in Example 1 of the present invention; Figure 10 This is a chromatogram of the monitoring channel m / z 289.1 (parent ion) → 199.1 (daughter ion) in Example 1 of the present invention; Figure 11 This is a chromatogram of the monitoring channel m / z 243.3 (parent ion) → 199.1 (daughter ion) in Example 1 of the present invention. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0025] The technical solution of the present invention is further described below with reference to specific examples. The present invention has no special restrictions on the sources of the reagents used in the following examples, and commercially available products familiar to those skilled in the art can be used. The standard flurbiprofen in the following examples of the present invention was purchased from the China Food and Drug Inspection Institute; the trade name of flurbiprofen gel patch is Zepusi, produced by Mikasa Seiyaku Co., Ltd. of Japan and packaged by Beijing Taide Pharmaceutical Co., Ltd.; tolbutamide was purchased from Shanghai McLean Biochemical Technology Co., Ltd. The biological homogenizer is an OMNI multi-sample grinding bead homogenizer (Bead Ruptor Elite). In the expressions "0.1% formic acid" and "70% acetonitrile aqueous solution" involved in the following examples, unless otherwise specified, "%" refers to the volume fraction.

[0026] The pig epidermal tissue, pig dermal tissue and pig muscle tissue after administration of flurbiprofen used in the following examples were prepared by the following method: (1) Preparation: Bama miniature pigs, 12-16 months old, half male and half female, were selected as experimental subjects. Before dosing, the marked dosing area was shaved to avoid trauma. If the shaved area exceeded the marked area, the dosing area was marked with a marker. Before dosing, the pigs were cleaned with soapy water and purified water and wiped clean with dry gauze.

[0027] (2) Administration: Flurbiprofen gel patches were applied in reverse order, with medication administered to multiple dosing areas (corresponding to multiple time points) on the same pig. Finally, skin tissue samples were collected from multiple dosing areas (corresponding to multiple time points) after dissection to obtain tissue concentrations at different time points. Dosing was performed on the same pig at six time points: 48 hours, 12 hours, 8 hours, 6 hours, 4 hours, and 2 hours. The area of ​​a single dosing area was 5 × 6.8 cm. 2 Apply the flurbiprofen gel patch to the area at 24h and 48h, and replace it every 12h. After 24h, remove the flurbiprofen gel patch and cover it with disposable gauze. The dosing areas at each time point need not overlap with each other to avoid mutual penetration. The dosing area diagram is as follows Figure 1 shown.

[0028] (3) Materials taken: At the scheduled sampling time, the animals were anesthetized and euthanized by exsanguination after administering a 5:1 mixture of Shutai® 50 and xylazine hydrochloride injection at a volume ratio of 0.2 mL / kg to the hindlimb muscles. The skin surface of each dosing area was gently wiped clean with wet gauze and discarded. The dosing area was then wiped dry with disposable dry gauze. For each dosing area, medical tape was applied 12 times in a row to remove the entire stratum corneum and prevent drug residue. The tape was applied for approximately 15 seconds before being removed. After each application, a new area was replaced with a new tape area before the next application. After removing the stratum corneum, the marked skin and muscle tissue were removed en bloc with a scalpel. Each bag was placed in a sealed, labeled ziplock bag, temporarily placed on dry ice, and then frozen in an ultra-low temperature freezer.

[0029] (4) Layered sample preparation: The skin biopsy tissue was taken out of the frozen environment and thawed and softened at room temperature. 2 Skin layers were separated and sampled at each dosing area, with the epidermis layer sampled at a thickness of 0.1 mm and the dermis layer sampled at a thickness of 1.4 mm.

[0030] The sampling operation of blank pig tissue samples was the same as the above steps, except that flurbiprofen was not administered.

[0031] Example 1 This example provides a method for detecting the concentration of flurbiprofen in pig dermis.

[0032] 1. Preparation of relevant solutions (1) Flurbiprofen standard solution: Accurately weigh an appropriate amount of flurbiprofen standard, dissolve in acetonitrile by ultrasonication, and quantitatively dilute to a 1 mg / mL standard stock solution. Dilute the standard stock solution with 70% acetonitrile in water to prepare a series of working solutions for a standard curve, with concentrations of 0.2, 0.4, 1.0, 2.0, 4.0, 10, 16, and 20 µg / mL, respectively.

[0033] (2) Quality control working solution: Accurately weigh an appropriate amount of flurbiprofen standard, dissolve in acetonitrile by ultrasonication, and quantitatively dilute to a 1 mg / mL standard stock solution. Dilute the standard stock solution with 70% acetonitrile in water to prepare quality control working solutions at concentrations of 0.6, 3, and 15 µg / mL, respectively.

[0034] (3) Internal standard working solution: Accurately weigh an appropriate amount of tolbutamide standard, dissolve it in acetonitrile by ultrasonication, and quantitatively dilute it to a 1 mg / mL standard stock solution. Then, dilute it with 0.1% formic acid in acetonitrile to prepare an internal standard working solution at a concentration of 10 ng / mL.

[0035] (4) Homogenate solvent: Phosphate buffered saline (PBS) pH 7.4.

[0036] 2. Processing of pig dermal tissue samples The porcine dermal tissue sample was thawed and softened at room temperature. Approximately 0.20 g of the sample was weighed and placed in a 7 mL tissue grinding tube. The sample was minced with ophthalmic scissors. A homogenate solution (PBS solution) was added at a ratio of 1 g:10 mL. Three spoonfuls of 3 mm diameter zirconium beads were added and placed in the sample chamber of a biohomogenizer, which was kept below -10°C. The sample was then ground at a speed of 5 m / s for 30 seconds per cycle for three cycles, with 30 seconds between each cycle, to obtain a porcine dermal tissue homogenate.

[0037] The above-mentioned porcine dermal tissue homogenate was vortex-mixed at 1500 rpm for 10 min, and then 50 μL of each of the upper, middle and lower layers was taken, and 200 μL of the internal standard working solution was added to each of them. The mixture was vortex-mixed at 500 rpm for 30 min, and centrifuged at 3500 rpm and 8°C for 15 min. 100 μL of the supernatant after centrifugation was added to a 96-well sample plate, and 100 μL of 0.1% formic acid aqueous solution was added. The mixture was vortex-mixed at 500 rpm for 30 min, and then sampled and analyzed.

[0038] 3. High performance liquid chromatography-mass spectrometry analysis (HPLC-MS / MS) The SCIEX Exion LC-TQ5500 liquid chromatography-mass spectrometry system and Analyst data acquisition software were used.

[0039] The liquid phase conditions are as follows: The chromatographic column was an Agilent ZORBAX Extend C18 (5 μm, 2.1 × 50 mm); the mobile phases were 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The elution gradient was: 10% mobile phase B (0–0.3 min); 10% mobile phase B (0.3–0.8 min); 45%–90% mobile phase B (0.8–1.5 min); 90% mobile phase B (1.5–2.1 min); and 10% mobile phase B (2.2–3.0 min). The sample run time was 3 min. The flow rate was 0.5 mL / min, the injection volume was 5 μL, the column temperature was 40°C, and the injector temperature was 15°C.

[0040] Mass spectrometry conditions are as follows: Mass spectrometry detection was performed with an electrospray ionization (ESI) source in negative ion mode. The key parameters were set as follows: curtain gas pressure of 35 psi, collision gas pressure of 8 psi, ionization voltage of 4500 V, ion source temperature of 350°C, nebulizer gas pressure of 50 psi, and auxiliary heating gas pressure of 55 psi. The monitoring mode was multiple reaction monitoring (MRM), with the monitoring ion transition of flurbiprofen being m / z 289.1 (parent ion)→199.1 (daughter ion) and that of tolbutamide being m / z 268.9 (parent ion)→170.1 (daughter ion).

[0041] 4. Method reliability verification experiment (1) Exclusivity Determination of blank matrix chromatogram: The chromatogram was obtained using the method in steps 1 to 3 above, with the only difference being that the dermis of a blank porcine tissue sample (i.e., no flurbiprofen was administered) was used and tolbutamide was not added to the internal standard working solution. The blank matrix chromatogram is shown in Figure 2. Figure 2 and Figure 3 As shown, the detection results of the flurbiprofen monitoring channel and the internal standard tolbutamide monitoring channel are respectively.

[0042] Determination of the chromatogram of the blank matrix containing the internal standard: The chromatogram was obtained using the method in steps 1 to 3 above, with the only difference being that the dermis of the blank pig tissue sample (i.e., no flurbiprofen was administered) was used. The chromatogram of the blank matrix containing the internal standard is shown in FIG. Figure 4 and Figure 5 The following are the detection results of the flurbiprofen monitoring channel and the internal standard tolbutamide monitoring channel, respectively. The retention time of the internal standard tolbutamide is 1.53 min.

[0043] Determination of the chromatogram of the blank matrix containing flurbiprofen: The chromatogram was obtained using the method in steps 1 to 3 above, with the only difference being that the dermis tissue of a blank porcine tissue sample (i.e., no flurbiprofen was administered) was processed this time; the porcine dermal tissue homogenate was mixed with a 20 μg / mL flurbiprofen standard solution at a volume ratio of 1:19, and tolbutamide was not added to the internal standard working solution. The chromatogram of the blank matrix containing flurbiprofen is shown in Figure 1. Figure 6 and Figure 7 The following are the detection results of the flurbiprofen monitoring channel and the internal standard tolbutamide monitoring channel, respectively. The retention time of flurbiprofen is 1.67 min.

[0044] Determination of the blank matrix chromatogram containing internal standard and flurbiprofen: The chromatogram was obtained using the method in steps 1 to 3 above, with the only difference being that the dermis tissue of a blank porcine tissue sample (i.e., no flurbiprofen was administered) was processed this time; the porcine dermal tissue homogenate was mixed with a 0.2 μg / mL flurbiprofen standard solution at a volume ratio of 1:19. The blank matrix chromatogram containing internal standard and flurbiprofen is shown in Figure 1. Figure 8 and Figure 9 As shown, the detection results of the flurbiprofen monitoring channel and the internal standard tolbutamide monitoring channel are respectively.

[0045] The results showed that endogenous substances in the dermis did not interfere with flurbiprofen and tolbutamide, and there was no mutual interference between flurbiprofen and the internal standard tolbutamide, indicating that the method of this example has good specificity.

[0046] (2) Linearity and lower limit of quantification 10 μL of flurbiprofen standard curve working solutions at concentrations of 0.2, 0.4, 1.0, 2.0, 4.0, 10, 16, and 20 μg / mL were added to 190 μL of blank porcine dermal tissue homogenate and vortexed to obtain simulated dermal standard samples at concentrations of 10, 20, 50, 100, 200, 500, 800, and 1000 ng / mL. The samples were processed and analyzed according to the methods described in "2. Porcine Dermal Tissue Sample Processing" and "3. HPLC-MS Analysis." The flurbiprofen concentration was plotted as the X coordinate, and the peak area ratio of flurbiprofen to the internal standard, tolbutamide, was plotted as the Y coordinate. The weighted least squares method (weight 1 / X) was used to calculate the peak area ratio. 2 ) was used for regression calculation, and the resulting linear equation became the standard curve. The regression equation was Y = 0.124X - 0.000792, r = 0.9995. Flurbiprofen dermal homogenate samples showed good linearity over the range of 10 to 1000 ng / mL, with a lower limit of quantification of 10 ng / mL.

[0047] (3) Residual effect After the injection of the highest concentration of the simulated dermal standard sample, the blank matrix chromatogram was measured according to the method provided in "(1) Specificity" to evaluate the residue of the method and instrument. The results showed that the residual amount of flurbiprofen was less than 10% of the lower limit of quantification, indicating that the residue did not affect the accurate quantification of flurbiprofen.

[0048] (4) Accuracy and precision For each analytical batch, 10 μL of flurbiprofen quality control working solution at concentrations of 0.6, 3, and 15 μg / mL was added to 190 μL of blank porcine dermal tissue homogenate. Vortexing was performed to obtain simulated dermal accuracy and precision samples at concentrations of 30, 150, and 750 ng / mL. These samples were processed and analyzed according to the methods described in "2. Porcine Dermal Tissue Sample Processing" and "3. HPLC-MS Analysis." Six samples were tested in parallel at each concentration, for a total of three analytical batches. The concentrations of the accuracy and precision samples were calculated based on the standard curve. Intra- and inter-batch accuracy and precision of flurbiprofen were also calculated. The results showed that the intra-batch accuracy (RE) ranged from -10.6% to 7.8%, the inter-batch accuracy ranged from -9.5% to 6.4%, the intra-batch relative standard deviation (RSD) for precision was ≤6.0%, and the inter-batch RSD was ≤8.6%. This demonstrates that the method of this example exhibits good accuracy and precision.

[0049] (5) Matrix effect To prepare 380 μL of blank porcine dermal tissue homogenate from 6 different individuals (three parallel samples per individual), 20 μL of flurbiprofen quality control working solution at concentrations of 0.6, 3, and 15 μg / mL were added. The homogenate was processed and analyzed according to the methods of "2. Processing of porcine dermal tissue samples" and "3. High performance liquid chromatography-mass spectrometry analysis".

[0050] The accuracy and RSD calculated based on the standard curve were between -6.5% and 10.2%, with an RSD of ≤4.3%, indicating that matrix effects had no impact on quantification.

[0051] (6) Extraction recovery rate Preparation of blank matrix extract samples containing standard solutions: Quality control working solutions at concentrations of 0.6, 3, and 15 µg / mL were diluted 20-fold with 70% acetonitrile to prepare recovery working solutions. Blank porcine dermal tissue homogenate was added to 0.1% formic acid-acetonitrile at a volume ratio of 1:2.6 to precipitate protein. Vortex and centrifuge, and the supernatant was used as the blank matrix solution. To this solution, 50 µL was added to 20 µL of the 100 ng / mL internal standard working solution. 180 µL of each blank matrix solution was then added, vortexed, and centrifuged (3500 rpm, 8°C, 15 min). 100 µL of the supernatant was added to 100 µL of 0.1% formic acid in water. Six replicates were measured at each concentration.

[0052] Preparation of samples for normal processing: Prepare quality control working solutions with concentrations of 0.6, 3, and 15 µg / mL, and process and analyze according to the methods of "2. Porcine dermal tissue sample processing" and "3. HPLC-MS analysis". Measure six samples in parallel at each concentration.

[0053] Recovery = peak area of ​​blank matrix extract containing standard solution / peak area of ​​normally treated sample × 100%. The results showed that recoveries for low (0.6 µg / mL), medium (3 µg / mL), and high (15 µg / mL) concentrations were 106.3%, 110.3%, and 98.5%, respectively. The RSD for all concentrations was 5.7%, indicating high and consistent recoveries across different concentrations.

[0054] (7) Stability The stability of porcine dermal tissue homogenate samples at room temperature (25°C) before processing, after three freeze-thaw cycles, in a cryogenic freezer (-80°C), and in a sampler (15°C) after processing were investigated. For each stability test, stability samples at concentrations of 30 ng / mL and 750 ng / mL were prepared according to the method described in "Accuracy and Precision." Six samples were analyzed at each concentration. After storage under these stability conditions, sample concentrations were measured according to the methods described in "2. Porcine Dermal Tissue Sample Processing" and "3. High-Performance Liquid Chromatography-Mass Spectrometry Analysis," and relative errors were calculated. The results showed that the porcine dermal tissue homogenate samples were stable at room temperature for 6 hours before processing, stable after three freeze-thaw cycles, stable after long-term freezing for 58 days, and stable after storage in a sampler at 15°C for 61 hours after processing.

[0055] (8) Determination of flurbiprofen concentration and homogeneity in pig dermal tissue Flurbiprofen concentrations in porcine dermal tissue were determined using the methods described in "2. Porcine Dermal Tissue Sample Processing" and "3. High-Performance Liquid Chromatography-Mass Spectrometry Analysis." Flurbiprofen concentrations in porcine dermal tissue were quantified using standard curve samples and quality control samples. Six Bama miniature pigs were assayed, numbered "Individual 1," "Individual 2," and so on. Three parallel samples of homogenate prepared from the sampling area at specific times were collected from each Bama miniature pig and numbered "Individual 1-1," "Individual 1-2," and so on. The results are shown in Table 1.

[0056] Table 1 Flurbiprofen concentration (unit: ng / g) and RSD (unit: %) in porcine dermis

[0057] As can be seen from Table 1, the concentration of flurbiprofen in the dermis of miniature pigs showed a trend of first increasing and then decreasing 2 to 48 hours after administration of the patch. The relative standard deviation (RSD) of the precision of parallel sampling of homogenate at different time points of each individual was ≤10.0%, and the uniformity was good.

[0058] (9) Effect of homogenate solvent on flurbiprofen concentration and uniformity in pig dermal tissue The homogenate ratio of 1 g:10 mL (abbreviated as 1:10) in "2. Pig Dermal Tissue Sample Preparation" was replaced with 1 g:3 mL (abbreviated as 1:3) and 1 g:20 mL (abbreviated as 1:20). The homogenate solvent was replaced with PBS containing 20% ​​acetonitrile. The effects of the homogenate ratio and solvent on the concentration and homogeneity of flurbiprofen in pig dermal tissue were determined using the same testing method as described in "3. High-Performance Liquid Chromatography-Mass Spectrometry Analysis." Three Bama miniature pigs were tested, numbered "Individual 1," "Individual 2," and so on. Three parallel samples of the homogenate prepared from the sampling area after a 2-hour application period were collected from each Bama miniature pig and numbered "Individual 1-1," "Individual 1-2," and so on. The test results are shown in Table 2.

[0059] Table 2 Flurbiprofen concentration (unit: ng / g) and RSD (unit: %) data in different homogenate solvents and different homogenate ratios

[0060] As can be seen from Table 2, when PBS solution containing 20% ​​acetonitrile is used as the homogenate solvent, the RSD is high, with most of the values ​​above 20% and even reaching nearly 40%, indicating that the sampling repeatability of the homogenate prepared under this homogenization condition is poor, further affecting the precision and accuracy of the final test results.

[0061] (10) Optimization and comparison of flurbiprofen mass spectrometry monitoring channels Flurbiprofen contains a carboxyl group (-COOH), which readily forms deprotonated ions [MH] in negative ion mode. - (m / z 243.1), but its response is weak. After adding formic acid aqueous solution, formic acid (HCOOH) dissociates into formate (HCOO-), and flurbiprofen forms a stable adduct ion [M+HCOO] with formate. -(m / z 289.1). A simulated dermis standard sample with a concentration of 100 ng / mL was prepared according to the method described in "Linearity and Lower Limit of Quantitation." The sample was injected and analyzed according to the methods described in "2. Processing of Porcine Dermis Tissue Samples" and "3. HPLC-MS Analysis." Chromatograms were obtained under different monitoring channels. The only difference was that the monitoring channels for flurbiprofen were m / z 289.1 (parent ion) → 199.1 (daughter ion) and m / z 243.3 (parent ion) → 199.1 (daughter ion). The chromatogram with the monitoring channel of m / z 289.1 (parent ion) → 199.1 (daughter ion) is shown in the figure below. Figure 10 As shown in the figure, the chromatogram with the monitoring channel of m / z 243.3 (parent ion) → 199.1 (daughter ion) is as follows Figure 11 As shown, when other conditions remain unchanged, the signal response value of the mass spectrometry monitoring channel m / z 289.1 (parent ion) → 199.1 (daughter ion) used in the present invention is 4 times that of m / z 243.3 (parent ion) → 199.1 (daughter ion).

[0062] Example 2 This example provides a method for detecting the concentration of flurbiprofen in pig epidermis.

[0063] 1. Preparation of relevant solutions (1) Flurbiprofen standard solution: Accurately weigh an appropriate amount of flurbiprofen standard, dissolve in acetonitrile by sonication, and quantitatively dilute to a 1 mg / mL standard stock solution. Dilute the standard stock solution with 70% acetonitrile in water to prepare a series of working solutions for a standard curve, with concentrations of 2, 4, 10, 20, 40, 100, 160, and 200 µg / mL, respectively.

[0064] (2) Quality control working solution: Accurately weigh an appropriate amount of flurbiprofen standard, dissolve in acetonitrile by ultrasonication, and quantitatively dilute to a 1 mg / mL standard stock solution. Dilute the standard stock solution with 70% acetonitrile in water to prepare quality control working solutions at concentrations of 6, 30, and 150 µg / mL, respectively.

[0065] (3) Internal standard working solution: Accurately weigh an appropriate amount of tolbutamide standard, dissolve it in acetonitrile by ultrasonication, and quantitatively dilute it to a 1 mg / mL standard stock solution. Then, dilute it with 0.1% formic acid in acetonitrile to prepare an internal standard working solution at a concentration of 10 ng / mL.

[0066] (4) Homogenate solvent: Phosphate buffered saline (PBS) pH 7.4.

[0067] 2. Processing of pig epidermal tissue samples The porcine epidermal tissue sample was thawed and softened at room temperature. Approximately 0.80 g of the sample was weighed and placed in a 7 mL tissue grinding tube. The sample was minced with ophthalmic scissors. A homogenate solution (PBS solution) was added at a ratio of 1 g:20 mL. Three spoonfuls of 3 mm diameter zirconium beads were added and placed in the sample chamber of a biohomogenizer, which was kept below -10°C. The homogenate was then ground at a speed of 5 m / s for 20 s per cycle, with 10 s between cycles. This yielded a porcine epidermal tissue homogenate.

[0068] The above-mentioned pig epidermal tissue homogenate was vortexed at 1500 rpm for 10 min, and then 50 μL of each of the upper, middle and lower layers was taken, and 200 μL of the internal standard working solution was added to each of them. The mixture was vortexed at 500 rpm for 30 min, and centrifuged at 3500 rpm and 8°C for 15 min. 100 μL of the supernatant after centrifugation was added to a 96-well sample plate, and 100 μL of 0.1% formic acid aqueous solution was added. The mixture was vortexed at 500 rpm for 30 min, and then sampled and analyzed.

[0069] 3. High performance liquid chromatography-mass spectrometry analysis (HPLC-MS / MS) The SCIEX Exion LC-TQ5500 liquid chromatography-mass spectrometry system and Analyst data acquisition software were used.

[0070] The liquid phase conditions are as follows: The chromatographic column was an Agilent ZORBAX Extend C18 (5 μm, 2.1 × 50 mm); the mobile phases were 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The elution gradient was: 10% mobile phase B (0–0.3 min); 10% mobile phase B (0.3–0.8 min); 45%–90% mobile phase B (0.8–1.5 min); 90% mobile phase B (1.5–2.1 min); and 10% mobile phase B (2.2–3.0 min). The sample run time was 3 min. The flow rate was 0.5 mL / min, the injection volume was 5 μL, the column temperature was 40°C, and the injector temperature was 15°C.

[0071] Mass spectrometry conditions are as follows: Mass spectrometry detection was performed with an electrospray ionization (ESI) source in negative ion mode. The key parameters were set as follows: curtain gas pressure of 35 psi, collision gas pressure of 8 psi, ionization voltage of 4500 V, ion source temperature of 350°C, nebulizer gas pressure of 50 psi, and auxiliary heating gas pressure of 55 psi. The monitoring mode was multiple reaction monitoring (MRM), with the monitoring ion transition of flurbiprofen being m / z 289.1 (parent ion)→199.1 (daughter ion) and that of tolbutamide being m / z 268.9 (parent ion)→170.1 (daughter ion).

[0072] 4. Method reliability verification experiment (1) Linearity and lower limit of quantification 10 μL of flurbiprofen standard curve working solutions at concentrations of 2, 4, 10, 20, 40, 100, 160, and 200 μg / mL were added to 190 μL of blank porcine epidermal tissue homogenate and vortexed to obtain simulated epidermal standard samples at concentrations of 100, 200, 500, 1000, 2000, 5000, 8000, and 10000 ng / mL. The samples were processed and analyzed according to the methods described in "2. Porcine Epidermal Tissue Sample Preparation" and "3. HPLC-MS Analysis." The flurbiprofen concentration was used as the horizontal axis (X) and the peak area ratio of flurbiprofen to the internal standard (tolbutamide) was used as the vertical axis (Y). The weighted least squares method (weight 1 / X) was used to calculate the peak area ratio. 2 ) was used for regression calculation, and the resulting linear equation became the standard curve. The regression equation was Y = 0.131X + 0.00715, with r = 0.9990. Flurbiprofen epidermal homogenate samples showed good linearity within the range of 100 to 10,000 ng / mL.

[0073] (2) Accuracy and precision For each analytical batch, 10 μL of flurbiprofen quality control working solution at concentrations of 6, 30, and 150 μg / mL was added to 190 μL of blank porcine epidermal tissue homogenate and vortexed to obtain simulated epidermal accuracy and precision samples at concentrations of 300, 1500, and 7500 ng / mL. These samples were processed and analyzed according to the methods described in "2. Porcine Epidermal Tissue Sample Processing" and "3. HPLC-MS Analysis." Six samples were tested in parallel at each concentration, for a total of three analytical batches. The concentrations of the accuracy and precision samples were calculated based on the standard curve. Intra- and inter-batch accuracy and precision of flurbiprofen were also calculated. The results showed that the intra-batch accuracy (RE) ranged from -12.4% to 10.6%, the inter-batch accuracy ranged from -10.8% to 13.2%, the intra-batch relative standard deviation (RSD) for precision was ≤7.9%, and the inter-batch RSD was ≤8.5%. This demonstrates that the method of this example exhibits good accuracy and precision.

[0074] (3) Stability Porcine epidermal tissue homogenate samples were tested for stability at room temperature (25°C) before processing, after three freeze-thaw cycles, in a cryogenic freezer (-80°C), and in a sampler (15°C) after processing. For each stability test, stability samples were prepared at concentrations of 300 ng / mL and 7500 ng / mL according to the method described in "Accuracy and Precision." Six samples were analyzed at each concentration. Sample concentrations were measured according to the methods described in "2. Porcine Epidermal Tissue Sample Processing" and "3. High-Performance Liquid Chromatography-Mass Spectrometry Analysis," and relative errors were calculated. The results showed that porcine epidermal tissue homogenate samples were stable at room temperature for 6 hours before processing, stable after three freeze-thaw cycles, stable after long-term freezing for 55 days, and stable after 58 hours in a sampler at 15°C after processing.

[0075] (4) Determination of flurbiprofen concentration and uniformity in pig epidermal tissue The samples were processed and analyzed according to the methods described in "2. Pig Epidermal Tissue Sample Processing" and "3. High-Performance Liquid Chromatography-Mass Spectrometry Analysis." Flurbiprofen concentrations in pig epidermal tissue were quantified using standard curve samples and quality control samples. Six Bama miniature pigs were assayed, numbered "Individual 1," "Individual 2," and so on. Three samples were collected from each Bama miniature pig within the designated sampling area at specific times and numbered "Individual 1-1," "Individual 1-2," and so on. The results are shown in Table 3.

[0076] Table 3 Flurbiprofen concentration (unit: ng / g) and RSD (unit: %) in pig epidermis

[0077] As can be seen from Table 3, the concentration of flurbiprofen in the epidermis of miniature pigs showed a trend of first increasing and then decreasing 2 to 48 hours after administration of the patch. The relative standard deviation (RSD) of the precision of parallel sampling of homogenate at different time points of each individual was ≤10.0%, and the uniformity was good.

[0078] Example 3 This example provides a method for detecting the concentration of flurbiprofen in pig muscle.

[0079] 1. Preparation of relevant solutions (1) Flurbiprofen standard solution: Accurately weigh an appropriate amount of flurbiprofen standard, dissolve in acetonitrile by ultrasonication, and quantitatively dilute to a 1 mg / mL standard stock solution. Dilute the standard stock solution with 70% acetonitrile in water to prepare a series of working solutions for a standard curve, with concentrations of 0.02, 0.04, 0.1, 0.2, 0.4, 1.0, 1.6, and 2.0 µg / mL, respectively.

[0080] (2) Quality control working solution: Accurately weigh an appropriate amount of flurbiprofen standard, dissolve in acetonitrile by ultrasonication, and quantitatively dilute to a 1 mg / mL standard stock solution. Dilute the standard stock solution with 70% acetonitrile in water to prepare quality control working solutions at concentrations of 0.06, 0.3, and 1.5 µg / mL, respectively.

[0081] (3) Internal standard working solution: Accurately weigh an appropriate amount of tolbutamide standard, dissolve it in acetonitrile by ultrasonication, and quantitatively dilute it to a 1 mg / mL standard stock solution. Then, dilute it with 0.1% formic acid in acetonitrile to prepare an internal standard working solution at a concentration of 10 ng / mL.

[0082] (4) Homogenate solvent: Phosphate buffered saline (PBS) pH 7.4.

[0083] 2. Processing of pig muscle tissue samples Thaw and soften the porcine muscle tissue sample at room temperature. Approximately 0.20 g of the sample was weighed and placed in a 7 mL tissue grinding tube. Mince the sample with ophthalmic scissors and add a homogenate solvent (PBS solution) at a ratio of 1 g:5 mL. Three spoonfuls of 3 mm diameter zirconium beads were added and placed in the sample chamber of a biohomogenizer, which was kept below -10°C. The sample was then ground at a speed of 5 m / s for 30 seconds per cycle. Two cycles of grinding were performed, with 30 seconds between each cycle, to obtain a porcine muscle tissue homogenate.

[0084] The above-mentioned pig muscle tissue homogenate was vortexed at 1500 rpm for 10 min, and then 50 μL was taken from each of the upper, middle and lower layers, and 200 μL of the internal standard working solution was added to each of them. The mixture was vortexed at 500 rpm for 30 min, and centrifuged at 3500 rpm and 8°C for 15 min. 100 μL of the supernatant after centrifugation was added to a 96-well sample plate, and 100 μL of 0.1% formic acid aqueous solution was added. The mixture was vortexed at 500 rpm for 30 min, and then sampled and analyzed.

[0085] 3. High performance liquid chromatography-mass spectrometry analysis (HPLC-MS / MS) The SCIEX Exion LC-TQ5500 liquid chromatography-mass spectrometry system and Analyst data acquisition software were used.

[0086] The liquid phase conditions are as follows: The chromatographic column was an Agilent ZORBAX Extend C18 (5 μm, 2.1 × 50 mm); the mobile phases were 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The elution gradient was: 10% mobile phase B (0–0.3 min); 10% mobile phase B (0.3–0.8 min); 45%–90% mobile phase B (0.8–1.5 min); 90% mobile phase B (1.5–2.1 min); and 10% mobile phase B (2.2–3.0 min). The sample run time was 3 min. The flow rate was 0.5 mL / min, the injection volume was 5 μL, the column temperature was 40°C, and the injector temperature was 15°C.

[0087] Mass spectrometry conditions are as follows: Mass spectrometry detection was performed with an electrospray ionization (ESI) source in negative ion mode. The key parameters were set as follows: curtain gas pressure of 35 psi, collision gas pressure of 8 psi, ionization voltage of 4500 V, ion source temperature of 350°C, nebulizer gas pressure of 50 psi, and auxiliary heating gas pressure of 55 psi. The monitoring mode was multiple reaction monitoring (MRM), with the monitoring ion transition of flurbiprofen being m / z 289.1 (parent ion)→199.1 (daughter ion) and that of tolbutamide being m / z 268.9 (parent ion)→170.1 (daughter ion).

[0088] 4. Method reliability verification experiment (1) Linearity and lower limit of quantification 10 μL of flurbiprofen standard curve working solutions at concentrations of 0.02, 0.04, 0.1, 0.2, 0.4, 1.0, 1.6, and 2.0 μg / mL were added to 190 μL of blank porcine muscle tissue homogenate and vortexed to obtain simulated muscle standard samples at concentrations of 1.0, 2.0, 5.0, 10, 20, 50, 80, and 100 ng / mL. The samples were processed and analyzed according to the methods described in "2. Porcine Muscle Tissue Sample Processing" and "3. HPLC-MS Analysis." The peak area ratio of flurbiprofen to the internal standard, tolbutamide, was used as the horizontal coordinate (X) and the peak area ratio of flurbiprofen to tolbutamide as the vertical coordinate (Y). The weighted least squares method (weight 1 / X) was used to calculate the peak area ratio. 2 ) was used for regression calculation, and the resulting linear equation was used as the standard curve. The regression equation was Y = 0.13X - 0.000411, with r = 0.9991. Flurbiprofen muscle homogenate samples showed good linearity within the range of 1 to 100 ng / mL.

[0089] (2) Accuracy and precision For each analytical batch, 10 μL of flurbiprofen quality control working solution at concentrations of 0.06, 0.3, and 1.5 μg / mL was added to 190 μL of blank porcine muscle homogenate. The solution was vortexed to obtain simulated muscle accuracy and precision samples at concentrations of 3, 15, and 75 ng / mL. These samples were processed and analyzed according to the methods described in "2. Porcine Muscle Tissue Sample Processing" and "3. HPLC-MS Analysis." Six samples were tested in parallel at each concentration, for a total of three analytical batches. The concentrations of the accuracy and precision samples were calculated based on the standard curve. Intra- and inter-batch accuracy and precision of flurbiprofen were also calculated. The results showed that the intra-batch accuracy (RE) ranged from -6.7% to 8.6%, the inter-batch accuracy ranged from -10.2% to 9.7%, the intra-batch relative standard deviation (RSD) for precision was ≤5.8%, and the inter-batch RSD was ≤4.3%. This demonstrates that the method of this example exhibits good accuracy and precision.

[0090] (3) Stability The stability of porcine muscle tissue homogenate samples was investigated at room temperature (25°C) before processing, after three freeze-thaw cycles, in a cryogenic freezer (-80°C), and in the injector (15°C) after processing. For each stability test, two stability samples at low and high concentrations (3 and 75 ng / mL) were prepared according to the method described in "Accuracy and Precision." Six samples were analyzed at each concentration. Sample concentrations were measured according to the methods described in "2. Porcine Muscle Tissue Sample Processing" and "3. High-Performance Liquid Chromatography-Mass Spectrometry Analysis," and relative errors were calculated. The results showed that the porcine muscle tissue homogenate samples were stable at room temperature for 6 hours before processing, stable after three freeze-thaw cycles, stable after long-term freezing for 56 days, and stable after 49 hours in the injector at 15°C after processing.

[0091] (4) Determination of flurbiprofen concentration and homogeneity in pig muscle tissue The samples were processed and analyzed according to the methods described in "2. Pig Muscle Tissue Sample Processing" and "3. High-Performance Liquid Chromatography-Mass Spectrometry Analysis." Flurbiprofen concentrations in pig muscle tissue were quantified using standard curve samples and quality control samples. Six Bama miniature pigs were assayed, numbered "Individual 1," "Individual 2," and so on. Three samples were collected from each Bama miniature pig within the designated sampling area at specific times and numbered "Individual 1-1," "Individual 1-2," and so on. The results are shown in Table 4.

[0092] Table 4 Flurbiprofen concentration (unit: ng / g) and RSD (unit: %) in pig muscle

[0093] As can be seen from Table 3, the concentration of flurbiprofen in miniature pig muscle showed a trend of first increasing and then decreasing 2 to 48 hours after administration of the patch. The relative standard deviation (RSD) of the precision of parallel sampling of homogenate at different time points of each individual was ≤10.0%, and the uniformity was good.

[0094] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for detecting the concentration of flurbiprofen in pig tissue, characterized in that: The steps include: The pig tissue sample treated with flurbiprofen patch was minced and added with PBS buffer, and then ground using a biological homogenizer to obtain a homogenate. The homogenate is vortexed to obtain a vortexed homogenate, and the vortexed homogenate is then mixed with an internal standard working solution, vortexed, and centrifuged to obtain a supernatant, and then an aqueous formic acid solution is added, vortexed, and analyzed by liquid chromatography-tandem mass spectrometry after mixing; the internal standard working solution is a formic acid-acetonitrile solution containing tolbutamide; In the liquid chromatography-tandem mass spectrometry method, the mass spectrometry system adopts the multiple reaction monitoring mode, and the parent ion of flurbiprofen is the formate adduct ion [M+HCOO] - .

2. The method according to claim 1, wherein The porcine tissue samples include porcine epidermal tissue samples, porcine dermal tissue samples and porcine muscle tissue samples.

3. The method according to claim 1, wherein The mass-to-volume ratio of the porcine epidermal tissue sample to the PBS buffer is 1 g: (15-25) mL, the mass-to-volume ratio of the porcine dermal tissue sample to the PBS buffer is 1 g: (5-15) mL, and the mass-to-volume ratio of the porcine muscle tissue sample to the PBS buffer is 1 g: (2-5) mL.

4. The method according to claim 1, wherein The specific steps of the grinding process using the biohomogenizer are: grinding at a speed of 3-7 m / s for 2-4 cycles, each cycle lasting 20-30 seconds, and each cycle interval being 10-30 seconds.

5. The method according to claim 1, wherein In the step of vortexing the homogenate to obtain a vortexed homogenate, the vortexing speed is 1000~2000rpm, and the vortexing time is 8~20min; in the step of mixing the vortexed homogenate with the internal standard working solution, the volume ratio of the homogenate to the internal standard working solution is 1:(3~5).

6. The method according to claim 1, wherein In the internal standard working solution, the concentration of tolbutamide is 8-12 ng / mL, and in the formic acid-acetonitrile solution, the volume fraction of formic acid is 0.08-0.12%.

7. The method according to claim 1, wherein In the steps of vortexing and centrifuging to obtain the supernatant, the vortex speed is 300-700 rpm, and the vortex time is 20-40 min.

8. The method according to claim 1, wherein The volume ratio of the supernatant to the formic acid aqueous solution is 1:(0.8-1.2), and the volume fraction of formic acid in the formic acid aqueous solution is 0.08-0.12%.

9. The method according to claim 1, wherein In the liquid chromatography-tandem mass spectrometry, the liquid chromatography conditions are as follows: The mobile phase includes mobile phase A and mobile phase B, mobile phase A is 0.08~0.12% formic acid aqueous solution, and mobile phase B is 0.08~0.12% formic acid-acetonitrile solution; Gradient elution was used, and the program of gradient elution was as follows: 0-0.3 min, 10% mobile phase B; 0.3-0.8 min, 10%→45% mobile phase B; 0.8-1.5 min, 45%→90% mobile phase B; 1.5-2.1 min, 90% mobile phase B; 2.2-3.0 min, 10% mobile phase B; The flow rate is 0.4~0.6mL / min; the column temperature is 30~45℃.

10. The method according to claim 1, wherein In the liquid chromatography-tandem mass spectrometry method, the mass spectrometry conditions are as follows: The negative ion mode of the electrospray ion source was used, with a curtain gas pressure of 30–40 psi, a collision gas of 6–10 psi, an ionization voltage of 4000–5000 psi, and an ion source temperature of 340–360°C. Atomizing gas: 45~55psi; auxiliary heating gas: 50~60psi.

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