A method for detecting loxoprofen and its active metabolite trans-OH in human plasma by HPLC-MS / MS

Abstract

The present application relates to a kind of HPLC-MS / MS detection method of loxoprofen and its active metabolite trans-OH body in human plasma, it includes the following steps: (1) human plasma sample pretreatment;(2) liquid chromatography-mass spectrometry detection, using mobile phase A and mobile phase B as mixed mobile phase is carried out gradient elution, wherein, mobile phase A is acetonitrile, mobile phase B is 0.05% acetic acid aqueous solution;(3) the determination of loxoprofen and its active metabolite trans-OH body concentration in human plasma.The present application uses ACE, Excel 3SuperC18 as chromatographic column, in the process of gradient elution, the time of optimization elution and the proportion of mobile phase, linear range is all 0.200-40.0ng / mL, lower limit of quantification is low, sensitivity is high, reproducibility, accuracy is all better, can be used to evaluate loxoprofen and its active metabolite trans-OH body each dosage form bioequivalence.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological analysis, and particularly relates to a method for detecting loxoprofen and its active metabolite trans-OH in human plasma by HPLC-MS / MS. BACKGROUND

[0002] Pain is the most common symptom of musculoskeletal system diseases, and usually requires the use of analgesics and non-steroidal anti-inflammatory drugs (NSAIDs) for treatment. NSAIDs exert antipyretic, analgesic and anti-inflammatory effects by inhibiting the biological activity of cyclooxygenase (COX) in the metabolic process of cell membrane arachidonic acid (AA), thereby reducing the synthesis and accumulation of prostaglandins (PG).

[0003] Systemic (oral or intravenous) application of NSAIDs has good efficacy for musculoskeletal pain, but its safety and tolerability issues have been widely concerned. Systemic application of NSAIDs has many adverse reactions, the most common of which is gastrointestinal reaction, and other adverse reactions include: cardiovascular adverse reactions, affecting platelet aggregation function, affecting kidney function (sodium and water retention, renal insufficiency), affecting liver function, etc. Compared with oral or intravenous administration, topical NSAIDs can be directly applied to the skin of the lesion site, penetrate through the skin to reach the painful tissue and exert analgesic effect, and has the advantages of fast onset, high local concentration, less systemic exposure and fewer systemic adverse reactions, and is more suitable for the treatment of acute and chronic pain of the musculoskeletal system.

[0004] Loxoprofen sodium is a propionic acid precursor type NSAID, which has strong and balanced analgesic, anti-inflammatory and antipyretic effects, and has rapid onset and fewer adverse reactions.

[0005] At present, although domestic literature has reported methods for determining the content of loxoprofen and its active metabolite trans-OH by liquid chromatography-mass spectrometry (LC-MS), there is a common problem of low sensitivity. For example, the method for detecting loxoprofen and its active metabolite trans-OH in plasma disclosed in HPLC-MS method for rapid determination of the concentration of loxoprofen sodium in human plasma and its pharmacokinetic study (Li Hao, China Pharmaceutical University, 2006) has a linear range of 0.10-10.0 g / ml and a detection limit of 0.10 g / ml, and the sensitivity is very low, which cannot meet the needs of the analysis of biological samples in new drug and bioequivalence tests with low pharmacodynamic range. In addition, during the pretreatment of plasma samples, 5 ml of n-hexane-ether is used as an extractant, the amount of extractant is large, batch sample analysis work cannot be performed using a conventional 96-well plate, and the pretreatment process is complex; at the same time, ether is a controlled reagent and is difficult to obtain. Moreover, the internal standard used is ibuprofen, which is difficult to control the same matrix effect of the analyte and the internal standard, and may cause a difference between the sample detection result and the true value.

[0006] Chinese patent CN 109991334 A discloses a method for detecting loxoprofen and its active metabolite trans-OH form in plasma. The limit of quantitation (LOQ) for loxoprofen is 50,000 ng / mL, and for its active metabolite trans-OH form, it is 10,000 ng / mL. This method has low sensitivity and cannot meet the analytical requirements of new drugs with low pharmacokinetic ranges or biological samples in bioequivalence studies. Furthermore, in gradient elution, isocratic elution results in broad chromatographic peaks with tailing, and consumes a large amount of solvent.

[0007] To meet the needs of large-scale clinical sample analysis for evaluating drug bioequivalence, it is necessary to develop simpler, more reliable, and higher-throughput sample pretreatment methods, as well as methods for detecting the concentration of loxoprofen and its active metabolite trans-OH in human plasma. Summary of the Invention

[0008] The purpose of this invention is to provide, based on existing technology, a method for detecting the concentration of loxoprofen and its active metabolite trans-OH in human plasma, which has good reproducibility, high sensitivity, fast analysis speed, and minimal matrix effect.

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

[0010] A method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS, comprising the following steps: (1) pretreatment of human plasma samples; (2) detection using HPLC-MS / MS, employing a gradient elution with mobile phase A and mobile phase B as mixed mobile phases, wherein mobile phase A is acetonitrile and mobile phase B is a 0.01–0.2% aqueous acetic acid solution; (3) determination of the concentration of loxoprofen and its active metabolite trans-OH form in human plasma, wherein the gradient elution process is as follows: at 0 Within 0-3.0 minutes, the volume ratio of mobile phase A to mobile phase B is 45:55; within 3.0-3.2 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 45:55 to 95:5; within 3.2-3.8 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5; within 3.8-4.1 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 95:5 to 30:70; within 4.1-4.5 minutes, the volume ratio of mobile phase A to mobile phase B is 30:70. The specific gradient elution process is shown in Table 1.

[0011] Table 1. Liquid Chromatographic Gradients of Loxoprofen and its Active Metabolite Trans-OH

[0012]

[0013] In one approach, step (1) of the pretreatment of human plasma samples includes: adding internal standard working solution and pH adjuster to the plasma sample, shaking, adding extraction solvent, vortexing and centrifuging, collecting the supernatant, blowing it dry with nitrogen, and then reconstituted with a rehydration solvent to obtain the sample to be tested. The internal standard working solution contains loxoprofen-d4 as the internal standard; the pH adjuster is 0.01–0.05 M disodium hydrogen phosphate buffer, with the pH adjusted to 1.5–3.5 using phosphoric acid.

[0014] In step (1), the present invention uses liquid-liquid extraction to pretreat human plasma samples, employing methyl tert-butyl ether as the extractant. This enhances the extraction effect of loxoprofen and its active metabolite trans-OH form. The extraction method is simple, the pretreatment is rapid, and it is easy to promote and use in clinical practice. During the experiment, it was found that using commonly used extractants, such as ethyl hexanoate and n-hexane, resulted in very low extraction efficiency for loxoprofen and its active metabolite trans-OH form, leading to low sensitivity and failing to meet the quantitative analysis requirements for low concentrations of loxoprofen and its active metabolite trans-OH form in plasma samples.

[0015] In step (1), the present invention pretreatment of human plasma samples using liquid-liquid extraction, employing 0.01–0.05 M disodium hydrogen phosphate buffer (adjusted to pH 1.5–3.5 with phosphoric acid) as a pH adjuster. During the experiment, it was found that other pH adjusters, such as 2 mol / L HCl solution, failed to enhance the signal of loxoprofen and its active metabolite trans-OH, leading to sensitivity deviations. In a preferred embodiment, the pH adjuster is 0.01 M disodium hydrogen phosphate buffer, adjusted to pH 2.1 with phosphoric acid.

[0016] In this invention, in step (1), the resolvent is a mixed solution of methanol and water. Preferably, the resolvent is a mixed solution of methanol and water with a volume ratio of 1:1 to 5. More preferably, the resolvent is a mixed solution of methanol and water with a volume ratio of 1:1.

[0017] In a more preferred embodiment, in step (1), the pretreatment of the human plasma sample includes: taking 50 μL of human plasma sample, adding 50 μL of internal standard working solution and 200 μL of pH adjuster, shaking, adding 800 μL of methyl tert-butyl ether, centrifuging by vortexing, taking 200 μL of supernatant, blowing it dry with nitrogen at 40-50℃, redissolving it with 100 μL of a 1:1 volume ratio of methanol and water, and shaking well to obtain the sample to be tested; wherein, the pH adjuster is 0.01M disodium hydrogen phosphate buffer, and the pH is adjusted to 2.1 with phosphoric acid.

[0018] In a preferred embodiment, 200 μL of the supernatant is taken and dried by nitrogen blowing at 45°C.

[0019] This invention uses liquid-liquid extraction to pretreat human plasma samples. After adding the extractant methyl tert-butyl ether, the conditions for vortexing and centrifugation are as follows: vortexing for 10 min, followed by centrifugation at 4000 rpm / min for 5 min at 4°C.

[0020] In this invention, the sample to be tested is placed in an autosampler for LC-MS / MS analysis during chromatographic detection. The injection volume is 10 μL and the autosampler temperature is 4 °C.

[0021] The detection method of the present invention further includes the preparation of an internal standard working solution: weigh loxoprofen-d4 reference standard, dissolve it in methanol to obtain a loxoprofen-d4 stock solution with a concentration of 1.00 mg / mL, and then dilute it with a methanol-water mixture with a volume ratio of 50:50 to obtain a loxoprofen-d4 internal standard working solution with a concentration of 500 ng / mL.

[0022] When using the internal standard method, the selection of the internal standard is crucial. An ideal internal standard should be able to be added to the sample in an accurate and known amount, and should have essentially the same or as closely as possible the same physicochemical properties, chromatographic behavior, and response characteristics as the analyte. Under chromatographic conditions, the internal standard must be able to completely separate from the components in the sample. In this invention, when using HPLC-MS / MS to detect loxoprofen and its active metabolite trans-OH in human plasma, loxoprofen-d4 is used as the internal standard. The deuterated internal standard and the analyte have the same retention time, chemical properties, and matrix effect, resulting in good reproducibility and accuracy in determining the concentration of loxoprofen and its active metabolite trans-OH in plasma.

[0023] This invention employs HPLC-MS / MS to detect loxoprofen and its active metabolite trans-OH form in human plasma, wherein mobile phase A is acetonitrile; and mobile phase B is a 0.01–0.2% aqueous acetic acid solution.

[0024] In a preferred embodiment, the mobile phase B is a 0.05% aqueous acetic acid solution; preferably, the mobile phase B contains 0.01 to 0.20% acetic acid by volume, based on 100% of the total volume of the aqueous acetic acid solution; more preferably, the mobile phase B contains 0.05% acetic acid by volume, based on 100% of the total volume of the aqueous acetic acid solution.

[0025] In this invention, when using HPLC-MS / MS to detect loxoprofen and its active metabolite trans-OH in human plasma, an ACE, Excel 3 SuperC18 column is used. Specifically, the column has a length of 50 mm, a diameter of 2.1 mm, and a packing particle size of 3 μm, i.e., ACE, Excel 3 SuperC18 (2.1 × 50 mm, 3 μm).

[0026] In chromatography, the selection of the chromatographic column is crucial, requiring high column efficiency, good selectivity, and fast analysis speed. This invention employs the aforementioned mobile phase for gradient elution, using an ACE, Excel 3 SuperC18 column. Under suitable conditions, endogenous substances do not interfere with sample determination, and the method exhibits good reproducibility, high sensitivity, fast analysis speed, and minimal matrix effect.

[0027] The detection method of the present invention, step (2) of using liquid chromatography-mass spectrometry for detection includes the following detailed chromatographic conditions: using ACE, Excel 3 SuperC18 (2.1×50mm, 3μm) as the chromatographic column, performing gradient elution according to the above-mentioned elution process, with a column temperature of 30~45℃, preferably 40℃; and a flow rate of 0.2~1.0mL / min, preferably 0.4mL / min.

[0028] In this invention, when using HPLC-MS / MS to detect loxoprofen and its active metabolite trans-OH form in human plasma, a preferred embodiment uses a methanol-water mixture as the weak washing buffer during gradient elution. To better implement this invention, the volume ratio of methanol to water in the weak washing buffer is 1–3:1. In a further preferred embodiment, without affecting the effectiveness of this invention, the volume ratio of methanol to water in the weak washing buffer is 1:1. During experiments, it was found that using commonly used strong washing buffers, such as methanol, acetonitrile, methanol-acetonitrile, methanol-ethanol, methanol-isopropanol, and methanol-acetonitrile-isopropanol, resulted in the peak area of ​​the analyte in the residual sample after high-concentration samples exceeding 20% ​​of the peak area of ​​the analyte in the lower limit of quantitation sample, affecting the accurate determination of samples after high-concentration samples. (Except for methanol, other solvents used as strong washing buffers all resulted in the peak area of ​​the analyte in the residual sample exceeding 20% ​​of the peak area of ​​the analyte in the lower limit of quantitation sample, affecting the accurate determination of samples after high-concentration samples.)

[0029] The mass spectrometry conditions of this invention include: electrospray ionization source, negative ion multiple reaction monitoring (MRM) scanning, spray voltage -4500V, and ion source temperature 550°C; loxoprofen, [MH]. - m / z 254.3→83.1, DP value -50V, CE value -16V; active metabolite trans-OH form, [MH] - m / z 247.2→202.9, DP value -50V, CE value -10V; loxoprofen-d4, [MH] - m / z 249.2→205.1, DP value -50V, CE value -9V.

[0030] The detection method of the present invention, step (3) of determining the concentration of loxoprofen and its active metabolite trans-OH in human plasma includes: preparing the plasma to be tested according to the sample pretreatment method in step (1), detecting it according to the liquid chromatography-mass spectrometry in step (2), recording the peak area corresponding to the trans-OH of loxoprofen and its active metabolite, and dividing the peak area ratio of loxoprofen and its active metabolite trans-OH to the internal standard by a weighting coefficient w = 1 / x. 2 Linear regression was performed, with the equation y = ax + b, to calculate the concentration of loxoprofen and its active metabolite trans-OH in the plasma to be tested.

[0031] The detection method of this invention can be used for clinical pharmacokinetic sample monitoring. The steps for calculating clinical drug metabolism kinetic parameters include: calculating pharmacokinetic parameters using WinNonlin 8.0, including: C max T max t 1 / 2 AUC 0-t At the same time, the mean and standard deviation of each parameter are calculated.

[0032] The advantages of using the technical solution of this invention are as follows:

[0033] (1) In this invention, deuterated loxoprofen is used as an internal standard. The deuterated internal standard and the analyte have the same retention time, chemical properties and matrix effect. The reproducibility and accuracy of determining the concentration of loxoprofen and its active metabolite trans-OH in plasma are both good.

[0034] (2) The detection method of the present invention uses methanol solution as a strong washing solution in the gradient elution process, which can effectively remove residues and make the detection results accurate and reliable.

[0035] (3) This method uses only 50 μL of sample, which is small and suitable for large-scale plasma sample detection. The linear range of loxoprofen and its active metabolite trans-OH is 0.200 to 40.0 ng / mL. The lower limit of quantification is low and it is suitable for analyzing plasma samples after administration of different specifications. It has a wide range of applications.

[0036] (4) The detection method of the present invention selects a specific mobile phase and optimizes the elution time and the ratio of the mobile phase during gradient elution. It has the advantages of good reproducibility, high sensitivity, fast analysis speed, small matrix effect and high recovery rate. The method has been fully validated, including specificity, accuracy, precision, matrix effect, extraction recovery rate and stability. It can be reliably used to evaluate the bioequivalence of various dosage forms of loxoprofen and its active metabolite trans-OH form in the human body. Attached Figure Description

[0037] Figure 1This is a scan of loxoprofen daughter ions;

[0038] Figure 2 This is a scan of the trans-OH fragment ion of the active metabolite;

[0039] Figure 3 This is a scan of the loxoprofen-d4 daughter ion;

[0040] Figure 4 is a specific chromatogram for the determination of loxoprofen in plasma by LC-MS / MS.

[0041] in, Figures 4-1 to 4-6 Chromatograms of blank plasma from 6 different individuals; Figures 4-1 to 4-6 In each figure, the chromatogram on the left is loxoprofen, and the chromatogram on the right is loxoprofen-d4;

[0042] Figure 5 is a specific chromatogram for the determination of trans-OH bodies of active metabolites in plasma by LC-MS / MS method;

[0043] in, Figures 5-1 to 5-6 Chromatograms of blank plasma from 6 different individuals; Figures 5-1 to 5-6 In each figure, the chromatogram on the left is the trans-OH form of the active metabolite, and the chromatogram on the right is loxoprofen-d4;

[0044] Figure 6 This is a chromatogram of loxoprofen in mixed blank plasma;

[0045] The chromatogram on the left is loxoprofen, and the chromatogram on the right is loxoprofen-d4;

[0046] Figure 7 This is a chromatogram of the trans-OH form of the active metabolite in mixed blank plasma;

[0047] The chromatogram on the left is the trans-OH form of the active metabolite, and the chromatogram on the right is loxoprofen-d4.

[0048] Figure 8 This is the chromatogram of a sample with the lower limit of quantification for loxoprofen;

[0049] The chromatogram on the left is loxoprofen, and the chromatogram on the right is loxoprofen-d4;

[0050] Figure 9 This is a chromatogram of a sample with the lower limit of quantification for the trans-OH form of the active metabolite;

[0051] The chromatogram on the left is the trans-OH form of the active metabolite, and the chromatogram on the right is loxoprofen-d4.

[0052] Figure 10 This is a chromatogram of a sample with the lower limit of quantification using 0.01M disodium hydrogen phosphate buffer (adjusted to pH 4.5 with phosphoric acid);

[0053] The chromatogram on the left is loxoprofen, and the chromatogram on the right is the trans-OH form of the active metabolite.

[0054] Figure 11 This is a chromatogram of a mixed double blank sample obtained using a ZORBAX Eclipse XDB-Phenyl (4.6mm*75mm, 3.5μm) column;

[0055] The chromatogram on the left is loxoprofen, and the chromatogram on the right is loxoprofen-d4;

[0056] Figure 12 This is a chromatogram of a mixed double blank sample when using ethyl acetate as the extractant;

[0057] The chromatogram on the left is loxoprofen, and the chromatogram on the right is loxoprofen-d4. Detailed Implementation

[0058] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0059] Example 1

[0060] I. Materials and Methods

[0061] 1. Instruments and reagents

[0062] High-performance liquid chromatography (Shimadzu LC-30AD series); mass spectrometry (API 5500, Applied Biosystems / Sciex); pure water system (MilliDirectQ, Millipore); microbalance (XP6, METTLER TOLEDO); centrifuge (Heraeus Muitifuge X1R, ThermoFisher); shaker (LPD2500, LE PARD).

[0063] Methanol (Merck, HPLC grade), acetonitrile (Merck, HPLC grade), water (ultrapure water, lab-prepared), acetic acid (Sigma, HPLC grade), methyl tert-butyl ether (Merck, HPLC grade), disodium hydrogen phosphate (Sigma, HPLC grade), phosphoric acid (Macklin, HPLC grade). Blank plasma was obtained from healthy subjects. Loxoprofen (China National Institutes for Food and Drug Control, batch number: 100638-202104), active metabolite trans-OH form (TLC, batch number: 2475-002A2), loxoprofen-d4 (TLC, batch number: 2569-058A6)

[0064] 2. Liquid chromatography-mass spectrometry (LC-MS) conditions

[0065] Liquid chromatography conditions: Column: ACE, Excel 3 SuperC18 (2.1×50mm, 3μm); Column temperature: 40℃; Injector temperature: 4℃; Mobile phase A: acetonitrile; Mobile phase B: 0.05% acetic acid-0.05% acetic acid aqueous solution (based on a total volume of 100% acetic acid aqueous solution, containing a volume ratio of 0.05% acetic acid); Gradient elution process as follows: Within 0.0–3.0 minutes, the volume ratio of mobile phase A to mobile phase B is 45:55. Within 3.0–3.2 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed from 45:55 to 95:5; within 3.2–3.8 minutes, the volume ratio of mobile phase A to mobile phase B remained at 95:5; within 3.8–4.1 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed from 95:5 to 30:70; within 4.1–4.5 minutes, the volume ratio of mobile phase A to mobile phase B remained at 30:70. Flow rate: 0.4 mL / min; Washing method: Before and after aspiration; Weak wash buffer: methanol:water (1:1, v / v); Washing volume: 500 μL; Strong wash buffer: methanol.

[0066] Mass spectrometry conditions: Ion detection mode: Multiple reaction monitoring (MRM); Ionization mode: Pneumatic-assisted electrospray ionization (ESI); Ion polarity: Negative; Detector: Loxoprofen, [MH] - m / z 254.3→83.1, DP value -50V, CE value -16V; active metabolite trans-OH form, [MH] - m / z 247.2→202.9, DP value -50V, CE value -10V; loxoprofen-d4, [MH] - m / z 249.2→205.1, DP value -50V, CE value -9V. Mass spectrometry parameters: IonSprayVoltage: -4500V; TEM: 550℃. Specific ion scans of loxoprofen and its active metabolite trans-OH form with loxoprofen-d4 are shown below. Figure 1 , Figure 2 and Figure 3 As shown.

[0067] 3. Preparation of standard solutions

[0068] Preparation of working solutions of loxoprofen and its active metabolite trans-OH body: Accurately weigh two portions of loxoprofen and its active metabolite trans-OH body reference standards, correct them with a mass correction factor, and dissolve them in methanol to obtain two stock solutions of loxoprofen and its active metabolite trans-OH body with concentrations of 0.100 / 0.100 mg / mL. The stock solutions are stored in a -20°C freezer. After the stock solution passes inspection, accurately measure one portion of the loxoprofen and its active metabolite trans-OH isomer stock solution and dilute it with a methanol and water mixture (50:50, v / v) to prepare a series of working solutions for the standard curve of loxoprofen and its active metabolite trans-OH isomer with concentrations of 800 / 800, 640 / 640, 400 / 400, 150 / 150, 60.0 / 60.0, 24.0 / 24.0, 8.00 / 8.00, and 4.00 / 4.00 ng / mL. Accurately measure another portion of the loxoprofen and its active metabolite trans-OH isomer stock solution and dilute it with a methanol and water mixture (50:50, v / v) to prepare QC working solutions with concentrations of 600 / 600, 240 / 240, 50.0 / 50.0, 12.0 / 12.0, and 4.00 / 4.00 ng / mL.

[0069] Preparation of loxoprofen-d4 working solution: Accurately weigh loxoprofen-d4 reference standard, correct for mass, and dissolve in methanol to obtain a loxoprofen-d4 stock solution with a concentration of 1.00 mg / mL. Store the stock solution at -20°C. Accurately measure a certain amount of the loxoprofen-d4 stock solution and dilute it with a methanol and water mixture (50:50, v / v) to prepare an internal standard working solution with a concentration of 500 ng / mL.

[0070] 4. Preparation of standard curve samples and quality control samples

[0071] The preparation process for standard curve samples and quality control samples at each concentration level is illustrated below: Add 10 μL of the corresponding working solution to 190 μL of blank plasma, mix thoroughly, and adjust the volume as needed. Prepare standard curve samples containing loxoprofen and its active metabolite trans-OH isomers at concentrations of 0.200 / 0.200, 0.400 / 0.400, 1.20 / 1.20, 3.00 / 3.00, 7.50 / 7.50, 20.0 / 20.0, 32.0 / 32.0, and 40.0 / 40.0 ng / mL, and standard curve samples and quality control samples at concentrations of 0.200 / 0.200 ng / mL (LLOQ) are prepared sequentially. Quality control samples with concentrations of 0.600 / 0.600 ng / mL (LQC), 2.50 / 2.50 ng / mL (GMQC), 12.0 / 12.0 ng / mL (MQC), and 30.0 / 30.0 ng / mL (HQC).

[0072] 5. Sample pretreatment

[0073] Add 50 μL of sample (biological sample to be tested, standard curve sample, quality control sample) to a 2.0 mL 96-well plate; for double blank samples and blank samples, add 50 μL of blank matrix. Add 50 μL of solvent methanol:water (1:1, v / v) to the double blank samples. Except for the double blank samples, add 50 μL of internal standard working solution to all wells, then add 200 μL of 0.01 M disodium hydrogen phosphate buffer (pH = 2.1), shake at 1000 rpm for 1 min, add 800 μL of extraction solvent methyl tert-butyl ether, then vortex the 96-well plate at 2000 rpm for 10 min, and continue to centrifuge the 96-well plate at 4000 rpm for 5 min at 4 °C. Add 200 μL of supernatant to a clean 96-well plate and blow it dry with nitrogen at 45 °C. Then add 100 μL of a mixed solution of methanol and water (1:1, v / v) to reconstitute the solution and place it in the injection chamber or a refrigerator at the same temperature for testing.

[0074] 6. Methodological Examination Content

[0075] The detection method was validated according to the "Guidelines for Validation of Quantitative Analysis Methods for Biological Samples" in the 2015 edition of the Chinese Pharmacopoeia to ensure the accuracy, repeatability, and stability of the assay. Validation included the following: specificity, standard curve, precision and accuracy, matrix effect, extraction recovery, and stability.

[0076] II. Results and Discussion

[0077] 1. Exclusivity

[0078] Under the chromatographic conditions used in this experiment, the retention time of loxoprofen was approximately 2.40 min. Figure 8 The retention time of the active metabolite trans-OH form is approximately 2.05 min. Figure 9 The retention time of the internal standard (loxoprofen-d4) is approximately 2.37 minutes. Figure 8 Take 50 μL of blank plasma from each of six different sources. Except for the absence of internal standards, perform sample pretreatment procedures to obtain the chromatograms of the blank plasma samples, as shown below. Figures 4-1 to 4-6 and Figures 5-1 to 5-6 The chromatogram of the sample with the lower limit of quantitation is shown in [reference needed]. Figure 8 , Figure 9 The results indicate that endogenous substances in plasma do not affect the detection of loxoprofen and its active metabolite trans-OH, and internal standards also do not affect the detection of loxoprofen and its active metabolite trans-OH.

[0079] 2. Accuracy and precision tests

[0080] Quality control samples containing loxoprofen and its active metabolite trans-OH isomer at concentrations of 0.200 / 0.200 ng / mL (LLOQQC), 0.600 / 0.600 ng / mL (LQC), 2.50 / 2.50 ng / mL (GMQC), 12.0 / 12.0 ng / mL (MQC), and 30.0 / 30.0 ng / mL (HQC). Six samples were prepared for each concentration, and two standard curves were prepared (obtained by regression analysis of two sets of standard curve samples). The ratio f of the peak area As of loxoprofen and its active metabolite trans-OH isomer to the peak area Ai of the internal standard was calculated and denoted as f. Substituting f into the standard curve of the day, the measured concentration, the average measured concentration, and the accuracy were obtained. Intra-batch precision and accuracy were calculated, and the results are shown in Table 2. The results showed that, except for the limit of quantitation (LLOQ), the precision RSD of the intra-batch quality control samples of loxoprofen and its active metabolite trans-OH form was less than 15%, the intra-batch accuracy (RE) was at least 67% not exceeding ±15%, and at least 50% of the quality control samples at each concentration level deviated from their theoretical values ​​by no more than ±15%. For the LLOQ samples of loxoprofen and its active metabolite trans-OH form, the precision RSD was less than 20%, the intra-batch accuracy (RE) was at least 67% not exceeding ±20%, and at least 50% of the quality control samples at each concentration level deviated from their theoretical values ​​by no more than ±20%. In summary, both precision and accuracy met the requirements.

[0081] Table 2 Precision and accuracy of intra-batch and inter-batch sample testing

[0082]

[0083]

[0084]

[0085] 3. Investigation of matrix effect

[0086] Matrix sample preparation: Six batches of blank plasma from different donors were used, and nine replicate double blank samples were prepared from each batch of blank plasma. The blank matrix extract was obtained by following the sample pretreatment procedure. After extraction, a certain amount of analyte and internal standard were added to make the final concentration consistent with the injection concentration of the low, medium and high concentration quality control samples (three replicates for each concentration level).

[0087] Preparation of solution samples: Pure water was used instead of blank plasma for the pretreatment step. The working solution was then diluted to prepare the corresponding concentration so that the concentration after adding 200 μL of blank matrix extract or 200 μL of pure water extract was consistent with the injection concentration after pretreatment of low, medium and high concentration quality control samples, and 3 replicate samples were prepared for each concentration.

[0088] The results showed that the total matrix effect factor of loxoprofen (calculated as peak area ratio) was 99.2–102.1, with a precision of less than 1.9%; the total matrix effect factor of the active metabolite trans-OH body (calculated as peak area ratio) was 99.8–100.6, with a precision of less than 1.4%. The plasma matrix did not affect the accurate quantification of loxoprofen and its active metabolite trans-OH body. The matrix effect data are shown in Tables 3-1 and 3-2.

[0089] Table 3-1 Matrix effects of the compound loxoprofen

[0090]

[0091]

[0092] Table 3-2 Matrix effects of the trans-OH forms of active metabolites of compounds

[0093]

[0094] 4. Analysis of extraction recovery rate

[0095] Matrix sample preparation: Plasma was prepared by mixing blank plasma from 6 batches of different donors to form 18 replicate double blank samples. The blank plasma extract was obtained by following the sample pretreatment procedure. After extraction, a certain amount of analyte and internal standard were added to make the final concentration consistent with the injection concentration of the low, medium and high concentration quality control samples (3 replicates for each concentration level).

[0096] Quality control sample preparation: Take quality control samples of low, medium and high concentrations and process them according to the sample processing method. Prepare 6 samples for each concentration level.

[0097] Recovery is evaluated by comparing the response values ​​of the analyte or internal standard in a single quality control sample with the mean response values ​​of a double blank sample after extraction with the addition of the analyte and internal standard.

[0098] The acceptance criteria for recovery were: the precision of the recovery at each concentration level and across all concentration levels should be within 15.0%. The recovery rate of loxoprofen (calculated as peak area ratio) was 57.52%, with recoveries of 56.49%, 58.46%, and 57.61% at low, medium, and high concentrations, respectively. The recovery rate of the trans-OH isoform of the active metabolite (calculated as peak area ratio) was 65.10%, with recoveries of 64.58%, 67.99%, and 62.77% at low, medium, and high concentrations, respectively. The results are shown in Tables 4-1 and 4-2.

[0099] Table 4-1 Extraction recovery rate of compound loxoprofen

[0100]

[0101] Table 4-2 Extraction and recovery rates of trans-OH forms of active metabolites of compounds

[0102]

[0103]

[0104] 5. Stability Study

[0105] Stability of processed samples: After the first injection analysis of the analytical batch to examine precision and accuracy, the samples were placed in the autosampler (4℃) for 193 hours. Freshly prepared standard curve samples and previously analyzed samples were then injected and analyzed, and the chromatograms were recorded. The results are shown in Tables 5-1 and 5-2. The injection solutions of loxoprofen and its active metabolite trans-OH plasma samples showed good stability after being placed in the autosampler for 193 hours, which meets the requirements for biological sample analysis.

[0106] Room temperature stability: The prepared low and high concentration levels of quality control samples, containing loxoprofen and its active metabolite trans-OH, were 0.600 / 0.600 ng / mL and 30.0 / 30.0 ng / mL, respectively. After each concentration level of the sample was mixed evenly, it was placed at room temperature for 22 h and then analyzed by LC-MS / MS. The chromatograms were recorded. The results are shown in Tables 6-1 and 6-2. The plasma samples showed good stability after being placed at room temperature for 22 h.

[0107] Freeze-thaw stability: Freshly prepared samples containing loxoprofen and its active metabolite trans-OH form at concentrations of 0.600 / 0.600 ng / mL and 30.0 / 30.0 ng / mL were placed in a -80°C freezer for 5 freeze-thaw cycles. Acceptance criteria were: the %RE (mean reactivity ratio) of the average measured value of the stable sample should not exceed ±15.0% from its theoretical value, and the %RSD (mean relative standard deviation) of the measured values ​​of the stable sample at each concentration level should be ≤15.0%. The results are shown in Tables 7-1, 7-2, 7-2, and 7-4. The samples showed good stability after 5 freeze-thaw cycles at -80°C.

[0108] Long-term stability: Freshly prepared samples containing loxoprofen and its active metabolite trans-OH isomer at concentrations of 0.600 / 0.600 ng / mL and 30.0 / 30.0 ng / mL were stored at -80°C for 52 days before testing. Acceptance criteria were: the %RE (mean reactivity ratio) of the average measured value of the stable samples should not exceed ±15.0% from its theoretical value, and the %RSD (mean relative standard deviation) of the measured values ​​of the stable samples at each concentration level should be ≤15.0%. The results are shown in Tables 8-1 and 8-2. The samples showed good stability after being stored at -80°C for 52 days.

[0109] Table 5-1 Stability of samples after preparation of the compound loxoprofen

[0110]

[0111]

[0112] Table 5-2 Stability of samples after preparation of trans-OH forms of active metabolites of compounds

[0113]

[0114] Table 6-1 Stability of the compound loxoprofen during sample pretreatment (room temperature stability)

[0115]

[0116]

[0117] Table 6-2 Stability of trans-OH forms of active metabolites of compounds during biological sample pretreatment (room temperature stability)

[0118]

[0119] Table 7-1 Freeze-thaw stability test conditions for compound loxoprofen: -20℃, 5 times

[0120]

[0121]

[0122] Table 7-2 Freeze-thaw stability test conditions for compound loxoprofen: -80℃, 5 times

[0123]

[0124] Table 7-3 Freeze-thaw stability test conditions for the trans-OH forms of active metabolites of compounds: -20℃, 5 times

[0125]

[0126]

[0127] Table 7-4 Freeze-thaw stability test conditions for the trans-OH forms of active metabolites of compounds: -80℃, 5 times

[0128]

[0129] Table 8-1 Experimental conditions for the long-term stability of compound loxoprofen: -80℃, 43 days

[0130]

[0131] Table 8-2 Long-term stability test conditions of trans-OH forms of active metabolites of compounds: -80℃, 43 days

[0132]

[0133] This invention establishes a method for the detection of loxoprofen and its active metabolite trans-OH form in plasma using HPLC-MS / MS. The method exhibits good specificity, and endogenous substances in plasma do not interfere with the determination of the samples. The linear range of the standard curves for loxoprofen and its active metabolite trans-OH form is 0.200–40.0 ng / mL, showing good linearity. The intra- and inter-assay precision of the detection results for quality control samples at three concentration levels (high concentration, 30.0 ng / mL, medium concentration, 12.0 ng / mL, and low concentration, 0.600 ng / mL) is less than 15.0%. The intra- and inter-assay precision of the detection results for the quality control sample at the limit of quantitation (0.200 ng / mL) is less than 20.0%. The total matrix effect factor (calculated as peak area ratio) for loxoprofen ranged from 99.2 to 102.1, with a precision of less than 1.9%; the total matrix effect factor (calculated as peak area ratio) for the active metabolite trans-OH body ranged from 99.8 to 100.6, with a precision of less than 1.4%. The plasma matrix did not affect the accurate quantification of loxoprofen and its active metabolite trans-OH body. The recovery rate of loxoprofen extraction was 57.52%; the recovery rate of the active metabolite trans-OH body extraction was 65.10%. Plasma samples containing loxoprofen and its active metabolite trans-OH body showed good stability after 22 hours at room temperature; good stability after 5 freeze-thaw cycles; good stability after 193 hours in an autosampler at 4°C; and good stability after 43 days at -80°C, meeting the requirements for biological sample analysis.

[0134] In summary, the HPLC-MS / MS method established in this invention for determining the concentration of loxoprofen and its active metabolite trans-OH in human plasma meets the relevant requirements of the 2020 edition of the Pharmacopoeia "Guiding Principles for Validation of Quantitative Analysis Methods for Biological Samples" and can be used for plasma sample analysis and detection in clinical trials.

[0135] Comparative Example 1

[0136] The liquid chromatography and mass spectrometry conditions were the same as in Example 1, except that during sample pretreatment, the pH adjuster was changed from 0.01M disodium hydrogen phosphate buffer (adjusted to pH 2.1 with phosphate) to 0.01M disodium hydrogen phosphate buffer (adjusted to pH 4.5 with phosphate).

[0137] The results showed that when the pH adjuster was changed from 0.01M disodium hydrogen phosphate buffer (adjusted to pH 2.1 with phosphoric acid) to 0.01M disodium hydrogen phosphate buffer (adjusted to pH 4.5 with phosphoric acid), the chromatographic signals of loxoprofen and the trans-OH form decreased, the limit of quantitation increased from 0.2 ng / mL to 1 ng / mL, the sensitivity decreased significantly, and many interfering peaks appeared near the retention time of the trans-OH form. Figure 10 .

[0138] Comparative Example 2

[0139] The mass spectrometry conditions and pretreatment were the same as in Example 1, except that the liquid chromatography conditions were changed by replacing the ACE, Excel3 SuperC18 (2.1×50mm, 3μm) column with ZORBAX Eclipse XDB-Phenyl (4.6mm*75mm, 3.5μm).

[0140] The results showed that when the chromatographic column was replaced by ZORBAX Eclipse XDB-Phenyl (4.6mm*75mm, 3.5μm) from ACE, Excel 3 SuperC18 (2.1×50mm, 3μm), large interference peaks appeared at the positions of loxoprofen and internal standard peaks in the double blank samples, such as... Figure 11 .

[0141] Comparative Example 3

[0142] The liquid chromatography and mass spectrometry conditions were the same as in Example 1, except that the extractant was replaced with ethyl acetate instead of methyl tert-butyl ether during sample pretreatment.

[0143] The results showed that when the extractant was replaced with ethyl acetate instead of methyl tert-butyl ether, the signals of loxoprofen, the trans-OH form, and the internal standard in the double blank samples all decreased, and the peak positions showed significant interference. Furthermore, the chromatograms of loxoprofen and the internal standard contained too many interfering peaks. Figure 12 Furthermore, the limit of quantification for loxoprofen and its trans-OH form increased from 0.2 ng / mL to 8 ng / mL, while the sensitivity decreased significantly.

[0144] Comparative Example 4

[0145] The mass spectrometry conditions and pretreatment were the same as in Example 1, except for the liquid phase conditions. The elution program was adjusted to a gradient elution as follows: within 0.0-1.0 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55; within 1.0-1.5 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed from 45:55 to 85:15; within 1.5-3.0 minutes, the volume ratio of mobile phase A to mobile phase B was 85:15; within 3.0-3.5 minutes, the volume ratio of mobile phase A to mobile phase B gradually changed from 85:15 to 45:55; and within 3.5-4.5 minutes, the volume ratio of mobile phase A to mobile phase B was 45:55.

[0146] The results showed that when the elution program changed, the signals of loxoprofen, trans-OH, and internal standard in the test sample all decreased. The limit of quantification for loxoprofen increased from 0.2 ng / mL to 1 ng / mL, and the limit of quantification for trans-OH increased from 0.2 ng / mL to 2 ng / mL, while the sensitivity decreased significantly.

[0147] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS, characterized in that, It includes the following steps: (1) Pretreatment of human plasma samples; wherein, the pretreatment of human plasma samples includes: adding internal standard working solution and pH adjuster to human plasma samples, shaking, adding methyl tert-butyl ether, vortexing and centrifuging, taking the supernatant, blowing to dry with nitrogen, and reconstituted with a rehydration solvent to obtain the sample to be tested; the internal standard working solution contains loxoprofen-d4; the pH adjuster is 0.01~0.05M disodium hydrogen phosphate buffer, and adjusting the pH to 1.5~3.5 with phosphoric acid; the rehydration solvent is a mixed solution of methanol and water with a volume ratio of 1:1~5; (2) Detection by liquid chromatography-mass spectrometry, using a mixed mobile phase A and mobile phase B for gradient elution, wherein mobile phase A is acetonitrile and mobile phase B is a 0.01~0.2% aqueous acetic acid solution; (3) Determination of the concentration of loxoprofen and its active metabolite trans-OH in human plasma, wherein the gradient elution process is as follows: within 0.0-3.0 minutes, the volume ratio of mobile phase A to mobile phase B is 45:55; within 3.0-3.2 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 45:55 to 95:5; within 3.2-3.8 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5; within 3.8-4.1 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 95:5 to 30:70; within 4.1-4.5 minutes, the volume ratio of mobile phase A to mobile phase B is 30:

70.

2. The method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS according to claim 1, characterized in that, In step (1), the pH adjuster is 0.01M disodium hydrogen phosphate buffer, and the pH is adjusted to 2.1 with phosphoric acid; the resolvent is a mixed solution of methanol and water with a volume ratio of 1:

1.

3. The method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS according to claim 1, characterized in that, In step (1), the pretreatment of human plasma samples includes: taking 50 μL of human plasma sample, adding 50 μL of internal standard working solution and 200 μL of pH adjuster, shaking, adding 800 μL of methyl tert-butyl ether, centrifuging by vortexing, taking 200 μL of supernatant, blowing it dry with nitrogen at 40~50℃, reconstituted with 100 μL of a 1:1 volume ratio of methanol and water, and shaking well to obtain the sample to be tested; the pH adjuster is 0.01M disodium hydrogen phosphate buffer, and the pH is adjusted to 2.1 with phosphoric acid.

4. The method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS according to claim 1, 2, or 3, characterized in that, In step (1), the internal standard working solution is prepared as follows: weigh loxoprofen-d4 reference standard, dissolve it in methanol to obtain a loxoprofen-d4 stock solution with a concentration of 1.00 mg / mL, and then dilute it with a methanol-water mixture with a volume ratio of 50:50 to obtain a loxoprofen-d4 internal standard working solution with a concentration of 500 ng / mL.

5. The method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS according to claim 4, characterized in that, The conditions for vortexing and centrifugation are as follows: vortex for 10 min, centrifuge at 4000 rpm / min for 5 min at 4℃; place the sample to be tested in an autosampler for LC-MS / MS analysis, with an injection volume of 10 μL and an autosampler temperature of 4℃.

6. The method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS according to claim 1, characterized in that, The mobile phase B is a 0.05% aqueous solution of acetic acid.

7. The method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS according to claim 1, characterized in that, The liquid chromatography conditions included: ACE column, Excel 3 SuperC18.

8. The method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS according to claim 7, characterized in that, The liquid chromatography conditions included a column length of 50 mm, a diameter of 2.1 mm, and a packing particle size of 3 µm.

9. The method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS according to claim 1, characterized in that, The liquid chromatography conditions include: column temperature of 30~45℃; flow rate of 0.2~1.0 mL / min.

10. The method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS according to claim 9, characterized in that, The liquid chromatography conditions included: column temperature of 40℃ and flow rate of 0.4 mL / min.

11. The method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS according to claim 1, characterized in that, Mass spectrometry conditions included: electrospray ionization source, negative ion multiple reaction monitoring (MRM), spray voltage -4500 V, ion source temperature 550 °C; loxoprofen, [M+H]. + m / z 245.3→ 83.1, DP value -50 V, CE value -16 V; its active metabolite is the trans-OH form, [M+H] + m / z 247.2→ 202.9, DP value -50 V, CE value -10 V; loxoprofen-d4, [M+H] + m / z 249.2→ 205.1, DP value -50 V, CE value -9 V.

12. The method for detecting loxoprofen and its active metabolite trans-OH form in human plasma using HPLC-MS / MS according to claim 1, characterized in that, This method can be used for clinical pharmacokinetic sample monitoring.

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