Method and kit for detecting blood concentration of compound sulfamethoxazole and metabolite thereof

Abstract

The invention provides a method and a kit for detecting blood concentration of compound sulfamethoxazole and metabolite thereof. According to the method, a methanol precipitator is combined with Oasis PRiME HLB reversed-phase solid-phase extraction, the centrifugal process of plasma treatment is reduced, traditional activation and balance steps are omitted, time is saved, meanwhile, the method is more environmentally friendly, and the extraction recovery rate of a to-be-detected substance can be improved; gradient elution is replaced by isocratic elution, the balance returning time is avoided, the overall operation time is shorter, the separation application is not influenced, the operation time is only 6 minutes, all the to-be-measured substances can be accurately and simultaneously measured only by 1 microliter, and the peak appearance time is 2 minutes. According to the invention, trimethoprim, sulfamethoxazole and metabolites thereof sulfamethoxazole-N-acetyl and 4-nitro sulfamethoxazole in human plasma can be monitored at the same time by using a treatment method and a computer method.

Description

Technical Field

[0001] The invention belongs to the technical field of testing and analysis, and specifically relates to a method and a kit for detecting the blood drug concentration of compound sulfamethoxazole and its metabolites. Background Art

[0002] Co-trimoxazole is the drug of choice for the treatment of Pneumocystis jiroveci, Stenotrophomonas maltophilia, Nocardia infections and melioidosis eradication period. High concentrations of trimethoprim or sulfamethoxazole may cause adverse reactions, such as hyperkalemia, renal damage or drug hypersensitivity. For example, high doses of trimethoprim may cause hyperkalemia; monitoring sulfamethoxazole levels can improve the clinical outcomes of patients with Pneumocystis carinii pneumonia and reduce adverse reactions; sulfamethoxazole metabolites lead to crystal formation, causing obstruction and renal damage. The severe skin adverse reactions of co-trimoxazole are related to the specific metabolite 4-nitrosulfamethoxazole, but the concentration of 4-nitrosulfamethoxazole in human plasma has not yet been detected. By monitoring blood drug concentrations, the dose can be detected and adjusted in time to prevent these reactions. In addition, there are significant inter-individual differences in co-trimoxazole. In order to ensure that patients receive effective therapeutic doses, avoid inadequacy or overdose, and thus improve the treatment effect, therapeutic drug monitoring of co-trimoxazole is necessary.

[0003] Fatica E et al. reported a method for quantifying sulfamethoxazole and trimethoprim in serum by ultra-fast solid-phase extraction-tandem mass spectrometry (Fatica E, Faber J, Gaffron C, et al. Quantification of Serum SuLfamethoxazole and Trimethoprim by ULtra-fast Solid-Phase Extraction-Tandem Mass Spectrometry [J]. Ther Drug Monit, 2020, 42(5):724-732), including: 1) collecting serum samples and performing necessary pretreatment steps, such as centrifugation and protein removal; 2) injecting the sample into the SPE column and enriching the target compound from the sample by solid phase extraction; 3) using the elution solution to elute the target compound from the SPE column; 4) introducing the target compound in the eluate into the mass spectrometer for analysis, and using tandem mass spectrometry (MS / MS) technology for quantitative analysis; 5) using the corresponding software for data processing and quantitative calculation based on the output results of the mass spectrometer. However, it has the following disadvantages: lack of chromatographic separation steps, which may lead to ion suppression and interference problems; long sample preparation time; only trimethoprim and sulfamethoxazole can be monitored simultaneously, and all related substances for efficacy and adverse reactions cannot be fully covered; lack of complete matrix effect and extraction recovery rate research may affect the reliability and accuracy evaluation of this method.

[0004] Xiangxiang Fu et al. reported a high-performance liquid chromatography (HPLC) method to simultaneously monitor the concentrations of sulfamethoxazole and trimethoprim in the plasma of patients with melioidosis during the intensive or eradication period (Xiangxiang Fu, Lili Zhong, Rong Wang, et al. Exploration of a high-performance liquid chromatography method for antibiotic drug monitoring in melioidosis patients and establishment of its sampling procedure [J]. ResuLts in Chemistry, 2023, 6: 2211-7156). The chromatographic column of this method is an Acutfex YS-C8 column (4.6×250mm, 5μm), and the mobile phase A is composed of 1000mL ultrapure water and 2mL triethylamine. The pH is adjusted to 6.0 with glacial acetic acid, and the mobile phase B is acetonitrile. This method uses gradient elution, with aminopyrine as the internal standard and trichloroacetic acid as the precipitant to pre-treat the sample and remove the supernatant for detection on the machine. The flow rate is 1.0mL min -1 , wavelength of 240nm, and then the established method was used to measure the plasma concentrations of SMZ and TMP in 11 patients with melioidosis in Hainan, China. However, it also has the following disadvantages: the sample preparation time is long and an online solid phase extraction step is required; only trimethoprim and sulfamethoxazole can be monitored simultaneously, and all related substances for efficacy and adverse reactions cannot be fully covered; the lack of complete matrix effect and extraction recovery research may affect the reliability and accuracy evaluation of the method; the HPLC (Waters e2695, Waters Corporation, USA) instrument is used for detection, the total running time is too long, the injection volume is too large; the amount of blood sample and working solution is large.

[0005] Merro Pharmaceutical Technology Co., Ltd. reported a method for simultaneously detecting multiple antimicrobial drugs in blood (CN116660413A). The pretreatment steps are as follows: take a 2mL centrifuge tube, add 50μL of the plasma sample to be tested, 5μL of the internal standard working solution and 200μL of 5% formic acid-acetonitrile solution as a precipitant in turn, vortex shake for 30s to mix, or use an oscillating incubator to mix at a speed of 1300r for 2 minutes, let stand for about 2min until the solution is separated, press into a Spectra Easy Fast Filtration Column, which takes 10s, take the 10s filtrate, dilute it with 0.1% ammonia aqueous solution at 90, and mix it; the detection conditions are as follows: the high performance liquid chromatograph is equipped with a triple quadrupole detector, C 18Chromatographic column, 3.0×50mm, 2.7um, mobile phase A: 0.1% formic acid + 5mM ammonium acetate aqueous solution; mobile phase B: 0.1% formic acid + 5mM ammonium acetate methanol solution, flow rate 0.6mL / min, column temperature 35℃, injection volume 5μL; mobile phase gradient: from 0 to 2min, the volume fraction of mobile phase A changes uniformly from 90% to 5%, and the volume fraction of mobile phase B changes uniformly from 10% to 95%; from 2 to 3min, the volume fraction of mobile phase A is 5%, and the volume fraction of mobile phase B is 95%; from 3 to 3.1min, the volume fraction of mobile phase A changes uniformly from 5% to 90%, and the volume fraction of mobile phase B changes uniformly from 95% to 10%; from 3.1 to 4.5min, the volume fraction of mobile phase A is 90%, and the volume fraction of mobile phase B is 10%. However, it also has the following disadvantages: it can only monitor trimethoprim and sulfamethoxazole at the same time, and cannot fully cover all relevant substances for efficacy and adverse reactions; the chromatograph injection volume is too large. Summary of the invention

[0006] The object of the present invention is to provide a method based on high performance liquid chromatography-tandem mass spectrometry for accurately and rapidly quantitatively analyzing the contents of trimethoprim, sulfamethoxazole and their metabolites sulfamethoxazole-N-acetyl and 4-nitrosulfamethoxazole.

[0007] The present invention provides a method for simultaneously detecting the blood drug concentration of sulfamethoxazole and its metabolites in blood, comprising:

[0008] Pre-treating the blood sample to be tested to obtain a target sample solution, subjecting it to liquid chromatography-mass spectrometry detection, and calculating the result in combination with a standard curve;

[0009] Among them, the test substances include trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl, and 4-nitrosulfamethoxazole.

[0010] In one embodiment, the pretreatment of the blood sample to be tested includes adding a precipitant and an internal standard mixed working solution to the blood sample to be tested, vortexing, taking the mixed solution and purifying it through an Oasis PRiME HLB solid phase extraction column, collecting the effluent, mixing it with ultrapure water, and vortexing it in a centrifuge tube to obtain a target sample solution.

[0011] In one embodiment, the blood sample is a human blood sample.

[0012] In one embodiment, the blood sample is plasma.

[0013] In one embodiment, the precipitation agent is methanol.

[0014] In one embodiment, the volume ratio of the precipitant to plasma is 5-15:1, preferably 7-9:1, and more preferably 8:1.

[0015] In one embodiment, the internal standard is trimethoprim-D9, sulfamethoxazole-D4, sulfamethoxazole-N-acetyl-D4, 4-nitrosulfamethoxazole-D4.

[0016] In one embodiment, the solvent of the internal standard mixed working solution is methanol.

[0017] In one embodiment, the concentration of the internal standard mixed working solution is 1-3 μg / mL, preferably 2 μg / mL.

[0018] In one embodiment, the volume ratio of the internal standard mixed working solution to the blood sample is 0.5 to 2:1, preferably 1:1.

[0019] In one embodiment, the volume ratio of ultrapure water to effluent is 0.2-0.6:1, preferably 0.4:1.

[0020] In one embodiment, the standard curve is drawn by the following method:

[0021] a. Mix the blank blood sample with the mixed standard curve working solution, add the precipitant and the internal standard mixed working solution thereto, vortex and oscillate, take the mixed solution and purify it through the Oasis PRiME HLB solid phase extraction column, collect the effluent, mix it with ultrapure water, and vortex in a centrifuge tube to obtain a mixed standard curve sample solution;

[0022] b. The mixed standard curve sample solution was tested using high performance liquid chromatography tandem mass spectrometry, the peak area ratios of trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl, 4-nitrosulfamethoxazole and the internal standard were calculated, and a standard curve was established by the internal standard method.

[0023] In one embodiment, the volume ratio of the blank blood sample to the mixed standard curve working solution is 1-2:2-1, preferably 1:1.

[0024] In the present invention, the blank blood sample refers to the plasma of healthy volunteers who do not take any drugs.

[0025] In one embodiment, the solvent of the mixed standard curve sample solution is methanol.

[0026] In one embodiment, in the liquid chromatography, the mobile phase comprises mobile phase A and mobile phase B, mobile phase A is a formic acid aqueous solution, and the mobile phase B is acetonitrile.

[0027] In one embodiment, the volume fraction of formic acid in the formic acid aqueous solution is 0.05-0.15%, preferably 0.1%.

[0028] In one embodiment, the liquid chromatography adopts an isocratic elution procedure, with mobile phase A being 30-50% and mobile phase B being 50-70%; preferably, mobile phase A being 40% and mobile phase B being 60%.

[0029] In one embodiment, the flow rate of the mobile phase is 0.2 to 0.5 mL min -1 , preferably 0.350 mL·min -1 .

[0030] In one embodiment, the chromatographic column of the liquid chromatography is ACQUITY UPLC HSS T3 (2.1×100 mm) with a particle size of 1.8 μm; and the pre-column is ACQUITY UPLC HSS T3 (2.1×5 mm) with a particle size of 1.8 μm.

[0031] In one embodiment, the column temperature is 30-50°C, preferably 40°C.

[0032] In one embodiment, the injection volume is 1 to 2 μL, preferably 1 μL.

[0033] In one embodiment, the liquid chromatography conditions are: chromatographic column ACQUITY UPLC HSS T3 (2.1×100 mm), particle size 1.8 μm; pre-column ACQUITY UPLC HSS T3 (2.1×5 mm), particle size 1.8 μm; mobile phase A is 0.1% formic acid water, mobile phase B is acetonitrile; an isocratic elution program is used, and the flow rate is 0.350 mL·min -1 , column temperature 40℃, injection volume 1μL, sample plate temperature 9℃; elution conditions: 0-6min mobile phase A 40%, mobile phase B 60%.

[0034] In one embodiment, the mass spectrometry conditions are: electrospray ionization source HESI source, positive ion scanning mode; spray voltage is set to 3.5 kV, ion transfer tube temperature: 350°C, auxiliary gas heating temperature: 300°C, sheath gas flow rate: 50 arb, auxiliary flow rate: 10 arb; scanning range: 100-800.

[0035] In one embodiment, the method further comprises a process of verifying the detection result according to the concentration of the quality control substance.

[0036] The present invention also provides the application of the method in clinical detection of blood drug concentration of compound sulfamethoxazole and its metabolites; wherein the test substances include trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl, and 4-nitrosulfamethoxazole.

[0037] The present invention also provides a test kit for simultaneously detecting the blood drug concentration of sulfamethoxazole and its metabolites in blood, wherein the kit comprises: an Oasis PRiME HLB solid phase extraction column, an internal standard mixed working solution, a precipitant, ultrapure water, a mobile phase A, and a mobile phase B;

[0038] Among them, the analytes include trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl, and 4-nitrosulfamethoxazole;

[0039] The precipitant is methanol;

[0040] The mobile phase A is a formic acid aqueous solution, and the mobile phase B is acetonitrile;

[0041] In the internal standard mixed working solution, the internal standards are trimethoprim-D9, sulfamethoxazole-D4, sulfamethoxazole-N-acetyl-D4, and 4-nitrosulfamethoxazole-D4.

[0042] In one embodiment, the test kit further comprises: a mixed standard curve working solution.

[0043] In one embodiment, the test kit further comprises: a blank plasma sample.

[0044] In one embodiment, the test kit further comprises: a mixed quality control working solution. The beneficial effects of the present invention are:

[0045] 1. Use chromatographic columns for chromatographic separation, reducing the problems of ion suppression and interference.

[0046] 2. The protein precipitation method is used, and the precipitant is methanol, which can be used to precipitate proteins in plasma. The volume ratio of the precipitant to plasma is about 8:1, and the amount of blood sample and working solution is small. Only 50μL of plasma is needed for accurate quantification. The method is simple to operate, the sample preparation time is often shortened, and the cost is low. A new type of PRiME HLB reversed solid phase extraction reduces the 15-minute centrifugation process in pretreatment and eliminates the traditional activation and equilibration steps. After extraction, the sample is directly passed through the column, which saves time and is more environmentally friendly. It can remove more than 95% of matrix interferences such as proteins, salts, and phospholipids, making the samples cleaner and the matrix effect smaller. Compared with the pretreatment method using 5% formic acid-acetonitrile as a precipitant and filtration with a Spectra Easy Fast Filtration Cartridge, the chromatograph injection volume is smaller, the peak shape of all analytes is better, the intensity is higher, and the results are more accurate, especially for sulfamethoxazole-N-acetyl-D4.

[0047] 3. Tests on matrix effect and extraction recovery were added to more comprehensively evaluate the reliability and stability of the method.

[0048] 4. Mobile phase A is 0.1% formic acid water, and mobile phase B is acetonitrile. Isocratic elution is used instead of gradient elution to avoid the return to equilibrium time, making the overall running time shorter without affecting the separation application. With only 6 minutes of running time and 1 μL of injection volume, trimethoprim, sulfamethoxazole and its metabolites, sulfamethoxazole-N-acetyl and 4-nitrosulfamethoxazole can be accurately determined simultaneously. The peak time of each analyte is within 2 minutes, which greatly shortens the peak time.

[0049] 5. Using a single processing method and an on-machine method, trimethoprim, sulfamethoxazole and its metabolites sulfamethoxazole-N-acetyl and 4-nitrosulfamethoxazole, two substances related to efficacy and adverse reactions, in human plasma can be monitored simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The standard curve is shown in Figure 1. A: trimethoprim; B: sulfamethoxazole; C: sulfamethoxazole-N-acetyl; D: 4-nitrosulfamethoxazole;

[0051] Figure 2 The chromatograms are as follows: A: blank matrix; B: blank matrix with only the analyte added but no internal standard; C: blank matrix with only the internal standard added but no analyte; D: sample after oral administration.

[0052] Figure 3 The chromatograms are: A: the chromatogram obtained by the comparative method; B: the chromatogram obtained by the method of the present application. DETAILED DESCRIPTION

[0053] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0054] Example 1

[0055] 1. Preparation of Stock and Working Solutions

[0056] Prepare the stock solutions, standard curve working solutions and quality control working solutions of the analytes trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl and 4-nitrosulfamethoxazole and the stock solutions and working solutions of their corresponding deuterated internal standards (trimethoprim-D9, sulfamethoxazole-D4, sulfamethoxazole-N-acetyl-D4 and 4-nitrosulfamethoxazole-D4) as follows:

[0057] Stock solutions of the test substances: accurately weigh trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl and 4-nitrosulfamethoxazole, respectively, and add a certain amount of methanol to prepare stock solutions with a concentration of 1 mg / mL trimethoprim, 1 mg / mL sulfamethoxazole, 1 mg / mL sulfamethoxazole-N-acetyl and 1 mg / mL 4-nitrosulfamethoxazole.

[0058] Standard curve working solution: 60 μL of trimethoprim stock solution, 400 μL of sulfamethoxazole stock solution, 200 μL of sulfamethoxazole-N-acetyl stock solution, and 200 μL of 4-nitrosulfamethoxazole stock solution were respectively taken from each stock solution to prepare mixed standard solutions with concentrations of 60, 400, 100, and 100 μg / mL, respectively, and then diluted stepwise with methanol to obtain mixed standard curve working solutions with concentrations of 0.3-60 μg / mL trimethoprim, 2-400 μg / mL sulfamethoxazole, 1-200 μg / mL sulfamethoxazole-N-acetyl, and 1-200 μg / mL 4-nitrosulfamethoxazole.

[0059] Quality control working solution: 45 μL of trimethoprim stock solution, 300 μL of sulfamethoxazole stock solution, 150 μL of sulfamethoxazole-N-acetyl stock solution and 150 μL of 4-nitrosulfamethoxazole stock solution were respectively taken from each stock solution to prepare mixed quality control solutions with concentrations of 45, 300, 150 and 150 μg / mL, respectively. By diluting with methanol, mixed quality control working solutions with low, medium and high concentrations of 1.5, 30 and 45 μg / mL trimethoprim, 10, 200 and 300 μg / mL sulfamethoxazole, 5, 100 and 150 μg / mL sulfamethoxazole-N-acetyl and 5, 100 and 150 μg / mL 4-nitrosulfamethoxazole were obtained.

[0060] Internal standard stock solution: Accurately weigh trimethoprim-D9, sulfamethoxazole-D4, sulfamethoxazole-N-acetyl-D4 and 4-nitrosulfamethoxazole-D4, respectively, and add a certain amount of methanol to prepare internal standard stock solutions with concentrations of 1 mg / mL trimethoprim-D9, 1 mg / mL sulfamethoxazole-D4, 1 mg / mL sulfamethoxazole-N-acetyl-D4 and 1 mg / mL 4-nitrosulfamethoxazole-D4.

[0061] Internal standard working solution: Take an appropriate amount of trimethoprim-D9 internal standard stock solution, sulfamethoxazole-D4 internal standard stock solution, sulfamethoxazole-N-acetyl-D4 internal standard stock solution, and 4-nitrosulfamethoxazole-D4 internal standard stock solution from each internal standard stock solution, and add a certain amount of methanol to prepare an internal standard mixed working solution with a concentration of 2 μg / mL.

[0062] 2. Establishment of conditions

[0063] Chromatographic and mass spectrometric conditions were established as follows:

[0064] 1) Chromatographic conditions

[0065] The chromatographic column was ACQUITY UPLC HSS T3 (2.1×100 mm), with a particle size of 1.8 μm; the pre-column was ACQUITY UPLC HSST3 (2.1×5 mm), with a particle size of 1.8 μm. Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. An isocratic elution procedure was used. Flow rate was 0.350 mL·min -1 , column temperature 40°C, injection volume 1 μL, sample plate temperature 9°C. Gradient elution is shown in Table 1.

[0066] Table 1 Isocratic elution table

[0067]

[0068] 2) Mass spectrometry conditions

[0069] Electrospray ionization source HESI source, positive ion scanning mode; spray voltage was set to 3.5 kV, ion transfer tube temperature: 350 ° C, auxiliary gas heating temperature: 300 ° C, sheath gas flow rate: 50 arb, auxiliary flow rate: 10 arb; scanning range: 100-800.

[0070] 3. Sample Pretreatment

[0071] Preparation of target sample solution: transfer 50 μL of plasma to be tested into a 1.5 mL Ep tube, add 400 μL of methanol, add 50 μL of internal standard solution (2 μg / mL), and vortex for 1 min. Take 200 μL of the mixed solution and purify it through an Oasis PRi ME HLB solid phase extraction column, collect the effluent, take 100 μL of the effluent and 400 μL of ultrapure water and transfer them into a 1.5 mL centrifuge tube, vortex for 1 minute, and take 1 μL for injection analysis.

[0072] Preparation of mixed standard curve sample solution: Take 25μL blank plasma, add 25μL standard curve working solution and transfer to 1.5mL Ep tube, add 400μL methanol, add 50μL internal standard solution (2μg / mL), and vortex for 1min. Take 200μL mixed solution and purify it through OasisPRiMEHLB solid phase extraction column, collect the effluent, take 100μL effluent and 400μL ultrapure water and transfer to 1.5mL centrifuge tube, vortex for 1 minute, and take 1μL for injection analysis.

[0073] Preparation of mixed quality control sample solution: Take 25μL blank plasma, add 25μL quality control working solution and transfer to a 1.5mL Ep tube, add 400μL methanol, add 50μL internal standard solution (2μg / mL), and vortex for 1min. Take 200μL of the mixed solution and purify it through the OasisPRiMEHLB solid phase extraction column, collect the effluent, take 100μL of the effluent and 400μL of ultrapure water and transfer to a 1.5mL centrifuge tube, vortex for 1 minute, and take 1μL for injection analysis.

[0074] 4. Methodological Validation

[0075] Methodological validation includes: linearity, selectivity, system adaptability, matrix effect, extraction recovery, accuracy, precision, stability at 4℃ for 12h, stability after repeated freeze-thaw at -20℃, stability at -20℃ for 15d, and stability at -80℃ for 15d, etc. After pretreatment, the samples to be tested are tested on the machine.

[0076] 1) Linear

[0077] Prepare the standard curve working solution according to the method in step 1, and prepare the drug-containing plasma standard curve with concentrations of 0.15-30 μg / mL trimethoprim, 1-200 μg / mL sulfamethoxazole, 0.5-100 μg / mL sulfamethoxazole-N-acetyl, and 0.5-100 μg / mL 4-nitrosulfamethoxazole. After sample pretreatment, test on the machine, with the concentration of the analyte as the horizontal axis and the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard as the vertical axis. Use the weighted least squares method (W=1 / X 2 ) to perform regression operation.

[0078] Acceptance criteria: Within the linear range, the correlation coefficient should be no less than 0.99. The back-calculated concentration of the calibration standard should generally be within ±15% of the labeled value, and the lower limit of quantification should be within ±20%. For unacceptable standard samples, the sample with the largest deviation value should be eliminated each time, and the remaining standard samples should be regressed. If one or more standard samples still exceed the acceptance criteria, continue to eliminate standard samples with relatively large deviation values, and regress the remaining standard samples. This process continues until no standard sample has a deviation value exceeding the standard, and at least 75% of the calibration standards, containing at least 6 valid concentrations, should meet the above criteria.

[0079] Experimental results: The concentration of trimethoprim in plasma was in the range of 0.15-30 μg / mL, the concentration of sulfamethoxazole was in the range of 1-200 μg / mL, the concentration of sulfamethoxazole-N-acetyl was in the range of 0.5-100 μg / mL, and the concentration of 4-nitrosulfamethoxazole was in the range of 0.5-100 μg / mL. The standard curve in each test had a good linear relationship, with a correlation coefficient of ≥0.99, which met the needs of analytical testing. See Table 2 and Figure 1 .

[0080] Table 2 Standard curve regression equations for each analyte

[0081]

[0082] 2) Selectivity

[0083] The interference of 6 different blank matrices on the analyte and internal standard was tested respectively. No analyte and internal standard were added to the blank matrix; only analyte was added to the blank matrix without internal standard; only internal standard was added to the blank matrix without analyte; samples of patients after oral administration were tested on the machine after sample pretreatment. Whether endogenous substances in plasma would affect the determination of analyte and internal standard was investigated.

[0084] Acceptance criteria: No interfering peaks appear at the corresponding retention times of the analyte and internal standard, or if a peak appears, its peak area is within 20% of the LLOQ of the component being tested and within 5% of the internal standard. At least 5 blank plasma samples meet the above requirements.

[0085] Experimental results: see Figure 2 , the peak areas at the corresponding retention times of the analyte and internal standard were within 20% of the LLOQ of the assay component and within 5% of the internal standard.

[0086] 3) System adaptability

[0087] Before batch analysis, take the lower limit of quantitation sample and inject it three times continuously to examine the system adaptability. The system adaptability should record the retention time of trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl, 4-nitrosulfamethoxazole and their corresponding internal standards, as well as the peak area ratio of trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl, 4-nitrosulfamethoxazole and their corresponding internal standards, and calculate the coefficient of variation.

[0088] Acceptance criteria: The coefficient of variation of the retention time of the analyte and the corresponding internal standard shall not exceed 5%, and the ratio of the peak area of ​​each standard to the peak area of ​​the internal standard shall not exceed 20%.

[0089] Experimental results: As shown in Table 3, the coefficient of variation of the retention time of each analyte and the corresponding internal standard is less than 5%, and the ratio of the peak area of ​​each standard to the peak area of ​​the internal standard is less than 20%.

[0090] Table 3 System adaptability test results

[0091]

[0092] 4) Matrix effect and extraction recovery

[0093] Six batches of blank matrices from different donors were used to prepare the following samples using working solutions of low, medium and high concentrations:

[0094] Plasma quality control samples with concentrations of 5, 100, and 150 μg / mL of sulfamethoxazole, 2.5, 50, and 75 μg / mL of sulfamethoxazole-N-acetyl and 4-nitrosulfamethoxazole, and 0.75, 15, and 22.5 μg / mL of trimethoprim were prepared respectively. After being treated according to the sample pretreatment method, peak area A was obtained by injection. Blank plasma (not containing the analyte and internal standard) was treated according to the sample pretreatment method, and plasma quality control samples with low, medium, and high concentrations of each analyte were prepared with the extracted supernatant. After being treated according to the sample pretreatment method, peak area B was obtained by injection. Plasma quality control samples with low, medium, and high concentrations of each standard were prepared with methanol solution. After being treated according to the sample pretreatment method, peak area C was obtained by injection.

[0095] Matrix effect ME = (B / C) / (B IS / C IS )*100%

[0096] Extraction recovery rate = (A / B) × 100%.

[0097] A: Response of the known concentration of the test component before extraction of the blank sample matrix; B: Response of the known concentration of the test component added to the pre-treated blank sample matrix; C: Response of the pure solution of the test component with equal concentration; IS: Internal standard

[0098] Acceptance criteria: Calculate the matrix factor normalized by the internal standard, preferably in the range of 0.8-1.2. The coefficient of variation of the matrix factor normalized by the internal standard and the coefficient of variation of the extraction recovery calculated from 6 batches of matrix should not be greater than 15%. The determination should be performed at low and high concentrations.

[0099] Experimental results: See Table 4. The results meet the acceptance criteria.

[0100] Table 4. Experimental results of matrix effect and extraction recovery of analytes at low, medium and high concentrations

[0101]

[0102]

[0103] 5) Accuracy and precision:

[0104] The quantitative limit and low, medium and high concentration quality control samples of an analytical batch were taken, and the samples were processed according to the sample pretreatment method and then put on the machine. Six samples were paralleled for each concentration to examine the intra-batch accuracy and precision. The quantitative limit and six parallel samples of low, medium and high concentration quality control samples of three independent analytical batches were processed on the machine within two days to examine the inter-batch accuracy and precision.

[0105] Acceptance criteria:

[0106] The intra-assay and inter-assay accuracy for each concentration level was between 85.0% and 115.0%, while that for LLOQ samples was between 80.0% and 120%.

[0107] The intra-assay precision (%CV) and inter-assay precision (%CV) at each concentration level should not exceed 15.0% and for samples at the lower limit of quantitation, should not exceed 20.0%.

[0108] Experimental results: see Table 5, the intra-batch accuracy of each analyte is between 87.19 and 107.38, and the inter-batch accuracy is between 88.49 and 103.96; the intra-batch precision (%CV) and inter-batch precision (%CV) are both below 15.0%.

[0109] Table 5. Accuracy and precision test results

[0110]

[0111]

[0112] 6) Stability

[0113] Stability after 12 hours at 4°C: Take low and high concentration quality control samples from qualified batches of precision and accuracy inspection, place at 4°C for no less than 12 hours, and inject 6 replicates for each concentration. Freshly prepare standard curve and inject to determine its concentration.

[0114] -20℃ for 12h stability: Take low and high concentration quality control samples and put them into a -20℃ refrigerator to freeze. Take them out after at least 12h (the samples are completely frozen at this time), put them into the refrigerator after they are completely thawed at room temperature, take them out after at least 12h and put them into the refrigerator after they are completely thawed at room temperature, freeze them again for at least 12h and take them out and put them into the refrigerator after they are completely thawed at room temperature, perform freeze-thaw cycles three times, and inject and analyze the samples after processing. 6 samples are processed for each concentration, and the concentration is determined by injecting freshly prepared standard curves.

[0115] Stability at -20℃ and -80℃ for 15 days: Take low and high concentration quality control samples stored in -80℃ and -20℃ refrigerators for at least 14 days, take low and high concentration quality control samples at two time points, process 6 samples in parallel for each concentration, and inject freshly prepared standard curves to determine their concentrations. Investigate the stability of long-term stored samples.

[0116] Acceptance criteria: The results should meet the requirements of precision and accuracy.

[0117] Experimental results: See Table 6. The results all meet the requirements of precision and accuracy.

[0118] Table 6. Stability test results under different conditions

[0119]

[0120]

[0121] Comparative Example:

[0122] Take a 2mL centrifuge tube, add 25μL blank plasma sample, 25μL high concentration mixed quality control working solution, 5μL internal standard working solution and 200μL 5% formic acid-acetonitrile solution as precipitant, vortex shake for 30s to mix, or use an oscillating incubator to mix at a speed of 1300r for 2 minutes, let stand for about 2min to allow the solution to separate, press into the Spectra Easy Fast Filtration Column, which takes 10s, take 10μL of the filtrate, dilute it with 90μL of 0.1% ammonia aqueous solution, mix well, inject 5μL, and test according to the detection conditions of this application. The results are as follows Figure 3 A is shown in FIG. 1. The results obtained by testing 25 μL of high-concentration mixed quality control working solution according to the method of the present application are compared ( Figure 3 Compare with B).

[0123] Comparing the spectra of the two methods: the peak shapes of trimethoprim-D9 and sulfamethoxazole-N-acetyl-D4 in A are poor; the retention times of trimethoprim and trimethoprim-D9 are delayed; the intensity of sulfamethoxazole-N-acetyl-D4 is only E4, and the peak area is only 255161; the peak intensities of trimethoprim-D9, sulfamethoxazole-D4, sulfamethoxazole-N-acetyl-D4, 4-nitrosulfamethoxazole and 4-nitrosulfamethoxazole-D4 in A are all lower than those in B.

[0124] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for simultaneously detecting the blood concentration of sulfamethoxazole and its metabolites in blood, comprising: Pre-treating the blood sample to be tested to obtain a target sample solution, subjecting it to liquid chromatography-mass spectrometry detection, and calculating the result in combination with a standard curve; Among them, the test substances include trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl, and 4-nitrosulfamethoxazole.

2. The method according to claim 1, characterized in that The pretreatment of the blood sample to be tested includes adding a precipitant and an internal standard mixed working solution to the blood sample to be tested, vortexing, taking the mixed solution and purifying it through an Oasis PRiMEHLB solid phase extraction column, collecting the effluent, mixing it with ultrapure water, and vortexing it in a centrifuge tube to obtain a target sample solution.

3. The method according to claim 2, characterized in that The precipitant is methanol; in one embodiment, the volume ratio of the precipitant to plasma is 5-15:1, preferably 7-9:1, and more preferably 8:

1.

4. The method according to claim 2, characterized in that: The internal standards are trimethoprim-D9, sulfamethoxazole-D4, sulfamethoxazole-N-acetyl-D4, and 4-nitrosulfamethoxazole-D4; the solvent of the internal standard mixed working solution is methanol; the concentration of the internal standard mixed working solution is 1 to 3 μg / mL, preferably 2 μg / mL; the volume ratio of the internal standard mixed working solution to the blood sample is 0.5 to 2:1, preferably 1:

1.

5. The method according to claim 1, characterized in that: The standard curve is drawn by the following method: a. Mix the blank blood sample with the mixed standard curve working solution, add the precipitant and the internal standard mixed working solution thereto, vortex and oscillate, take the mixed solution and purify it through the Oasis PRiME HLB solid phase extraction column, collect the effluent, mix it with ultrapure water, and vortex in a centrifuge tube to obtain a mixed standard curve sample solution; b. The mixed standard curve sample solution was tested using high performance liquid chromatography tandem mass spectrometry, the peak area ratios of trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl, 4-nitrosulfamethoxazole and the internal standard were calculated, and a standard curve was established by the internal standard method.

6. The method according to claim 5, characterized in that The volume ratio of the blank blood sample to the mixed standard curve working solution is 1-2:2-1, preferably 1:

1.

7. The method according to claim 1, characterized in that In the liquid chromatography, the mobile phase includes mobile phase A and mobile phase B, mobile phase A is formic acid aqueous solution, and mobile phase B is acetonitrile.

8. The method according to claim 7, characterized in that The liquid chromatography adopts an isocratic elution procedure, with mobile phase A being 30-50% and mobile phase B being 50-70%; preferably, mobile phase A being 40% and mobile phase B being 60%.

9. Application of the method according to any one of claims 1 to 8 in clinical detection of blood concentration of trimethoprim-sulfamethoxazole and its metabolites; wherein: The substances to be tested include trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl, and 4-nitrosulfamethoxazole.

10. A test kit for simultaneously detecting the blood concentration of sulfamethoxazole and its metabolites in blood, the kit comprising: O asis PRiME HLB solid phase extraction column, internal standard mixed working solution, precipitant, ultrapure water, mobile phase A, mobile phase B; Among them, the analytes include trimethoprim, sulfamethoxazole, sulfamethoxazole-N-acetyl, and 4-nitrosulfamethoxazole; The precipitant is methanol; The mobile phase A is a formic acid aqueous solution, and the mobile phase B is acetonitrile; In the internal standard mixed working solution, the internal standards are trimethoprim-D9, sulfamethoxazole-D4, sulfamethoxazole-N-acetyl-D4, and 4-nitrosulfamethoxazole-D4.

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