HPLC (High Performance Liquid Chromatography) method for detecting concentration of meropenem in plasma

Abstract

The invention discloses a method for determining the concentration of meropenem in plasma by using a high performance liquid chromatography (HPLC) method, and relates to the field of medical technology and in-vivo drug analysis. Comprising the following steps: preparing a meropenem reference substance solution, preparing a metronidazole internal standard solution, determining chromatographic conditions and a standard curve, determining a meropenem detection limit, a meropenem quantitation limit, a plasma recovery rate and stability, treating a blood sample, measuring and the like. The HPLC method disclosed by the invention is simple and convenient to operate, accurate in result, good in durability and low in cost, is suitable for detecting meropenem in plasma in various combined medication states, and can help a patient to adjust the dosage in time according to the plasma concentration monitored by the method through clinical use, so that poor curative effect or adverse reaction is avoided, and the medicine is safer and more effective to use. The HPLC method for detecting the concentration of meropenem in plasma meets the determination requirements of biological samples, and is suitable for clinical treatment drug concentration monitoring and pharmacokinetic research on animals and human bodies.

Description

Technical Field

[0001] The present invention relates to the fields of medical technology and in vivo drug analysis, and in particular to an HPLC method for detecting the blood drug concentration of meropenem in plasma. Background Art

[0002] Meropenem is a new generation of synthetic carbapenem antibiotics with ultra-broad-spectrum antibacterial activity. It is mainly used clinically to treat severe infections caused by Gram-negative bacteria, Gram-positive bacteria, and most anaerobic and aerobic bacteria. Due to changes in liver and kidney function and disease status in patients with severe infections, the pharmacokinetic parameters of meropenem change, resulting in a large variation in meropenem blood concentration, which affects the safety and efficacy of clinical medication. Therefore, the blood concentration and pharmacokinetic detection of meropenem are used to adjust the patient dose and formulate an individualized dosing regimen, which plays a key guiding role in medication. At present, many reports at home and abroad use HPLC-MS or HPLC methods. Although the precision and accuracy are high, the HPLC-MS instrument is expensive and difficult to popularize. In the reported methods, the mobile phase mostly uses a buffer salt system with two salts added for gradient elution. The preparation and use are inconvenient, and the blood sample processing is complicated. Therefore, in order to enable clinical practice to obtain rapid and effective guidance on the use of meropenem, it is of great significance to establish a rapid, simple, accurate and universal HPLC method for the determination of meropenem blood concentration and pharmacokinetics. Summary of the invention

[0003] The present invention aims to overcome the deficiencies of existing technologies and methods, and to provide a rapid, simple, accurate and popular HPLC method for determining the blood concentration and pharmacokinetics of meropenem, so as to provide a basis for rational clinical drug use.

[0004] In order to achieve the above object, the technical solution provided by the present invention is:

[0005] The method for determining the blood concentration and pharmacokinetics of meropenem by HPLC comprises the following steps:

[0006] (1) Preparation of reference solution: Take the meropenem reference, weigh it accurately, dissolve it in methanol and prepare a 1000 µg / mL reference solution. Accurately measure the solution and dilute it with water to prepare 200, 100 and 40 µg / mL reference solutions.

[0007] (2) Preparation of internal standard solution: Take the metronidazole reference substance, weigh it accurately, dissolve it in methanol and prepare a 1 mg / mL internal standard solution.

[0008] (3) Chromatographic conditions: Chromatographic column: Merck Purospher® STARLP RP-18 endcapped (250 mm × 4.6 mm, 5 µm); mobile phase: 0.025 mol / L KH2PO4 (pH = 6.0)-acetonitrile, volume ratio 92:8; flow rate 1.0 ml / mL; detection wavelength 299 nm; column temperature 35 °C; injection volume 20 µL.

[0009] (4) Blood sample processing: accurately measure 200 µL of plasma sample into a 1.5 mL EP tube, add 10 µL of internal standard solution, vortex for 30 seconds, add 400 µL of acetonitrile, vortex for 2 minutes, let stand for 10 minutes, centrifuge at 11,000 rpm for 5 minutes, take 400 µL of supernatant, add 400 µL of dichloromethane, vortex for 1 minute, centrifuge at 11,000 rpm for 5 minutes, take 20 µL of supernatant, and inject and measure according to the chromatographic conditions in step (3).

[0010] (5) Determination of standard curve, minimum detection limit and quantification limit: Take 7 portions of 200 µL of blank plasma, add 20 µL, 15 µL, and 10 µL of 1000 µg / mL meropenem control solution, 20 µL and 10 µL of 200 µg / mL control solution, 10 µL of 100 µg / mL control solution, and 10 µL of 40 µg / mL control solution, respectively, and then add 10 µL of internal standard solution to each, vortex for 30 seconds, and treat according to step (4) to prepare standard solutions of 100, 75, 50, 20, 10, 5, and 2 µg / mL, respectively. Inject and measure according to step (3). With the concentration C of meropenem as the horizontal axis and the correction factor f as the vertical axis, f is the peak area of ​​meropenem / peak area of ​​internal standard, and perform linear regression to obtain the regression equation f=0.1116C+0.0918, R 2 =0.9994, n = 7. When the signal-to-noise ratio S / N ≈10, the limit of quantification of meropenem was 0.75 ng, and when S / N ≈3, the limit of detection was 0.23 ng.

[0011] (6) Determination of precision and stability: Take 200 µL of blank plasma, add 10 µL of 1000 µg / mL meropenem control solution, and treat according to step (4) to make a 50 µg / mL sample. Inject the sample 6 times in succession, and calculate the RSD to be < 5.0%. The assay solution is placed at room temperature and tested at 0, 1, 2, 4, 8, and 18 hours on the same day. The patient plasma is placed at 4°C and sampled at 0, 1, and 2 days, and the RSD is calculated to be < 5.0%.

[0012] (7) Determination of extraction rate and recovery rate: Accurately measure 20 µL and 10 µL of 1000 µg / mL meropenem control solution and 10 µL of 40 µg / mL meropenem control solution, respectively, and then add 10 µL of internal standard solution to each, vortex for 30 seconds, and treat according to step (4) to prepare 3 portions of 100, 50, and 2 µg / mL meropenem test solutions, respectively, and inject and measure according to step (3). Take another meropenem control solution of the same concentration and inject and measure directly. Calculate the extraction recovery rate by the ratio of the measured concentration to the actual concentration. The extraction rate of low, medium, and high concentrations of meropenem should be >90%. At the same time, calculate the actual measurement using the standard curve, and calculate the recovery rate by the ratio of the actual measurement to the added amount. The result should be >95%.

[0013] (8) Repeatability and freeze-thaw test determination: Take 200 µL of blank plasma, add 10 µL of 1000 µg / mL meropenem control solution, and treat according to step (4) to prepare 5 50 µg / mL test solutions. Determine the meropenem concentration according to step (3). The RSD of the results should be <5.0%. Store the patient's plasma samples at -20°C, freeze-thaw repeatedly for 3 times, and take samples at 0, 3, 5, and 7 days. Treat according to step (4) to prepare test solutions. The RSD of the results should be <5.0%.

[0014] (9) Determination of durability: Take different blood samples, and the same tester changes different chromatographic columns and three random staff members measure according to steps (4) and (3). The RSD of the three blood samples should be <5.0%.

[0015] The beneficial effects of the present invention are:

[0016] (1) The mobile phase used in this method is a low-concentration KH2PO4 buffer salt-acetonitrile system. KH2PO4-acetonitrile does not damage the instrument and chromatographic column and is easy to prepare, store and use.

[0017] (2) The blood sample processing established in this method adopts direct precipitation by adding acetonitrile and treatment by adding dichloromethane, and the protein is completely processed. The whole sample processing process takes 25 minutes, which is fast, time-saving and easy to operate.

[0018] (3) This method uses HPLC to detect the blood concentration and pharmacokinetics of meropenem. The instrument can be popularized in various medical institutions, which is convenient for monitoring the blood concentration of meropenem and studying its pharmacokinetic properties. The linear relationship, minimum detection limit, extraction rate, recovery rate, precision, repeatability and stability tests all show that this method has specificity, stability, reliability and accuracy in determining meropenem, and meets the requirements for the determination of biological samples. This method is suitable for clinical therapeutic drug monitoring and pharmacokinetic research. Clinicians can guide patients to adjust the dosage of meropenem according to the blood drug concentration, improve drug efficacy, avoid the occurrence of adverse reactions, and promote the development of precision medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 HPLC spectra of meropenem reference solution, metronidazole internal standard solution and ranitidine internal standard solution using water:acetonitrile (88:12) as mobile phase;

[0020] Figure 2 HPLC spectra of meropenem reference solution, metronidazole internal standard solution and ranitidine internal standard solution using KH2PO4 (pH=6.0):acetonitrile (93:7) as mobile phase;

[0021] Figure 3 HPLC spectra of meropenem reference solution, metronidazole internal standard solution and ranitidine internal standard solution using KH2PO4 (pH=6.0):acetonitrile (92:8) as mobile phase;

[0022] Figure 4 This is the HPLC spectrum after the blood sample was processed by the blood sample processing method ①;

[0023] Figure 5 This is the HPLC spectrum after the blood sample was processed by the blood sample processing method ②;

[0024] Figure 6 This is the HPLC spectrum after the blood sample was processed by method ③;

[0025] Figure 7 is the HPLC spectrum of meropenem control solution;

[0026] Figure 8 is the HPLC spectrum of internal standard metronidazole solution;

[0027] Fig. 9 This is the HPLC spectrum of meropenem in standard control blood sample;

[0028] Fig.10 is the HPLC spectrum of the negative control (blank plasma);

[0029] Fig.11 This is the standard curve of meropenem;

[0030] Fig.12 This is the HPLC spectrum of meropenem detected in clinical patient No. 1;

[0031] Fig.13 This is the HPLC spectrum of meropenem detected in clinical patient No. 2;

[0032] Fig.14 This is the HPLC spectrum of meropenem detected in clinical patient No. 3; Figure 1-Figure 10 , Figure 12-14 Among them, 1 is meropenem, 2 is metronidazole, and 3 is ranitidine. DETAILED DESCRIPTION

[0033] 1. Instruments and reagents

[0034] 1.1 Instruments: Agilent 1260 high performance liquid chromatograph, including G1311C quaternary pump, G1329B autosampler, G1316A column oven and G1314F VWD detector (Agilent UV detector); Mettler Toledo TML303E electronic analytical balance; Eppendorf 5424R low temperature high speed centrifuge, Germany; Beckman Allegra X-30R high speed centrifuge, USA.

[0035] 1.2 Test drugs: Meropenem reference substance, batch number 130506-202004, content 86.8%, metronidazole reference substance, batch number 100191-201808, both from China Food and Drug Inspection Institutes; blank healthy human blood, centrifuged at 3000 rpm, plasma stored at -80℃; acetonitrile and methanol were chromatographically pure, from Merck, Germany; water was miliQ ultrapure water, and other reagents were of analytical grade.

[0036] 2 Methods and Results

[0037] 2.1 Chromatographic conditions Chromatographic column: Merck Purospher® STARLP RP-18 endcapped (250 mm×4.6 mm, 5 µm), guard column: WATCH C18 HPLC Guard column cartridges G101 (10 mm); mobile phase: 0.025 mol / LKH2PO4 (pH=6.0):acetonitrile, volume ratio of 92:8; flow rate 1.0 mL / min; detection wavelength 299 nm; column temperature 35°C; injection volume 20 µL.

[0038] 2.2 Preparation of meropenem control solution: Accurately weigh 10.75 mg of meropenem reference substance, place in a 10 mL volumetric flask, add water to dissolve and dilute to the mark, shake well, and prepare a 933.10 µg / mL solution ①. Accurately measure 2 mL of solution ①, place in a 10 mL volumetric flask, add water to dilute to the mark to prepare a 186.62 µg / mL solution ②, accurately measure 1 mL of solution ①, place in a 10 mL volumetric flask, add water to dilute to the mark to prepare a 93.310 µg / mL solution ③, accurately measure 2 mL of solution ②, place in a 10 mL volumetric flask, add water to dilute to the mark to prepare a 37.324 µg / mL solution ④.

[0039] 2.3 Internal standard solution 1: Accurately weigh 4.96 mg of metronidazole reference substance, place it in a 5 mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and prepare a 992.0 µg / mL metronidazole internal standard solution ①. Accurately weigh 1 mL of internal standard solution ①, place it in a 10 mL volumetric flask, add methanol to dilute to the mark, shake well, and prepare a 99.20 µg / mL metronidazole internal standard solution ②.

[0040] 2.4 Internal standard solution 2: Accurately weigh 4.67 mg of ranitidine hydrochloride reference substance, place it in a 5 mL volumetric flask, add water to dissolve and dilute to the scale, shake well to prepare a 93.40 µg / mL ranitidine internal standard solution.

[0041] 2.5 Pretreatment of plasma samples 2-3 mL of peripheral venous blood was drawn from patients taking meropenem 30 minutes before administration, placed in a blood collection tube containing EDTA anticoagulant, and centrifuged at 3000 rpm for 10 min. 200 µL of plasma sample was accurately measured, 10 µL of metronidazole internal standard solution① was added, vortexed for 30 s, 400 µL of acetonitrile was added, vortexed for 2 min, allowed to stand for 10 min, centrifuged at 11000 rpm for 5 min, 400 µL of supernatant was taken, 400 µL of dichloromethane was added, vortexed for 1 min, centrifuged at 11000 rpm for 5 min, 20 µL of supernatant was taken, and sampled and measured according to the method in "2.1".

[0042] 2.6 Selection of different mobile phase systems and ratios and screening of different internal standards Take meropenem control solution ③, metronidazole internal standard solution ② and ranitidine control solution, and use different mobile phases for sample injection according to the method of "2.1". When the water:acetonitrile system is used and the volume ratio is 88:12, the peak shapes of meropenem and ranitidine chromatographic peaks are very poor, as shown in Figure 2. Figure 1 A and C in the experiment; using 0.025mol / LKH2PO4 (pH=6.0):acetonitrile system, when the volume ratio was 93:7, the chromatographic peaks of meropenem, ranitidine and metronidazole were well improved, with symmetrical peak shapes and high theoretical plate numbers, but the retention times of the three peaks were very close, t RThey are 12.941, 9.355 and 10.881 points respectively. Figure 2 A, B and C in the analysis. The retention time also needs to be optimized for the analysis of in vivo drug samples. Therefore, it is necessary to further change the ratio of 0.025mol / L KH2PO4 (pH = 6.0): acetonitrile to adjust the retention time. When the volume ratio of 0.025mol / L KH2PO4 (pH = 6.0): acetonitrile system is 92:8, the t R The chromatographic peaks were all good, with the number of plates > 10,000. Figure 3 A, B and C in the figure have complex chromatographic peaks for in vivo sample analysis, but the t R T with meropenem R Too close is not conducive to analysis. In order to obtain good analysis of meropenem and internal standard with adjacent peaks, the mobile phase was finally selected as: 0.025 mol / L KH2PO4 (pH = 6.0): acetonitrile, the volume ratio was 92:8, and metronidazole was selected as the internal standard. See Table 1.

[0043] Table 1 Selection of different mobile phase systems and ratios and screening results of internal standards

[0044]

[0045] 2.7 Determination of blood sample pretreatment methods We selected three blood sample pretreatment methods: ① Take 200 µL of blank plasma, add 10 µL of meropenem solution ③ and 10 µL of metronidazole internal standard solution ①, vortex for 30 seconds, add 400 µL of acetonitrile, vortex for 2 minutes, let stand for 10 minutes, centrifuge at 11,000 rpm for 5 minutes, take 20 µL of the supernatant, and inject and measure according to the chromatographic conditions of "2.1". ② After treatment with the method of "① in 2.7", take 400 µL of the supernatant, add 400 µL of water, vortex for 2 minutes, centrifuge at 11,000 rpm for 5 minutes, take 20 µL of the supernatant, and inject and measure. ③ After treatment with the method in "2.7 ①", take 400 µL of the supernatant, add 400 µL of dichloromethane, vortex for 2 minutes, centrifuge at 11000 rpm for 5 minutes, take 20 µL of the supernatant, and inject for determination. Results: After treatment with method ①, the peak shapes of meropenem and metronidazole in the chromatogram were not good, and the extraction of meropenem was incomplete, as shown in Figure 1. Figure 4 In the chromatogram after treatment with method ②, the peak shapes of meropenem and metronidazole were significantly improved, but the extraction of meropenem was incomplete, as shown in Figure 5 In the chromatogram after treatment with method ③, the peaks of meropenem and metronidazole were symmetrical, the plate number was >10000, and the extraction rate of meropenem reached 98.8%. Figure 6Finally, method ③ was selected as the blood sample processing method.

[0046] 2.8 System suitability test In blank plasma, add 10 µL of meropenem solution ① and 10 µL of internal standard solution, vortex mix, and prepare plasma sample test solution according to the sample processing method. In addition, prepare blank plasma and plasma samples of patients taking meropenem according to "2.2". According to the chromatographic conditions of "2.1", the retention times of meropenem and metronidazole are 7.377 minutes and 11.994 minutes, respectively, and the theoretical plate numbers are 11050 and 14789, respectively. The separation between meropenem and metronidazole and adjacent chromatographic peaks is 4.76 and 4.36, respectively. Endogenous impurities in serum have no interference with the determination. The chromatograms of meropenem reference substance, metronidazole reference substance, control blood sample, blank plasma, and patient blood sample are shown in Figure 2. Figure 7-10 、12-14.

[0047] 2.9 Extraction recovery test Take 3 portions of meropenem solution ①20 µL, solution ①10 µL, and solution ④10 µL, add 10 µL of metronidazole internal standard solution ① to each portion, and add 200 µL of blank plasma to each portion. According to the method in "2.4", 3 portions of test solutions with meropenem blood concentrations of 84.830, 44.430, and 1.777 µg / mL are prepared. Samples are injected and measured according to "2.1". Another meropenem solution with the same concentration is directly injected and measured. The extraction recovery (mean ± standard deviation) was calculated. The results showed that the extraction recovery rates of high, medium, and low concentrations were 99.68% ± 1.31%, 97.87% ± 1.04%, and 99.48% ± 2.47%, respectively, which can meet the requirements of the determination.

[0048] 2.10 Preparation of standard curve Take 7 portions of 200 µL of blank plasma, add 20 µL, 15 µL, 10 µL of meropenem solution ①, 20 µL, 10 µL of solution ②, 10 µL of solution ③, and 10 µL of solution ④ to each portion, and then add 10 µL of metronidazole internal standard solution ① to each portion, so as to prepare a series of control solutions of 84.830, 65.100, 44.430, 16.965, 8.887, 4.443, and 1.777 µg / mL. Determine according to "2.1" and record the peak area of ​​each component. Perform linear regression with the plasma concentration C of meropenem and the correction factor f, where f is the peak area of ​​voriconazole / peak area of ​​internal standard. The results showed that meropenem had a good linear relationship with the correction factor f in the concentration range of 1.777~84.830 µg / mL, and the regression equation was f=0.1116C+0.0918, R 2 =0.9994, n=7. Linear relationship graph is shown in Fig.11 .

[0049] 2.11 Method recovery test According to the method in "2.8", 3 portions of the test solution with concentrations of 84.830, 44.430, and 1.777 µg / mL were prepared. The samples were injected and measured according to "2.1". The measured amount was calculated using the standard curve. Recovery = measured amount / added amount × 100%. The results showed a good recovery rate (see Table 2).

[0050] Table 2 Recovery test results

[0051]

[0052] 2.12 Precision test and stability test The 44.430 µg / mL test solution prepared in "2.9" was sampled 6 times in succession, and the RSD of the correction factor f was 1.02%. The test solution was placed at room temperature and sampled at 0, 1, 2, 4, 8, and 18 h on the same day. The RSD of the 0, 1, 2, and 4 h test was 2.36%, but the content decreased by about 7.68% after the test solution was placed for 8 h, and the content decreased by 20.47% after 18 h. The patient's plasma was placed at 4°C for 2 days, and the RSD was 2.29%. Therefore, it is recommended that after collecting the patient's plasma, it should be placed at room temperature for 4 h to complete the test, or placed at 4°C for 2 days to complete the test as soon as possible to avoid degradation of meropenem and inaccurate test results.

[0053] 2.13 Detection limit and quantification limit test Take the 1.777 µg / mL solution prepared in "2.8" and dilute it with methanol in turn. When S / N≈10, the quantification limit is 0.75 ng, and when S / N≈3, the detection limit is 0.23 ng.

[0054] 2.14 Repeatability and freeze-thaw test The same patient plasma sample was taken, and 5 test solutions were prepared according to "2.4". The test was performed according to "2.1". The RSD of blood drug concentration was 2.03%. The patient plasma sample was stored at -20℃, and repeatedly frozen and thawed 3 times. Samples were taken at 0, 3, 5, and 7 days. The samples were prepared and tested according to "2.4" and "2.1". The RSD of blood drug concentration was 2.30% and 2.57%, respectively.

[0055] 2.15 Durability experiment: Three blood samples were taken from patients. The same tester changed different chromatographic columns to test the three blood samples. At the same time, the laboratory randomly selected three people and processed their blood samples according to "2.4" and tested them according to "2.1". The RSD of the three blood samples were <5.0%, respectively. The durability of the experimental method was good.

[0056] Table 3 Durability test results of different inspectors (Chromatographic column: Merck Purospher® STARLP RP-18 endcapped, 250 mm×4.6 mm, 5 µm)

[0057]

[0058] Table 4 Durability test results of different chromatographic columns (the same tester A used different chromatographic columns)

[0059]

[0060] 2.16 Clinical Application of Therapeutic Drug Monitoring Blood drug concentrations of 125 patients who were clinically treated with meropenem were measured. The results showed that the meropenem and metronidazole chromatographic peaks in 125 plasma samples were well separated from adjacent peaks, with symmetrical peak shapes and high plate numbers. The measurement results were accurate and reliable, indicating that the detection conditions can meet the requirements of blood sample determination. The HPLC chromatograms of meropenem blood drug concentrations of 3 patients are shown in the table below. Figure 12-14 .

Claims

1. A HPLC method for detecting the concentration of meropenem in human plasma, characterized in that: The method comprises the following steps: (1) Preparation of reference solution: accurately weigh the meropenem reference substance, dissolve it in water and dilute it to make a 1000 μg / mL reference solution. Accurately measure the reference solution and dilute it with water to make 200, 100 and 40 μg / mL reference solutions respectively. (2) Preparation of internal standard solution: Take the metronidazole reference substance, weigh it accurately, dissolve it in methanol and prepare a 1 mg / mL internal standard solution; (3) Chromatographic conditions: Column: Merck Purospher® STARLP RP-18 endcapped, 250 mm × 4.6 mm, 5 µm; mobile phase: 0.025 mol / L KH2PO4:acetonitrile, volume ratio 92:8, 0.025 mol / L KH2PO4 adjusted to pH 6.0 with H3PO4; flow rate 1.0 mL / min; detection wavelength 299 nm; column temperature 35 °C; injection volume 20 µL; (4) Blood sample processing: accurately measure 200 µL of serum sample, add 10 µL of metronidazole internal standard solution, vortex for 30 seconds, add 400 µL of acetonitrile, vortex for 2 minutes, let stand for 10 minutes, centrifuge at 11,000 rpm for 5 minutes, take 400 µL of supernatant, add 400 µL of dichloromethane, vortex for 1 minute, centrifuge at 11,000 rpm for 5 minutes, take 20 µL of supernatant, and inject and measure according to step (3); (5) Determination of standard curve, detection limit and quantification limit: Take 7 portions of 200 µL of blank plasma, add 20, 15, and 10 µL of 1000 µg / mL meropenem control solution, 20 and 10 µL of 200 µg / mL control solution, 10 µL of 100 µg / mL control solution, and 10 µL of 40 µg / mL control solution to each portion, then add 10 µL of internal standard solution to each portion, vortex for 30 seconds, and process according to step (4) to prepare a series of standard solutions of 100, 75, 50, 20, 10, 5, and 2 µg / mL, respectively. Inject and measure according to step (3). With the concentration of meropenem C as the horizontal axis and the correction factor f as the vertical axis, f is the peak area of ​​meropenem / peak area of ​​internal standard, and perform linear regression to obtain the regression equation f=0.1116C+0.0918, R 2 =0.9994, n = 7. When the signal-to-noise ratio S / N ≈10, the limit of quantification of meropenem was 0.75 ng, and when S / N ≈3, the limit of detection was 0.23 ng; (6) Determination of precision and stability: Take 200 µL of blank plasma, add 10 µL of 1000 µg / mL meropenem control solution, take the sample after treatment in step (4), make a 50 µg / mL sample, inject it 6 times in a row, and calculate the RSD should be < 5.0%. The test solution is placed at room temperature and tested at 0, 1, 2, 4, 8, and 18 hours on the same day, and the patient plasma is placed at 4°C for injection and testing at 0, 1, and 2 days, and the RSD should be calculated to be < 5.0%; (7) Determination of recovery rate: Accurately measure 20 µL and 10 µL of 1000 µg / mL meropenem control solution and 10 µL of 40 µg / mL meropenem control solution, respectively, and then add 10 µL of internal standard solution to each, vortex for 30 seconds, and treat according to step (4) to prepare 3 portions of 100, 50, and 2 µg / mL meropenem test solutions, respectively. Sample injection and determination according to step (3), and take another meropenem control solution of the same concentration and directly inject and determine. Calculate the extraction recovery rate by the ratio of the measured concentration to the actual concentration. The extraction rate of low, medium, and high concentrations of meropenem should be >90%. At the same time, calculate the actual measurement using the standard curve, and calculate the recovery rate by the ratio of the actual measurement to the added amount. The result should be >95%; (8) Repeatability determination: Take 200 µL of patient plasma, process according to step (4), prepare 5 test solutions, and determine the meropenem concentration according to step (3). The RSD of the result should be < 5.0%; (9) Determination of durability: Take different blood samples, and the same tester changes different chromatographic columns and three random staff members measure according to steps (4) and (3). The RSD of the three blood samples should be <5.0%.

2. The method according to claim 1, characterized in that When measuring the control solution, internal standard solution and sample solution, the theoretical plate number calculated based on meropenem and metronidazole should not be less than 8000.

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