Abstract: Meropenem is a new generation of carbapenem antibiotics, with broad-spectrum antibacterial activity and high stability of β-lactamase, highly sensitive to common biliary system infection pathogenic bacteria. At present, there are only reports on the determination of the concentration of meropenem in blood and urine by high-performance liquid chromatography in China, and there are no reports on the determination of the concentration of meropenem in bile. The HPLC external standard method used in this study provides a reference for the detection method of the bile drug concentration determination and pharmacokinetic study of the drug.
High-performance liquid chromatography (HPLC) was used to determine the concentration of meropenem in bile. Methods Chromatography column: Waters Symmetry C18 (4.6mm × 150mm, 5μm); mobile phase: acetonitrile: 0.05mol / L potassium dihydrogen phosphate (pH6.5, 8:92); flow rate 1.5ml / min; detection wavelength 300nm; Sample volume 20μl; column temperature 22.5 ℃. Results The bile drug concentration of meropenem was in the range of 0.5-500.0 μg / ml, and the linear relationship between the concentration and peak area ratio was good (r = 0.9999), and the lowest limit of quantification in bile was 0.5 μg / ml. The recovery rate of HPLC method was over 96.87% (n = 15). RSD≤6.22% within the day, RSD≤3.69% during the day. Conclusion This method is simple, rapid and accurate, and can be used for the determination of meropenem concentration and pharmacokinetic studies.
1 Materials and methods
1.1 Instrument
Waters high-performance liquid chromatography system: Waters1525 dual pump, Waters2487 UV detector, SCL-10A injector, Breeze chromatography analysis software; XW-80A vortex mixer (Shanghai Medical University Instrument Factory); Flying Pigeon TGL-16C high-speed centrifuge Machine (Shanghai Anting Scientific Instrument Factory); micro sampler (Shanghai Qiujing Biochemical Reagent Factory); 0.45um microporous filter membrane (Tianjin Tengda Filter Device Factory).
1.2 Medicines and reagents
Meropenem (meropenem, trade name: Meiping, batch number FK174, 0.5g each) and meropenem reference (content 87.6%, batch number 80549A) are produced by Sumitomo Pharmaceutical Co., Ltd., Japan. Acetonitrile is first grade chromatographically pure; potassium dihydrogen phosphate and potassium hydroxide are analytically pure; double distilled water.
1.3 Chromatographic conditions
Column: Waters Symmetry C18 (4.6mm × 150mm, 5μm); mobile phase: acetonitrile: 0.05mol / L potassium dihydrogen phosphate (pH6.5, 8:92); flow rate 1.5ml / min; detection wavelength 300nm; injection Amount 20μl; column temperature 22.5 ℃ (room temperature).
1.4 Determination method of bile drug concentration
(1) Preparation of reference substance solution Weigh 11.42mg of meropenem reference substance accurately, place in a 10ml brown volumetric flask, dissolve with mobile phase, dilute to the mark, shake well to obtain 1000μg / ml of meropenem stock solution, precise amount Take 1ml and dilute to 10ml with mobile phase to get 100μg / ml stock solution two; then take precise 1ml of stock solution and dilute to 100ml with mobile phase to get 10μg / ml stock solution three.
(2) Pretreatment of bile [3] Precisely take 200μl of bile, add mobile phase to 2ml precisely, vortex and centrifuge (5000r / min) for 10min, take the supernatant and filter it with a microporous membrane for injection.
(3) Standard curve and detection limit Take blank bile and add meropenem standard stock solution to make it into a bile sample containing meropenem 500.0, 100.0, 50.0, 25.0, 10.0, 5.0, 1.0 and 0.5μg / ml, pretreated according to bile After the method was processed, the sample was injected, and the peak area was quantified using the external standard method. Results A linear regression of the chromatographic peak area (A) with the sample concentration (Ï).
(4) Recovery rate test Take blank bile and add meropenem standard solution to make each a bile solution of 500.0, 50.0 and 5.0μg / ml. Perform bile pretreatment according to the method described above and inject the sample. The recovery rate of meropenem in bile at the concentration.
(5) Precision test: Prepare 5 parts of bile solution of high, middle and low concentration in the same way as above, measure within the same day, and calculate the intra-day precision of the method. The rest of the samples were stored in a -20 ℃ refrigerator. Afterwards, the bile samples of high, medium and low concentrations were measured by the same method on different days, and the day-to-day precision was calculated by calculating the coefficient of variation during the day.
2 results
2.1 Selection of chromatographic conditions
The content of meropenem and related substances are mostly determined by HPLC method, which is still not included in the Chinese Pharmacopoeia, and there is no report on the determination of the concentration of the drug in bile samples in China. To ensure the versatility of the method, the C18 column was used as the experimental column. With reference to the HPLC determination method of this class of substance, under the condition of ensuring that all impurity peaks are completely separated from the main peak, acetonitrile: 0.05 mol / L potassium dihydrogen phosphate (pH6.5, 8:92) is used as the mobile phase. Meropenem's ultraviolet scanning results show that there is maximum absorption at the wavelength of 300nm, so 300nm is selected as the detection wavelength. The external standard method is simpler, more convenient and more intuitive than the internal standard method. It has been verified by a series of testing methodologies to meet the testing requirements (2.2 to 2.5 items).
2.2 Specificity of the determination method of meropenem
Under the above chromatographic conditions, the four test samples including blank bile, control solution, blank bile plus control solution, and bile after intravenous injection of meropenem in animals have good separation effect, and there is no endogenous substance interference (Fig. 1). Surgical clinical antimicrobial treatment is based on the principle of simplicity. Usually, only a single antibiotic is used. Meropenem is a new generation of carbapenem antibiotics with ultra-wide spectrum bactericidal activity. Patients rarely use combination drugs. The complexity of clinical medication and Other drugs are less likely to cause interference, so this specific test has practical value.
2.3 Standard curve and detection limit
Meropenem has a linear relationship between the bile concentration (Ï) and the chromatographic peak area (A) in the concentration range of 0.5 to 500.0 μg / ml. The regression equation:
A = 2217.49Ï-3215.42 r = 0.9999
The minimum quantitation limit of bile drug concentration is 0.5μg / ml, suggesting that this standard curve has a high correlation and accuracy in calculating the bile drug concentration of meropenem chromatographic peak area, and the bile drug concentration only needs to reach 0.5μg / ml Accurate quantification.
2.4 Recovery rate
The recovery rates of meropenem 500.0, 50.0 and 5.0 μg / ml were 99.58%, 96.87% and 110.28% respectively (Tab.1). The recovery rate is the ratio of the measured concentration and the standard concentration, which reflects the repeatability of the measurement method and the degree of conformity of the test results. The results show that the recovery rate of this experiment is very high and meets the test requirements.
2.5 Precision
The intra-day precision RSD (%) of the three concentrations of meropenem 500.0, 50.0 and 5.0 μg / ml were 0.69, 2.28 and 6.22, respectively; the intra-day precision RSD (%) were 1.33, 4.78 and 3.69 (Tab. 2, Tab .3). Precision is the precision of the HPLC measurement method. The smaller the RSD, the higher the precision within the day; otherwise, the precision is lower. The experimental results show that the method has a high degree of precision in the determination of meropenem, and the RSD is less than 10%, which meets the detection method requirements.
2.6 Determination of rabbit bile drug concentration
One healthy male rabbit was used for the experiment, fasted for 12 hours, and then anesthetized with 3% pentobarbital for choledochostomy. After leaving blank bile, intravenously inject meropenem 75mg / kg, and collect 1.5ml of bile at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, and 4h respectively after the administration (for the concentration of bile, see Tab.4 ). The results showed that the peak concentration time of the bile drug concentration in rabbits after intravenous injection of meropenem was very fast. The peak concentration reached 15 minutes after the end of the administration, and the peak concentration was 78.64 μg / ml. After that, the bile concentration of meropenem decreased rapidly with time, 30 minutes At this time, the concentration of bile drugs has dropped to less than half of the peak value. After 4 hours of administration, the peak of meropenem could not be detected in the bile of rabbits, suggesting that meropenem was eliminated in the bile faster. However, due to the small number of experimental samples, the results of this concentration measurement can only be used as a reference; if you need to further understand the pharmacokinetics of meropenem in bile, you must increase the sample size.
3 Discussion
(1) Treatment of biliary tract infections, especially acute suppurative severe cholangitis, must use antibiotics that can reach and maintain a high effective concentration in the biliary tract [1, 3, 4]. Meropenem is highly sensitive to biliary tract pathogens, but at present there is no data on the determination method of the concentration of meropenem in bile. At present, the determination methods of meropenem mainly include microbiological method [5], optical rotation method [6], high performance capillary electrophoresis method [7] and HPLC method [8]. The microbial method has a large error, and the relative error can reach 10% to 25%. It is time-consuming and complicated to operate; the optical rotation method has low sensitivity and large interference; the high-efficiency capillary electrophoresis method has poor sensitivity and is not suitable for the determination of drug concentration in biological samples; The three determination methods have great limitations, and the accuracy is limited. The HPLC method is efficient, fast, accurate, highly sensitive, and reproducible [9].
(2) This article uses HPLC method to determine the concentration of meropenem in bile. The bile pretreatment of the determination method is simple and requires no special treatment. Since the mobile phase uses acetonitrile, the mobile phase containing acetonitrile is also used to precipitate and remove impurities such as proteins in the bile when pretreating bile samples. Results The peak of meropenem in the bile was clean and neat, the peak shape was good, the separation was complete, and there was no interference of miscellaneous peaks. This treatment method is significantly simpler and faster than other methods (such as distillation, concentration, and adsorption methods), and also makes the determination method more specific. In this study, the external standard method is used to quantify the peak area, which is economical and simple, and has high recovery and precision. It has the advantages of high efficiency, fast, accurate, high sensitivity and good reproducibility. Reliable testing methods for scientific research.
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