美罗培南治疗药物监测的HPLC方法探索及其临床采样流程建立

目的:建立HPLC法测定人血浆中美罗培南的血药浓度,结合其稳定性研究制定临床采样流程,并应用到治疗药物监测个体化给药方案设计。方法:血浆样品经甲醇沉淀蛋白,取上清加水稀释后进样分析;色谱柱为Venusil HILIC(4.6 mm×250 mm,5.0μm);流动相为50 mmol·L^-1磷酸二氢钾含0.05%甲酸-乙腈(30∶70,V/V);流速0.8 mL·min^-1;检测波长为299 nm;柱温40℃;分别考察美罗培南全血、溶血样本在不同温度、不同采血管等条件下的稳定性。结果:美罗培南在0.38~71.30μg·mL^-1范围内线性关系良好(r=0.999 9),定量下限为0.38μg·mL^-1,低、中、高浓度的萃取回收率在101.87%~107.89%,日内、日间RSD均小于2.81%;以EDTA-K2为最佳的临床采血管,EDTA-K2管和肝素钠管中的美罗培南能在室温(19~22℃)下稳定4 h;溶血样本在2~6℃冰箱存放7 h稳定,但相对于EDTA-K2管和血浆质控,肝素钠管会使美罗培南检出率偏高;血浆样本在室温(19~22℃)、2~6℃冰箱内6 h、-40℃冻存20 d、-40℃反复冻融3次均稳定。结论:建立的TDM临床采样流程充分考虑了临床采样的实际情况并能确保美罗培南药物的稳定性及分析检测结果的准确性,可满足临床血药浓度监测需求。

Exploration of HPLC method for therapeutic drug monitoring of meropenem and its establishment of clinical sampling procedure

OBJECTIVE To establish a HPLC method for the determination of meropenem concentration in human plasma and clinical sampling process based on its stability study,which can be applied to the design of individualized dosing regimen for therapeutic drug monitoring.METHODS Protein in plasma was precipitated by methanol,and supernatant was taken and diluted with water.The chromatographic column was Venusil HILIC (4.6 mm×250 mm,5.0 μm).The mobile phase composition included solvent A (50 mmol·L^-1 potassium dihydrogen phosphate containing 0.05% formic acid) and solvent B (acetonitrile) (30:70,V/V).Flow rate was set at 0.8 mL·min^-1,the detection wavelength at 299 nm and column temperature at 40℃.The stability of whole blood and hemolytic samples of meropenem at different temperatures and under different conditions were investigated.RESULTS The linear range of meropenem was good at 0.38-71.30 μg·mL^-1 (r=0.999 9),and the lower limit concentration was 0.38 μg·mL^-1.The extraction recovery of low,medium and high concentration were 101.87%-107.89%,the relative standard deviation of intra-day precision and inter-day precision were less than 2.81%.With EDTA-K2 tubes as the optimal clinical blood collection,meropenem in EDTA-K2 tubes and heparin sodium tubes were stable at 19-22℃for 4 h.Hemolysis sample was stable in 2-6℃ for 7 h.Compared with meropenem in EDTA-K2 tubes and in plasma quality control samples,the component of heparin tubes may make the detection rate higher of meropenem.The plasma samples could maintain stable at 19-22℃ and 2-6℃ conditions for 6 h,in long-term stability of frozen samples after 42 days and in two freeze-thaw cycle at-40℃,respectively.CONCLUSION The established TDM clinical sampling process fully considered the actual situation of clinical sampling and could ensure the stability of meropenem and the accuracy of analysis results,which could satify the needs of clinical blood drug concentration determination.