高效液相色谱-质谱联用技术测定儿童血浆中厄他培南的浓度并考察溶血效应

Quantitative Analysis of Ertapenem in Children′s Plasma by HPLC-MS/MS and Hemolytic Effect Evaluation

目的建立基于高效液相色谱-质谱联用技术(HPLC-MS/MS)测定儿童血浆中厄他培南浓度的方法并考察溶血效应。方法样品经沉淀蛋白法进行前处理,以美罗培南为内标,采用Hypersil GOLD^(TM)柱(2.1 mm×50 mm,3μm)分离;以0.1%甲酸-水和0.1%甲酸-乙腈为流动相,梯度洗脱;质谱检测采用电喷雾离子源正离子扫描方式,多反应监测(MRM)模式;模拟不同程度(0.5%、2%、5%)的溶血样品进行溶血效应考察。结果厄他培南的血药浓度在0.1~50μg·mL^(-1)内呈良好的线性关系(r>0.998 0),方法学验证均符合要求,溶血效应符合标准。结论本方法快速、准确、灵敏、稳定、专属性强,且不受溶血程度为5%以下的溶血效应影响,可应用于厄他培南儿童药动学研究及治疗药物监测。

OBJECTIVE To establish a high performance liquid chromatography-tandem mass spectrometry method for the quantitative analysis of ertapenem in children′s plasma and evaluate the influence caused by hemolytic effect.METHODS Hypersil GOLDTM column(2.1 mm×50 mm,3μm)was used and the column temperature was mainteined at 40℃.The mobile phases was composed of water(A)and acetonitrile(B),each containing 0.1%formic acid.Gradient elution was conducted at a flow rate of 0.5 mL·min-1.The injection volume was 3 mL.Total run time was 4 min.For mass spectrometry,electrospray ionization source was chosen,and positive ion monitoring mode was used with multi-reaction monitoring(MRM)mode.The precursor-to-product ion pairs of ertapenem and meropenem(IS)for quantitation were 476.2→432.2 and 384.1→141.2,respectively.Acetonitrile containing 0.1%formic acid was used as protein precipitation.RESULTS The calibration curve was linear over a range of 0.1 to 50μg·mL-1(r>0.9980,n=6).The within-run and between-run accuracy RSDs were both less than±15%.The within-run and between-run variable coefficients(CV)were both less than 8.5%.No significant matrix effect was observed for ertapenem and the hemolytic effect was acceptable.Ertapenem was stable for 72 h in whole blood at 5℃.Analytes in plasma were stable after three freeze-thaw cycles and robust for 4 h(at room temperature),8 h(in autosampler,5℃)and 30 d(-20℃),respectively.Incurred sample analysis was satisfactory within Ch.P guideline limits.CONCLUSION This rapid,sensitive,robust and exclusive HPLC-MS/MS assay requires small-volume of samples and simple preparation without interference by hemolytic effect,which can be applied in pharmacokinetic studies and therapeutic drug monitoring in children.