用高效液相色谱-质谱联用法测定新生儿血浆中苯巴比妥的浓度

Determination of phenobarbital in neonatal plasma by HPLC-MS/MS method

目的建立高效液相色谱-质谱联用(HPLC-MS/MS)法测定新生儿微量血浆中苯巴比妥(PHB)的浓度。方法用有机溶剂沉淀蛋白进行前处理,以PHB-D5为内标,反相色谱柱,流动相:0.05%甲酸-1 mmoL·L^(-1)乙酸铵-水(A)和甲醇(B),梯度洗脱,流速为0.5 mL·min^(-1),柱温为50℃,进样量为2μL。用负离子电喷雾离子源负,多反应监测(MRM)模式扫描。考察该方法的专属性、标准曲线与定量下限、残留和稀释效应、精密度与回收率、基质效应和稳定性。结果血浆样品中PHB的线性方程式为y=109.38×10^(-3)x+19.57×10^(-3)(r=0.9996),PHB在1~75μg·mL^(-1)线性关系良好,定量下限为1μg·mL^(-1)。批内和批间精密度相对标准偏差≤5.74%,准确度为91.29%~103.31%,提取回收率为102.83%~111.22%,稳定性良好,不受血浆基质的影响。结论本方法快速、简便、灵敏且专属性强,适用于新生儿血浆中PHB的治疗药物监测和药代动力学-药效学研究。

Objective To establish a method for determination of phenobarbital(PHB)in epileptic neonatal plasma by high performance liquid chromatography-mass spectrometry(HPLC-MS/MS).Methods The samples were pre-treated by precipitation protein method with organic solvent.PHB-D5 was used as isotope internal standards(SIL-IS).Chromatographic column:flow phase:0.05%formic acid-1 mmol·L^(-1)ammonium acetate-water(A)and methanol(B),gradient elution;flow speed:0.5 mL·min^(-1).Ion source is electric spray ion source,multireaction monitoring mode for quantitative analysis,using negative ion monitoring mode.The specificity,lower limit of quantitation,standard curve,residual effect,dilution effect,precision,matrix effects,extraction recovery and stability were investigated.Results The linear equation for PHB in plasma samples:y=109.38×10^(-3)x+19.57×10^(-3)(r=0.9996).The linear range of PHB was 1-75μg·mL^(-1),and lower limit of quantitation was 1μg·mL^(-1).The intra-day and inter-day precision relative standard deviation was equal or lesser than 5.74%,and the accuracy ranges from 91.29%-103.31%,the recovery ranges from 102.83%-111.22%,with good stability,and this method was not affected by common matrix.Conclusion This study determined the blood concentration of PHB in epileptic neonatal with HPLC-MS/MS,which is rapid,simple,stable,economical,and high specificity.It can be applied to therapeutic drug monitoring of PHB in clinical neanates.