HPLC测定人血清苯巴比妥、卡马西平、苯妥英钠浓度及其与FPIA法测定结果的比较

Determination and Comparison of Phenobarbital,Carbamazepine and Phenytoin in Human Serum by HPLC and FPIA Methods

目的建立能同时测定人血清苯巴比妥（PB）、卡马西平（CBZ）、苯妥英钠（PT）3种药物浓度的高效液相色谱（HPLC）,并与常规荧光偏振免疫法（FPIA）测定结果进行相关性分析。方法以乙酸乙酯-二氯甲烷（4∶1）为提取溶剂,固定相Diamon-sil C18柱（4.6 mm×250 mm,5μm）,流动相为乙腈-水（40∶60）,紫外检测波长285 nm,流速1.0 mL·min^-1。收集癫痫患者服药后稳态谷浓度血样,分别用HPLC和FPIA法测定,考察2种方法的相关程度。结果PB,CBZ,PT的标准曲线范围分别为1.0～45.0,0.1～15.0,1.0～25.0 mg·L^-1,最低检测浓度分别为0.5,0.05,0.5 mg·L^-1,提取回收率分别为80.2%,78.2%,86.2%,日内和日间RSD均小于9%。PB、PT2种方法的测定值无统计学差异（P〉0.05）,但CBZ测定值FPIA法高于HPLC（P〈0.01）。结论HPLC操作简单、灵敏、准确,适用于治疗药物监测;PB和PT2种测定方法有良好的相关性,CBZ不同测定方法,结果存在差异。

OBJECTIVE To establish a HPLC method for simultaneous determination of phenobarbital （PB）, carbamazepine （CBZ） and phenytoin（PT） concentrations in human serum, and to evaluate its relationship to concentrations determined by Fluorescene Polarization Immunoassay（ FPIA ） method. METHODS Serum samples were collected from well controlled epilepsy patients treated with PB, CBZ and PT in Qilu Hospital of Shandong University,and determined by HPLC and FPIA methods respectively. The samples were extracted with acetoacetate：dichlormethane （4：1 ）, separated on a C18 reversed-phase column （4. 6 mm × 250 mm,5 μm）, eluted with mobile phase of 40：60 acetonitril-water, and detected at 285 nm. RESULTS The calibration curves were linear within the range of 1.0 -45. 0 mg·L^-1 for PB, 0. 1 - 15.0 mg·L^-1 for CBZ ,1.0 -25. 0 mg·L^-1 for PT ,the limits of determination were 0. 5, 0. 05, 0. 5 mg·L^-1 , and the average recoveries were 80. 2% ,78. 2% ,86.2% respectively. The inter- and intra-day RSDs were less than 9%. No significant statistic differences existed between FPIA and HPLC method for PB and PT（ P 〉 0. 05）, but for CBZ, the concentration determined by FPIA were higher than that by HPLC. CONCLUSION The HPLC method for PB, PT and CBZ determination was convenient, sensitive and accurate, and suitable for therapeutic monitoring , but more attention should be paid to the difference of CBZ concentration determined by HPLC and FPIA methods in its dosage modification.