HPLC同时测定血浆中头孢氨苄及甲氧苄啶浓度.docVIP

HPLC同时测定血浆中头孢氨苄及甲氧苄啶浓度 　 作者：夏登宁 夏艳姣 尹佳 贺英菊 【摘要】 采用高效液相色谱紫外检测器同时检测血浆中头孢氨苄和甲氧苄啶的浓度。以0.5倍血浆体积，浓度为10%(v/v)的高氯酸为蛋白沉淀剂。色谱条件：Diamonsil C18(250mm×4.6mm，5μm)，柱温：30℃，流动相：pH3.0的0.05mol/L的磷酸盐缓冲液∶乙腈∶甲醇(76∶16∶8)，流速1.0ml/min，进样量20μl，检测波长240nm。头孢氨苄、甲氧苄啶及内源性物质分离良好。头孢氨苄血药浓度线性范围为0.5～50.0μg/ml，检测限为50ng/ml，高、中、低浓度的相对回收率在97.3%～100.5%之间(n=5)，日内RSD≤6.5%(n=5)，日间RSD≤8.3%(n=5)。甲氧苄啶血药浓度线性范围为0.25～25.0μg/ml，检测限为25ng/ml，高、中、低浓度的相对回收率在96.5%～99.3%之间(n=5)，日内RSD≤6.9%(n=5)，日间RSD≤8.6%(n=5)。本法样品处理简单、灵敏、准确，适合于同时测定两者的体内浓度。 【关键词】 头孢氨苄； 甲氧苄啶； 血药浓度； 高效液相色谱法。 ABSTRACT To establish an HPLC method for the simultanecous determination of cefalexin and trimethoprin in rabbits′ plasma. The protein was precipitated by perchloric acid. Chromatographic separation of analytes was achieved on a C18 column at the temperature of 30℃, using a mixture of 0.05mol/L PBS (pH=3.0), acetonirile and methanol (76∶16∶8, v/v/v) as mobile phase and detection was performed at 240 nm. The linearity was obtained over the concentration range of 0.5～50.0μg/ml for cefalexin and 0.25～25.0μg/ml for trimethoprim. The relative recoveries of cefalexin and trimethoprim at high, medium and low concentrations were 97.3%～100.5% and 96.5%～99.3% respectively. Both interand intraday RSD (n=5) were ≤8.3% for cefalexin and ≤8.6% for trimethoprim. The limits of detection for cefalexin and trimethoprim were 50ng/ml and 25ng/ml respectively. The present HPLC method is simple, sensitive, accurate, and was successfully applied to the simultaneous determination of cefalexin and trimethoprim in rabbits′ plasma. KEY WORDS Cefalexin; Trimethoprim; Plasma drug concentration; HPLC 头孢氨苄为口服头孢菌素类药物，作用机制主要是通过干扰细菌细胞壁主要成分肽聚糖的合成发挥抗菌作用。甲氧苄啶作用机制为干扰细菌的叶酸代谢，选择性抑制细菌的二氢叶酸还原酶的活性，使二氢叶酸不能还原为四氢叶酸，阻止细菌核酸和蛋白质的合成。头孢氨苄与甲氧苄啶联用(5∶1)能扩大抗菌谱，提高抗菌活性［1］，现有相应产品上市。国内外有较多的文献报道了高效液相紫外检测器检测头孢氨苄的血药浓度［2，3］，和使用紫外检测器［4］、质谱检测器［5］、高效毛细管电泳法［6］检测甲氧苄啶的血药浓度。由于两者的物理化学性质差异较大，以及生物样品内源物质干扰较多，高效液相色谱紫外检测器同时测定两者血药浓度有一定的困难，国内外文献报道较少。本实验采用外标法，建立了一个简单、灵敏、准确，可同时测定两者血药浓度的方法。 1 材料与方法 1.1 仪器与试药 高效液相色谱仪：LC10ATv