脊柱结核病椎骨中利福平浓度HPLC测定方法研究

Study on the affected vertebrae level determination of rifampicin in spinal tuberculosis by HPLC

目的建立利福平在脊柱结核髂骨与病椎骨中药物浓度的反相高效液相色谱分析方法。方法以利福喷丁为内标,采用氯仿为萃取剂处理骨匀浆样品,加入二乙胺解除利福平对骨基质的吸附。分析柱为HypersilBDSC18柱(150mm×4.6mm),以0.2mol·L-1KH2PO4(pH=4.0)-乙腈(65:35)为流动相,流速1.0ml·min-1,检测波长254nm,内标法定量。结果在0.0560～1.875μg·g-1骨匀浆浓度(相当于骨中浓度0.5600～18.75μg·g-1)范围内利福平和内标峰面积比与骨样本利福平浓度呈良好线性关系(r=0.9994),萃取回收率在88.47%～96.51%(n=5),方法回收率为98.85%～103.51%(n=5),日内、日间RSD分别为2.34%～4.58%,3.38%～5.79%,(n=5)。结论本方法准确、精密度高、重复性好,是一可靠的测定骨中利福平浓度的方法,适用于脊柱结核手术患者体内正常及病椎骨组织中利福平浓度的定量分析,以便于个体化抗结核化疗方案的判定与改进。

Objective To establish high performance liquid chromatography (HPLC) method for determination of rifampicin in vertebrae and ilium of spinal tuberculosis. Methods The chloroform was used as extraction solution and ethylenediamine was added to eliminate rifampicin’s adsorption. The chromatography condition was Hypersil BDS C_ 18 column(150 mm×4.6 mm) and 0.2 moL·L -1 phosphate buffer-acetonitrile (65:35, adjusted pH to 4.0 with H_ 3PO_ 4) as a mobile phase at 1.0 ml·min -1. Detection wavelength was 254 nm. Rifapentine was used as an internal standard. Results Methodology evaluation showed that bone homogenates concentration between 0.0560-1.8750μg·g -1(correspond to 0.560-18.75μg·g -1 in bone) had a good linear coefficient relation(r=0.9994), the detection limit concentration was 0.028μg·g -1, the extraction recovery rate was 88.47%-96.51%(n=5) , the method recovery rate was 98.85%-103.51%(n=5),and the within-day RSD and between-day RSD were 2.34%-4.58% and 3.38%-5.79% respectively (n=5) . ConclusionThe method possesses merits of sensitivity, good accuracy, high precision and fine reproducibility,which is suitable for the study of rifampicin in veterbral focus of patients for the sake of evaluating and adjusting the individual chemotherapy regimen.