固相萃取-HPLC测定人血清万古霉素、去甲万古霉素浓度及其与荧光偏振免疫法测定结果的比较

Determination and Comparison of Vancomycin and Norvancomycin Concentration in Human Serum by HPLC with Solid Phase Extraction and Fluorescence Polarization Immunoassay

关键词:  高效液相色谱法  固相萃取  荧光偏振免疫法  万古霉素  去甲万古霉素

Key words:  HPLC  solid phase extraction  fluorescence polarization immunoassay  vancomycin  norvancomycin

摘要:

目的 建立固相萃取-高效液相色谱法(SPE-HPLC)测定人血清中万古霉素和去甲万古霉素浓度，并与荧光偏振免疫法(FPIA)测定结果进行相关性分析。方法 血清样品经C₁₈固相萃取小柱净化后进样。色谱柱为HyperSil C₁₈柱(250 mm × 4.6 mm，5 μm)，流动相为0.05 mol?L^-1磷酸二氢钾-乙腈(91︰9)，柱温25 ℃，流速1 mL?min^-1，检测波长236 nm，进样量30 μL；28例患者的血浆分别用SPE-HPLC和FPIA测定，考察2种方法的相关程度。结果 万古霉素、去甲万古霉素的标准曲线范围分别为0.4~80 mg?L^-1和1~50 mg?L^-1，回收率分别为87.2%和89.8%，日内、日间精密度均<7%；SPE-HPLC和FPIA 2种方法的测定值差异无统计学意义。结论 SPE-HPLC法快速简便，准确，灵敏度高，适用于万古霉素和去甲万古霉素血药浓度检测；2种测定方法有良好的相关性。

Abstract:

OBJECTIVE To estabish a solid phase extraction(SPE)-HPLC method for the determination of vancomycin and norvancomycin in human serum, and to evaluate its relationship to concentrations determined by fluorescence polarization immunoassay(FPIA). METHODS Serum samples were collected from well controlled patients treated with vancomycin and norvancomycin, and determined by SPE-HPLC and FPIA methods respectively. The serum samples were cleaned up by SPE. A reversed-phase HyperSil C₁₈ column(250 mm×4.6 mm, 5 μm) was used, and the mobile phase consisted of 0.05 mol?L^-1 phosphate buffer-acetonitrile (91︰9). The flow rate was 1 mL?min^-1. The detection wavelength was 236 nm, and the column temperature was 25 ℃. RESULTS The calibration curves were linear within the range of 0.4?80 mg?L^-1 for vancomycin and 1-50 mg?L^-1 for norvancomycin, and the method recovery was 87.2% and 89.8%, respectively. The RSDs of inter-day and intra-day were both <7%. No significant statistic differences existed between SPE-HPLC and FPIA methods for vancomycin and norvancomycin. CONCLUSION The rapid, sensitive and accurate method was suitable for the assay of vancomycin and norvancomycin in serum. The determination results by the two methods do not need the mutual correction.