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

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.