HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY COUPLED TO ION-SPRAY MASS-SPECTROMETRY FOR THE DETERMINATION OF COLCHICINE AT PPB LEVELS IN HUMAN BIOFLUIDS

An original method based upon high-performance liquid chromatography c oupled to ion spray mass spectrometry (HPLC-ISP-MS) has been developed for the identification and quantification of colchicine (COL) in huma n blood, plasma or urine. After single-step liquid-liquid extraction b y dichloromethane at pH 8.0 using tofisopam (TOF) as an internal stand ard, solutes are separated on a 5-mu m C-18 Microbore (Alltech) column (250X1.0 mm, LD.), using acetonitrile-2 mM NH4COOH, pH 3 buffer (75: 25, v/v) as the mobile phase (how-rate 50 mu l/min). Detection is done by a Perkin-Elmer Sciex AP1-100 mass analyzer equipped with a ISP int erface (nebulizing and curtain gas: N-2, quality U; main settings: ISP , +4.0 kV; OR, +50 V; QO, -10 V; Q1, -13 V; electron multiplier, +2.2 kV); MS data are collected as either total ion current (TIC, m/z 100-5 00 or 380-405), or selected ion monitoring (SIM) at m/z 400 and 383 fo r COL and TOF, respectively. COL mass spectrum shows a prominent molec ular ion [M + H](+) at m/z 400. Increasing OR potential fails to provi de a significant fragmentation. Retention times are 2.70 and 4.53 min for COL and TOF, respectively. The quantification method shows a good linearity (r=0.998) over a concentration range from 5 to 200 ng/ml. Th e lower limit of detection in SIM mode is 0.6 ng/ml COL, making the me thod convenient for both clinical and forensic purposes.