Clinical assay of nicotine and its metabolite, cotinine, in body fluids by HPLC following solid phase extraction

A rapid and accurate reversed phase high performance liquid chromatographic (RP-HPLC) method, is described for the simultaneous determination of nicotine and cotinine in body fluids. The analytical column, an MZ Kromasil C-8, 250 x 4 mm, 5 mum, was operated at ambient temperature with backpressure values of 290 kg/cm(2). The mobile phase consisted of A: 0.05 M ammonium acetate and phase B: CH3OH at a volume ratio 60:40, delivered at a flow rate of 1.4 mL/min. Scopolamine was used as internal standard at a concentration of 85 ng/muL. Detection was performed with a variable wavelength UV-visible detector at 262 nm, resulting in a detection limit of 0.2 ng per 20 muL injection and a quantitation limit of 1.0 ng, while linearity held up to 20 ng/muL for nicotine and 30 ng/muL for cotinine. The statistical evaluation of the method was examined by performing intra-day (n = 8) and inter-day calibration (n = 8) and was found to be satisfactory, with high accuracy, and precision results. High relative extraction recoveries from biological matrices: blood serum and urine, ranging from 93.3% to 106.4% for cotinine and from 94.2% to 115.5% for nicotine, were achieved using Lichrolut RP-select B (Merck) SPE cartridges with tetrahydrofuran as eluent, requiring small volumes, 200 muL of blood serum, and 200 muL of urine.