Optimized Method of High Performance Liquid Chromatography using Octyl Silica Fully Endcapped Residual Silanol Column on Separation of Cotinine and 3-Hydroxycotinine in Urine Sample
Cotinine (COT) and 3-hydroxycotinine (3-HCOT) are nicotine metabolite excreted in urine. Mediated by the enzyme cytochrome P450 2A6 (CYP 2A6), nicotine will be metabolized to COT and 3-HCOT. The activity of CYP 2A6 can be predicted from the ratio 3-HCOT to the COT, therefore the ratio of 3-HCOT and COT can be used as phenotyping and polymorphism studies of the enzyme. In this study, isolation COT and 3-HCOT of urine samples was carried out by liquid-liquid back extraction. Simultaneous analysis of COT and 3-HCOT using High Performance liquid Chromatography (HPLC) was performed by a reversed-phase octyl silica column (C8; Shimadzu 250 × 4.6 mm, 5 μm) fully endcapped residual silanol. The internal standard solution (SI) was acetanilide. The mobile phase which separate COT, 3-HCOT and SI was methanol : ammonium acetate 5 mM (50:50) at a flow rate 0.8 mL/min. Retention time (tR) of the three compounds was less than 10 minutes, with peak tailing factor (tf) was less than 2. The resolution (Rs) 3-HCOT to COT was 2.67, while the Rs COT to SI was 8.836.
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