Method Development and Validation for Determination of Cocaine, its Main Metabolites and Pyrolytic Products by HPLC–UV–CAD

A method was developed using hydrophilic interaction liquid chromatography (HILIC), a versatile mode of liquid chromatography, to determine cocaine, its main degradation products, benzoylecgonine and ecgonine, main pyrolytic products, anhydroecgonine and anhydroecgonine methyl ester, to be used as standards, and a frequent contaminant, levamisole. Detection of all compounds was achieved using two detectors connected in series: charged aerosol detector (CAD) and ultraviolet detector (UV). No method was found in the literature using HILIC coupled to CAD and UV to determine these compounds. The method was carried out with a Phenomenex Kinetex HILIC (150 mm × 4.6 mm ID; 2,6 µm) column with mobile-phase acetonitrile: 10 mM ammonium acetate pH 6.3 (75:25 v/v) with flow rate 0.8 mL min−1 and oven temperature set at 30 °C. CAD detection was performed using a nitrogen pressure of 35 psi, range of 100 pA and the UV detection at 200 nm. Validation parameters such as selectivity, linearity, precision and accuracy were evaluated. Methods were successfully validated to detect and quantify all related substances.