Development and Validation of a Method for Identifying Pregabalin in Urine by Thin-Layer Chromatography

A simple, rapid, and cost-effective thin-layer chromatographic (TLC) method has been developed and validated for the identification of pregabalin (PGB) in urine. The method involves liquid-liquid extraction of PGB from urine using ethyl acetate under acidic conditions. Chromatographic separation was performed on silica gel 60 F254 TLC plates with methanol as the mobile phase. Detection was achieved using ninhydrin reagent (2% and 5%) with heating, resulting in a characteristic violet spot. The method was validated for selectivity, specificity, detection limit, and stability. The method demonstrated high selectivity, with no interference from endogenous substances or a panel of 30 common drugs, psychotropic substances, and drugs of abuse, except for furosemide and azithromycin, whose interference was resolved using a methanol-ammonia mobile phase. The detection limit was established at 50 µg/L, which is significantly lower than the cut-off values of common urinary immunoassays. Stability studies indicated that PGB is stable in urine after single and double freezing and storage at -20°C for up to 7 days, but unstable at + 4°C. This validated TLC method is suitable for reliable and precise detection of pregabalin in urine, offering a valuable tool for toxicological routine in resource-limited settings.