Validated High-Performance Thin-Layer Chromatography Method for the Determination of Nicotine in Tobacco (Nicotiana tabacum L.) Extracts

A new, simple, sensitive, selective, precise, and robust high-performance thin-layer chromatography (HPTLC) method was developed and validated for the determination of nicotine in tobacco extracts. The analysis of nicotine was performed on thin-layer chromatography (TLC) glass plates precoated with silica gel 60 F254 as stationary phase. Development was carried out in a horizontal chamber under sandwich configuration with the mobile phase ethyl acetate-methanol-ammonia 2.8% (v/v) (20:10:4, v/v/v) at room temperature (25 ± 2°C). A TLC scanner was used for spectrodensitometric scanning and analysis in the absorbance mode at 263 nm. The system was found to give compact spots for nicotine (RF value of 0.20 ± 0.02). The calibration plots showed good linear relationships with r2 = 0.9998 ± 0.0001 in the concentration range 90–900 ng/spot with respect to the peak area. According to the International Conference of Harmonization (ICH) guidelines, the TLC method was validated for precision, recovery, robustness, and ruggedness. The detection and quantification limits were determined. Statistical analysis of the data revealed that the method is reproducible and selective for the determination of nicotine content in tobacco extracts.