Development and validation of a high-performance thin-layer chromatography based method for the quantification of trigonelline in fenugreek (Trigonella foenum-graecum) seeds

Abstract

The present research work was undertaken to develop and validate a sensitive, fast, and reproducible high-performance thin-layer chromatography (HPTLC) method for the analysis of trigonelline in fenugreek seeds. Chromatographic analysis was carried out using thin-layer chromatography (TLC) aluminum plates pre-coated with silica gel G60 F 254 as the stationary phase, and the samples were spotted with the help of a CAMAG Linomat 5 TLC applicator. For optimization of the method, different combinations of extraction solvents and mobile phases were tested, and methanol was found to be the ideal extract ion solvent; the best separation was achieved using n-propanol-methanol-water-ammonia (10:1.5:15:0.25, v/v) as the mobile phase. Densitometric scanning of the plates was used for the quantification of trigonelline. This method gave compact spots with retardation factor (RF) value corresponding to 0.40 ± 0.02 for trigonelline. The method was validated as per the International Conference on Harmonization (ICH) guidelines in terms of limit of detection (LOD), limit of quantification (LOQ), specificity, precision (intra-day and inter-day), accuracy, and robustness. The accuracy of the method was checked by recovery study of 4 different levels with average percentage recoveries of 100.077 ± 0.41. While employing this method, dried fenugreek seed samples were found to contain trigonelline in the range of 0.262% to 0.386% (w/w). This method is being reported for the fi rst time and could be successfully applied for the quantification and routine quality control of this marker compound in several plant samples, herbal extracts, and market preparations.