Development and validation of a densitometric TLC method for analysis of trigonelline and 4-hydroxyisoleucine in fenugreek seeds

Abstract

A simple, sensitive, and reproducible high-performance thin-layer chromatographic method has been established for simultaneous analysis of trigonelline and 4hydroxyisoleucine from fenugreek seeds (Trigonella foenum-graceum). Chromatography was performed on silica gel GF 254 with n-butanol-methanol-acetic acid-water 4:1.5:1:1 (v/v) as mobile phase. Densitometric scanning of the plates at 266 nm was used for analysis of trigonelline, and plates were scanned at 395 nm after derivatisation with ninhydrin reagent for analysis of 4-hydroxyisoleucine. The method was validated for specificity, precision (intraday and interday), accuracy, and robustness. Response was a linear function of the concentration of standard solutions in the range 100 to 1000 ng per band and 50 to 500 ng per band, respectively, with correlation coefficients of 0.9992 ± 0.04 and 0.9986 ± 0.06, for trigonelline and 4-hydroxyisoleucine. The accuracy of the method was checked by determination of recovery at three different levels; average percentage recovery was 99.39 ± 0.34 for trigonelline and 99.03 ± 0.26 for 4-hydroxyisoleucine. The method was found to be simple, precise, specific, sensitive, and accurate and can be used for routine quality control of fenugreek seeds and also for quantitative analysis of these marker compounds in processed fenugreek and its marketed preparations.

Comparison between high performance liquid chromatography and high performance thin layer chromatography determination of Diosgenin from fenugreek seeds

“…There are numerous other uses of fenugreek, including treatment of indigestion and baldness. [1,2] Seeds are known for their pungent aromatic properties. [3] The seeds, available in whole and ground forms, are used to flavour many foods including curry powders, spice blends and teas.…”

“…[10] The literature search showed that few analytical methods have been published for fenugreek such as high performance thin layer chromatography (HPTLC) determination of trigonelline and 4-hydroxyisoleucine from fenugreek seed. [2] But Comparison between high performance liquid chromatography and high performance thin layer chromatography determination of Diosgenin from fenugreek seeds there is no analytical method available for the estimation of Diosgenin from fenugreek seeds.…”

Trigonella foenum‐graecum L. Seed Oil Obtained by Supercritical CO2 Extraction

“…In China, it is a medicinal and edible plant for making pancakes, and its seeds are included in the ''Chinese Pharmacopoeia''. Saponins [1], flavonoids [2,3], galactomannans [4], trigonelline, and 4-hydroxyisoleucine [5] were isolated from fenugreek seeds in previous phytochemical research studies. The seeds are used in folk medicine because of their reported hypoglycemic [6], hypolipidemic, antidiabetic [7], liver-protective [8], renoprotective, anticancer [9], and antioxidant [10] effects.…”

Development and validation of HPTLC-densitometric method for estimation of trigonelline in leaves ofAbrus precatoriusL. and its formulation

“…Pharmacological activities such as antiinflammatory [4], antimicrobial [5], neuromuscular [6], and toxicity studies [7] are reported on leaves. It is also a major ingredient of many marketed polyhedral digestive mixtures such as Chatak ® . Leaves are foremost source of an alkaloid trigonelline [8] having diverse pharmacological activities such as hypoglycemic, hypocholesterolemic, antiseptic, antimigraine, antitumor, mutagenic, and osmoregulatory [9]. Hence, active principle trigonelline has been used as a marker compound for quality control and standardization of the A. precatorius leaves and its formulations.…”