A validated HPTLC method for quantification of cordifolioside A, 20-β-hydroxyecdysone and columbin with HPTLC–ESI–MS/MS characterization in stems of Tinospora cordifolia

The objective of the present work was to develop a simple, specific, and fast high-performance thin-layer chromatographic (HPTLC) method identify quantify cordifolioside A, 20-β-hydroxyecdysone columbin with HPTLC‒electrospray ionization‒tandem mass spectrometry (ESI‒MS/MS) for characterization in Tinospora cordifolia stem extracts. Chromatographic development performed using HPTLC aluminum plate, pre-coated silica gel 60 F254 hexane‒chloroform‒methanol‒formic acid as mobile phase. Densitometric quantification A at 254 nm 600 after derivatization anisaldehyde‒sulfuric acid. optimized phase resulted separation peaks 20-β-hydroxyecdysone, RF 0.12, 0.47, 0.86, respectively. linear concentration range found be 750‒2250 ng/band 675‒1875 (r2 > 0.99). methodology showed good recoveries 98.96‒101.43% 98.15‒101.56% 98.06‒98.80% columbin. limit detection columbin, 53.86 ng/band, 40.90 107.05 while 163.21 123.94 324.38 relative standard deviation precision robustness study all markers within 2%. Three were identified confirmed T. extracts by ESI‒MS/MS. Compounds assigned norditerpene furan glycosides, ecdysteroids, diterpenoid furanolactone: (m/z = 527 [M + Na]+; UV λmax 221 nm), 481.30 H]+; 247 359 210 nm). accurate, reproducible, robust, specific can applied quality control cordifolia.