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