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.

本工作的目的是开发一种简单、特异且快速的高性能薄层色谱 (HPTLC) 方法，以使用 HPTLC-电喷雾电离-串联质谱来鉴定和量化心叶苷 A、20-β-羟基蜕皮激素和科伦宾(ESI-MS/MS) 用于表征心叶青霉茎提取物。使用 HPTLC 铝板进行色谱显影，预涂硅胶 60 F ₂₅₄以己烷-氯仿-甲醇-甲酸为流动相。用茴香醛-硫酸衍生后，在 254 nm 处对 20-β-羟基蜕皮激素和心叶苷 A 进行光密度定量，在 600 nm 处对 Columbin 进行光密度定量。优化后的流动相在R _F分别为 0.12、0.47 和 0.86处实现了心叶苷 A、20-β-羟基蜕皮激素和 Columbin 峰的色谱分离。发现 20-β-羟基蜕皮激素和心叶苷 A 的线性浓度范围为 750-2250 ng/条带，而具有 ( r ² > 0.99）。该方法显示出良好的回收率，心叶苷 A 为 98.96-101.43%，20-β-羟基蜕皮激素为 98.15-101.56%，哥伦宾为 98.06-98.80%。发现 Columbin、20-β-羟基蜕皮激素和心叶苷 A 的检测限为 53.86 ng/条带、40.90 ng/条带和 107.05 ng/条带，而定量限为 163.21 ng/条带、123.94 ng/band 和 324.38 ng/band。发现所有标记的精密度和稳健性研究的相对标准偏差在 2% 以内。通过 ESI-MS/MS在T.cordifolia茎提取物中鉴定并确认了三个标志物。化合物被指定为降二萜呋喃糖苷、蜕皮激素和二萜呋喃内酯：cordifolioside A ( m/z  = 527 [M + Na] ⁺ ; UVλ _max 221 nm）、20-β-羟基 蜕皮激素（m/z = 481.30 [M + H] ⁺；UV λ _max 247 nm）和 columbin（m/z  = 359 [M + H] ⁺；UV λ _max 210纳米）。优化的方法被发现准确的，可重复，可靠和特定和可应用于cordifolioside A，20-β羟基蜕皮，和columbin的定量为提取物的质量控制T.茜草。