[Analysis of chemical components of Chinese medicine Ligustici Radix by achiral-chiral liquid chromatography-predictive multiple reaction monitoring]

Ligustici Radix (Chinese name: maoqianhu) consists of the dried roots of Ligusticum brachylobum Franch., which is mainly distributed in the Yunnan and Sichuan provinces. This herbal medicine has been primarily used for the treatment of cough in traditional Chinese medicine. Ligustici Radix is rich in coumarin derivatives. Interestingly, enantiomers and diastereomers are widely used for these Coumarins, thus posing a great challenge for in-depth chemical profile characterization. In the present study, a new analytical platform, achiral-chiral liquid chromatography-tandem mass spectrometry (achiral-chiral LC-MS/MS) was configured to profile the chemical composition of Ligustici Radix. Because achiral and chiral columns were serially coupled, especially enantiomers, both chemically and enantiomerically selective separations could be accomplished simultaneously. The newly configured achiral-chiral LC-MS/MS platform did not require any electronic valve; hence, it could overcome the drawbacks of heart-cutting achiral-chiral two-dimensional LC, i. e., sophisticated instrumentation and limited reproducibility due to the use of electronic valve(s) and the undesired retention time shift across different analytical runs. Some available candidates for chemically selective or enantiomerically selective separation were assayed; then, Capcell core RP-C₁₈ column that was packed with core-shell type particles, and AD-RH column embedding amylose coated particles were employed the achiral and the chiral columns, respectively. The narrow-bore core-shell RP-C₁₈ column served as the front tool to achieve efficient chemoselective separation of coumarin analogs, and enantioselective enantiomers were obtained by using a wide-bore AD-RH chiral column. The predictive multiple reaction monitoring (predictive MRM) mode allowed for the sensitive detection of potential components, and an enhanced product ion (EPI) scan, which was a unique function of Qtrap-MS, was programmed to record the MS² spectra for all captured signals and thus aid structural annotation. Online energy-resolved mass spectrometry (online ER-MS) was introduced to pursue the suitable collision energy for each compound; in particular, inferior collision energy instead of the optimal one was utilized to suppress the response of the primary components such as praeruptorin A, B and pteryxin. The criteria to judge enantiomers or not included identical quantitative and qualitative precursor-to-product ion transitions, identical quantitative versus qualitative responses, and longer retention times from achiral-chiral LC over single-column achiral LC. As a result, a total of sixty components were observed and structurally identified. In particular, enantiomerically selective separations were achieved for eight enantiomers, cis-khellactone (CKL), qianhucoumarin G (QC-G), pteryxin (Pte), praeruptorin A (PA), cis-3'-isovaleryl-4'-acetylkhellactone (IAK), praeruptorin B (PB), praeruptorin E (PE), and cis-3',4'-diisovalerylkhellactone (DIK). Notably, none of the enantiomers were present as racemates; instead, the proportion of one enantiomer in each pair was greater than the other. Achiral-chiral LC-predictive MRM is a feasible choice for the quantitative and qualitative analyses of Ligustici Radix as well as other herbal medicines characterized by enantiomers and diastereomers.

Ligustici Radix; achiral-chiral liquid chromatography (achiral-chiral LC); angular-type pyranocoumarins (APs); enantioseparation; predictive multiple reaction monitoring (predictive MRM).