Multiple ionization patterns enable UHPLC-Q-Orbitrap MS/MS to quantify 80 compounds in Tongmai Granule and recommend new anti-counterfeiting Q-markers for Pharmacopoeia

To explore the enabling role of multiple ionization patterns in UHPLC-Q-Orbitrap MS/MS analysis of complicated traditional Chinese herbal medicines, Tongmai Granule (TMG) composed of Salviae Miltiorrhizae Radix et Rhizoma (SM), Chuanxiong Rhizoma (CR), and Puerariae Lobatae Radix (PL), is introduced in the study. The Granule is systematically analyzed under five ionization patterns, including [M − H]⁻, [M + H]⁺, [M + NH₄]⁺, [M + H − H₂O]⁺, and [M − CH₃]⁺. A total of 80 compounds were putatively identified and subsequently quantified, including 28 isomers (e.g., senkyunolide I vs. senkyunolide H). Fifty "unexpected" compounds were also discovered in TMG for the first time. Quantification experiments revealed that the contents of all 80 compounds ranged widely from 0.0002 ± 0.0001 to 2,600.46 ± 27.99 µg/g. Based on their contents and relevant principles, seven compounds were recommended as new anti-counterfeiting quality markers (Q-markers), namely puerarin, 3'-methoxypuerarin, mirificin, senkyunolide I, xanthotoxin, danshensu, and salvianolic acid B. Seven validation experiments using lower-resolution LC–MS analysis confirmed that these Q-markers successfully detected counterfeiting TMG products from PL-shortage, CR-shortage, and SM-shortage, respectively. This success not only promotes the Q-markers update for Pharmacopoeia inclusion in the future, but also demonstrates the enabling role of multiple ionization patterns in UHPLC-Q-Orbitrap MS/MS analysis. This systematic ‘multi-ionization plus comprehensive analysis plus anti-counterfeiting Q-marker screening’ strategy also provides a reproducible blueprint for analyzing other TCM formulations.