LCW Content Determination

High performance liquid chromatography (HPLC)-grade acetonitrile and HPLC grade formic acid were obtained from Thermo-Fisher (USA). Reference standards provided by the National Institute on Drug Abuse of China:Hydroxysafflor-Yellow-A (batch number: 111637-200502), amygdalin (batch number: 110820-200403), paeoniflorin (batch number: 0905-9805), caffeic acid (batch number: 110728-200506), phillyrin (batch number: 120908-200914), liquiritin (batch number:111610-200503), peimisine (batch number: 0750-9303), harpagoside (batch number: 712-9403), rhein (batch number: 0902-200207), Z-Ligustilide (batch number: 927-9908), berberine (batch number: 0713-200107), tanshinone II a (batch number: 0766-200011), catalpol (batch number: 0808-9602). The purities of all standards were no less than 98% and suitable for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. LCW was purchased from Changchun Overseas Pharmaceutical Co. Ltd.

LCW (batch number: 20190601) was grinded into powder. Each sample of LCW powder (0.5 g) was weighed precisely and ultrasonically extracted in 50 ml hydrochloric-acid methanol (1:100) for 30 min. Supplement the weight lost with hydrochloric acid-methanol (1:100) solution and then filtered through 0.22 μm nylon membrane filters. The filtrate was analyzed directly by UPLC-ESI-MS/MS. At the same time, a stock solution containing 13 reference standards was prepared in methanol. All solutions were stored at 4°C prior to analysis.

Chemical profiling of LCW and reference standards were detected by an Agilent ultra-performance liquid chromatography system (UHPLC) (Agilent, USA) coupled to a Q-trap 3200 (AB SCIEX, USA). Chromatography separation was carried out on a Waters ACQUITYUPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) maintained at 40°C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), and run under the following program: 0 ~ 1 min, 15% B; 1 ~ 4 min, 15 ~ 45% B; 4 ~ 10 min, 45 ~ 60% B, 10 ~ 15 min, 60 ~ 90% B; 15 ~ 18 min, 90 ~ 95% B. The sample injection volume was 5 μl and the flow rate was set at 0.2 ml/min. The mass spectrometer was fitted with an electrospray ionization source and the mass detection was operated in both positive and negative ion modes with the following setting: ion source temperature, 500°C; Sheath gas velocity, 50 psi; Auxiliary gas flow rate, 12 L/min; scan range, m/z 150–900.