Chromeleon version 7

FA composition of erythrocytes was analysed using ultra-fast gas chromatography, as described by Sissener et al.^{(7 )}. In this method, MUFA is not separated according to the position of their double bond and these FA are stated as 16:1, 18;1, 20:1 and 22:1. In brief, samples were thawed and weighed. Nonadecanoic acid (19:0) was added as an internal standard to the samples, and then the samples were saponified and methylated by adding 1 ml NaOH (0·5M) and 2 ml BF₃ in methanol. The samples were evaporated and then purified with hexane. The final concentration of the samples was adjusted to 0·2–0·3 mg/ml and injected into FA detection system. The system used for FA detection was a Trace GC Ultra (Thermo Corporation) with SSL injector, flame Ionization Detector, and the column was a Wax column (P/N UFMC00001010501, 5-m long, 0·1-mm. Id., 0·1-μm film thickness). Chromeleon^® version 7.2 was the integrator used (Thermo Scientific).

Catalpol has been reported to ameliorate the pathological process of EAE mice (Yang et al., 2017 (link); Li et al., 2018 (link)). Thus, we selected Catalpol as mark compound for quantitative quality control of RR. Catalpol was determined at 210 nm wavelength using UHPLC UltiMate 3000 (Thermo Fisher Scientific, USA). The chromatographic conditions were the same with qualitative experiment described as previous. RR extract powder was accurately weighed, dissolved in 70% MeOH by sonication and filtrated through 0.45 μm filter for quantitative analysis.
For validation of the quantitative methodology, linearity, sensitivity, precision, accuracy, and stability were detected as previous described (Liu et al., 2013 (link)). Briefly, stock solution (1 mg/mL) of Catalpol was prepared in 50% MeOH. Six concentrations of Catalpol stander were analyzed in triplicates in HPLC to prepare calibration curves. Accuracy and precision were evaluated by measuring the intra-day and inter-day variabilities and recovery of standard compounds. Stability was conducted by analyzing RR extract over a period of 2, 4, 6, 8, 12, and 24 h. The limits of detection (LOD) and limits of quantitation (LOQ) under the present conditions were determined at an S/N (signal/noise) of about 3 and10, respectively. The data were monitored, recorded and analyzed by Chromeleon Version 7.2 (Thermo, USA).

Analytical reversed-phase chromatography was performed with a TSKgel Protein C4-300 column (3 µm, 4.6 × 150 mm) from Tosoh Bioscience (Tokyo, Japan) on a Vanquish UHPLC system, controlled by Chromeleon version 7.2 (both Thermo Fisher Scientific, Waltham, MA, USA). Chromatography was carried out at 50 °C with mobile phase A (mpA) containing 0.5% TFA in water and mobile phase B (mpB) containing 0.4% TFA in ACN. Samples were injected without prior adjustment to the mobile phase composition during injection and adsorption. The chromatographic separation was carried out with a 10 min adsorption step applying 92% mpA and 8% mpB, followed by an 18 min linear AB gradient elution to 100% mpB and a 7 min re-equilibration with 92% mpA and 8% mpB at a flow rate of 0.5 mL/min. Eluting components were detected with a diode array detector, evaluating peak areas at 260 nm and 280 nm.