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Objective: To establish an HPLC method for the simultaneous determination of six components (uridine, adenine, adenosine (R, S)-goitrine, guanosine, clemastanin B) in Radix Isatidis, and to investigate the linear calibration with two reference substances (LCTRS) method for the qualitative analysis of multiple components in Radix Isatidis. Methods: HPLC method was used, with methanol as mobile phase A and water as mobile phase B. Gradient elution (0-3 min, 3% A; 3-18 min, 3% A→14% A; 18-25 min, 14% A→26% A; 25-34 min, 26% A; 34-40 min, 26% A→46% A; 40-60 min, 46% A→90% A) was performed at a flow rate of 0.8 mL·min-1. The column temperature was 30 ℃, the detection wavelengths were 254 nm (0-32 min) and 230 nm (32-60 min). The injection volume was 10 μL. The actual retention time of 6 components in Radix Isatidis was determined on 13 C18 chromatographic columns of different brands and models. Guanosine and clemastanin B were used as double reference compounds, and LCTRS method was used to locate the chromatographic peak of each component. Three unknown chromatographic columns were used for method validation. Using guanosine as a reference substance, the relative retention time method was used to predict the retention time of the other 5 components. The predictive accuracy and column coincidence of these two methods were compared. Results: The LCTRS method could effectively predict and qualitatively analyze the retention time of six indicator components. Compared with the relative retention time method, the LCTRS method had higher accuracy in predicting results and better column universality. Conclusion: The LCTRS method for simultaneous determination of multiple components in Radix Isatidis is feasible and accurate, with simple operation and good durability, and has promotional value.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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