Abstract: Simultaneous separation and determination of four vinca indole alkaloids by reversed-phase high performance liquid chromatography. Water: diethylamine = 986: 14, pH = 7.2 with phosphoric acid (solution A), methanol: acetonitrile: 4: 1 (solution B), 380 ml A and 620 ml B are mixed as mobile phase, flow rate 2 ml / min, the injection volume was 10 μL, and the detection was carried out at 220 nm. Four components, including vinblastine, were separated by constant current elution in 25 minutes. At the same time, the effect of mobile phase composition, pH value, etc. on the separation effect was also studied. Good results have been obtained by analyzing actual samples.
Keywords: reversed-phase high performance liquid chromatography, indole alkaloids
1 Introduction Vinblastine (Vinblastine), Vincristine (Vincristine), Vinblastine (Catharanthine), and Vindoline (Vindoline) are included in the oleander plant Catharanthus (Catharanthus roseus L.G. Don) 4 An important indole alkaloid. At present, vinblastine alkaloids are mainly extracted from periwinkle plants, and the establishment of rapid detection of these four alkaloids in vinca is of great significance for vinca GAP planting and the establishment of a quality evaluation system. The reported methods for determining the content of vinblastine alkaloids include thin layer scanning, high performance liquid chromatography, and radioimmunoassay. However, the simultaneous determination of four alkaloids has not been reported in China. Based on the previous literature, this study established a reversed-phase high-performance liquid chromatography method for the rapid determination of 4 indole alkaloids, and achieved satisfactory results in the analysis of actual samples.
2 Experimental part
2.1 Instruments and reagents JASCO high-performance liquid chromatograph (13 JASCO companies), 1580 pump, 1575 type ultraviolet detector, Version 6.1 chromatography workstation. Vinblastine, vincristine (purity ≥99%, Sigma), vinblastine, and vindolin (purity> 195%, Zhejiang Haizheng Pharmaceutical Co., Ltd.); vinca samples were collected from Haikou City, Hainan Province; acetonitrile And methanol are chromatographically pure; phosphoric acid and diethylamine are analytically pure; water is double distilled water.
2.2 Preparation of reference substance and sample Accurately weigh the appropriate amount of vinblastine, vincristine, vinblastine, and vindolin reference substance, dissolve in 5 mL methanol and dissolve it in a 25 mL volumetric flask, shake well, and prepare the concentration Respectively 0.5, 0.5, 0.5 and 1.0 g / L. Take 10 g of vinca whole plant powder, ultrasonically extract with 60, 40, and 20 mL of methanol at room temperature for 40, 30, and 20 min, filter, combine the extracts, evaporate at 50 ° C under reduced pressure, and filter through a 0.45 μm microporous membrane , The filtrate was made up to 50 mL. All samples are stored in the refrigerator at 4 ℃ for use.
2.3 Chromatographic conditions Chromatography column: DiamonsilTM Cl8 ODS (250 mm × 4.6 mm, 5 μm); mobile phase is water: diethylamine = 986: 14, pH is adjusted with phosphoric acid = 7.2 (solution A); methanol: acetonitrile = 4 : 1 (Solution B), 380 mL A and 620 mL B are mixed as mobile phase; flow rate is 2 mL / min; injection volume: 10 L; column temperature: 23 ° C; detection wavelength: 220 nm.
References
1 Pennanen S, Huhtikangas A. Photochem. and Photobiology, 1990, 51 (5): 515 ～ 518
2 Tam MN, Nikolova-Damyanora B, Pyuskyuhr B. Liq. Chromatogr. , 1995, 18: 849 ～ 852
3 Ding Ruxian (ä¸ å„’ è´¤), Ni Ku (倪 å¤ ä»ª), Cap Hai (曹 æµ·), Chen Wenjia (陈文佳). Journd of China Pharmaceuti—cal Univers ~ y (Journal of China Pharmaceutical University), 1995, 26 (3): 157 ～ 159
4 Mandal S, Maheshwari ML. Indian Journal of Pharmaceutical Sciences, 1987, 49 (6): 205 ～ 209
5 Huang Zongyu (黄宗玉), Gong Qing (Gong Qing), Lin Ltiji (林露基), Tang Xiangjiang (å”湘江). Chinese Traditional and Herbal DrH ~ s (Chinese herbal medicine), 1992, 23 (4): 185 ～ 186
Keywords: reversed-phase high performance liquid chromatography, indole alkaloids
1 Introduction Vinblastine (Vinblastine), Vincristine (Vincristine), Vinblastine (Catharanthine), and Vindoline (Vindoline) are included in the oleander plant Catharanthus (Catharanthus roseus L.G. Don) 4 An important indole alkaloid. At present, vinblastine alkaloids are mainly extracted from periwinkle plants, and the establishment of rapid detection of these four alkaloids in vinca is of great significance for vinca GAP planting and the establishment of a quality evaluation system. The reported methods for determining the content of vinblastine alkaloids include thin layer scanning, high performance liquid chromatography, and radioimmunoassay. However, the simultaneous determination of four alkaloids has not been reported in China. Based on the previous literature, this study established a reversed-phase high-performance liquid chromatography method for the rapid determination of 4 indole alkaloids, and achieved satisfactory results in the analysis of actual samples.
2 Experimental part
2.1 Instruments and reagents JASCO high-performance liquid chromatograph (13 JASCO companies), 1580 pump, 1575 type ultraviolet detector, Version 6.1 chromatography workstation. Vinblastine, vincristine (purity ≥99%, Sigma), vinblastine, and vindolin (purity> 195%, Zhejiang Haizheng Pharmaceutical Co., Ltd.); vinca samples were collected from Haikou City, Hainan Province; acetonitrile And methanol are chromatographically pure; phosphoric acid and diethylamine are analytically pure; water is double distilled water.
2.2 Preparation of reference substance and sample Accurately weigh the appropriate amount of vinblastine, vincristine, vinblastine, and vindolin reference substance, dissolve in 5 mL methanol and dissolve it in a 25 mL volumetric flask, shake well, and prepare the concentration Respectively 0.5, 0.5, 0.5 and 1.0 g / L. Take 10 g of vinca whole plant powder, ultrasonically extract with 60, 40, and 20 mL of methanol at room temperature for 40, 30, and 20 min, filter, combine the extracts, evaporate at 50 ° C under reduced pressure, and filter through a 0.45 μm microporous membrane , The filtrate was made up to 50 mL. All samples are stored in the refrigerator at 4 ℃ for use.
2.3 Chromatographic conditions Chromatography column: DiamonsilTM Cl8 ODS (250 mm × 4.6 mm, 5 μm); mobile phase is water: diethylamine = 986: 14, pH is adjusted with phosphoric acid = 7.2 (solution A); methanol: acetonitrile = 4 : 1 (Solution B), 380 mL A and 620 mL B are mixed as mobile phase; flow rate is 2 mL / min; injection volume: 10 L; column temperature: 23 ° C; detection wavelength: 220 nm.
References
1 Pennanen S, Huhtikangas A. Photochem. and Photobiology, 1990, 51 (5): 515 ～ 518
2 Tam MN, Nikolova-Damyanora B, Pyuskyuhr B. Liq. Chromatogr. , 1995, 18: 849 ～ 852
3 Ding Ruxian (ä¸ å„’ è´¤), Ni Ku (倪 å¤ ä»ª), Cap Hai (曹 æµ·), Chen Wenjia (陈文佳). Journd of China Pharmaceuti—cal Univers ~ y (Journal of China Pharmaceutical University), 1995, 26 (3): 157 ～ 159
4 Mandal S, Maheshwari ML. Indian Journal of Pharmaceutical Sciences, 1987, 49 (6): 205 ～ 209
5 Huang Zongyu (黄宗玉), Gong Qing (Gong Qing), Lin Ltiji (林露基), Tang Xiangjiang (å”湘江). Chinese Traditional and Herbal DrH ~ s (Chinese herbal medicine), 1992, 23 (4): 185 ～ 186
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