Abstract: An analytical method of dual wavelength high performance liquid chromatography with fluorescence detector was developed for simultaneous determination of melatonin and vitamin B6 in health products. The effects of different ultrasonic time and cleaning time of melatonin and vitamin B6 in health products were investigated, and the chromatographic conditions were optimized. The assay was carried out- DOI:10.3969/j.issn.1000-1565.2019.05.008双波长HPLC-荧光法同时测定褪黑素和维生素B6刘丽艳¹,张宏馨^1,2,黄珊²,廉海晨¹,白洁^1,2,韩艳梅¹,吴素焕¹(1. 河北大学 医学综合实验中心,河北 保定 071000;2.河北大学 公共卫生学院,河北 保定 071000)摘 要:建立了双波长高效液相色谱-荧光检测器同时检测保健品中褪黑素和维生素B6含量的方法. 考察了不同超声时间、清洗次数对保健品中褪黑素和维生素B6溶出量的影响,并对色谱条件进行了优化. 采用Inertsil-100A C18色谱柱(4.6 mm×250 mm,5 μm),以甲醇-水(65∶35,体积比)为流动相,流速1.0 mL/min,柱温25 ℃,进样量10 μL. 采用双波长法,0~4 min,λ_ex=332 nm,λ_em=397 nm;4 min后,λ_ex=286 nm,λ_em=352 nm. 在优化的样品预处理条件、色谱条件下,维生素B6和褪黑素分别在0.1~100 μg/L和1.0~1 000 μg/L内线性关系良好(r≥0.998),最低检出限分别为3×10^-3 μg/L和3×10^-2 μg/L;定量限分别为1×10^-2 μg/L和1×10^-1 μg/L,平均回收率分别为99.07%和97.97%(n=3),相对标准偏差为0.04%~0.98%. 本方法检测快速,灵敏,重现性较好,可用于保健品中褪黑素和维生素B6的同时测定以及市售保健品的质量控制.关键词:褪黑素;维生素B6;高效液相色谱;荧光检测;双波长中图分类号:O657.39 文献标志码:A 文章编号:1000-1565(2019)05-0491-07Simultaneous determination of melatonin and vitamin B6 by dual wavelength high performance liquid chromatography with fluorescence detectionLIU Liyan¹, ZHANG Hongxin^1,2, HUANG Shan², LIAN Haichen¹, BAI Jie^1,2, HAN Yanmei¹, WU Suhuan¹ (1.Medical Comprehensive Experimental Center of Hebei University, Hebei University, Baoding 071000, China; 2. College of Public Health, Hebei University, Baoding 071000, China)Abstract: An analytical method of dual wavelength high performance liquid chromatography with fluorescence detector was developed for simultaneous determination of melatonin and vitamin B6 in health products. The effects of different ultrasonic time and cleaning time of melatonin and vitamin B6 in health products were investigated, and the chromatographic conditions were optimized. The assay was carried out- 收稿日期:2018-06-29 基金项目:河北省卫计委医学重点项目(20170189);河北大学医学专科专项资金项目(2014A2001);河北大学校内引进人才科研启动项目(2014-284);国家自然科学基金资助项目(21605035);河北省自然科学基金资助项目(B2014201162);保定市科学技术与发展指导计划项目(12ZF054);河北大学中西部提升综合实力专项资金资助项目(20150505);河北大学实验室开放基金项目(sy201672);河北大学医学部第一批教育教学改革研究课题项目(YXB01-F-05) 第一作者:刘丽艳(1984—),女,河北唐山人,河北大学实验师,主要从事色谱分析、荧光分析、生化分析、生物传感器等研究. E-mail:liyanliu.ok@163.com 通信作者:张宏馨(1971—),女,河北保定人,河北大学副教授,主要从事预防医学与实验动物学教学与研究.E-mail:lzwen5653@163.com韩艳梅(1964—),女,河北保定人,河北大学教授,主要从事生理学等研究.E-mail:hanyanmei6465@sina.com第5期刘丽艳等:双波长HPLC-荧光法同时测定褪黑素和维生素B6on a Inertsil-100A C₁₈ column(4.6 mm ×250 mm, 5 μm)with methanol-water(65∶35, V/V)as the mobile phase, the flow rate was 1.0 mL/min, the column temperature was 25 ℃, and the injection volume was 10 μL. The dual wavelength method was used to make accurate quantitative analysis at the best wavelength of vitamin B6 and melatonin. At the time of 0~4 min, the excitation wavelength and emission wavelength were placed at 332 nm and 397 nm respectively, and the vitamin B6 was detected. After 4 min, the excitation wavelength and the emission wavelength were converted to 286 nm and 352 nm respectively, and melatonin was detected. Under the optimized sample pretreatment conditions and chromatographic conditions, the calibration curves of vitamin B6 and melatonin showed good linearities within the mass concentrations of 0.1~100 μg/L and 1.0~1 000 μg/L(r > 0.998), respectively. The detection limits for vitamin B6 and melatonin were 3×10^-3 μg/L and 3×10^-2 μg/L. The average recoveries was 99.07% and 97.97%(n=3), respectively. The relative standard deviations(RSDs)varied from 0.04% to 0.98%. This method is rapid, sensitive, reproducible, and requires short detection time. A sample can be tested in 6min. The method can be employed for the simultaneous determination of melatonin and vitamin B6 in health care products and the quality control of marketed healthcare products.

Key words: melatonin, vitamin B6, high performance liquid chromatography, fluorescence detection, double wavelength