双波长HPLC-荧光法同时测定褪黑素和维生素B6

Simultaneous determination of melatonin and vitamin B6 by dual wavelength high performance liquid chromatography with fluorescence detection

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,5μm),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10μL.采用双波长法,0~4 min,λex=332 nm,λem=397 nm;4 min后,λex=286 nm,λem=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0.1~100μg/L和1.0~1 000μg/L内线性关系良好(r≥0.998),最低检出限分别为3×10^-3μg/L和3×10^-2μg/L;定量限分别为1×10^-2μg/L和1×10^-1μg/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.

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 on a Inertsil-100 AC18 column(4.6 mm×250 mm,5μm)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 10μL.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 0.1~100μg/L and 1.0~1 000μg/L(r>0.998),respectively.The detection limits for vitamin B6 and melatonin were 3×10^-3μg/L and 3×10^-2μg/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 6 min.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.