高效液相色谱分离-在线光化学衍生/荧光光谱法测定5种水溶性维生素的研究

Simultaneous Determination of 5 Water-soluble Vitamins by HPLC with On-line Photochemical Derivatization and Fluorescence Detection

建立了高效液相色谱分离-在线光化学衍生/荧光光谱法测定水溶性维生素烟酸(NIA)、烟酰胺(NIC)、B1、B12及B2的新方法。以含有0.018 mol/L三乙胺、0.002 mol/L庚烷磺酸钠的0.05 mol/L磷酸盐缓冲液(A相,pH 5.8)和甲醇(B相)为流动相(85∶15),等度洗脱分离5种水溶性维生素;将自制的程序控制时间/光强光化学反应器接在二极管阵列检测器(DAD)和荧光检测器(FLD)之间,进行光化学衍生。优化了光化学衍生的条件,并用于复合维生素片中水溶性维生素的测定。在优化实验条件下,5种水溶性维生素的线性范围分别为:NIA、NIC为0.1～10.0 mg/L,B1、B12为0.2～20.0 mg/L,B2为0.01～1.0 mg/L,方法的检出限(LOD,3σ)和定量下限(LOQ,10σ)分别为1.5～25.4μg/L和5.0～84.7μg/L,优于传统的高效液相色谱分离/二极管阵列检测方法。方法的相对标准偏差(n=7)不大于7.8%,样品的加标回收率为98%～103%。

A high performance liquid chromatographic （HPLC） method using on-line photochemical derivatization and fluorescence detection was developed for the simuhaneous determination of nicotinic acid（NIA）, nieotinamide （ NIC）, thiamine hydrochloride （ B, ）, riboflavin （ B2 ） and cyanocobalamin（B12）. A self-buih time/energy programmed photochemical reactor （PCR） with an ultraviolet mercury lamp was installed between the diode-array detector（DAD） and fluorescence detector（FLD） to convert the non- or weakly fluorescent vitamins into fluorescence compounds. The separation of NIA, NIC, B1, B12 and B2 was achieved within 12 min using 0.05 mol/L phosphate buffer（pH 5.8） - 0. 018 mol/L triethylamine - 0. 002 mol/L sodium heptane sulfonate （A） and methanol（B） as mobile phase by isocratic elution（A ： B, 85 ： 15）. The separation and photochemical derivatization conditions were optimized. Under the optimal conditions, the calibration curves of five vitamins were linear in the range of 0. 1 - 10.0 mg/L for NIA and NIC, 0.2 - 20. 0 mg/L for B1 and B12, 0.01 - 1.0 mg/L for B2 with correlation coefficient （r） more than 0. 996. The limits of detection （LOD, 3σ） ranged front 1.5 μg/L to 25.4 μg/L and the limits of quantitation（LOQs, 10σ） were 5.0 -84.7 μg/L, which were better than those of the traditional HPLC/DAD method. The relative standard deviations（RSDs, n = 7） were not more than 7.8% and the spiked recoveries were between 98% and 103%. The developed method was successfully applied in the determination of vitamins NIC, B1 and B2 in muhivitamin tablets.