| A high performance liquid chromatographic（HPLC） method using on-line photochemical derivatization and fluorescence detection was developed for the simultaneous determination of nicotinic acid（NIA）,nicotinamide（NIC）,thiamine hydrochloride（B1）,riboflavin（B2） and cyanocobalamin（B12）.A self-built 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 from 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 multivitamin tablets. |