Determination of vitamin B1 and vitamin B12 in grass carp by HPLC-DAD
High performance liquid chromatography-diode array detection(HPLC-DAD)was proposed for simultaneous separation and determination of vitamin B1 and vitamin B12 in grass carp in this paper.After acid digestion and enzyme extraction,the protein-bound and phosphorylated vitamin B1 and vitamin B12 in the grass carp samples were released,and then purified by solid phase extraction.The two analytes were determined by HPLC-DAD.The effect of mobile phase composition on the separation effect of HPLC and the effects of pH of sample solution and the kind of eluent on the recovery of sample in solid phase extraction were studied.Grass carp samples were purified by HLB solid phase extraction column,and then vitamin B1 and vitamin B12 were eluted by the mixture of methanol/water(v/v=1∶1).The mobile phases were 85%methanol and 50mmol/L sodium dihydrogen phosphate(pH 3.00,containing 5mmol/L hexanesulfonic acid),respectively.The mobile phase was gradient elution.The column temperature was 40℃,the detection wavelength was 255nm,and the inject sample volume was 20μL.Vitamin B1 and B12 were separated within 15 minutes.Under the optimal isolation and detection conditions,the detection limit(S/N=3)of vitamin B1 and vitamin B12 were 0.21～0.36μg/mL,the standard recovery rate was 87.9%～98.1%.The method established in this paper could accurately quantify the content of vitamin B1 and vitamin B12 in the grass carp.
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