Determination and uncertainty analysis of B vitamins in infant formula by HPLC-DAD/FLD
A method was developed for the simultaneous determination of vitamin B2,niacin,nicotinamide,pyridoxine and pyridoxal in infant formula by HPLC.The sample was extracted by 0.1％phosphoric acid aqueous solution of 1％methanol and precipitated protein,separated by Thermo Hypersil GOLD™ aQ(5 μm,4.6 mm×250 mm)and detected using diode array detector(DAD)and fluorescence detector(FLD)in series mode.At the same time,an uncertainty evaluation model was constructed to examine the sources of uncertainty.The results showed that calibration of five vitamins were linear in the range of 0.025-4.000 μg/ml for vitamin B2,pyridoxine,pyridoxal and 0.125-20.00 μg/ml for niacin acid,nicotinamide.The standard curve presented a good linearity(R2＞0.999),the adding standard recovery was between 93.20％and 108.00％(n=6),and the relative standard deviation(RSD)was less than 8％.When the content of niacin and niacinamide in infant formula was 4 mg/100 g,the expanded uncertainty was 0.300 mg/100 g(k=2)and 0.182 mg/100 g(k=2);When the content of pyridoxol,pyridoxal,and VB2 was 800 μg/100 g,the expanded uncertainty was 100 μg/100 g(k=2),71.5 μg/100 g(k=2)and 41.9 μg/100 g(k=2).The main source of uncertain was the preparation of standard solutions and the fitting of standard curves.The method was simple,rapid,sensitive and accurate,and could meet the determination requirements of B vitamins in infant formula.
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