Development of a simple and sensitive HPLC-DAD method for quantification of vitamin B12 fortified in infant food

To prevent infants from vitamin B12 deficiency, infant food is designed based on cow's milk or cereal with the fortification of vitamin B12. A method for quantitative determination of vitamin B12 in infant food was developed with hydrophilic high performance liquid chromatography (HPLC) coupled with a diode array detector (DAD). The sensitivity of the detector was enhanced by implementing a 60 mm high-sensitivity LightPipe flow cell, and the limit of detection (LOD) and limit of quantification (LOQ) were improved as low as 0.006 μg 100 g^-1 and 0.02 μg 100 g^-1 respectively. The effect of sample extraction and enrichment, chromatography separation parameters on the analyte, were studied in detail and optimized. Under these conditions, the method performed a good linear analytical range of 0.3-50 μg L^-1, and a good repeatability with % RSD below 2.8% and recovery of 90.2-96.5% (n = 6). To the best of our knowledge, for the first time, 60 mm high-sensitivity LightPipe flow cell was included in the HPLC-DAD method for determination of the trace amount of vitamin B12 in infant food. The proposed method was further validated by analysis of FAPAS QC samples (T21120 and T21118), and it was specific and precise for the intended use.