| Biotin is one of numerous nutritive material in dairy products. Biotin is an indispensable water-soluble B vitamins, which take part in three significant nutritive material metabolism of organism. Biotin have special importance for infants and pregnant women, so it is very necessary to add biotin into infant dairy products, pregnant women products and boosting nutrition food. Current GB (GB/T 5413.19-1997) is a unique detection method for detect biotin in dairy products. Though the Microbiological method is high-sensitivity and low detection limit, the problems of slow analysis procedure, complex operation, more interference factors and worse repeatability has limited the method development in dairy products.Therefore, the purpose of this research was toestablish a stable and universal HPLC analysis method to quantitate biotin in dairy products.Formula milk powder was investigated from several aspects in this paper, which included extraction of biotin in samples, solid-phase extraction, derivative reaction condition of biotin and condition of HPLC. The objective of the experiment is to establish a rapid detection method of biotin in dairy products by HPLC, and the mothod was fit for quantitative measurement and qualitative analysis of biotin. Two important indicators of precision and regression coefficient can testify the feasibility of the method. Another objective of experiment is to establish verification of promotion of the method by detecting other dairy products. But the trace quantity, dynamic state, the lagre density movement scope of target analytes and complicated matrix make the determination of biotin very difficult and complex. So in this research adopted solid-phase extraction technology to preconcentration, purification samples. This research optimized the conditions of solid-phase extraction, derivative reaction, HPLC, and obtained preferably results.The results were as follow:1. Optimization of extraction conditionsThe optimal temperature and time of ultrasonic extraction was 50℃×15min; and the optimal volume of precipitation solvent was 0.8～0.9mL 10%HCl.2. Optimization of SPE conditionsThe optimal ultrasonic extraction conditions were as follow: the supernatant (5ml) was applied to SPE column-C18, which had previously been washed with 6ml of methanol, 2ml of water and 5ml of 1% aqueous acetic acid. Velocity of flow was 60d/min. After washing the SPE column-C18 with 5ml of 1% acetic acid and 2ml of water, the biotin was eluted with 10ml of methanol. 3. Establishment of derivative conditionsThe optimal temperature and time of derivatization reaction was 50℃×60min, and lucifuge.4. Establishment of chromatographic conditionsThe chromatographic separations were performed on BDS Hypersil C18 (250×4.6mm i.d. 5μm) column, the biotin derivative was separated on the BDS column kept at 50℃by using water-acetonitrile (procedures of grads elution, see table3-2) at a constant flow-rate of 1.0 ml/min. It was measured fluorometricaly at the excitation and florescence wavelengths of 340 and 395 nm and a 10μL aliquot of the mixture was directly injected in the chromatograph.5. Methodology evaluation of SPE-HPLC (FLD) method for enumeration biotin in dairy productsMethodology evaluation included precision, stability, sensitivity as well as recovery rate. The relative coefficient was 0.9996 for biotin. The linearity ranged from 25 to 300ng/mL for biotin. At a signal-to-noise ratio of 3:1, the minimum detectable concentration was 25ng/mL. The extraction recoveries of biotin was 90-100% and method recoveries was also.The precision (RSD) of within-day and between-day were all less than 3%.SPE-HPLC detection method could be used to detect biotin in dairy products quantificationally. The method is simple, rapid, good selectivity while traditional Microbiological method can not. By now, these methods we established are unique and not only have a significant value in the field of biotin separation and accurate quantitation but also have a very broad application prospect. |
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