Heudi O, Kilinç T, Fontannaz P, Marley E
Quality and Safety Department, Nestlé Research Center, Nestec Ltd., Vers-Chez-les-Blanc, 1000 Lausanne 26, Switzerland.
J Chromatogr A. 2006 Jan 6;1101(1-2):63-8. doi: 10.1016/j.chroma.2005.09.059. Epub 2005 Oct 10.
A new, faster and simple method to quantify Vitamin B12, both in foods and in premixes, by reversed-phase liquid chromatography with UV detection has been developed. Vitamin B12 was extracted from food products with 50 mM sodium acetate buffer pH 4.0 (at 100 degrees C for 35 min) in the presence of sodium cyanide, followed by a purification step on an immunoaffinity column prior to the LC analysis. An enzymatic hydrolysis (pepsin at 37 degrees C and pH 4 for 3 h) prior to the purification step efficiently released the bound Vitamin B12, and thus, allowed obtaining total Vitamin B12 content in food products. Vitamin B12 was monitored by UV at 361 nm after its separation on a reversed-phase narrow-bore column with a gradient of mobile phase made of water/acetonitrile and trifluoroacetic acid (TFA) 0.025%. The specificity of the method was demonstrated by the retention characteristics, UV spectra and by comparing the peak purity with the Vitamin B12 standard. The calibration graphs plotted with six concentrations of Vitamin B12 was linear with a regression coefficient R2 > 0.9997. The repeatability of the method was evaluated at different levels of concentration on six fortified products and the relative standard deviation (RSDr) was below 3.2%. The value of the relative standard deviation of the intermediate precision was below 5.6% (n = 4). The method was successfully applied to several food products and consistent results were obtained in comparison with microbiological assay (MBA). Our data demonstrate that the immunoaffinity columns are highly efficient for the purification of Vitamin B12 and that our HPLC could be used as an alternative method to the microbiological assay for the determination of Vitamin B12 in food products.
已开发出一种新的、更快且简单的方法，通过带有紫外检测的反相液相色谱法对食品和预混物中的维生素B12进行定量。在氰化钠存在的情况下，用pH 4.0的50 mM醋酸钠缓冲液（在100℃下35分钟）从食品中提取维生素B12，然后在液相色谱分析之前在免疫亲和柱上进行纯化步骤。在纯化步骤之前进行酶促水解（在37℃和pH 4下用胃蛋白酶处理3小时）可有效释放结合的维生素B12，从而能够获得食品中维生素B12的总含量。维生素B12在由水/乙腈和0.025%三氟乙酸（TFA）组成的流动相梯度的反相窄孔柱上分离后，于361 nm处通过紫外进行监测。该方法的特异性通过保留特性、紫外光谱以及将峰纯度与维生素B12标准品进行比较得以证明。用六种浓度的维生素B12绘制的校准曲线呈线性，回归系数R2>0.9997。在六种强化产品的不同浓度水平下评估了该方法的重复性，相对标准偏差（RSDr）低于3.2%。中间精密度的相对标准偏差值低于5.6%（n = 4）。该方法已成功应用于多种食品，并与微生物测定法（MBA）相比获得了一致的结果。我们的数据表明，免疫亲和柱对维生素B12的纯化效率很高，并且我们的高效液相色谱法可作为食品中维生素B12测定的微生物测定法的替代方法。
