School of Science, University of Waikato, Private Bag 3105, Hamilton, 3240, New Zealand.
Fonterra Co-operative Group Limited, P.O. Box 7, Waitoa, 3341, New Zealand.
Talanta. 2025 Jan 1;282:126970. doi: 10.1016/j.talanta.2024.126970. Epub 2024 Sep 28.
The analysis of vitamin B in infant formulas typically requires the use of cyanide during sample preparation to convert the unstable vitamers (hydroxocobalamin, methylcobalamin and adenosylcobalamin) to cyanocobalamin, the most stable form of vitamin B. To eliminate the risk to laboratory analysts in handling cyanide, alternative strategies are preferred for the analysis of vitamin B. This research demonstrates the use of cobalamin-derived α-ribazole (a nucleoside moiety of vitamin B) to determine total vitamin B content. Infant formula samples underwent protein denaturation and sugar removal with subsequent acidic hydrolysis and dephosphorylation employed to release α-ribazole, which was isolated by boronate affinity chromatography then analysed by hydrophilic interaction liquid chromatography with fluorescence detection. The method was validated using bovine- and ovine milk-based infant formula samples. The newly developed method was linear over the range of 0.65-6.48 ng mL with repeatability of 3.78-5.47% relative standard deviation (RSD, n = 10) and an intermediate precision of 3.59-10.0% RSD (n = 10). The limits of detection and quantitation (LOD and LOQ) were 0.4 and 1.2 μg 100 g of dry weight, respectively. Accuracy was 68.9-76.4% and 68.7-80.0% at 50 and 150% of typical B concentrations in infant formula, respectively. The validated method was applied to eleven infant formulas and no statistical difference (p = 0.45, α = 0.05) was found when comparing with the results obtained using the AOAC Official Method 2014.02 high performance liquid chromatography with ultraviolet detection that requires the use of cyanide. These results indicate that the newly validated method is not only reliable but also offers a safer alternative for routine vitamin B determination in infant formula while maintaining high accuracy and precision.
婴儿配方食品中维生素 B 的分析通常需要在样品制备过程中使用氰化物将不稳定的维生素（羟钴胺素、甲钴胺素和腺苷钴胺素）转化为氰钴胺素，这是维生素 B 最稳定的形式。为了消除实验室分析人员在处理氰化物时的风险，人们更喜欢使用替代策略来分析维生素 B。本研究展示了使用钴胺素衍生的α-核糖唑（维生素 B 的核苷部分）来测定总维生素 B 含量。婴儿配方食品样品经过蛋白质变性和糖去除，随后采用酸性水解和去磷酸化释放α-核糖唑，该核糖唑通过硼酸盐亲和色谱法分离，然后通过亲水相互作用液相色谱法与荧光检测分析。该方法使用牛和羊乳基婴儿配方食品样品进行了验证。新开发的方法在 0.65-6.48ng/mL 范围内呈线性，重复性为 3.78-5.47%相对标准偏差（RSD，n=10），中间精密度为 3.59-10.0%RSD（n=10）。检测限和定量限（LOD 和 LOQ）分别为 0.4 和 1.2μg/100g 干重。在婴儿配方食品中典型 B 浓度的 50%和 150%时，准确度分别为 68.9-76.4%和 68.7-80.0%。验证后的方法应用于 11 种婴儿配方食品，与使用 AOAC 官方方法 2014.02 高效液相色谱法与紫外检测（需要使用氰化物）得到的结果相比，无统计学差异（p=0.45，α=0.05）。这些结果表明，新验证的方法不仅可靠，而且在保持高精度和高精确度的同时，为婴儿配方食品中常规维生素 B 测定提供了更安全的替代方案。
