Abbott Laboratories, 3300 Stelzer Road, Columbus, Ohio 43219, USA.
J AOAC Int. 2020 Jul 1;103(4):1060-1072. doi: 10.1093/jaoacint/qsaa012.
BACKGROUND/OBJECTIVE: A multi-laboratory study was completed for AOAC First Action Method 2015.14. Ten laboratories from eight countries participated. Each laboratory analyzed (in duplicate) a placebo and 14 fortified nutritionals. Product matrices analyzed included milk, soy, partially hydrolyzed milk, partially hydrolyzed soy, and elemental-based infant formula powders, milk based infant formula ready-to-feed liquids, adult low-fat powders, and adult high fat and high protein ready-to-drink nutritionals. Data was then compared to standard method performance requirements (SMPR).
Samples were prepared by enzymatic digestion with papain, α-amylase, and phosphatase to hydrolyze protein and complex carbohydrate and to free phosphorylated vitamin forms respectively. Stable-isotope labeled internal standards were incorporated into the sample preparation to correct for variability in both the sample preparation and instrument response. Prepared samples and working standard solutions were injected onto an ultra-high-pressure liquid chromatograph, interfaced to a triple-quadrupole mass spectrometer (MS/MS). Reverse phase gradient chromatography, using mobile phases of methanol and 20 mM ammonium formate in water on a C-18 column, were used for the analysis. The MS/MS was configured to monitor precursor-fragment ion pairs for each analyte and internal standard. Vitamins B1 (thiamine), B2 (riboflavin), B3 (niacin), and B6 (pyridoxine) were quantified by least squares regression using the response ratio of the analyte to its internal standard.
RESULTS/CONCLUSIONS: Total vitamins B1, B2, B3, and B6 had average repeatability of 2.3%, 3.9%, 2.7%, and 2.2% RSD, and reproducibility of 8.2%, 6.9%, 6.7%, and 5.8% RSD, respectively. Repeatability and reproducibility SMPR were met for 53/56 and 50/56 fortified-product/analyte pairs analyzed, respectively.
背景/目的：完成了 AOAC 第一行动方法 2015.14 的多实验室研究。来自八个国家的十个实验室参加了该研究。每个实验室都对（一式两份）安慰剂和 14 种强化营养品进行了分析。分析的产品基质包括牛奶、大豆、部分水解牛奶、部分水解大豆、基于元素的婴儿配方奶粉粉末、基于牛奶的婴儿配方奶粉即饮液体、成人低脂粉末以及成人高脂肪和高蛋白即饮营养品。然后将数据与标准方法性能要求（SMPR）进行比较。
通过木瓜蛋白酶、α-淀粉酶和磷酸酶的酶解消化制备样品，分别水解蛋白质和复杂碳水化合物，并使磷酸化维生素形式游离。在样品制备中加入稳定同位素标记的内标物，以校正样品制备和仪器响应的变异性。制备好的样品和工作标准溶液注入超高效液相色谱仪，与三重四极杆质谱仪（MS/MS）相连。使用甲醇和 20 mM 甲酸铵在 C-18 柱上的反相梯度色谱法对样品进行分析。MS/MS 配置为监测每个分析物和内标的前体-片段离子对。使用分析物与其内标物的响应比，通过最小二乘法回归定量维生素 B1（硫胺素）、B2（核黄素）、B3（烟酸）和 B6（吡哆醇）。
结果/结论：B1、B2、B3 和 B6 总维生素的重复性平均为 2.3%、3.9%、2.7%和 2.2%RSD，再现性分别为 8.2%、6.9%、6.7%和 5.8%RSD。分析的 53/56 个强化产品/分析物对和 50/56 个重复性和再现性 SMPR 分别符合要求。
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