Nestlé Research, Route du Jorat 57, Vers-chez-les-Blanc, 1000, Lausanne, Switzerland.
Anal Bioanal Chem. 2020 Jan;412(2):365-375. doi: 10.1007/s00216-019-02248-5. Epub 2019 Dec 12.
A novel method to quantitate vitamin D and its main metabolites (vitamin D3, vitamin D2, and their 25-hydroxy metabolites) in breast milk by supercritical fluid chromatography has been developed and fully validated. A small volume of sample (1 mL) is subjected to ethanolic protein precipitation and liquid-liquid extraction. Final extracts are derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione and vitamin D derivatives analyzed by supercritical fluid chromatography hyphenated to tandem mass spectrometry with atmospheric pressure chemical ionization. Multiple reaction monitoring is used for quantitation. Separation conditions were optimized using a gradient of methanol-water-ammonium formate into carbon dioxide. Make-up solvent was methanol containing ammonium formate. The quantitation limit reached levels as low as 50 pmol/L, with intra- and inter-day relative standard deviations lower than 15% and 20% for all analytes. Accuracy was evaluated by spiking experiments and was well within acceptability ratios (± 15%). The method was then applied to a subset of commercially available human milk samples. The newly developed method provides opportunities to determine the nutritional status of mother-infant dyads from a non-invasive measure, or for interventional or observational studies building knowledge on the composition of human milk. Graphical abstract.
一种新的方法来定量维生素 D 及其主要代谢物（维生素 D3、维生素 D2 和它们的 25- 羟代谢物）在母乳中通过超临界流体色谱已经被开发并充分验证。一个小体积的样本（1 毫升）进行乙醇蛋白质沉淀和液液萃取。最终提取物用 4-苯基-1,2,4-三唑-3,5-二酮衍生化，并用超临界流体色谱与大气压化学电离串联质谱联用进行维生素 D 衍生物分析。多重反应监测用于定量。使用甲醇-水-甲酸铵梯度进入二氧化碳优化分离条件。补偿溶剂是含有甲酸铵的甲醇。定量限达到 50pmol/L 以下，所有分析物的日内和日间相对标准偏差均低于 15%和 20%。通过添加实验评估了准确性，并且在可接受的比例（±15%）范围内。该方法随后应用于一组商业上可获得的人乳样本。新开发的方法提供了从非侵入性测量来确定母婴对的营养状况的机会，或者用于干预或观察性研究，以建立关于人乳成分的知识。
