Laboratory of Toxicology, Department of Bioanalysis, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium.
Laboratory of Functional Plant Biology, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
Anal Chem. 2020 Mar 3;92(5):4073-4081. doi: 10.1021/acs.analchem.9b05717. Epub 2020 Feb 24.
serves as a model plant for genetic research, including vitamin research. When aiming at engineering the thiamine (vitamin B1) pathway in plants, the availability of tools that allow the quantitative determination of different intermediates in the biosynthesis pathway is of pivotal importance. This is a challenge, given the nature of the compounds and the minute quantities of genetically engineered material that may be available for analysis. Here, we report on the first LC-MS/MS method for the simultaneous quantification of thiamine, its mono- and diphosphate derivatives and its precursors 4-methyl-5-(2-hydroxyethyl) thiazole (HET) and 4-amino-2-methyl-5-hydroxymethylpyrimidine (HMP). This method was optimized and validated for the quantitative determination of these analytes in . All analytes were chromatographically separated within less than 2.5 min during an 8 min run. No unacceptable interferences were found. The method was fully validated based on international guidelines. Accuracy (%bias) and total imprecision (%CV) were within preset acceptance criteria for all analytes in both QC and real samples. All analytes were stable in extracted samples when stored for 48 h at 4 °C (autosampler stability) and when reanalyzed after storage at -80 °C and -20 °C for 2 weeks (freeze/thaw stability). We demonstrated the start material should be stored at -80 °C to ensure stability of all analytes during short- and long-term storage (up to 3 months). The validity and applicability of the developed procedure was demonstrated via its successful application on lines, genetically engineered to enhance thiamine content.
它是遗传研究的模式植物，包括维生素研究。当目标是在植物中工程化硫胺素（维生素 B1）途径时，拥有允许定量测定生物合成途径中不同中间产物的工具至关重要。鉴于化合物的性质和可能用于分析的遗传工程材料的微量，这是一个挑战。在这里，我们报告了第一个用于同时定量测定硫胺素、其单磷酸和二磷酸衍生物以及其前体 4-甲基-5-(2-羟乙基)噻唑（HET）和 4-氨基-2-甲基-5-羟甲基嘧啶（HMP）的 LC-MS/MS 方法。该方法经过优化和验证，可用于定量测定 中的这些分析物。在 8 分钟的运行中，所有分析物在不到 2.5 分钟内实现色谱分离。未发现不可接受的干扰。该方法完全按照国际指南进行验证。在 QC 和实际样品中，所有分析物的准确度（%偏差）和总不精密度（%CV）均符合预设接受标准。在 4°C 下提取样品中储存 48 小时（自动进样器稳定性）和在-80°C 和-20°C 下储存 2 周后重新分析时（冻融稳定性），所有分析物在提取样品中均稳定。我们证明，为了确保在短期和长期储存（长达 3 个月）期间所有分析物的稳定性，起始材料应储存在-80°C。通过成功应用于基因工程以提高硫胺素含量的 株系，证明了所开发程序的有效性和适用性。
