Gmelch Lena, Wirtz Daniela, Witting Michael, Weber Nadine, Striegel Lisa, Schmitt-Kopplin Philippe, Rychlik Michael
Chair of Analytical Food Chemistry, Technical University of Munich, 85354 Freising-Weihenstephan, Germany.
Research Unit BioGeoChemistry, Helmholtz Zentrum Munich, 85764 Neuherberg, Germany.
Metabolites. 2020 Jul 23;10(8):301. doi: 10.3390/metabo10080301.
Folates are a group of B vitamins playing an important role in many metabolic processes such as methylation reactions, nucleotide synthesis or oxidation and reduction processes. However, humans are not able to synthesize folates de novo and thus rely on external sources thereof. Baker's yeast () has been shown to produce high amounts of this vitamin but extensive identification of its folate metabolism is still lacking. Therefore, we optimized and compared different sample preparation and purification procedures applying solid phase extraction (SPE). Strong anion exchange (SAX), C18 and hydrophilic-lipophilic-balanced (HLB) materials were tested for their applicability in future metabolomics studies. SAX turned out to be the preferred material for the quantitative purification of folates. Qualification of several folate vitamers was achieved by ultra-high pressure liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-Q-ToF-MS) measurements and quantification was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS) applying stable isotope dilution assays (SIDAs). The oxidation product -pyrazino-triazine (MeFox) was included into the SIDA method for total folate determination and validation. Applying the best protocol (SAX) in regard to folate recovery, we analyzed 32 different vitamers in different polyglutamate states up to nonaglutamates, of which we could further identify 26 vitamers based on tandem-MS (MS) spectra. Total folate quantification revealed differences in formyl folate contents depending on the cartridge chemistry used for purification. These are supposedly a result of interconversion reactions occurring during sample preparation due to variation in pH adjustments for the different purification protocols. The occurrence of interconversion and oxidation reactions should be taken into consideration in sample preparation procedures for metabolomics analyses with a focus on folates.
叶酸是一组B族维生素，在许多代谢过程中发挥重要作用，如甲基化反应、核苷酸合成或氧化还原过程。然而，人类无法从头合成叶酸，因此依赖于外部来源。已证明面包酵母（）能产生大量这种维生素，但仍缺乏对其叶酸代谢的广泛鉴定。因此，我们优化并比较了应用固相萃取（SPE）的不同样品制备和纯化程序。测试了强阴离子交换（SAX）、C18和亲水亲油平衡（HLB）材料在未来代谢组学研究中的适用性。结果表明，SAX是定量纯化叶酸的首选材料。通过超高压液相色谱四极杆飞行时间质谱（UHPLC-Q-ToF-MS）测量实现了几种叶酸维生素异构体的鉴定，并通过应用稳定同位素稀释分析（SIDA）的液相色谱串联质谱（LC-MS/MS）进行了定量。氧化产物-吡嗪三嗪（MeFox）被纳入SIDA方法用于总叶酸的测定和验证。应用关于叶酸回收率的最佳方案（SAX），我们分析了多达九谷氨酸状态的不同聚谷氨酸状态下的32种不同维生素异构体，其中我们可以基于串联质谱（MS）光谱进一步鉴定26种维生素异构体。总叶酸定量显示，根据用于纯化的柱化学性质，甲酰叶酸含量存在差异。这些可能是由于不同纯化方案的pH调节变化导致样品制备过程中发生相互转化反应的结果。在以叶酸为重点的代谢组学分析的样品制备过程中，应考虑相互转化和氧化反应的发生。
