Lehrstuhl für Lebensmittelchemie, Technische Universität München, Garching, Germany.
Anal Biochem. 2010 Mar 15;398(2):150-60. doi: 10.1016/j.ab.2009.11.007. Epub 2009 Nov 10.
New stable isotope dilution assays were developed for the simultaneous quantitation of the folates 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, tetrahydrofolic acid, 10-formylfolic acid, and folic acid as well as for their catabolites para-aminobenzoylglutamate (pABG) and acetyl-para-aminobenzoylglutamate (ApABG) in clinical samples. The methods were based on cleanup by strong anion exchange followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. Deuterated analogues of the folates and [(13)C(5)]-labeled isotopologues of the catabolites were applied as the internal standards in stable isotope dilution assays. Extraction in 4-morpholineethanesulfonic acid (MES) buffer at pH 5.0 ensured the optimum stability of folates and, in combination with solid-phase extraction (SPE) based on strong anion exchange, resulted in higher recoveries compared with other combinations of extraction buffers and SPE. The method was sensitive enough to detect pABG in plasma generally and unmetabolized folic acid in the plasma of a volunteer after oral dosage of an aqueous folic acid solution. The sum of folate catabolites increased by a factor of 2 in the urine of the latter volunteer, compared with that resulting when only water was dosed.
新的稳定同位素稀释分析方法被开发出来，用于同时定量测定临床样本中的叶酸代谢物 5-甲基四氢叶酸、5-甲酰基四氢叶酸、四氢叶酸、10-甲酰基叶酸以及叶酸，以及它们的代谢物对氨基苯甲酰谷氨酸（pABG）和乙酰对氨基苯甲酰谷氨酸（ApABG）。该方法基于强阴离子交换的净化，然后进行液相色谱-串联质谱（LC-MS/MS）检测。采用叶酸的氘代类似物和代谢物的 [(13)C(5)] 标记同量异位体作为稳定同位素稀释分析中的内标。在 pH 值为 5.0 的 4-吗啉乙磺酸（MES）缓冲液中进行提取，确保了叶酸的最佳稳定性，与基于强阴离子交换的固相萃取（SPE）相结合，与其他提取缓冲液和 SPE 的组合相比，提高了回收率。该方法足够灵敏，能够检测血浆中的 pABG 以及口服叶酸水溶液后志愿者血浆中的未代谢叶酸。与仅给予水的情况相比，后者志愿者尿液中的叶酸代谢物总量增加了两倍。
