Midttun Øivind, Hustad Steinar, Ueland Per M
Bevital A/S, Armauer Hansens Hus, N-5021 Bergen, Norway.
Rapid Commun Mass Spectrom. 2009 May;23(9):1371-9. doi: 10.1002/rcm.4013.
Vitamins B2 and B6 serve as cofactors in enzymatic reactions involved in tryptophan and homocysteine metabolism. Plasma concentrations of these vitamins and amino acids are related to smoking and inflammation, and correlate with other markers of immune activation. Large-scale studies of these relations have been hampered by lack of suitable analytical methods. The assay described includes riboflavin, five vitamin B6 forms (pyridoxal 5'-phosphate, pyridoxal, 4-pyridoxic acid, pyridoxine and pyridoxamine), tryptophan and six tryptophan metabolites (kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, xanthurenic acid and 3-hydroxyanthranilic acid), cystathionine, neopterin and cotinine. Trichloroacetic acid containing 13 isotope-labelled internal standards was added to 60 microL of plasma, the mixture was centrifuged, and the resulting supernatant used for analysis. The analytes were separated within 5 min on a stable-bond C8 column by a gradient-type mobile phase containing acetonitrile, heptafluorobutyric acid and high concentration (650 mmol/L) of acetic acid, and detected using electrospray ionization tandem mass spectrometry (ESI-MS/MS). The mobile phase ensured sufficient separation and high ionization efficiency of all analytes. Recoveries were 75-123% and within-day and between-day coefficients of variance (CVs) were 2.5-9.5% and 5.4-16.9%, respectively. Limits of detection ranged from 0.05 to 7 nmol/L. The method enables quantification of endogenous plasma concentrations of 16 analytes related to B-vitamin status and inflammation, and may prove useful in large-scale epidemiological studies.
维生素B2和B6在色氨酸和同型半胱氨酸代谢所涉及的酶促反应中作为辅因子。这些维生素和氨基酸的血浆浓度与吸烟和炎症有关，并与免疫激活的其他标志物相关。由于缺乏合适的分析方法，对这些关系的大规模研究受到了阻碍。所描述的测定方法包括核黄素、五种维生素B6形式（磷酸吡哆醛、吡哆醛、4-吡哆酸、吡哆醇和吡哆胺）、色氨酸和六种色氨酸代谢物（犬尿氨酸、犬尿喹啉酸、邻氨基苯甲酸、3-羟基犬尿氨酸、黄尿酸和3-羟基邻氨基苯甲酸）、胱硫醚、新蝶呤和可替宁。将含有13种同位素标记内标的三氯乙酸加入60微升血浆中，混合液离心，所得上清液用于分析。分析物在5分钟内在稳定键合C8柱上通过含有乙腈、七氟丁酸和高浓度（650毫摩尔/升）乙酸的梯度型流动相进行分离，并使用电喷雾电离串联质谱（ESI-MS/MS）进行检测。流动相确保了所有分析物的充分分离和高电离效率。回收率为75%-123%，日内和日间变异系数（CV）分别为2.5%-9.5%和5.4%-16.9%。检测限范围为0.05至7纳摩尔/升。该方法能够定量测定与B族维生素状态和炎症相关的16种分析物的内源性血浆浓度，可能在大规模流行病学研究中有用。
