Forteschi Mauro, Zinellu Angelo, Assaretti Stefano, Mangoni Arduino A, Pintus Gianfranco, Carru Ciriaco, Sotgia Salvatore
Department of Biomedical Sciences, School of Medicine, University of Sassari, Viale San Pietro 43/B, 07100, Sassari, Italy.
Department of Clinical Pharmacology, School of Medicine, Flinders University, Bedford Park, Adelaide, South Australia, 5042, Australia.
Anal Bioanal Chem. 2016 Oct;408(26):7505-12. doi: 10.1007/s00216-016-9848-6. Epub 2016 Aug 8.
Plasma concentrations of choline, betaine, and dimethylglycine provide valuable information on the flow of methyl groups in key biological processes, particularly during folate deficiency states. We developed a new method to simultaneously measure these analytes in human plasma. Following sample deproteinization using acetonitrile, an aliquot was evaporated to dryness under vacuum to be then taken up by water. Finally, analytes were separated by capillary electrophoresis and detected by electrospray ionization triple-quadrupole mass spectrometry, in multiple reaction monitoring mode, using two stable isotope-labeled internal standards. Linearity of the calibration curves of each analyte was good (R(2) > 0.99). Average limits of detection (LODs) and limits of quantification (LOQs) for choline, betaine, and dimethylglycine were, respectively, 0.43, 0.62, and 0.31 μmol/L and 1.52, 2.11, and 0.97 μmol/L. Mean recovery of three replicates of two spiked concentrations levels was close to 100 % for all of the analytes. Repeatability and intermediate precision, expressed as %RSD of measurements, were <9 %. The method, applied to measure analytes in samples from 30 patients with chronic kidney disease and 30 age- and sex-matched healthy controls, was able to detect differences between groups and the sexes.
血浆中胆碱、甜菜碱和二甲基甘氨酸的浓度为关键生物过程中甲基的流动提供了有价值的信息，尤其是在叶酸缺乏状态下。我们开发了一种新方法来同时测量人血浆中的这些分析物。使用乙腈对样品进行脱蛋白处理后，取一份等分试样在真空下蒸发至干，然后用水复溶。最后，通过毛细管电泳分离分析物，并使用两种稳定同位素标记的内标物，在多反应监测模式下通过电喷雾电离三重四极杆质谱进行检测。每种分析物校准曲线的线性良好（R(2) > 0.99）。胆碱、甜菜碱和二甲基甘氨酸的平均检测限（LOD）和定量限（LOQ）分别为0.43、0.62和0.31 μmol/L以及1.52、2.11和0.97 μmol/L。所有分析物在两个加标浓度水平下进行三次重复测定的平均回收率接近100%。以测量的相对标准偏差（%RSD）表示的重复性和中间精密度均<9%。该方法应用于测量30例慢性肾病患者和30例年龄及性别匹配的健康对照者的样品中的分析物，能够检测出组间和性别间的差异。
