Bornø Andreas, Foged Lene, van Hall Gerrit
Clinical Metabolomics Core Facility (CMCF), Rigshospitalet, section 7652, Ole Maaløesvej 26, 2100, Copenhagen Ø, Denmark.
J Mass Spectrom. 2014 Oct;49(10):980-8. doi: 10.1002/jms.3407.
The present study describes a new liquid chromatography tandem mass spectrometry method for high-throughput quantification of glucose and glycerol in human plasma using stable isotopically labeled internal standards and is suitable for simultaneous measurements of glucose and glycerol enrichments in connection to in vivo metabolic studies investigating glucose turnover and lipolytic rate. Moreover, in order to keep up with this new fast analysis, simple derivatization procedures have been developed. Prior to analysis, glucose and glycerol were derivatized using benzoyl chloride in order to form benzoylated derivatives via new simplified fast procedures. For glucose, two internal standards were evaluated, [U-(13) C(6)]glucose and [U-(13) C(6), D(7)]glucose, and for glycerol, [U-(13) C(3), D(8)]glycerol was used. The method was validated by means of calibration curves, quality control samples, and plasma samples spiked with [6,6-D(2)]glucose, [U-(13) C(6)]glucose, and [1,1,2,3,3-D(5)]glycerol in order to test accuracy, precision, and recovery of the method. Moreover, post preparative and freeze-thaw sample stability were tested. The correlation of calibration curves for the glucose concentration were r(2) = 0.9998 for [U-(13) C(6)]glucose and r(2) = 0.9996 for [U-(13) C(6), D(7)]glucose, and r(2) = 0.9995 for the glycerol concentration. Interday accuracy for glucose using [U-(13) C(6)]glucose and glycerol determined in spiked plasma were respectively 103.5% and 106.0%, and the coefficients of variation were 2.0% and 9.7%, respectively. After derivatization, plasma samples were stable for at least 14 days. In conclusion, we have developed and validated a novel, accurate, and sensitive high-throughput liquid chromatography tandem mass spectrometry method for simultaneous determination of glucose and glycerol concentrations and enrichment of infused tracers most commonly used in human metabolic kinetic studies.
本研究描述了一种新的液相色谱串联质谱法，该方法使用稳定同位素标记的内标物对人血浆中的葡萄糖和甘油进行高通量定量，适用于与研究葡萄糖周转率和脂解速率的体内代谢研究相关的葡萄糖和甘油富集的同时测量。此外，为了跟上这种新的快速分析方法，已开发出简单的衍生化程序。在分析之前，使用苯甲酰氯对葡萄糖和甘油进行衍生化，以便通过新的简化快速程序形成苯甲酰化衍生物。对于葡萄糖，评估了两种内标物，即[U-(13)C(6)]葡萄糖和[U-(13)C(6),D(7)]葡萄糖，对于甘油，使用了[U-(13)C(3),D(8)]甘油。通过校准曲线、质量控制样品以及添加了[6,6-D(2)]葡萄糖、[U-(13)C(6)]葡萄糖和[1,1,2,3,3-D(5)]甘油的血浆样品对该方法进行验证，以测试该方法的准确性、精密度和回收率。此外，还测试了制备后和冻融样品的稳定性。葡萄糖浓度校准曲线的相关性对于[U-(13)C(6)]葡萄糖为r(2)=0.9998，对于[U-(13)C(6),D(7)]葡萄糖为r(2)=0.9996，对于甘油浓度为r(2)=0.9995。在加标血浆中使用[U-(13)C(6)]葡萄糖和甘油测定的葡萄糖日间准确度分别为103.5%和106.0%，变异系数分别为2.0%和9.7%。衍生化后，血浆样品至少稳定14天。总之，我们已经开发并验证了一种新颖、准确且灵敏的高通量液相色谱串联质谱法，用于同时测定人代谢动力学研究中最常用的输注示踪剂的葡萄糖和甘油浓度及富集情况。
