Nonesterified Fatty Acid Determination for Functional Lipidomics: Comprehensive Ultrahigh Performance Liquid Chromatography鈥揟andem Mass Spectrometry Quantitation, Qualification, and Parameter Predicti

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• 作者：Christian Hellmuth ; Martina Weber ; Berthold Koletzko ; Wolfgang Peissner
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：February 7, 2012
• 年：2012
• 卷：84
• 期：3
• 页码：1483-1490
• 全文大小：280K
• 年卷期：v.84,no.3(February 7, 2012)
• ISSN：1520-6882

文摘

Despite their central importance for lipid metabolism, straightforward quantitative methods for determination of nonesterified fatty acid (NEFA) species are still missing. The protocol presented here provides unbiased quantitation of plasma NEFA species by liquid chromatography鈥搕andem mass spectrometry (LC鈥揗S/MS). Simple deproteination of plasma in organic solvent solution yields high accuracy, including both the unbound and initially protein-bound fractions, while avoiding interferences from hydrolysis of esterified fatty acids from other lipid classes. Sample preparation is fast and nonexpensive, hence well suited for automation and high-throughput applications. Separation of isotopologic NEFA is achieved using ultrahigh-performance liquid chromatography (UPLC) coupled to triple quadrupole LC鈥揗S/MS detection. In combination with automated liquid handling, total assay time per sample is less than 15 min. The analytical spectrum extends beyond readily available NEFA standard compounds by a regression model predicting all the relevant analytical parameters (retention time, ion path settings, and response factor) of NEFA species based on chain length and number of double bonds. Detection of 50 NEFA species and accurate quantification of 36 NEFA species in human plasma is described, the highest numbers ever reported for a LC鈥揗S application. Accuracy and precision are within widely accepted limits. The use of qualifier ions supports unequivocal analyte verification.