MSACL 2019 EU Abstract

Development and Validation of a LC-MS/MS-based Assay for Quantification of Polyunsaturated Fatty Acids from Human Plasma and Red Blood Cells

Vlad Serafim (1), Diana-Andreea Tiugan (1,2), Nicoleta Andreescu (1,2), Adela Chirita-Emandi (1,2), Alexandra Mihailescu (1), Corina Paul (3), Iulian Velea (3), Maria Puiu (1,2), and Mihai Dinu Niculescu (1)
(1) Genetics Discipline, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania (2) “Louis Turcanu” Clinical Emergency Hospital for Children, Timisoara, Romania (3) Paediatric Department, “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania.

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Vlad Serafim (Presenter) 'Victor Babeş' University of Medicine and Pharmacy >> POSTER (PDF)

Presenter Bio: The area that I am involved in is development of mass spectrometry-based analytical methods for biomolecule analysis. For over 15 years I have worked in various positions in both routine laboratories and academic institutions. Since 2016 I have been working in NUTRIGEN project at Victor Babeş University of Medicine and Pharmacy Timisoara, being involved in measurement of several metabolites involved in obesity. My main interest is LC-MS/MS for both targeted and non-targeted analysis of metabolites.

Relevant Financial Disclosures (within past 24 months) Salary \'Victor Babeş\' University of Medicine and Pharmacy, Timisoara (Romania) Conflict mitigation NOT REQUIRED.

INTRODUCTION: Polyunsaturated fatty acids (PUFAs) play essential roles in human physiology. Changes in PUFAs metabolism may have implications in obesity, type 2 diabetes, insulin resistance, cardiovascular diseases, and also interrelationship with other metabolic pathways. Considering their importance, accurate quantification of n-3 and n-6 PUFAs is required.
Until recently, PUFAs were quantified using gas chromatography coupled with mass spectrometry (GC-MS) or flame ionization detector (GC-FID); and by liquid chromatography–mass spectrometry (LC-MS) following derivatization. Chemical derivatization requires additional sample preparation steps, which adds extra-complexity to the method.
OBJECTIVE: As few methods have been developed for the full quantitation of fatty without derivatization, we aimed to develop a method that requires fewer sample preparation steps which can be used for the quantitation of several PUFAs from human plasma and red blood cells.
METHODS: The lipids were extracted from plasma and red blood cell samples. The fatty acids were released by alkaline hydrolysis. In plasma samples, the selected fatty acids were from both free and total forms. Chromatographic separation was achieved using a reversed phase C18 column with isocratic flow using 90% acetonitrile with ammonium acetate. Mass detection was performed in multiple reaction monitoring (MRM) mode, and deuterated internal standards were used for each targeted compound. The targeted fatty acids were: alfa-linolenic, arachidonic, docosahexaenoic, linoleic, and eicosapentaenoic. The method was validated according to the U.S. Department of Health and Human Services guidelines, addressing calibration curve, accuracy, precision, recovery, quality control samples, and sensitivity.
RESULTS: The limits of quantification were situated in the low nanomolar range, excepting linoleic acid, for which the limit was in the high nanomolar range. Elution started at 3.83 min with eicosapentaenoic acid, while the last was linoleic acid at 5.02 min. The HPLC method ended at 6.5 min. All acceptance criteria found in the validation guideline were met.
CONCLUSION:
Since derivatization was not employed, the sample preparation protocol was less time-consuming and labour-intensive. The LC-MS/MS proved significantly faster than previously published GS-MS methods. The proposed method offers a fast, sensitive, and reliable quantification of selected omega 3 and 6 fatty acids in human plasma and red blood cells.

Acknowledgement: This work was performed at The Centre of Genomic Medicine, POSCCE Project, SMIS:48749, and funded by POC Project NutriGen, SMIS:104852.