Nonesterified Fatty Acid Determination for Functional Lipidomics: Comprehensive Ultrahigh Performance Liquid Chromatography–Tandem Mass Spectrometry Quantitation, Qualification, and Parameter Prediction

Abstract

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-tandem mass spectrometry (LC-MS/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-MS/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-MS application. Accuracy and precision are within widely accepted limits. The use of qualifier ions supports unequivocal analyte verification.

Influence of moderate maternal nutrition restriction on the fetal baboon metabolome at 0.5 and 0.9 gestation

“…Analysis of NEFA was performed as previously reported [20]. Briefly, serum, liver extract, and standard solutions were mixed with isopropanol.…”

The Placental Exposome: Placental Determinants of Fetal Adiposity and Postnatal Body Composition

“…It is now possible to make in utero measurements of fetal and placental blood flow and growth [50,51]. The use of stable isotopes provides an important means to study placental transfer in vivo avoiding the drawbacks of ex vivo systems [52] when coupled with increasingly powerful biochemical and molecular approaches to study plasma and tissues samples [25,53]. An example of the use of these techniques in a longitudinal study comes from the Southampton Women's Survey, where fetal growth measures have been determined by ultrasound and related to placental gene expression and post-natal bone density [54].…”

Association of maternal prepregnancy BMI with metabolomic profile across gestation

“…While the impact of maternal obesity on adverse pregnancy and 71 offspring outcomes is well documented, a more in depth study of the maternal metabolome may highlight 72 biomarkers of gestational metabolic disturbances and potential causal pathways for fetal programming of 73 adult disease risks. 8 Metabolomics facilitates a detailed investigation of the metabolic state by 74 determining single molecular species, for example the determination of non-esterified fatty acids 9 and 75 glycerophospholipids 10 allows a differentiated view on fatty acid status . Such new insights among 76 pregnant populations are important to assist our efforts in adapting nutrition, lifestyle or other factors in 77 pregnancy for more favorable outcomes.…”