Robust Method for High-Throughput Screening of Fatty Acids by Multisegment Injection-Nonaqueous Capillary Electrophoresis–Mass Spectrometry with Stringent Quality Control

Abstract

High-throughput screening methods for fatty acid (FA) determination are urgently needed due to their critical biochemical roles in human health while serving as biomarkers of habitual diet and chronic disease risk assessment. Herein, we introduce multisegment injection-nonaqueouscapillary electrophoresis-mass spectrometry (MSI-NACE-MS) as a multiplexed separation platform for analysis of more than 20 nonesterified FAs in human serum or plasma. Optimization of experimental conditions was required to overcome major technical hurdles in MSI-NACE-MS prior to a rigorous method validation and intermethod comparison with gas chromatography/mass spectrometry (GC/MS). Following a simple methyl-tert-butyl ether extraction, seven serum extracts were analyzed directly by MSI-NACE-MS within a single run (<4 min/sample) under negative ion mode detection that incorporates stringent measures for quality control, including batch correction adjustment. Overall, excellent technical variance (RSD = 10%) and good mutual agreement was demonstrated for 12 nonesterified FAs consistently measured in 50 serum samples analyzed independently by MSI-NACE-MS and GC/MS within the same laboratory (mean bias = 24%, n = 600). Also, total hydrolyzed plasma FAs using MSI-NACE-MS was compared to mean concentrations reported from a NIST standard reference material as an interlaboratory method validation (mean bias = 15%, n = 20). Accurate prediction of ion migration behavior in CE also supports structural elucidation of FAs in conjunction with high resolution MS. For the first time, we demonstrate that MSI-NACE-MS offers a rapid yet robust platform for direct quantification of circulating FAs using volume-restricted blood specimens that expands metabolome coverage to encompass anionic classes of lipids as required for large-scale epidemiological studies.

Placental Metabolomics for Assessment of Sex-specific Differences in Fetal Development During Normal Gestation

“…Figure 2(B) shows an extracted ion electropherogram for placenta derived acetylcarnitine (204.1230:0.787:p), an important mediator of fatty acid metabolism in the mitochondria. Similarly, intracellular anionic metabolites from placental extracts were also measured in this work under negative ion mode detection, including polar/hydrophilic metabolites, such as uric acid (167.0211:0.960:n), and non-polar lipids using non-aqueous buffer conditions 28 , such as the omega-3 fatty acid, docosahexaenoic acid or DHA (327.2330:0.988:n).…”

“…Prior to MSI-CE-MS analysis, 5 µL of the internal standards (25 µM), 3-chloro-L-tyrosine (Cl-Tyr) and 2-napthalenesulfonic acid (NMS), were added to an aliquot of 20 µL of placenta extract. For the analysis of total (hydrolyzed) fatty acids and bile acids in placental tissue (~1-2 mg dried tissue), a hydrolysis reaction combined with a modified methyl-tert-butyl ether (MTBE) extraction procedure was employed 28,56 . First, hydrolysis of lipids was performed by the addition of 25 μL of butylated hydroxytoluene (BHT) in 0.1% vol toluene and 25 μL of 2.5 M of sulfuric acid to freeze-dried placental tissue.…”

The Function and Mechanism of Lipid Molecules and Their Roles in The Diagnosis and Prognosis of Breast Cancer

“…The fourth method is nonaqueous capillary electrophoresis/mass spectrometry (NACE), which has a high separation efficiency. This technique does not require the preparation of a complicated sample, it is relatively low cost, and it is suitable for inactivated fatty acids [ 52 ] and phospholipids [ 53 ]. The fifth is MALDI imaging [ 54 ], which can be utilized to determine the spatial distribution of lipids in tissue samples; it displays the density of various lipid molecules as a heat map and retains their anatomical structures.…”

Rapid biomonitoring of perfluoroalkyl substance exposures in serum by multisegment injection‐nonaqueous capillary electrophoresis‐tandem mass spectrometry

“…Improved solubilization and specific solvent-solute interactions have long been attributed to the unique selectivity in NACE 52 with PFOS being bulkier, less volatile, and more hydrophobic than PFOA. 23 We recently demonstrated that a homologous series of fatty acids are accurately modeled in NACE based on their characteristic mobilities reflecting differences in carbon chain length and degree of unsaturation supporting their unambiguous identification complementary to high resolution MS. 43 Other experimental variables also impacted PFAS separation performance, such as drying gas flow rate, nebulizer gas pressure, as well as a hydrodynamic pressure applied during electromigration (i.e., pressure-assisted NACE). Nebulizer gas pressure is required in the CE-MS interface to stabilize spray formation, and higher nebulizer gas velocities contributed to faster migration times due to a greater siphoning effect, 53 which in turn gave rise to sharper peaks and a threefold higher SNR as depicted for PFOA in Figure 1C.…”