Multidimensional Liquid Chromatography Coupled with Tandem Mass Spectrometry for Identification of Bioactive Fatty Acyl Derivatives

Abstract

Recognition of the contributions of lipids to cellular physiology, both as structural components of the membrane and as modulatory ligands for membrane proteins, has increased in recent years with the development of the biophysical and biochemical tools to examine these effects. Their modulatory roles in ion channels and transporters function have been extensively characterized, with the molecular mechanisms of these activities being the subject of intense scrutiny. The physiological significance of lipids in biochemistry is expanding as numerous fatty acyls are discovered to possess signaling properties. These bioactive lipids are often found in quantities of pmol/g of tissue and are co-extracted with numerous lipophilic molecules, making their detection and identification challenging. Common analytical methodologies involve chromatographic separation and mass spectrometric techniques; however, a single chromatographic step is typically ineffective due to the complexity of the biological samples. It is, therefore, essential to develop approaches that incorporate multiple dimensions of separation. Described in this manuscript are normal phase and reversed phase separation strategies for lipids that include detection of the bioactive primary fatty acid amides and N-acyl glycines via tandem mass spectrometry. Concerted utilization of these approaches are then used to separate and sensitively identify primary fatty acid amides extracted from homogenized tissue, using mouse brains as a test case.

Keywords: N-acyl ethanolamines; N-acyl glycines; bioactive lipids; fatty acyls; lipid-protein interactions; multidimensional liquid chromatography; primary fatty acid amides.

Figure 1

Chromatogram of seven fatty acyl subclasses separated by normal phase chromatography using a YMC PVA-Sil (4.6 × 250 mm, 5 μm particle size) on a Waters ZMD MS with an ESI probe with polarity switching. Separation was achieved with mobile phase A (heptane with 0.5% methyl-tert-butyl ether) and mobile phase B (10% 2-propanol, 0.2% acetic acid in methyl-tert-butyl ether) run in gradient mode from 95 to 50% A over 40 min. The monoacylglycerols were monitored as the [M+Na]⁺ peak, PFAM as the [M+H]⁺ peak, diacylglycerols as [M+Na]⁺ peak, NAE as [M+Na]⁺ peak, fatty acids as [M−H]⁻ peak, NAG as [M−H]⁻ peak, and triacylglycerols as [M+Na]⁺ peak. Each class was monitored on a different channel on the MS.

Figure 2

Separation of NAGs on (A) a C30 YMC carotenoid column (4.6 × 150 mm, 5 um particles size) and (B,C) Phenomenex C18 column (4.6 × 100 mm, 2.6 μm particles size) using two different gradient separations. Ionized by ESI and detected in MRM mode as the [M−H]⁻ parent and 74 m/z glycine head group product at collision energy of 20 V. Flow rate was 1 mL/min. The right axis shows the gradient elution profile for % methanol. Peak identities in (A,B) are (1) linoleoylglycine C18:2, (2) palmitoylglycine C16:0, (3) oleoylglycine C18:1, (4) arichidoylglycine C20:0, and (C) are (1) stearidonoylglycine C18:4, (2) linoleoylglycine C18:2, (3) arachidonoylglycine C20:4, (4) palmitoylglycine C16:0, (5) oleoylglycine C18:1, (6) arachidoylglycine C20:0.

Figure 3

Separation of very long chain PFAMs with Agilent RP C18 column (2.1 × 50 mm, 1.8 μm particle size) using a (A) steep and (B) shallow gradient (% methanol on the right axis). Peak identities were identified by MS/MS, with fractions ionized by APCI and identified in MRM mode. The peaks were identified as (1) lauramide, (2) myristamide, (3) linoleamide, (4) palmitamide, (5) oleamide, (6) elaidamide, (7) petroselaidamide, (8) heptadecanoamide, (9) stearamide, (10) erucamide, (11) arachidamide, (12) behenamide.

Figure 4

Isolation and identification of PFAMs in brain. The equivalent of 10 Swiss-Webster mouse brains (with mid brain removed) were Folch-Pi extracted. Samples were then subjected to MDLC using normal phase chromatography, and the peak corresponding to PFAM class of lipids were then separated by reversed phase chromatographs (as described in the text). PFAM substituents where identified by MS/MS as described Figure 3. PFAM substituents corresponding to 14:0, 19:2^9,12, 16:0, 18:1⁹, 18:0, and 22:0 PFAMs were identified in mouse brain tissue by MRM (as described in the text).