Determining double‐bond positions in monoenoic 2‐hydroxy fatty acids of glucosylceramides by gas chromatography‐mass spectrometry

Abstract

We applied a gas chromatography-mass spectrometry (GC-MS) method using dimethyl disulfide (DMDS) adducts and were able to determine the double-bond positions in monounsaturated 2-hydroxy fatty acids (2-HFA). 2-HFA methyl esters, prepared from the hydrolysate of Arabidopsis thaliana leaf glucosylceramides, were acetylated and methylthiolated. GC-MS analysis of the resulting DMDS adducts showed simple mass spectra with recognizable molecular ions and a series of key fragment ions indicating the original double-bond positions in the aliphatic chain. Based on this GC-MS elucidation, we confirmed that Arabidopsis leaf glucosylceramides have C22, C23, C24, C25, and C26 chain length 2-HFA with monounsaturation, and all their double bonds are placed at the n-9 position. This procedure is simple, time efficient, and highly sensitive.

Arabidopsis Sphingolipid Fatty Acid 2-Hydroxylases (AtFAH1 and AtFAH2) Are Functionally Differentiated in Fatty Acid 2-Hydroxylation and Stress Responses

“…Total lipids including total sphingolipids were extracted from the aboveground parts in 3-week-old plants according to Markham et al (2006), and these 2-HFA methyl esters were detected with GC-MS after derivatization. In this study, we analyzed five major 2-HFAs (16h:0, 22h:0, 24h:0, 24h:1, and 26h:0) of 13 2-HFAs in Arabidopsis (Imai et al, 2000).…”

“…2-HFA methyl esters with 2-hydroxy pentadecanoic acid (15h:0) methyl ester as an internal standard were prepared and separated by silica gel TLC using n-hexane/diethyl ether (4:1, vol/vol) as the mobile phase, as described by Imai et al (2000).…”

Characterization of Glucosylceramides in thePolygonaceae,Rumex obtusifoliusL. Injurious Weed

“…2A), this being distinct from the 2-hydroxy fatty acid composition of Arabidopsis leaves that demonstrated a near absence of 22h:1. 19) To determine the double-bond positions in the monoenoic 2-hydroxy fatty acids of R. obtusifolius glucosylceramides, the dimethyl disulfide adducts were analyzed by GC/MS. The key fragment ion at m=z 173 and the characteristic fragment ions ½M þ À 173 were detected (Supplemental Fig.…”

“…Total lipids were extracted according to the method of Bligh and Dyer. 18) The glucosylceramides were purified essentially according to the method previously described, 19) but without acetylating the crude glucosylceramides. In brief, the total lipids were subjected to mild alkaline hydrolysis with 0.4 M KOH for 2 h at 37 C. The alkaline-stable lipid fraction was separated in a Sep-Pak Plus silica cartridge (Waters, Milford, MA, USA) by eluting with chloroform/acetic acid (100/1, v/v) and chloroform/methanol (2/1, v/v).…”

“…19) The dimethyl disulfide adducts prepared from the O-acetylated 2-hydroxy fatty acid methyl esters were analyzed by a GC-2010 instrument (Shimadzu Scientific). Each analyte was fractionated in an RTX-1 column (100% dimethyl polysiloxane; 0.25-mm internal diameter, 0.25-mm film thickness, 30-m long; Restek), the temperature of the injection port and transfer line being maintained at 250 C. The column temperature was programmed from 100 C (2-min hold) to 280 C, with a 10 C per min temperature gradient, and was maintained at 280 C for another 45 min.…”