Sensitive analysis of fatty acid esters of hydroxy fatty acids in biological lipid extracts by shotgun lipidomics after one-step derivatization

Abstract

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are an important family of endogenous lipids, possessing antidiabetic and anti-inflammatory functions. Therefore, analysis of FAHFAs in biological samples obtained under healthy and disease states can uncover underlying mechanisms of various relevant disorders (e.g., diabetes and autoimmune diseases). Up to now, due to their extremely low abundance, the determination of the changed levels of these species is still a huge challenge, even though great efforts have been made by utilizing liquid chromatography-tandem mass spectrometry with or without derivatization. Herein, we described a novel method for analysis of FAHFAs present in lipid extracts of biological examples after solid-phase extraction and chemical derivatization with one authentic FAHFA specie as an internal standard based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. The approach possessed marked sensitivity, high specificity, and broad linear dynamic range of over 3 orders without obvious matrix effects. Moreover, after chemical derivatization, the molecular masses of FAHFAs shift from an overlapped region with ceramide species to a new region without overlaps, removing these contaminating signals from ceramides, and thereby reducing the false results of FAHFAs. Finally, this novel method was successfully applied for determining FAHFAs levels in varieties of representative biological samples, including plasma from lean and overweight/obese individuals of normoglycemia, and tissue samples (such as liver and white adipose tissue from diabetic (db/db) mice). We revealed significant alterations of FAHFAs in samples under patho(physio)logical conditions compared to their respective controls. Taken together, the developed method could greatly contribute to studying altered FAHFA levels under a variety of

Analytical Methods for the Determination of Fatty Acid Esters of Hydroxy Fatty Acids (FAHFAs) in Biological Samples, Plants and Foods

“…Two years later, the same group described a faster protocol for the endogenous FAHFA measurements [17], employing positive pressure (nitrogen) to push solvents though a Strata SI-1 silica SPE cartridge (500 mg silica, 3 mL, Phenomenex, Torrance, CA, USA). By this modification, they shortened the SPE step time from 4 h to 1 h. Other methods described the use of a Hysphere C8 catridge [18], a HyperSep silica SPE column (500 mg, 6 mL, Thermo Scientific, Waltham, MA, USA) [15,[19][20][21], or a strong anion exchange (SAX) SPE-cartridge (1 mL, 50 mg, Weltech, Wuhan, China) [22][23][24].…”

“…This derivatization approach has been used by the same group in their more recent studies [23,24]. Most recently, Han and co-workers reported the sensitive analysis of FAHFAs in biological lipid extracts by shotgun lipidomics after one-step derivatization with N-(4-aminomethylphenyl) pyridinium (AMPP) [21]. AMPP is one of the most representative derivatization reagents used for charge reversal of carboxylic acids [26].…”

“…In positive ionization mode, the most abundant fragment ions in the product-ion MS spectrum of AMPP-derivatized FAHFAs were [(FA2-2H) + AMPP] + ions generated from the neutral losses of side aliphatic chains ( Figure 6B). Most recently, Han and co-workers reported the sensitive analysis of FAHFAs in biological lipid extracts by shotgun lipidomics after one-step derivatization with N-(4-aminomethylphenyl) pyridinium (AMPP) [21]. AMPP is one of the most representative derivatization reagents used for charge reversal of carboxylic acids [26].…”

“…FAHFA spectra are recorded in ESI negative mode, since they contain a free carboxyl group. Positive mode is employed when a derivatization step is performed before the analysis [21][22][23][24]. Triple quadrupole (tandem) mass spectrometers have been mainly employed in FAHFA analysis (Table 1).…”

Chemical Labeling Assisted Detection and Identification of Short Chain Fatty Acid Esters of Hydroxy Fatty Acid in Rat Colon and Cecum Contents

“…However, FAHFAs are low abundant and these techniques are limited by the low detection sensitivity and poor isomer separation. An effort to overcome these problems by chemical labeling experiments using n-[4-(aminomethyl) phenylpyridinium] (AMPP) [ 15 ], 2-dimethylaminoethylamine (DMED) [ 10 , 16 ] were established. In particular, DMED labeling of carboxyl moiety of FAHFAs was found to be advantageous because of enhanced detection sensitivities from 7–72 folds [ 10 ].…”