Quantification of diacylglycerol molecular species in biological samples by electrospray ionization mass spectrometry after one-step derivatization.

Diacylglycerols (DAGs) are important lipid intermediates in cellular trafficking and signaling. Their concentrations are altered in diabetes, cancer, and other disease states. Quantification of DAGs in biological samples may provide critical information to uncover molecular mechanisms leading to various cellular functional disorders. Recent advances in lipidomics using mass spectrometry have greatly accelerated global lipid analysis and quantification. Quantification of DAGs by electrospray mass spectrometry (ESI/MS), however, is challenged by the absence of a permanent charge on the molecule, its low proton affinity and acidity, and its low abundance under normal biological conditions. We describe here the introduction of a quaternary ammonium cation to DAG molecules, using N-chlorobetainyl chloride, to afford a derivatized DAG that gives 2 orders of magnitude higher signal intensities than their underivatized sodium adducts. A linear calibration curve in which peak intensity ratios are plotted versus molar ratios can be achieved by using ESI/MS with dilauroyl glycerol as the internal standard. Employing this new approach to this analyte, we found a 9-fold increase of total DAGs in the livers of obese db/db mice as compared to their heterozygous lean controls. This proven strategy can be used to detect and quantify DAG molecular species from biological samples using ESI/MS after one-step derivatization.

Liquid chromatography with particle-beam electron-impact mass spectrometry of diacylglycerol nicotinates.

Several synthetic diacylglycerols and natural mixtures derived from soybean, egg yolk, and bovine liver phosphatidylcholines were examined in the form of nicotinate derivatives by liquid chromatography with particle-beam electron-impact mass spectrometry. High-performance liquid chromatography was carried out in the reversed-phase mode with a base-deactivated octyl-/octadecylsilyl stationary phase. The nature (size and degree of unsaturation) of the acyl residues was readily determined from the mass spectra. Uniquely, the positions of the double bonds could be deduced, although this became increasingly difficult as the degree of unsaturation of diacylglycerols containing mixed acyl residues increased. Reverse 1,2-diacylglycerol isomers could be distinguished by their mass spectra.

Quantification of diacylglycerols by capillary gas chromatography-negative ion chemical ionization-mass spectrometry.

We describe a method for quantifying diacylglycerols as their 1-pentafluorobenzoyl-2-acyl-3-acetyl-glycerol derivatives by capillary gas chromatography-negative ion chemical ionization-mass spectrometry. The basis of the method resides in the sequential treatment of diacylglycerols with acetic anhydride, pancreatic lipase, and pentafluorobenzoyl chloride. Cultured rat mesenteric artery vascular smooth muscle cells (VSMC) were incubated for 20 min in the presence of vehicle or vasopressin (10(-7) M). The incubations were stopped by aspirating the medium and adding 2 ml of methanol containing 790 pmol of internal standard 1-stearoyl-2-(10,13)-nonadecadienoyl- glycerol. After extraction, diacylglycerols were isolated by thin-layer chromatography, acetylated, and treated with pancreatic lipase. The resulting 2-acyl-3-acetylglycerols were then purified by thin-layer chromatography, transformed into their 1-pentafluorobenzoyl-derivatives, and monitored by capillary gas chromatography-negative ion chemical ionization-mass spectrometry on the selected ion-monitoring mode (m/z 614 and 604 for 2-arachidonoyl and 2-nonadecadienoyl species, respectively). The levels of diacylglycerols bearing an arachidonoyl moiety were 128 +/- 26 pmol/100 nmol lipid phosphorus in resting cells and 333 +/- 28 in stimulated cells (mean +/- SD, n = 3, P < 0.01). The presence of diacylglycerol species bearing an oleoyl or a linoleoyl group at the second position could also be detected in VSMC preparations by this approach. This new method can be applied to quantitate various diacylglycerol species bearing distinct acyl moieties at the second position of the glycerol molecule.