Application of tandem mass spectrometry for the analysis of long-chain carboxylic acids

The application of MS-MS for the analysis of long-chain carboxylic acids and their esters has proved enormously successful but expensive. It is discussed mainly on basis of results obtained with different instruments with lesser attention to principles of the method, which have been adequately reviewed elsewhere. The use of electrospray ionization (ESI) has greatly increased the sensitivity of the method and has permitted assay of total lipid extracts. The combination of HPLC with electrospray and single quadrupole mass spectrometry, LC-ESI-CID-MS, rivals the triple quadrupole MS-MS application in many instances at considerably lower cost. However, LC-ESI-MS-MS remains the most desirable system at the present time for lipid ester analyses.     

Analysis of diastereomeric DAG naphthylethylurethanes by normal-phase HPLC with on-line electrospray MS

Normal-phase HPLC resolution of sn-1,2(2,3)- and x-1,3-DAG generated by partial Grignard degradation from natural TAG was carried out with both (R)-(−) and (S)-(+)-1-(1-naphthyl)ethylurethane derivatives. The diastereomeric sn-1,2- and sn-2,3-DAG derivatives were resolved using two Supelcosil LC-Si (5 μm, 25 cm × 4.6 mm i.d.) columns in series and an isocratic elution with 0.37% isopropanol in hexane at a flow rate of 0.7 mL/min. The DAG were detected by UV absorption at 280 nm and were identified by electrospray ionization MS in the positive ion mode following postcolumn addition of chloroform/methanol/30% ammonium hydroxide (75∶24.5∶0.5, by vol) at 0.6 mL/min. Application of the method to a stereospecific analysis of the molecular species of TAG of rat VLDL showed that the TAG composition of VLDL circulating under basal conditions differs markedly from that of VLDL secreted by the liver during inhibition of serum lipases. The inhibition of serum lipases resulted in a significant proportional decrease in 16∶0 and PUFA and an increase in 18∶0 and oligoenoic FA in the sn-1-position, whereas the FA compositions in the sn-2- and sn-3-positions were much less affected.     

Comparative study of the molecular species of chloropropanediol diesters and triacylglycerols in milk fat

In an effort to establish the origin of the fatty acid esters of 3-chloropropanediol, which recently have been isolated in small amounts from goat milk, we compared the molecular species composition of the chlorohydrin diesters and of goat milk triacylglycerols. The chloropropanediol diesters were found to be composed of molecular species containing C₁₀−C₁₈ fatty acids and corresponded closely in carbon number to those calculated for the long chain sn-1,2-diacyl-glycerol moieties of goat milk triacylglycerols. The molecular species of goat milk total triacylglycerols contained C₄−C₁₈ fatty acids. It is suggested that triacylglycerols and chloropropanediol diesters are derived from the same pool of long chain fatty acids. A molecular distillate of bovine milk fat did not contain chloropropanediol diesters, while the available samples of human milk fat were shown to contain alkyldiacylglycerols as the major components of a neutral lipid fraction corresponding in polarity to the chloropropanediol diesters.     

Lipid methodology — Chromatography and beyond. Part I. GC/MS and LC/MS of glycerolipids

This paper reviews recent examples of the application of combined high temperature gas-liquid chromatography (GLC) and reversed phase high pressure liquid chromatography (HPLC) with electron impact and chemical ionization mass spectrometry for structural studies of natural diacyl and triacylglycerols. It was concluded that the combination of reversed phase HPLC with direct liquid inlet chemical ionization mass spectrometry provides the most complete resolution and most reliable identification of natural acylglycerols, far exceeding the capabilities of either technique alone. The LC/MS method is suitable for quantitative analysis following appropriate calibration of the total or fragment ion response.     

Preparation of schiff base adducts of phosphatidylcholine core aldehydes and aminophospholipids, amino acids, and myoglobin

We have prepared Schiff base adducts of the core aldehydes of phosphatidylcholine and aminophospholipids, free amino acids, and myoglobin. The Schiff bases of the ethanolamine and serine glycerophospholipids were obtained by reacting sn-1-palmitoyl(stearoyl)-2-[9-oxo]nonanoyl-glycerophosphocholine (PC-Ald) with a twofold excess of the aminophospholipid in chloroform/methanol 2∶1 (vol/vol) for 18 h at room temperature. The Schiff bases of the amino acids and myoglobin were obtained by reacting the aldehyde with an excess of isoleucine, valine, lysine, methyl ester lysine and myoglobin in aqueous methanol for 18 h at room temperature. Prior to isolation, the Schiff bases were reduced with sodium cyanoborohydride in methanol for 30 min at 4°C. The reaction products were characterized by normal-phase high-performance liquid chromatography and on-line mass spectrometry with electrospray ionization. The amino acids and aminophospholipids yielded single adducts. A double adduct was obtained for myoglobin, which theoretically could have accepted up to 23 PC-Ald groups. The yields of the products ranged from 12 to 44% for the aminophospholipids and from 15–57% for the amino acids, while the Schiff base of the myoglobin was estimated at 5% level. The new compounds are used as reference standards for the detection of high molecular weight Schiff bases in lipid extracts of natural products. Based on presentation at the AOCS Annual Meeting & Expo in Indianapolis, Indiana, April 28–May 1, 1996.     

Release of chylomicrons by isolated cells of rat intestinal mucosa

Fat-laden mucosal cells were isolated by flotation from fed male rats after digesting scrapings of washed jejunum with collagenase in bicarbonate buffer. About 50–60 million cells were obtained per preparation, which were 95–100% viable as assessed by Trypan Blue. The isolated cells were capable of effective incorporation of labeled fatty acids and glucose into triglycerides and phospholipids, and of labeled leucine and glucosamine into the protein envelope of the released chylomicrons. The secretion of the labeled protein paralleled the release of the labeled fat, both of which were linear with the concentration of the albumin in the incubation mixture. About 80% of the total fat of the cell was released as chylomicrons within 30 min when incubated in the presence of albumin-bicarbonate buffer. Injection of puromycin 24 hr prior to harvesting of cells led to a complete inhibition of chylomicron release. Addition of puromycin to the incubation medium gave 50–80% inhibition of release. No inhibition of release of chylomicrons resulted from a treatment with ethionine. The released chylomicrons were separated from the cells by Millipore filtration. Presented in part at the AOCS Meeting, Chicago, September 1970.     

Effect of temperature on the stereoselectivity of phospholipase D toward glycerol in the transphosphatidylation of phosphatidylcholine to phosphatidylglycerol

In this study, the effect of temperature on the stereoselectivity of phospholipase D (PLD) toward the two primary hydroxyl groups of glycerol in the transphosphatidylation reaction of phosphatidylcholine to phosphatidylglycerol (PtdGro) was investigated. For this purpose, PLD from bacteria (Streptomyces septatus TH-2, S. halstedii subsp. scabies K6, and Actinomadura sp.) and cabbage were tested. At the reaction temperatures employed (0–60°C), the proportions of the two PtdGro diastereomers, namely, 1,2-dioleoyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration) and 1,2-dioleoyl-sn-glycero-3-phosphol-1′-sn-glycerol (R,S configuration), which were produced with PLD from Streptomyces TH-2 and Actinomadura sp., changed gradually from 50% R,R and 50% R,S at 50–60°C to 70% R,R and 30% R,S at O°C. These alterations suggested that the stereoselectivity of the bacterial PLD toward the two primary hydroxyl groups of prochiral glycerol was significantly influenced by reaction temperature. PLD from Streptomyces K6 showed relatively little effect of temperature on stereoselectivity, giving 65–69% R,R in the temperature range of 60–10°C examined. The plots of In ([R,R]/[R,S]) vs. 1/T gave good linear fits for these three bacterial PLD. No temperature effect was observed for cabbage PLD, which gave an almost equimolar mixture of the R,R and R,S diastereomers in the range from 0 to 40°C. The temperature-dependent change in enantiomeric selectivity of the bacterial PLD promises potentially profitable commercial exploitation.     

Phospholipids and oxophospholipids in atherosclerotic plaques at different stages of plaque development

We identified and quantified the hydroperoxides, hydroxides, epoxides, isoprostanes, and core aldehydes of the major phospholipids as the main components of the oxophospholipids (a total of 5–25 pmol/μmol phosphatidylcholine) in a comparative study of human atheroma from selected stages of lesion development. The developmental stages examined included fatty streak, fibrous plaque, necrotic core, and calcified tissue. The lipid analyses were performed by normal-phase HPLC with on-line electrospray MS using conventional total lipid extracts. There was great variability in the proportions of the various oxidation products and a lack of a general trend. Specifically, the early oxidation products (hydroperoxides and epoxides) of the glycerophosphocholines were found at the advanced stages of the plaques in nearly the same relative abundance as the more advanced oxidation products (core aldehydes and acids). The anticipated linear accumulation of the more stable oxidation products with progressive development of the atherosclerotic plaque was not apparent. It is therefore suggested that lipid infiltration and/or local peroxidation is a continuous process characterized by the formation and destruction of both early and advanced products of lipid oxidation at all times. The process of lipid deposition appears to have been subject to both enzymatic and chemical modification of the normal tissue lipids. Clearly, the appearance of new and disproportionate old lipid species excludes randomness in any accumulation of oxidized LDL lipids in atheroma.     

Simultaneous analysis of low plasma levels of deuterium-labeled saturated and unsaturated fatty acids ast-butyldimethylsilyl esters

A sensitive and accurate method for detection and quantitation of deuterated fatty acids in the presence of large amounts of unlabeled fatty acids is described using mass fragmentography in combination with the preparation of tertiarybutyldimethylsilyl esters (t-BDMS). The method has been applied to determination of deuterated stearic, oleic, elaidic and linoleic acids in human plasma lipoproteins following duodenal perfusion with a micellar mixture of acids. Over a concentration range of 10–1000 ng/ml, the average coefficient of variation for the linoleate was 3% and for the oleate (elaidate) ester was 2%.     

Liquid Chromatography–Light Scattering Detector–Mass Spectrometric Analysis of Digested Oxidized Rapeseed Oil

Rapeseed oil was oxidized chemically and thermally to produce two distinct oxidized oils. These oils, along with unoxidized oils, were subjected to an artificial digestion model to simulate the digestive processes in humans. Lipid digestion involves lipases that break down the intact triacylglycerol (TAG) molecules first to diacylglycerols, and eventually to sn-2-monoacylglycerols (MAG) and free fatty acids. A high performance liquid chromatography-evaporative light scattering detector-electrospray ionization-mass spectrometric (HPLC–ELSD–ESI–MS) method was developed to monitor the lipolysis and the presence of oxidized lipids. The HPLC–ELSD–ESI–MS analysis enabled the separation and detection of nearly all the lipid species present in the sample after TAG hydrolysis. The HPLC–MS analyses of digestion products revealed that oxidized triacylglycerols are hydrolyzed by the digestive enzymes in a manner similar to that of native, unoxidized molecules. Significant amounts of sn-1(3)-MAG were found in all the samples after lipolysis, however, more of these were found in unoxidized rapeseed oil samples than in the oxidized oils. Several oxidized molecules were identified with the aid of synthesized oxylipids. This novel method is scalable to small-scale preparative fractionation of oxidized lipid molecules from a complex digestion sample. Also, the fingerprint-like, diagnostic, MS profiles of oxidized oils, reference compounds, and digestion products may be a great aid in comprehensive analysis of lipid oxidation and lipolysis.