Gas chromatography/tandem mass spectrometry as an analytical tool for the identification of fatty acids

Structures of fatty acids present at very low quantities in mycobacteria are difficult to determine. A commonly used strategy is to introduce heteroatoms into functional groups by chemical means before subjecting them to gas chromatography/tandem mass spectrometry (GC/MS/MS) analysis. Routinely used methods give very low abundance diagnostic ions leading to ambiguities in structural conclusions. GC/MS/MS associated with electron capture ionization of pentafluorobenzyl esters was used to study very complex mixtures of fatty acids fromMycobacterium fallax andM. aurum. The charge-remote fragmentation of fatty acid carboxylate anions was used for structure determination at the nanogram level of a large number of unsaturated, branched, and cyclopropane-containing fatty acids. Some of them have not been observed previously in these Mycobacteria. On the basis of these studies, biosynthetic pathways of unsaturated, branched, and cyclopropane-containing fatty acid are proposed.     

Sensitivity of positive ion mode electrospray ionization mass spectrometry (ESI MS) in the analysis of purine bases in ESI MS and on-line electrochemistry ESI MS (EC/ESI MS)

A cone-shaped MS inlet and on-line electrochemistry (EC) were used to enhance the ionization efficiency in electrospray ionization mass spectrometry (ESI MS) of purine bases. A pathway of positive ion mode ESI may involve oxidation of purine bases, guanine, adenine, xanthine and hypoxanthine, by 1e⁻, 1H⁺ processes. The electrospray process generates dimers of purine bases that are detected in ESI MS as protonated ions, except for xanthine, for which a protonated radical dimer is detected. Thus electrochemical oxidation of purine bases during ESI may generate reactive radicals that can subsequently dimerize. Dimer formation is facilitated in ESI MS when the carrier solution pH is high. The positive ion mode ESI MS ionization is consistent with the reactivity of the bases toward oxidation. Furthermore, the formation of the protonated ions, and Na⁺ and K⁺ adducts of the bases, expected in positive ion ESI MS, are observed. In addition, unusual H-bonding of purine bases guanine and xanthine is confirmed by ESI MS. Application of low EC voltage to the on-line EC cell in EC/ESI MS improves the sensitivity and correlates with the decrease of the intensity of the dimers, possibly as a result of their further oxidation.     

Characterization of N-acylphosphatidylethanolamine and acylphosphatidylglycerol in oats

Two polar lipid classes, both with three acyl groups, were isolated from an extract of oats and characterized by nuclear magnetic resonance spectroscopy, electrospray mass spectrometry (MS), and electron ionization MS (EIMS). Distortionless enhancement by polarization transfer (DEPT) and the two-dimensional correlation experiments 1H-detected heteronuclear multiple quantum coherence spectroscopy, heteronuclear multiple bond correlation spectroscopy, double quantum filtered correlation spectroscopy, and total correlation spectroscopy provided sufficient information for determination of the structure of the two lipid classes. The polar lipid classes were found to be N-acylphosphatidylethanolamine [1,2-diacyl-sn-glycero-3-phospho-(N-acyl)-1'-ethanolamine; N-acyl-PE] and acylphosphatidylglycerol [1,2-diacyl-sn-glycero-3-phospho-(3'-acyl)-1'-sn-glycerol]. High-performance liquid chromatography with electrospray ionization MS (HPLC-ESMS) and with electrospray ionization tandem MS (HPLC-MS/MS) were utilized for the separation and subsequent determination of molecular species. With HPLC-ESMS, ions of deprotonated molecules were obtained and with HPLC-MS/MS carboxylate ions (representing acyl groups) were obtained as well as other structurally significant ions. Fifty molecular species of N-acylphosphatidylethanolamine and 24 molecular species of acylphosphatidylglycerol were found, with a molecular mass range of 924-1032 Da and 959-1035 Da, respectively. Identification of the fatty acid isomers, as picolinyl ester derivatives, was done with gas chromatography with EIMS. Three isomers of 16:1 fatty acids were found in N-acyl-PE, and their double bond positions were determined to 6, 9, and 11 with a relative abundance of 4:10:1.     

Characterization of castor bean neutral lipids by mass spectrometry/mass spectrometry

A method has been developed for the characterization of intact neutral lipids isolated from castor bean (Ricinus communis L.) by mass spectrometry/mass spectrometry (MS/MS). The molecular weights of the trimethylsilyl (TMS) derivatives of the neutral lipids are determined by using both electron impact and chemical ionization (ammonia). Collision-induced dissociation daughter spectra of the (M-CH₃)⁺ ions yield fragment ions that allow easy determination of the acyloxy groups present. The chainlength and degree of unsaturation for each acyloxy group are indicated by R in the ion represented by the general formula (RCO + 74)⁺. Other ions of diagnostic value include (M-RCOO)⁺, (M-RCOOH)⁺, [(M-CH₃-RCOOH]⁺ and [(M-RCOOH)-16]⁺. The presence of a TMS group in any of these fragments results in the formation of ions representing the loss of OTMSH. Prior to MS/MS analysis, partial fractionation by high-performance liquid chromatography (according to degree of unsaturation in the neutral lipids) is useful because daughter spectra are generated free of any isotopic contamination, and minor components are concentrated in single fractions, which aids their characterization. By using this method, 11 neutral lipids were characterized in castor bean.     

Fast GC—MS in Supersonic Molecular Beams

The features of supersonic molecular beams (SMB) are used to improve gas chromatography—mass spectrometry (GC–MS) performance and establish a new method of fast GC–MS. In SMB, the sample compounds are vibrationally cooled such that their electron impact mass spectra are characterized by enhanced M⁺ peaks, together with library-searchable fragments. A new ionization method, hyperthermal surface ionization (HSI), provides ultrasensitive ionization coupled with a tunable degree of selectivity for nitrogen-containing drugs. SMB enables the use of very high carrier gas flow rates which, when a short megabore column is used, results in ultra-fast GC–MS having conventional chromatographic peak widths. Thus, fast GC–MS in SMB can be performed with conventional quadrupole analyzers. The slightly reduced GC resolution can, in many cases, be compensated for by the selectivity of hyperthermal surface ionization or by the increased EI selectivity through enhanced M⁺. “Fast”, “very-fast”, and “ultra-fast” GC–MS are defined and demonstrated with drugs, and the ability to analyze underivatized steroids is shown. Practical examples are shown including ultra-fast GC–MS of lidocaine in human plasma extract achieved in a few seconds and screening of other drugs, without any sample preparation or extraction, achieved in less than three minutes. We conclude that GC–SMB–MS exhibits a combination of faster analysis with improved sensitivity and selectivity, a wider range of molecules amenable to GC–MS, improved MS information, and higher a degree of flexibility.     

Isoprenoid Alcohols are Susceptible to Oxidation with Singlet Oxygen and Hydroxyl Radicals

Isoprenoids, as common constituents of all living cells, are exposed to oxidative agents—reactive oxygen species, for example, singlet oxygen or hydroxyl radicals. Despite this fact, products of oxidation of polyisoprenoids have never been characterized. In this study, chemical oxidation of isoprenoid alcohols (Prenol‐2 and ‐10) was performed using singlet oxygen (generated in the presence of hydrogen peroxide/molybdate or upon photochemical reaction in the presence of porphyrin), oxygen (formed upon hydrogen peroxide dismutation) or hydroxyl radical (generated by the hydrogen peroxide/sonication, UV/titanium dioxide or UV/hydrogen peroxide) systems. The structure of the obtained products, hydroxy‐, peroxy‐ and heterocyclic derivatives, was studied with the aid of mass spectrometry (MS) and nuclear magnetic resonance (NMR) methods. Furthermore, mass spectrometry with electrospray ionization appeared to be a useful analytical tool to detect the products of oxidation of isoprenoids (ESI–MS analysis), as well as to establish their structure on the basis of the fragmentation spectra of selected ions (ESI–MS/MS analysis). Taken together, susceptibility of polyisoprenoid alcohols to various oxidizing agents was shown for the first time.     

ELECTROSPRAY AS AN IONISATION METHOD FOR MASS SPECTROMETRY

One of the methods by which large and non-volatile molecules, especially those of biochemical interest, can be made amenable to mass spectrometry (MS) is electrospray (ES) ionisation. In ES MS the technique of electrohydrodynamic spraying of liquids is used to produce gas-phase ions from sample molecules present in a diluted solution, which are subsequently transferred into a mass analyser. The special conditions required for effective ion formation (voltages, flow rates, solution parameters such as conductivity, sample concentration, solvent composition) are described, as well as the instrumentation used to transfer and mass-analyse the gas-phase ions. Several variants of the ES MS technique are presented which have been developed to meet certain analytical requirements (such as spraying of highly conductive or aqueous solutions, coupling to separation techniques as liquid chromatography or capillary zone electrophoresis, or the analysis of very small sample quantities). The mechanisms of liquid charging, aerosol formation and ion release from the charged droplets (as far as they are understood) are briefly discussed, and the question is addressed in how far the ions observed in the ES mass spectrum reflect the state of the sample molecules in solution. Some examples of applications from the field of peptide/protein analysis are given, covering molecular weight determination, sequence and spatial structure determination, and studies on non-covalent molecular interactions. Finally, a short comment is made on the limitations of ES MS and, from a practitoner’s point of view, which problems need still to be solved.     

中药黄芪中异黄酮苷类化合物的高效液相色谱-串联质谱分析

利用高效液相色谱-大气压化学电离质谱联用技术,建立了黄芪提取物中的有效成分毛蕊异黄酮-7-O-β-D-葡萄糖苷和芒柄花素-7-O-β-D-葡萄糖苷的定性分析方法。这两个化合物在大气压化学电离质谱条件下,质谱裂解行为的研究表明,二者具有相似的特征质谱裂解行为,即在正离子模式下的一级质谱中均产生强的准分子离子[M+H]+峰和苷元离子[M+H-G lu]+峰;且苷元离子经碰撞诱导解离均产生.CH3(15 Da)、CH3OH(32Da)和2CO(56 Da)的中性丢失;同时发生Retro-D iels A lder(RDA)裂解反应,导致在毛蕊异黄酮-7-O-β-D-葡萄糖苷和芒柄花素-7-O-β-D-葡萄糖苷的二级质谱图中分别产生相对分子质量为m/Z148和m/Z133的特征碎片离子峰。这些特征碎片可以作为这两个化合物定性的依据。该方法已成功应用于黄芪注射液中的这两种物质的定性分析。     

Rapid complexing of oxoacylglycerols with amino acids peptides and aminophospholipids

We prepared model Schiff bases from 2-[9-oxo]nonanoyl glycerol (2-MAG-ALD) and various amino compounds. 2-MAG-ALD was obtained by pancreatic lipase hydrolysis of trioleoyl glycerol and reductive ozonolysis of the resulting 2-monooleoyl glycerol. The reaction products were purified by thin-layer chromatography. Schiff bases were synthesized in greater than 50% yield by reacting 2-MAG-ALD with twofold molar excess of valine, Nα-acetyl-l-lysine methyl ester and the tripeptides glycyl-glycyl-glycine, glycyl-glycyl-histidine, and glycyl-histidyl-lysine in aqueous methanol and with 1-palmitoyl-2-stearoyl glycerophosphoethanolamine (PE) in chloroform.methanol for 16 h at room temperature. Prior to analysis the bases were reduced with sodium cyanoborohydride in methanol for 30 min at 4°C. Reaction products were analyzed by high-performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC/ESI/MS). Reduced Schiff bases of 2-MAG-ALD with PF and amino acids were analyzed by normal-phase HPLC/ESI/MS and those with peptides by reversed-phase HPLC/ESI/MS. Single adducts were obtained in all cases and both the α-amino group of valine and the ε-amino group of Nα-acetyl-l-lysine methyl ester were reactive. Molecular ions of reaction products were the only detected ions in the negative ionization mode, whereas in the positive ion mode sodiated molecular ions were also detected. The present study suggests that 2-MAG-ALD may form Schiff base adducts with amino compounds in other aqueous media, such as the intestinal lumen and in the hydrophobic environment of cell membranes.     

Direct Analysis in Real Time (DART) of an Organothiophosphate at Ultrahigh Resolution by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry

Direct analysis in real time (DART) is a recently developed ambient ionization technique for mass spectrometry to enable rapid and sensitive analyses with little or no sample preparation. After swab-based field sampling, the organothiophosphate malathion was analyzed using DART-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Mass resolution was documented to be over 800,000 in full-scan MS mode and over 1,000,000 for an MS/MS product ion produced by collision-induced dissociation of the protonated analyte. Mass measurement accuracy below 1 ppm was obtained for all DART-generated ions that belonged to the test compound in the mass spectra acquired using only external mass calibration. This high mass measurement accuracy, achievable at present only through FTMS, was required for unequivocal identification of the corresponding molecular formulae.     

Rapid characterization of lipids by MALDI MS. Part 2: Artifacts,ion suppression,and TLC MALDI imaging

Several new methods have been developed recently that allow the direct detection of lipids without resorting to derivatization or chromatographic separation. The simplest of these is direct MALDI (matrix‐assisted laser desorption/ionization) mass spectrometry. This approach is most useful for mixtures that contain minimal amounts of ion‐suppressing interfering components. However, when such components are present, their effects can often be minimized by using simple separation techniques beforehand, such as solid phase extraction or thin layer chromatography. For example, direct MALDI has been used for rapid screening of lipids and taxonomic identification of the source organisms with no sample pretreatment. Collecting fractions from solid phase extraction cartridges have also been used to avoid the most extreme effects of ion suppression from more complex lipid mixtures. More recently, direct MALDI has been applied to the analysis of TLC plates allowing the detection of TLC‐separated lipids from the complex lipidome. Herein, we briefly describe the application of rapid MALDI MS to some typical research problems involving the characterization of lipids. In Part 1 [1] we covered bacterial taxonomy by direct analysis of intact lipids and the analysis of food oil triacylglycerols . Part 2 will address ion suppression, spontaneous fragmentation, and coupling MALDI with chromatography. The spontaneous fragmentation of protonated lipids in oils by direct MALDI produces artifactual diacylglycerol‐like ions. An understanding of this process and its minimization facilitates monitoring the decomposition of lipids by direct analysis. Suppression also has an impact on direct analysis of lipids, especially when mixtures contain both polar and non‐polar lipids. We demonstrate the use of solid phase extraction and thin layer chromatography to produce fractions or substrates from complex biological samples in which lipids can be detected by direct and rapid MALDI MS analysis.     

Capillary supercritical fluid chromatography-atmospheric pressure chemical ionization mass spectrometry of triacylglycerols in berry oils

A capillary supercritical fluid chromatograph (SFC), combined with a triple-quadrupole mass spectrometer (MS) via a liquid chromatography-atmospheric pressure chemical ionization (LC-APCI) interface, was utilized in the analysis of berry oil triacylglycerols. No modification of the commercially available interface was required. Vapor of different solvents, such as methanol, isopropanol, water, or ammonium hydroxide in methanol, was introduced in the sheath gas flow in the APCI source to achieve adequate ionization of triacylglycerols. The separation of triacylglycerols according to acyl carbon number and degree of unsaturation was accomplished on a 20 m × 50 μm i.d. SB-Cyanopropyl-25 column. The resolution of triacylglycerols in the reconstructed ion chromatogram and the sensitivity of the SFC-(APCI)MS system was comparable to or slightly better than that obtained with a flame ionization detector. No baseline drifting was observed during the SFC density programming. Triacylglycerols formed diagnostic [M + H]⁺ and [M - RCOO]⁺ ions with all tested reactant ion solvents except with ammonium hydroxide in methanol, which formed abundant [M + 18]⁺ ions instead of [M + H]⁺ ions. The abundance of the [M + H]⁺ ion increased with increasing degree of unsaturation of a triacylglycerol, whereas the abundance of the [M - RCOO]⁺ ion depended on the regiospecific distribution of the fatty acid moiety between the sn-1/3 positions and the sn-2 position and on the number of double bonds.     

Confirmation of identity of aflatoxins

Aflatoxins are detected and determined by analytical procedures based on physical and chemical properties, e.g., ultraviolet absorbance, fluorescence, solubility and Chromatographie retention times during thin layer (TLC) or liquid chromatography. For acceptance of analytical results based on these properties, especially for regulatory purposes, proof of identity of the compound being measured is essential. Numerous tests have been devised for confirmation of identity. Included are tests based on toxicological effects observed in the duckling, zebrafish, chick embryo,Bacillus megaterium and many other species; chemical tests based on formation of derivatives such as the acetates and water adducts; and tests based on color changes of TLC spots after contact with spray reagents, e.g., sulfuric acid. All of the foregoing have inherent uncertainties in interpretation of identity. On the other hand, mass spectrometry (MS) is one of the most specific methods of identification available; however, it has been difficult to apply at the low concentrations at which aflatoxins are routinely detected. In this paper, the confirmation techniques for aflatoxins are placed in historical perspective and are reviewed and evaluated A recently developed procedure for the application of negative ionization MS for the confirmation of identity of aflatoxins in foods or feeds at concentrations as low as 10 ng/g is described. This procedure consists of isolation of the aflatoxin by Association of Official Analytical Chemists’ methods, purification by preparatory 2-dimensional TLC, in situ elution of the aflatoxin TLC spot and analysis of the sample by negative ion chemical ionization MS using a direct insertion probe.     

Analysis of solvent dyes in refined petroleum products by electrospray ionization mass spectrometry

Solvent dyes are used to color refined petroleum products to enable differentiation between gasoline, diesel, and jet fuels. Analysis for these dyes in the hydrocarbon product is difficult due to their very low concentrations in such a complex matrix. Flow injection analysis/electrospray ionization/mass spectrometry in both negative and positive mode was used to optimize ionization of ten typical solvent dyes. Samples of hydrocarbon product were analyzed under similar conditions. Positive electrospray ionization produced very complex spectra, which were not suitably specific for targeting only the dyes. Negative electrospray ionization produced simple spectra because aliphatic and aromatic moieties were not ionized. This enabled screening for a target dye in samples of hydrocarbon product from a spill.     

Analysis of the polyethylene glycol glucosides and FA esters thereof by atmospheric-pressure ionization MS

Polyethylene glycol (PEG) glucosides (PEGG) and the PEGG esters of lauric acid were analyzed by atmospheric-pressure ionization MS (API-MS) with electrospray ionization. Straightforward mass characterization of the complex mixtures could be achieved without prior chromatographic separation. The constituents were identified on the basis of quasi-molecular ions. Individual components could be observed as protonated molecular ions [M+H]⁺ and/or as their NH₄ ⁺, Na⁺, or K⁺ adducts in positive ion mode. The mass spectrometric investigation showed that mixtures of PEGG consisted of monoglucoside, diglucoside, polyglucoside, and free PEG. The esterification product is a mixture of two types of nonionic surfactants: PEG-laurates and PEGG-laurates. The reasons for distortion of the quasi-molecular ion intensities and the stabilization of adduct ions were discussed. The rapid and highly sensitive API-MS analysis technique proposed here is well suited for direct characterization of complex mixtures and suitable for development as a routine analytical method.     

Rapid characterization of lipids by MALDI MS. Part 1: Bacterial taxonomy and analysis of food oils

Several new methods have been developed recently that allow the direct detection of lipids without resorting to derivatization or chromatographic separation. The simplest of these is direct MALDI (matrix‐assisted laser desorption/ionization) mass spectrometry. This approach is most useful for mixtures that contain minimal amounts of ion‐suppressing interfering components. However, even when such components are present, their effects can often be minimized by using simple separation techniques beforehand, such as solid phase extraction or thin layer chromatography. For example, direct MALDI has been used for rapid screening of lipids and for taxonomic identification of the source organisms with no sample pretreatment. Fractions collected from solid phase extraction cartridges have also been used to avoid the most extreme effects of ion suppression from more complex lipid mixtures. More recently, direct MALDI has been applied to the analysis of TLC plates allowing the detection of TLC‐separated lipids from the complex lipidome. Herein, we briefly describe the application of rapid MALDI MS to some typical research problems involving the characterization of lipids. In Part 1 these include bacterial taxonomy by direct analysis of intact lipids in simple extracts rather than by conversion to fatty acid methyl esters. Food oils such as triacylglycerols can be characterized simply and easily by direct MALDI MS without resort to any sort of separation. Part 2 (in the next issue of Lipid Technology) will cover the spontaneous fragmentation of protonated lipids, ion suppression and the use of solid phase extraction and thin layer chromatography with MALDI MS to characterize complex biological samples.     

Using MALDI MS for rapid analysis of food lipids

Matrix‐assisted laser desorption/ionization (MALDI) is an ionization method that produces intact lipid molecule species that can be separated and characterized by mass spectrometry. Recent research has concentrated on using rapid MALDI MS lipid analysis that employs little sample cleanup, simple sample preparation, no chemical derivatization, and minimal chromatography. This article takes a look at selected MALDI MS applications for edible oils and lipid rich foods, such as meat and eggs, and explores potential uses in food and lipid science.     

Electrospray Ionization Tandem Mass Spectrometry of Sodiated Adducts of Cholesteryl Esters

Cholesteryl esters (CE) are important lipid storage molecules. The present study demonstrates that sodiated adducts of CE molecular species form positive ions that can be detected in both survey scan mode as well as by exploiting class-specific fragmentation in MS/MS scan modes. A common neutral loss for CE is the loss of cholestane (NL 368.5), which can be used to specifically quantify tissue CE molecular species. Using this MS/MS technique, CE molecular species were quantified in mouse monocyte-derived macrophages (J774 cells) incubated with either linoleic (18:2) or arachidonic acid (20:4). These studies revealed that arachidonic acid was not only incorporated into the CE pool, but also was elongated resulting in the accumulation of 22:4 and 24:4 CE molecular species in macrophages. Additionally, this technique was used to quantify CE molecular species present in crude lipid extracts from plasma of female mice fed a Western diet, which led to an enrichment in CE molecular species containing monounsaturated fatty acids compared to female mice fed a normal chow diet. Last, NL 368.5 spectra revealed the oxidation of the aliphatic fatty acid residues of CE molecular species containing polyunsaturated fatty acids. Taken together, these studies demonstrate the utility of using sodiated adducts of CE in conjunction with direct infusion electrospray ionization tandem mass spectrometry to rapidly quantify CE molecular species in biological samples.     

A Computational Drug Metabolite Detection Using the Stable Isotopic Mass-Shift Filtering with High Resolution Mass Spectrometry in Pioglitazone and Flurbiprofen

The identification of metabolites in drug discovery is important. At present, radioisotopes and mass spectrometry are both widely used. However, rapid and comprehensive identification is still laborious and difficult. In this study, we developed new analytical software and employed a stable isotope as a tool to identify drug metabolites using mass spectrometry. A deuterium-labeled compound and non-labeled compound were both metabolized in human liver microsomes and analyzed by liquid chromatography/time-of-flight mass spectrometry (LC-TOF-MS). We computationally aligned two different MS data sets and filtered ions having a specific mass-shift equal to masses of labeled isotopes between those data using our own software. For pioglitazone and flurbiprofen, eight and four metabolites, respectively, were identified with calculations of mass and formulas and chemical structural fragmentation analysis. With high resolution MS, the approach became more accurate. The approach detected two unexpected metabolites in pioglitazone, i.e., the hydroxypropanamide form and the aldehyde hydrolysis form, which other approaches such as metabolite-biotransformation list matching and mass defect filtering could not detect. We demonstrated that the approach using computational alignment and stable isotopic mass-shift filtering has the ability to identify drug metabolites and is useful in drug discovery.     

UPLC-ESI-MS/MS法快速测定烟草中几种潜香物质

使用超高效液相色谱-电喷雾串联四级杆质谱(UPLC-ESI-MS/MS)在多反应离子监测(MRM)模式下分离鉴定了烟草中7种潜香物质。烟样经甲醇提取,XAD-2柱分离纯化,选择中性丢失162扫描确定母离子,通过对母离子二级质谱扫描(MS2),并根据193、179、163、134.7、133等特征碎片离子峰分析结果确定了7种潜香型物质,分别为熊果苷、香豆酸-葡萄糖苷、香豆素奎尼酸、绿原酸、隐绿原酸、葡萄糖基阿魏酸、3-氧化紫罗兰醇葡萄糖苷。最后以苯酚-β-D-吡喃葡萄苷为内标,测定了烟叶不同部位的7种潜香型物质的相对含量。该方法快速、简便,适合于不同烟叶中潜香型物质的分析研究。