Hydrogenation of unsaturated fatty esters during isobutane chemical ionization mass spectrometry

Hydrogenation of double bonds was observed to occur during the isobutane chemical ionization mass spectrometry (MS) of unsaturated fatty esters. Chemical ionization (CI) spectra of a series of methyl esters in the C16–C20 carbon range containing 0–4 double bonds showed a variety of ionization characteristics in the molecular ion cluster, including hydride abstraction, charge exchange, protonation and, for the unsaturated fatty acids (FA), hydrogenation of the double bond followed by protonation. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Detection of hydroxy fatty acids in biological samples using capillary gas chromatography in combination with positive and negative chemical ionization mass spectrometry

The most common method for use in the structural analysis of hydroxy fatty acids in biological samples is the gas chromatography-mass spectrometry (GC-MS) analysis of trimethylsilyl ethers of the methyl esters using electron impact ionization. A comparison of electron impact (EI) with chemical ionization mass spectrometry (CI-MS) shows that CI-MS is the superior technique. All ions necessary for structural analysis are observed at sufficiently high levels of intensity when methane or isobutane are used as reactant gases. The molecular weight can be determined from the ion group M+H, M-15 and M+H−90. The ionic series M+H−n×90 enables one to determine the number of hydroxyl groups. The position of the hydroxyl groups can be derived from the fragments of the α-cleavage of the fatty acid chain. The application of heptafluorobutyrates as derivatives for hydroxy fatty acid methyl esters shows advantages in the trace analysis of these compounds. Heptafluorobutyrates exhibit useful mass fragmentation patterns in the positive as well as in the negative CI mode. With methane as the reactant gas, M+H usually is base peak in positive mass spectra. The ionic series M+H−n×214 leads to the number of hydroxy groups in the molecule. In the negative mass spectra, M and M-20 are indicative for the molecular weight. The ion group m/z 213, 194 and 178 at high levels of intensity is typical for heptafluorobutyrates. The advantage of the application of heptafluorobutyrates is the high sensitivity which can be obtained in trace analysis using negative MS. Heptafluorobutyrates of hydroxy fatty acids gave a 20-fold higher response in the negative scan mode compared to that of the positive. The detection limit for heptafluorobutyrates in negative CI-MS was on the order of 1 fg (10⁻¹⁵ g).     

Photoelectron resonance capture ionization mass spectrometry of fatty acids in olive oil

Photoelectron resonance capture ionization (PERCI) mass spectrometry has been developed for the direct online analysis of organics, including lipids. Analysis is conducted without the need for sample preparation or chemical derivatization such as methylation, foregoing the use of harmful or toxic chemicals. PERCI is currently being adapted towards the analysis of edible oils. Herein, as a proof of principle of the simplicity and potential utility of this method towards the analysis of edible oils, we present the analysis of the prevalent fatty acids (FA) in Tuscan extra‐virgin olive oil along with triolein and linolenic acid (LNA) standards. Ionization of olive oil results in little fragmentation of the prevalent FA, which are measured as their molecular ions, [FA–H]^?. The relative concentrations of these FA determined by PERCI were in good agreement with established values. Further utility of PERCI was demonstrated by interrogation of ozonized LNA and olive oil, with 13 of the 18 anticipated products of the ozonolysis of LNA measured as their molecular anions, [M–H]^?. Similarly, the PERCI mass spectrum of ozonized olive oil showed all the anticipated ions of the predominant FA, oleic acid, as well as many molecular ions arising from less abundant unsaturated FA.     

Analysis of triglycerides using atmospheric pressure chemical ionization mass spectrometry

Atmospheric pressure chemical ionization (APCI) mass spectrometry was investigated as a new method for analysis of a mixture of triglycerides separated by reverse-phase high-performance liquid chromatography. A mixture of homogeneous (monoacid) triglyceride standards containing fatty acids with zero to three double bonds was analyzed to demonstrate the quality of mass spectra obtained by using the APCI interface. The mass spectra showed that minimal fragmentation occurs, resulting primarily in diglyceride [M−RCOO]⁺ ions and [M+1]⁺ protonated molecular ions. The degree of unsaturation within the acyl chains had a marked effect on the proportion of diglyceride ions vs. the [M+1]⁺ ions formed in the APCI source. The mass spectra of triglycerides containing fatty acids with two or three double bonds showed predominantly protonated triglyceride ions, with diglyceride peaks representing 13 to 25% of the base peak. The triglycerides containing singly unsaturated fatty acids gave diglyceride ions as the base peak, and [M+1]⁺ ions with an intensity 20 to 28% that of the base peak. Only diglyceride ions were observable in the spectra of triglycerides containing saturated fatty acids.     

Identification of steroids by chemical ionization mass spectrometry

Chemical ionization (CI) mass spectra of various natural and synthetic steroids have been studied using methane, isobutane, ammonia, trideuterioammonia and hydroxy anion as reagent gases. The CI spectra of steroids give simple and well characterized ions, which provide information about molecular weight as well as functionalities in the molecules. Trideuterioammonia exchanges rapidly with active hydrogens (e.g., OH, SH, COOH, NH₂) in steroid molecules in the CI reaction and thus provides a convenient means of active hydrogen determination by mass spectrometry. Application of various CI processes to the analysis of steroids and conjugates have been made. Low levels of hydroxycholesterols in biological samples and in cholesterol autoxidation products were identified by the 4 ion patterns, [M+NH₄]⁺, [M−OH+NH₃]⁺, [M−OH]⁺ and [M−H₂ O−OH]⁺, in ammonia CI. The position of hydroxy functions in the cholesterol side chain can be identified from the methane CI of hydroxycholesterol trimethylsilyl (TMS) derivatives. Sterol carboxylic esters can be identified as the ammonium adduct ion of the intact molecule, [M+NH₄]⁺, in ammonia CI. Isobutane and hydroxy anion CI spectra of the steroid esters give abundant ion fragments of both steroids and carboxylic acid moieties. Identification of free bile acids and steroid glycosides without derivatization is also feasible with the CI process when ammonia is used as reagent gas.     

Field ionization mass spectrometry of long chain fatty methyl esters

Mass spectrometry is particularly useful for identifying lipid materials. One primary factor in the interpretation of mass spectra is the recording of the molecular ion peak giving the molecular weight of the compound. Regrettably many compounds, including hydroxy compounds, do not give significant molecular ion peaks; consequently their identification is difficult. A mass spectrometer equipped with a field ionization source produces a greatly different mass spectrum consisting almost entirely of the molecular ion peak. This new source was used to measure the mass spectra of methyl esters of saturated, unsaturated and hydroxy fatty acids. Saturated esters gave the molecular ion peak almost exclusively; unsaturated esters yielded molecular plus metastable ion peaks; whereas the hydroxy esters had molecular, M-18, meaastable and fragment ion peaks. Presented at the AOCS Meeting, Chicago, October 1967. No. Mktg. and Nutr. Res. Div., ARS, USDA.     

The bottom-up solution to the triacylglycerol lipidome using atmospheric pressure chemical ionization mass spectrometry

Presented here is an approach to representing the data from atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) of triacylglycerols (TAG) using a set of one, two, or three Critical Ratios. These Critical Rations may be used directly to provide structural information concerning the regioisomeric composition of the triacylglycerols (TAG), and about the degree of unsaturation in the TAG. An AAA-type, or Typel, TAG has only one Critical Ratio, the ratio of the protonated molecule, [M+H]⁺, to the DAG fragment ion, [AA]⁺. The Critical Ratio for a Type I TAG is [MH]⁺/Σ[DAG]⁺, and the mass spectrum of a Type I TAG can be reproduced from only this one ratio. An ABA/AAB/BAA, or Type II, TAG has two Critical Ratios, the [MH]⁺/Σ[DAG]⁺ ratio and the [AA]⁺/[AB]⁺ ratio. The [AA]⁺/[AB]⁺ ratio for a single TAG or TAG mixture can be compared with the [AA]⁺/[AB]⁺ ratios of pure regioisomeric standards, and the percentage of each regioisomer can be estimated. The abundance of the protonated molecule and the abundances of the two [DAG]⁺ fragment ions can be calculated from the two Critical Ratios for a Type II TAG. To calculate the abundances, the Critical Ratios are processed through the Bottom-Up Solution to the TAG lipidome. First, Critical Limits are calculated from the Critical Ratios, and then the Critical Ratios are classified into Cases by comparison with the Critical Limits. Once the Case classification is known, the equation for the abundance of each ion in the mass spectrum is given by the Bottom-Up Solution. A Type III TAG has three different FA and three Critical Ratios. The [MH]⁺/Σ/[DAG]⁺ ratio is the first Critical Ratio, the [AC]⁺/([AB]⁺+[BC]⁺) ratio is the second Critical Ratio, and the [BC]⁺/[AB]⁺ ratio is the third Critical Ratio. The second critical ratio for a Type III TAG can be compared with regioisomeric standards to provide an estimate of the percentage composition of the regioisomers. The three Critical Ratios for a Type III TAG can be processed through the Bottom-Up Solution to calculate the four ion abundances that make up the APCI-MS mass spectrum. The Critical Ratios constitute a reduced data set that provides more information in fewer values than the raw abundances.     

Quantitative,multicomponent analysis of fatty acids from cholesteryl esters by chemical ionization reconstructed mass chromatography

Reconstructed mass chromatography using methane as a carrier and reagent gas for chemical ionization gas chromatography-mass spectrometry of the derived methyl esters allows rapid, quantitative microdeterminations of complete cholesteryl ester fatty acid profiles. The sensitivity of this method is consistent with completely specific, multicomponent assay at the picomole level. Introduction of two homologues as internal standards, one into the intact biological specimen and the other after derivatization, provides a measure of the net efficiency of the processes of extraction and derivatization. This procedure may be extended readily to the determination of fatty acid profiles in most biological fluids.     

Atmospheric pressure chemical ionization mass spectrometry for analysis of lipids

Atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) has proven to be a very valuable technique for analysis of lipids from a variety of classes. This instrumental method readily produces useful ions with gentle fragmentation from large neutral molecules such as triacylglycerols and carotenoids, which are often difficult to analyze using other techniques. Molecules that are easily ionized, such as phospholipids, produce molecular ions and diagnostically useful fragment ions that are complementary to those produced by methods such as electrospray ionization MS with collision-induced dissociation. The simplicity and versatility of APCI-MS make it an ideal tool for use in solving hitherto very difficult analytical problems.     

The mass spectrometry of iso and anteiso monoenoic fatty acids

The normal, iso, and anteiso Δ⁸- and Δ⁹-17:1 fatty acid methyl esters were synthesized and their electron impact-induced fragmentation was studied by mass spectrometry. The mass spectra of the preterminal branched monoenoic fatty acid methyl esters present characteristic fragment ions, now understood to be indicative of the position of the methyl group. These fragment ions are in the iso compound m/e 227 [M-55]⁺, m/e 195 [M-87]⁺, and m/e 177 [M-105]⁺, while in the anteiso compound these fragments are shifted by 14 mass units to m/e 213, m/e 181, and m/e 163. The 15-D-iso Δ⁸- and Δ⁹-17:1 methyl esters were synthesized because the characteristic fragment ions in the methyl branched compounds indicated a key role of the tertiary hydrogen atom in the rearrangement process. A fragmentation mechanism consisting of a double bond migration triggered by the tertiary hydrogen and an allylic cleavage assuming a displacement mechanism is proposed.     

The analysis of rigid polyurethanes by chemical ionization mass spectrometry

Methods for the elucidation of the chemical components of polyurethane foams are described. Foam samples of 50 mg were hydrolyzed in aqueous base and the resulting mixture of polyols and polyamines was analyzed by chemical ionization mass spectrometry (CI-MS) and high-pressure liquid chromatography (HPLC). The aromatic polyamines, which were separated by HPLC, produced few fragment ions under methane chemical ionization and were identified without difficulty. Each propoxylated homolog in the mixture of polyols was detected by CI-MS techniques, and approximate molecular weight profiles are presented for each polyol studied. Chemical ionization spectra are listed for samples of standard polyols and of base-hydrolyzed isocyanates. The hydrolysis products from urethane foam formulations were easily related to the standard compounds via CI-MS. These methods should be applicable to polymeric materials containing urethane, urea, and ester link-ages.     

Analysis of deuterium labeled blood lipids by chemical ionization mass spectrometry

A quantitative analytical method has been developed to analyze methyl esters of blood fatty acids derived from human subjects fed deuterium-labeled fats. The GCMS computer method provides for the analysis of the fed deuterium-labeled fatty acids, the naturally occurring blood fatty acids and new fatty acids formed by chain elongation or shortening of the fed labeled fats. Approximately 20 fatty acids including 16, 17, 18 and 20 carbon chain acids were analyzed with a relative standard deviation of 0.02 at the microgram level and a sensitivity of less than one nanogram. The method uses capillary GC to separate the fatty acid esters and isobutane chemical ionization mass spectrometry with multiple ion detection to determine the isotopic constituents of the GC peaks. The technique provides for the determination of overlapping GC peaks labeled with 2, 4 and 6 deuterium atoms and makes extensive use of computers both for data acquisition and processing.     

Gas chromatographic quantification of fatty acid methyl esters: Flame ionization detection vs. Electron impact mass spectrometry

The determination of FAME by GC is among the most commonplace analyses in lipid research. Quantification of FAME by GC with FID has been effectively performed for some time, whereas detection with MS has been used chiefly for qualitative analysis of FAME. Nonetheless, the sensitivity and selectivity of MS methods advocate a quantitative role for GC-MS in FAME analysis—an approach that would be particularly advantageous for FAME determination in complex biological samples, where spectrometric confirmation of analytes is advisable. To assess the utility of GC-MS methods for FAME quantification, a comparative study of GC-FID and GC-MS methods has been conducted. FAME in prepared solutions as well as a biological standard reference material were analyzed by GC-FID and GC-MS methods using both ion trap and quadrupole MS systems. Quantification by MS, based on total ion counts and processing of selected ions, was investigated for FAME ionized by electron impact. Instrument precision, detection limits, calibration behavior, and response factors were investigated for each approach, and quantitative results obtained by each technique were compared. Although there were a number of characteristic differences between the MS methods and FID with respect to FAME analysis, the quantitative performance of GC-MS compared satisfactorily with that of GC-FID. The capacity to combine spectrometric examination and quantitative determination advances GC-MS as a powerful alternative to GC-FID for FAME analysis.     

Analysis of lipoxygenase-derived fatty acid hydroperoxides by electrospray ionization tandem mass spectrometry

A rapid method is described for the identification of fatty acid hydroperoxides by electrospray ionization-tandem mass spectrometry coupled to liquid chromatography without any derivatization required prior to analysis. Localization of fatty acid hydroperoxides in complex mixtures was achieved by monitoring the loss of hydrogen peroxide using constant neutral loss scanning. In the presence of 5 mM NH₄OAc in methanol-water, adductions [M+NH₄]⁺ were formed almost exclusively, directly revealing the molecular mass of the thermolabile hydroperoxides. In addition, low energy collision-induced dissociation of precursor ions [M+NH₄]⁺ led to characteristic product ions from both the 9- and 13-regioisomers. Thus, electrospray ionization-tandem mass spectrometry provides a straightforward approach for the study of the regioselectivity of lipoxygenase catalysis without any derivatization step required prior to analysis.     

Regioisomers of octanoic acid-containing structured triacylglycerols analyzed by tandem mass spectrometry using ammonia negative ion chemical ionization

Tandem mass spectrometry based on ammonia negative ion chemical ionization and sample introduction via direct exposure probe was applied to analysis of regioisomeric structures of octanoic acid containing structured triacylglycerols (TAG) of type MML, MLM, MLL, and LML (M, medium-chain fatty acid; L, long-chain fatty acid). Collision-induced dissociation of deprotonated parent TAG with argon was used to produce daughter ion spectra with appropriate fragmentation patterns for structure determination. Fatty acids constituting the TAG molecule were identified according to [RCO₂]⁻ ions in the daughter ion spectra. With the standard curve for ratios of [M-H-RCO₂H-100]⁻ ions corresponding to each [RCO₂]⁻ ion, determined with known mixtures of sn-1/3 and sn-2 regioisomers of structured TAG, it was possible to determine the proportions of different regioisomers in unknown samples. The method enabled quantification of MML- and MLM-type structured TAG. In the case of MLL- and LML-type TAG, it was possible to determine the most abundant regioisomer in the unknown mixture and estimate the proportions of regioisomers when there were more than 50% MLL-type isomers in the mixture.     

Analysis of α-branched fatty acids by gas chromatography and mass spectrometry

Gas liquid chromatography and mass spectrometry were utilized in combination to identify isomeric α-branched chain fatty acid methyl esters. In a given isomeric series equivalent chain length, values decreased with increase in the number and size of α-alkyl substituents. Mass spectra of the α-monoalkyl derivatives are characterized by prominent McLafferty rearrangement ion peaks, whereas those of the α,α-dialkyl isomers contain the former ions plus an α-cleavage ion. Presented at the AOCS Meeting, Chicago, September 1973.     

Double-bond location in long-chain polyunsaturated fatty acids by chemical lonization-mass spectrometry

For determination of the double-bond position in polyunsaturated C_24–30 fatty acids from marine organisms, methoxy derivatives were prepared. Diagnostic mass spectral fragment as well as molecular ion intensities were obtained by adjusting the ion source optics in the presence of ammonia at a lower source pressure than used conventionally. A lower detection limit was observed compared to conventional methane chemical ionization, which is a more favorable condition for capillary gas chromatography. Analysis of fatty acids from the spongeCalyx niceaensis showed the double-bond position of 8 unsaturated fatty acids, including two new ones. In addition, structural proof is provided for the presence of a new cyclopropane-containing fatty acid: 19,20-methylene-hexacosanoic acid. “Mass Spectrometry in Structural and Stereochemical Problems 262.” For preceding paper in this series, see Patterson, D.G., Haley, M.J., Midgley, J., and Djerassi, C., Org. Mass. Spectrom., submitted for publication. Recipient of a travel grant from the Queen Wilhelmina Fund, The Netherlands Cancer Foundation     

Comparison of catabolic rates of fatty acids using stable isotope and isotope‐ratio mass spectrometry

The catabolic rates of individual fatty acids in mice were compared using stable isotope (¹³C)‐labeled fatty acids and isotope‐ratio mass spectrometry (IRMS). The catabolic rates were evaluated from the ratio of ¹³C and ¹²C in carbon dioxide expired by mice. The results showed that the catabolic rate of octanoic acid is three times faster than that of palmitic acid. This result is consistent with previous reports using radioisotope ¹⁴C showing that medium‐chain fatty acids are more easily beta‐oxidized than long‐chain fatty acids. The catabolic rates of odd‐numbered fatty acids such as pentadecanoic acid and heptadecanoic acid were significantly lower compared to those of even‐numbered fatty acids such as palmitic acid. These findings support previous reports that show odd‐numbered fatty acids easily accumulating in body fat. The high accumulation of odd‐numbered fatty acids in body fat thus directly reflects a low degree of beta‐oxidization. The combination of stable isotope‐labeled compounds and IRMS serves as a powerful tool in lipid analysis.