Ammonia Negative Ion Chemical Ionization Research Articles

Strategy for stereospecific characterization of natural triacylglycerols using multidimensional chromatography and mass spectrometry

Stereoisomeric determination of individual triacylglycerols (TAGs) in natural oils and fats is a challenge due to similar physicochemical properties of TAGs with different fatty acid combinations. In this study, we present a strategy to resolve the enantiomeric composition of nutritionally important TAGs in sea buckthorn (Hippophaë rhamnoides) as an example food matrix. The targeted strategy combines 1) fatty acid profiling with GC, 2) separation of TAGs with RP-HPLC, 3) stereospecific separation with chiral-phase HPLC and 4) structural characterization with MS. Three major asymmetric diacid- and triacid-TAG species were analyzed in sea buckthorn pulp oil. Off-line coupling of RP-HPLC and chiral-phase HPLC allowed separation of several TAG regioisomers and enantiomers, which could not be resolved using one-dimensional techniques. Enantiomeric ratios were determined and specific structural analysis of separated TAGs was performed using direct inlet ammonia negative ion chemical ionization method.Of the TAG 16:0/16:1/16:1 palmitic acid (C16:0) was located predominantly in a primary position and the enantiomeric ratio of TAG sn-16:1-16:1-16:0 to sn-16:0-16:1-16:1 was 70.5/29.5. Among the TAGs 16:0/16:0/18:2 and 16:0/16:0/16:1, only ca 5% had C16:0 in the sn-2 position, thus, ca 95% were symmetric sn-16:0-18:2-16:0 and sn-16:0-16:1-16:0. The enantiomeric ratio of triacid-TAGs containing C16:0 and two unsaturated fatty acids (palmitoleic C16:1, oleic C18:1 or linoleic acids C18:2) could not be resolved due to lack of commercial enantiopure reference compounds. However, it became clear that the targeted strategy presented offer unique and convenient method to study the enantiomeric structure of individual TAGs.

Quantification of triacylglycerol regioisomers by ultra‐high‐performance liquid chromatography and ammonia negative ion atmospheric pressure chemical ionization tandem mass spectrometry

The regioisomer composition of triacylglycerols (TAGs) in various vegetable oils was determined with a new liquid chromatography/tandem mass spectrometry (LC/MS/MS method). A direct inlet ammonia negative ion chemical ionization (NICI) MS/MS method was improved by adapting it to LC negative ion (NI) atmospheric pressure chemical ionization (APCI) MS/MS system using ammonia as nebulizer gas. The method is based on the preferential formation of [M-H-RCOOH-100](-) ions during collision-induced dissociation by loss of sn-1/3 fatty acids from [M-H](-) ions. Calibration curves were created from nine reference TAGs: Ala/L/L, Gla/L/L, L/L/O, L/O/O, P/O/O, P/P/O, Po/Po/V, Po/Po/O, and C/O/O. The calibration curves were used to quantify the regioisomer compositions of selected TAGs in rapeseed oil, sunflower seed oil, palm oil, black currant seed oil, and sea buckthorn pulp oil. The method discriminates the different regioisomers and the results obtained by this method were in good agreement with previous results. This proves that this new method can be used for the determination of regiospecific distribution of fatty acids in TAGs.

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.

Quantitation of acyl migration during lipase‐catalyzed acidolysis, and of the regioisomers of structured triacylglycerols formed

AbstractVarious MLM‐type (M, medium‐chain fatty acids; L, long‐chain fatty acids) structured triacylglycerols were produced in pilot‐ or small‐scale packed‐bed reactors by lipasecatalyzed acidolysis. The incorporation and acyl migration of octanoic acid were measured by gas chromatography and Grignard degradation, and ranged from 39.0 to 48.7% and 0.6 to 9.3%, respectively. Quantitation of triacylglycerol molecular species was performed by ammonia negative ion chemical ionization (NICI) mass spectrometry (MS). The proportion of ACN (acyl carbon number) 34 species that contained one C18 fatty acid and two C8∶0′ in samples analyzed, varied from 12.5 to 23.2%. The selected regioisomers MLM and MML within the ACN 34 species group were quantified by NICI tandem MS (MS/MS) and were in the range of 97.1 to 98.4% and 1.6 to 2.9%, respectively. There was no correlation between the level of acyl migration during lipase‐catalyzed esterification and the level of regioisomers of the selected MLM‐type triacylglycerols in the structured lipid samples.

Software (MSPECTRA) for automatic interpretation of triacylglycerol molecular mass distribution spectra and collision induced dissociation product ion spectra obtained by ammonia negative ion chemical ionization mass spectrometry.

Rapid analysis of molecular mass distributions of triacylglycerol (TAG) mixtures and regioisomeric structures of selected molecular mass species is possible using ammonia negative ion chemical ionization mass spectrometry utilizing sample introduction by direct exposure probe. However, interpretation of spectra and calculation of results is time consuming, thus lengthening the total analysis time. To facilitate result calculation a software package (MSPECTRA 1.3) was developed and applied to automatic processing of triacylglycerol molecular mass distribution spectra and collision induced dissociation (CID) product ion spectra. The program is capable of identifying triacylglycerol molecular mass species possessing different ACN:DB (acyl carbon number:number of double bonds) ratios on the basis of m/z values of [M - H](-) ions. In addition to such identification the program also corrects spectra for abundances of naturally occurring (13)C isotopes and calculates relative proportions of triacylglycerol molecular species in the analyzed samples. If several replicate spectra are processed simultaneously the program automatically calculates an average and standard deviation of relative proportions of molecular species. In the case of CID spectra the program identifies fatty acid fragment ions [RCO(2)](-) and the corresponding [M - H - RCO(2)H - 100](-) ions, and calculates the relative proportions of ions in both groups. These proportions are then used automatically to calculate the fatty acid combinations comprising the parent triacylglycerol molecule and the regiospecific positions of fatty acids. Processing of several replicate product ion spectra simultaneously produces averaged proportions of regioisomers comprising the parent triacylglycerol molecular species and the standard deviation of the analysis. The performance of the program was tested by analyzing triacylglycerol samples of human milk, human milk substitutes, human chylomicron and cocoa butter, and by comparing results obtained by automated processing of the data with manually calculated results.

Optimization of the mass spectrometric analysis of triacylglycerols using negative-ion chemical ionization with ammonia.

Conditions for the mass spectrometric analysis of triacylglycerols, via direct exposure probe, with ammonia negative-ion chemical ionization were optimized. Triacylglycerols were most favorably ionized, using the reactant gas pressure of approximately 8500 mtorr at the ion source temperature of 200 degrees C with the instrumentation used. Abundant [M-H]- ions were produced under these conditions without the formation of [M + 35]- cluster ions, which would interfere with the molecular weight region of triacylglycerols in the spectra. A rapid desorption of the sample from the probe wire is recommended, using a relatively high heating rate (approximately 40 mA s-1), to minimize thermal degradation of unsaturated molecules and the reducing effect of double bonds on the mass spectrometric response of triacylglycerols. Furthermore, the abundance of [M-H]- ion was enhanced by rapid heating, which we found to be important to improve the sensitivity. The appropriate amount of sample applied to the rhenium wire was in the region of 50-300 ng for one determination, i.e., only a few nanograms of a single triacylglycerol is required for production of a reliable spectrum. The reproducibility of the method was good as demonstrated with standards and a raspberry seed oil sample. The described mass spectrometric method is a fast and potentially useful tool for semiquantitative determination of triacylglycerol mixtures of various fats and oils. The discrimination, caused by differences in molecular size and unsaturation of triacylglycerols with 50 to 56 acyl carbons, was negligible under our optimized ionization conditions, thus, no correction factors were needed. These findings have not yet been verified with instruments in other laboratories. However, the present study shows how the analysis of triacylglycerols can be improved, regardless of the instrument, by optimization of the analytical conditions.

Analysis of low erucic acid turnip rapeseed oil (Brassica campestris) by negative ion chemical ionization tandem mass spectrometry. A method giving information on the fatty acid composition in positionssn‐2 andsn‐1/3 of triacylglycerols

AbstractA tandem mass spectrometric method is described for the rapid analysis of fatty acid combinations in mixtures of triacylglycerols. Triacylglycerols were introduced into a triple quadrupole mass spectrometervia a direct exposure probe and deprotonated using ammonia negative ion chemical ionization. Collisionally activated spectra were obtained and the resulting fragments used to identify the fatty acid constituents, and the fatty acids preferentially located at thesn‐2 position of the triacylglycerols. Fourteen major molecular weight species of purified triacylglycerols of a supercritical fluid extract of low erucic acid turnip rapeseed oil (Brassica campestris) were analyzed. The five major combinations of fatty acids comprised two thrids of the total triacylglycerols and contained oleic, linoleic and α‐linolenic acids with linoleic acid favoring thesn‐2 position.

Triacylglycerols of human milk: Rapid analysis by ammonia negative ion tandem mass spectrometry

Human milk triacylglycerols (TAG) were analyzed by ammonia negative ion chemical ionization tandem mass spectrometry. The deprotonated molecular ions of triacylglycerols were fractionated at the first mass spectrometry (MS) stage. Twenty-nine of the deprotonated TAG ions were further analyzed based on their collisionally activated (CA) spectra. The tandem MS analysis covered eleven major acyl carbon number fractions, two of which contained odd carbon number fatty acids. Fatty acids of 28 different molecular weights were recorded from the daughter spectra. Hexadecanoic acid was present in all CA spectra, octadecenoic acid in the CA spectra of all mono- and higher unsaturated TAG, and octadecadienoic acid in the CA spectra of all di- and higher unsaturated TAG. The major fatty acid combinations in triacylglycerols were: with 0 double bonds (DB), 12:0/12:0/16:0; with 1 DB, 12:0/16:0/18:1; with 2 DB, 16:0/18:1/18:1; with 3 DB, 16:0/18:2/18:1; with 4 DB, 18:2/18:1/18:1; and with 5 DB, 18:2/18:2/18:1; hexadecanoic acid typically occupied the sn-2 position. The most abundant TAG was shown to be sn-18:1-16:0-18:1, comprising about 10% of all triacylglycerols.