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Sample preparation and instrument parameters have regularly been demonstrated to impact upon the observed results in atmospheric pressure photoionization, mass spectrometry (MS), and analytical techniques in general but may be overlooked when such methods are applied to the characterization of real-world samples. An initial investigation into different solvent systems demonstrated that the inclusion of ethyl acetate inverted the ratio of relative intensities of radical and protonated species (R/P). Design of experiments was performed and indicated that the injection flow rate is also a significant factor. The impact of the solvent system and flow rate on signal intensity, the observed compositional profile, and R/P of selected molecular groups is demonstrated further. An inversion of R/P is observed at higher flow rates in solvent systems commonly used in petroleomics studies, effecting a loss of molecular speciation. The findings presented reiterate the critical importance in considering experimental parameters when interpreting the results of analytical procedures.

Ru-based catalysis results in highly unsaturated fatty acid (HUFA) ethyl esters (EE) deuterated to various extents. The products carry

The use of cosmogenically produced sulfur-35 (T1/2 = 87 days) and sodium-22 (T1/2 = 2.6 years) as intrinsic tracers can provide valuable information on catchment hydrology, flow paths, and subsurface storage. A new and straightforward method was created to determine the activities of both 35S and 22Na in various water sources by pumping large volumes (up to 1000 L) of water through cation- and anion-exchange resin columns in the field to collect sodium and sulfate ions and simple chemistry in the lab. Samples are counted for 35S using liquid scintillation counting (LSC) and for 22Na via γ spectroscopy. Our novel in situ method provides faster sample throughput as well as better counting statistics and lower detection limits. Both methods were successfully applied at the Southern Sierra Critical Zone Observatory.

Pine nuts and other edible gymnosperm seeds contain unusual, bioactive polymethylene-interrupted polyunsaturated fatty acids (PMI–PUFAs), a subset of nonmethylene-interrupted PUFA with (−CH2−)n≥2 i...

Monounsaturated normal fatty acids (n-MUFA) and saturated branched chain fatty acids (BCFA) are structurally characterized by separate tandem mass spectrometry methods for double bond localization and for chain branching in their respective fatty acid methyl ester (FAME) derivatives; however, these methods have never been applied to branched monounsaturated FAME. Here, we report application of electron ionization (EI)-MS/MS and solvent-mediated covalent adduct chemical ionization (CACI)-MS/MS of monounsaturated BCFA methyl esters (MUBCFAME) of a chain length of 15–20 carbons. A novel system was used to implement CI with low vapor pressure reagents in a tabletop triple quadrupole MS. Anteiso-MUBCFA EI-MS/MS of the molecular ion (M) yields a characteristic diagnostic ion [M-29]+. iso-MUBCFA can be distinguished from n-MUFA by an ion intensity ratio of [M-32]+/[M-43]+, with iso-MUBCFA yielding a ratio greater than 1.7, while n-MUFA yields a ratio less than 1.0. Chain branching at the iso and anteiso position...

Metal oxide-filled reactors constructed with ceramic tubes or fused silica capillary are widely used for combustion in gas chromatography combustion isotope ratio mass spectrometry (GCC-IRMS). However, they tend to be easily cracked or broken and prone to leaks at operating temperatures of ∼950 °C. Here we introduce a modified commercially available catalytic combustion/reduction methanizer to quantitatively convert organics to CO2 for δ13C analysis while retaining chromatographic resolution. These modified “ARC” reactors operate with a transition-metal catalyst that requires a flowing O2 gas to enable complete conversion to CO2 at lower temperature (620 °C) with acceptable reactor life, reduced complexity, and improved robustness. Performance of two versions of the ARC reactors with different combustion volumes was characterized by analysis of steroid and alkane isotopic standard materials. Linearity of steroid isotopic standards ranged from 0.02 to 0.60 ‰/V in the range of 25 to 200 ng of each steroid i...

The use of hyphenated Fourier transform mass spectrometry (FTMS) methods affords additional information about complex chemical mixtures. Co-eluted components can be resolved thanks to the ultra-high resolving power, which also allows extracted ion chromatograms (EICs) to be used for the observation of isomers. As such datasets can be large and data analyses laborious, improved tools are needed for data analyses and extraction of key information. The typical work-flow for this type of data is based upon manually dividing the total ions chromatogram (TIC) into several windows of usually equal retention time, averaging the signal of each window to create a single mass spectrum, extracting a peak list, performing the compositional assignments, visualizing the results, and repeating the process for each window. By removing the need to manually divide a data set into many time windows and analyze each one, a time-consuming work-flow has been significantly simplified. An environmental sample from the oil sands region of Alberta, Canada, and dissolved organic matter samples from the Suwannee River Fulvic Acid (SRFA) and marine waters (Marine DOM) were used as a test-bed for the new method. A complete solution named KairosMS, was developed in the R language utilizing the Tidyverse packages and Shiny for the user interface. KairosMS imports raw data from common file types, processes it and exports a mass list for compositional assignments. KairosMS then incorporate those assignments for analysis and visualization. The present method increases the computational speed while reducing the manual work of the analysis when compared to other current methods. The algorithm subsequently incorporates the assignments into the processed data set, generating a series of interactive plots, EICs for individual components or entire compound classes, and can export raw data or graphics for off-line use. Using the example of petroleum related data, it is then visualized according to heteroatom class, carbon number, double bond equivalents (DBE), and retention time. The algorithm also gives the ability to screen for isomeric contributions and to follow homologous series or compound classes, instead of individual components, as a function of time.

Self-association of proteins is important in a variety of processes ranging from acquisition of native quaternary structure (where the association is tightly controlled and proceeds in a highly ordered fashion) to aggregation and amyloidosis. The latter is frequently accompanied (or indeed triggered) by the loss of the native structure, but a clear understanding of the complex relationship between conformational changes and protein self-association/aggregation remains elusive due to the great difficulty in characterizing these complex and frequently heterogeneous species. In this study, size exclusion chromatography (SEC) was used in combination with online detection by native electrospray ionization mass spectrometry (ESI MS) to characterize a commercial protein sample (serum albumin) that forms small aggregates. Although noncovalent dimers and trimers of this protein are readily detected by native ESI MS alone, combination of SEC and ESI MS allows a distinction to be made between the oligomers present in solution and those formed during the ESI process (artifacts of ESI MS). Additionally, native ESI MS detection allows a partial loss of conformation integrity to be detected across all albumin species present in solution. Finally, ESI MS detection allows these analyses to be carried out readily even in the presence of other abundant proteins coeluting with albumin. Native ESI MS as an online detection method for SEC also enables meaningful characterization of species representing different quaternary organization of a recombinant glycoprotein human arylsulfatase A even when their rapid interconversion prevents their separation on the SEC time scale.

Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-α-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve quantitative results compared to use of a single standard.

We report the first coupling of comprehensive two-dimensional gas chromatography (GC x GC) to online combustion isotope ratio mass spectrometry (C-IRMS). A GC x GC system, equipped with a longitudinally modulated cryogenic system (LMCS), was interfaced to an optimized low dead volume combustion interface to preserve300 ms full width at half-maximum (fwhm) fast GC peaks generated on the second GC column (GC2). The IRMS detector amplifiers were modified by configuration of resistors and capacitors to enable fast response, and a home-built system acquired data at 25 Hz. Software was home-written to handle isotopic time shifts of less than one bin (40 ms) and to integrate peak slices to recover isotope ratios from cryogenically sliced peaks. The performance of the GC x GCC-IRMS system was evaluated by isotopic analysis of urinary steroid standards. Steroids were separated by a nonpolar GC1 column (30 m x 0.25 mm, 5% phenyl), modulated into multiple 4- or 8-s cryogenic slices by the LMCS, and then separated on a polar GC2 column (1 or 2 m x 0.1 mm, 50% phenyl). GC2 peak widths from a 1-m column averaged 276 ms fwhm. Steroid standard sliced peaks were successfully reconstructed to yield delta(13)C VPDB values with average precisions of SD(delta(13)C) = 0.30 per thousand and average accuracies within 0.34 per thousand, at 8 ng on column. Two steroids, coeluting in GC1, were baseline separated in GC2 and resulted in delta(13)C VPDB values with average precisions of SD(delta(13)C) = 0.86 per thousand and average accuracies within 0.26 per thousand, at 3 ng on column. Results from this prototype system demonstrate that the enhanced peak capacity and signal available in GC x GC is compatible with high-precision carbon isotope analysis.

We report here the first coupling of fast GC to IRMS, in a system capable of 250 ms peak widths (fwhm) at 1 mL/min flow rates, one-fifth as narrow as any previously reported GCC-IRMS system. We developed an optimized postcolumn interface that results in minimal peak broadening, using a programmable temperature vaporization injector in place of a rotary valve or backflush system to divert solvent, a narrow capillary combustion reactor followed by a cryogenic water trap with narrow-bore (0.20 mm i.d.) transfer lines, and a narrow i.d. open split to the IRMS directly inserted into the column effluent. Quantitative combustion was demonstrated with CH4 injections. A comparison of CO2 injections with different fwhm peak widths (250, 2500, and 7500 ms) showed similar precisions, SD(delta13C)=0.2-0.3 per thousand, for injections of600 pmol C on column; precision for the narrow peaks (250 ms) was considerably better for injections150 pmol C on column. SD(delta13C)1 per thousand was achievable for injections of 5-15 pmol on column for 250 ms wide peaks, 10-fold better precision than 2500 ms wide peaks, and within a factor of 3 of the counting statistics limit. For a mixture of 15 fatty acid methyl esters (FAME), 1.5 nmol C of each on column yielded typical SD(delta13Cpdb)=0.4 per thousand for fast GC and 0.5 per thousand for conventional GC. For 14 of the 15 FAME, delta13C values between the two systems were within+/-1.5 per thousand and not significantly different. Fast GCC-IRMS required one-third the run time (450 s vs 1400 s) to achieve comparable resolution. Mean peak widths for fast GCC-IRMS of the FAME were 720 ms, compared to 650 ms by fast GC with flame ionization detection. At a 15-fold dilution (100 pmol C on column for each FAME), fast GCC-IRMS achieved approximately 2-fold better precision and accuracy than similar injections on conventional GCC-IRMS. Finally, a mixture of 10 steroids (approximately 7 nmol C (100 ng) each on column) was analyzed with mean precision of SD(delta13C)=0.2 per thousand in 620 s by fast GCC-IRMS, while conventional GCC-IRMS required 1200 s and achieved poorer resolution. delta13C values for the two system were similar (Deltadelta13Cor=2 per thousand for all steroids), indicating that accuracy is not compromised. In summary, fast GCC-IRMS can achieve similar precision to conventional GC with considerable time savings for standard sample sizes (1 nmol C) and achieves modest precision (approximately 1 per thousand) near the counting statistics limit on low level components.

Trace elemental speciation in complex, real-world matrixes is a daunting task because of the low concentration of metals/metalloids and the correspondingly high molecular chemical noise. We constructed a liquid chromatography parallel elemental and molecular mass spectrometry (PEMMS) system and evaluated the use of peak elution profiles to identify trace molecular species containing specific heteroatoms, using the case of Se in yeast. We demonstrate that it is possible to use the HPLC-inductively coupled plasma (ICP)MS peak profile (retention time, width) to identify candidate ions with matching peak profiles in the molecular MS data. Proof of principle was demonstrated by C18 separation of three Se-amino acid standards (0.005-15 ppm as Se). The molecular MS (atmospheric pressure chemical ionization time-of-flight, APCI-TOF-MS) data set was converted into selected ion chromatograms of 0.05 Th width. ICPMS and APCI-TOF-MS ion chromatograms were fit by the Haarhoff-VanderLinde function using the following parameters: area, retention time, width, and skew. The ICPMS fit parameters were more reproducible than the APCI-TOF-MS fit parameters from run to run, and the APCI-TOF-MS signal was expected to limit correlation in most circumstances. Retention time and width were found to correlate well between the two MS systems for APCI-TOF-MS peaks with signal-to-fit-error (S/FE) of25. Correction factors for differences in flow path length and peak broadening were required. The normalized correction factors were species and concentration independent and were stable from run to run. The skew parameter was found to be highly susceptible to noise and was not generally useful in matching ICPMS and APCI-TOF-MS peaks. An artificially noisy sample was generated by spiking 30 ppb Se-methionine (SeMet) and 5 ppb Se-methylselenocysteine (SeMSC) with unselenized yeast extract and run by PEMMS. The PEMMS software was able to detect four molecular MS peaks associated with SeMet and two for SeMSC, while filtering out40 coeluting spectral peaks associated with chemical noise in each sample. In summary, we have demonstrated that correlated information in peak shape between parallel detectors can facilitate detection of trace elemental species in complex matrixes.

Intramolecular carbon isotope ratios reflect the source of a compound and the reaction conditions prevailing during synthesis and degradation. We report here a method for determination of relative (Deltadelta13C) and absolute (delta13C) intramolecular isotope ratios using the volatile lactic acid analogue propylene glycol as a model compound, measured by on-line gas chromatography-pyrolysis coupled to GC-combustion-isotope ratio mass spectrometry. Pyrolytic fragmentation of about one-third of the analyte mass produces optimal fragments for isotopic analysis, from which relative isotope ratios (Deltadelta13C) are calculated according to guidelines presented previously. Calibration to obtain absolute isotope ratios is achieved by quantifying isotope fractionation during pyrolysis with an average fractionation factor, alpha, and evaluated by considering extremes in isotopic fractionation behavior. The method is demonstrated by calculating ranges of absolute intramolecular isotope ratios in four samples of propylene glycol. Relative and absolute isotope ratios were calculated with average precisions of SD(Deltadelta13C)0.84 per thousand and SD(delta13C)3.0 per thousand, respectively. The various fractionation scenarios produce an average delta(13)C range of 2 per thousand for each position in each sample. Relative isotope ratios revealed all four samples originated from unique sources, with samples A, B, and D only distinguishable at the position-specific level. Regardless of pyrolysis fractionation distribution, absolute isotope ratios showed a consistent pattern for all samples, with delta13C(3)delta13C(2)delta13C(1). The validity of the method was determined by examining the difference in relative isotope ratios calculated through two independent methods: Deltadelta13C calculated directly using previous methods and Deltadelta13C extracted from absolute isotope ratios. Deviation between the two Deltadelta13C values for all positions averaged 0.1-0.2 per thousand, with the smallest deviation obtained assuming equal fractionation across all fragment positions. This approach applies generally to all compounds analyzed by pyrolytic PSIA.

We report an automated method for high-precision position-specific isotope analysis (PSIA) of carbon in amino acid analogues. Carbon isotope ratios are measured for gas-phase pyrolysis fragments from multiple sources of 3-methylthiopropylamine (3MTP) and isoamylamine (IAA), the decarboxylated analogues of methionine and leucine, using a home-built gas chromatography (GC)-pyrolysis-GC preparation system coupled to a combustion-isotope ratio mass spectrometry system. Over a temperature range of 620-900 degrees C, the characteristic pyrolysis products for 3MTP were CH4, C2H6, HCN, and CH3CN and for IAA products were propylene, isobutylene, HCN, and CH3CN. Fragment origin was confirmed by 13C-labeling, and fragments used for isotope analysis were generated from unique moieties with > 95% structural fidelity. Isotope ratios for the fragments were determined with an average precision of SD(delta13C) < 0.3% per thousand, and relative isotope ratios of fragments from different sources were determined with an average precision of SD(delta(delta)13C) < 0.5% per thousand. Delta(delta)13C values of fragments were invariant over a range of pyrolysis temperatures. The delta(delta)13C of complementary fragments in IAA was within 0.8% per thousand of the delta(delta)13C of the parent compounds, indicating that pyrolysis-induced isotopic fractionation is effectively taken into account with this calibration procedure. Using delta(delta)13C values of fragments, delta(delta)13C values were determined for all four carbon positions of 3MTP and for C1, C2, and the propyl moiety of IAA, either directly or indirectly by mass balance. Large variations in position-specific isotope ratios were observed in samples from different commercial sources. Most dramatically, two 3MTP sources differed by 16.30% per thousand at C1, 48.33% per thousand at C2, 0.37% per thousand at C3, and 5.36% per thousand at C(methyl). These PSIA techniques are suitable for studying subtle changes in intramolecular isotope ratios due to natural processes.

Fatty acids with conjugated double bonds have attracted great interest because of their reported potent bioactivities. However, there are currently no rapid methods for their structural characterization. We report here a convenient mass spectrometry-based strategy to establish double bond geometry by analysis of collisional dissociation products of cis/trans and trans/cis conjugated linoleic acids (CLAs), as methyl esters, and to distinguish CLAs from homoallylic (methylene-interrupted) fatty acids in a single-stage mass spectrum. A series of CLA standards with double bond positions 6,8; 7,9; 8,10; 9,11; 10,12; 11,13; 12,14; and 13,15, with all four possible geometries (cis/trans; trans/cis; cis/cis; trans/trans) were analyzed. The m/z 54 (1-methyleneimino)-1-ethenylium ion, generated by self-reaction of acetonitrile under chemical ionization conditions, reacts with unsaturated fatty acids to yield an [M + 54]+ ion, which decomposes in the single-stage mass spectrum by loss of neutral methanol to form [M + 54 - 32]+. The ratio of [M + 54]+/[M + 54 - 32]+ in the single-stage mass spectra of CLA isomers is 1 order of magnitude less than for homoallylic diene FAME. Collisional dissociation of the [M + 54]+ ion yields two diagnostic ions that contain the alpha- and omega-carbon atoms and is characteristic of double bond position in the analyte. The fragment vinylic to the trans double bond is significantly more abundant than that for the cis double bond, revealing double bond geometry. The ratio of alpha to we diagnostic ion abundances is >4.8 for cis/trans isomers

We present a rapid and selective method to increase the volatility of tyrosine and phenylalanine without adding derivative C for high-precision gas chromatography-continuous-flow isotope ratio mass spectrometry (GCC-IRMS) based on enzymatic decarboxylation to yield alkylamines and evaluated for 15N isotopic integrity. Purified tyrosine and phenylalanine were converted to tyramine and phenethylamine by tyrosine and phenylalanine decarboxylases, respectively. GC separation was achieved using a thick stationary phase (5-microm) capillary column. Recoveries were 95 +/- 2%. The reproducibility of delta15N of tyramine and phenethylamine measured by GCC-IRMS averaged SD(delta15N) = 0.33 per thousand. The absolute differences between delta15N of amino acids measured by elemental analyzer-IRMS and the alkylamines measured by GCC-IRMS was not significant. Phenethylamine and tyramine prepared from a mixture of 18 amino acids were extracted by ethanol with 95% recovery, and analysis yielded clean chromatograms and equivalent precision. These data indicate that enzymatic decarboxylation of phenylalanine and tyrosine is a convenient method to increase their volatility for continuous-flow isotopic analysis without introducing extraneous C or significant isotopic fractionation.

Desorption/ionization on porous silicon mass spectrometry (DIOS-MS) is a novel method for generating and analyzing gas-phase ions that employs direct laser vaporization. The structure and physicochemical properties of the porous silicon surfaces are crucial to DIOS-MS performance and are controlled by the selection of silicon and the electrochemical etching conditions. Porous silicon generation and DIOS signals were examined as a function of silicon crystal orientation, resistivity, etching solution, etching current density, etching time, and irradiation. Pre-and postetching conditions were also examined for their effect on DIOS signal as were chemical modifications to examine stability with respect to surface oxidation. Pore size and other physical characteristics were examined by scanning electron microscopy and Fourier transform infrared spectroscopy, and correlated with DIOS-MS signal. Porous silicon surfaces optimized for DIOS response were examined for their applicability to quantitative analysis, organic reaction monitoring, post-source decay mass spectrometry, and chromatography.

Arylsulfatase A is an endogenous enzyme that is responsible for the catabolism and control of sulfatides in humans. Its deficiency results in the accumulation of sulfatides in the cells of the central and peripheral nervous system leading to the destruction of the myelin sheath and resulting in metachromatic leukodystrophy (MLD), a neurodegenerative lysosomal storage disease. A recombinant human form of this glycoprotein (rhASA) is currently under development as an enzyme replacement therapy. At neutral and alkaline pH, this protein exists as a homodimer but converts to an octameric state in the mildly acidic environment of the lysosome, and a failure to form an octamer results in suboptimal catalytic activity (most likely due to a diminished protection from lysosomal proteases). Despite the obvious importance of the rhASA oligomerization process, its mechanistic details remain poorly understood. In this work, we use size exclusion chromatography (SEC) and electrospray ionization mass spectrometry (ESI MS) to monitor the dimer-to-octamer transition as a function of both solution pH and protein concentration. While SEC clearly shows different profiles (i.e., retention time differences) for rhASA when the chromatography is performed at neutral and lysosomal pH, consistent with changing oligomerization states, no resolved peaks could be observed for either octamer or dimer when analyzed at intermediate pH (5.5-6.5). This could be interpreted either as the result of a rapid dimer-to-octamer interconversion on the chromatographic time scale or as a consequence of the presence of previously unidentified intermediate species (e.g., tetramer and/or hexamer). In contrast, ESI MS provides strong evidence of the dimer-to-octamer transition state that occurs when the analysis is performed within a narrow pH range (6.0-7.0). Octamer assembly was shown to be a highly cooperative process with no intermediate states that are populated to detectable levels. A tetrameric state of rhASA exists at equilibrium with a dimer at neutral pH but does not appear to be involved in the octamer assembly process.

A review of Instrumental Methods in Food Analysis.

Direct profiling of total lipid extracts on a hybrid LTQ Orbitrap mass spectrometer by high-resolution survey spectra clusters species of 11 major lipid classes into 7 groups, which are distinguished by their sum compositions and could be identified by accurately determined masses. Rapid acquisition of survey spectra was employed as a "top-down" screening tool that, together with the computational method of principal component analysis, revealed pronounced perturbations in the abundance of lipid precursors within the entire series of experiments. Altered lipid precursors were subsequently identified either by accurately determined masses or by in-depth MS/MS characterization that was performed on the same instrument. Hence, the sensitivity, throughput and robustness of lipidomics screens were improved without compromising the accuracy and specificity of molecular species identification. The top-down lipidomics strategy lends itself for high-throughput screens complementing ongoing functional genomics efforts.

We report a method to elucidate the structure of triacyl-glycerols (TAGs) containing monoene or diene fatty acyl groups by atmospheric pressure covalent adduct chemical ionization (APCACI) tandem mass spectrometry using acetonitrile as an adduct formation reagent. TAGs were synthesized with the structures ABB and BAB, where A is palmitate (C16:0) and B is an isomeric C18 monoene unsaturated at position 9, 11, or 13 or an isomeric diene unsaturated at positions 9 and 11, 10 and 12, or 9 and 12. In addition to the species at m/z 54 observed in previous CI studies of fatty acid methyl esters, we also found that ions at m/z 42, 81, and 95 undergo covalent reaction with TAGs containing double bonds to yield ions at m/z 40, 54, 81, and 95 units greater than that of the parent TAG: [M + 40]+, [M + 54]+, [M + 81]+, and [M + 95]+ ions. When collisionally dissociated, these ions fragment to produce two or three diagnostic ions that locate the double bonds in the TAG. In addition, ions [RCH=C=O + 40]+ and [RCH=C=O + 54]+ formed from collisional dissociation are of strong abundance in MS/MS spectra, and collisional activation of these ions produces two intense confirmatory diagnostic ions in the MS3 spectra. Fragment ions reflecting neutral loss of an sn-1-acyl group from [M + 40]+ and [M + 54]+ are more abundant than those reflecting neutral loss of an sn-2-acyl group, analogous to previous reports for protonated TAGs. The position of each acyl group on the glycerol backbone is thus determined by the relative abundances of these ions. Under the conditions in our instrument, the [M + 40]+ adduct is at the highest signal and also yields all information about the double bond position and TAG stereochemistry. With the exception of geometries about the double bonds, racemic TAG isomers containing two monoenes or dienes and a saturate can be fully characterized by APCACI-MS/MS/MS.