Supercritical fluid extracts of New Zealand green-lipped mussels (NZGLM) have been suggested to have therapeutic properties related to their oil components. The large number of minor FA in NZGLM extract was characterized by a GC-CIMS/MS method that excels at identification of double-bond positions in FAME. The extract contained five major lipid classes: sterol esters, TAG, FFA, sterols, and polar lipids. The total FA content of the lipid extract was 0.664 g/mL. Fifty-three unsaturated FA (UFA) were fully identified, of which 37 were PUFA, and a further 21 UFA were detected for which concentrations were too low for assignment of double-bond positions. There were 17 saturated FA, with 14:0, 16:0, and 18:0 present in the greatest concentration. The 10 n-3 PUFA detected included 20:5n-3 and 22:6n-3, the two main n-3 FA; n-3 PUFA at low concentrations were 18:3, 18:4, 20:3, 20:4, 21:5, 22:5, 24:6, and 28:8. There were 43 UFA from the n-4, n-5, n-6, n-7, n-8, n-9, n-10, n-1 families, with 16:2n-4, 16:1n-5, 18:1n-5, 18:2n-6, 20:4n-6, 16:1n-7, 20:1n-7, 16:1n-9, 18:1 n-9, and 20:1 n-9 being the most abundant. In general, we estimated that FAME concentrations greater than 0.05% (w/w) were sufficient to assign double-bond positions. In total, 91 FA were detected in an extract of the NZGLM, whereas previous studies of fresh flesh from the NZGLM had reported identification of 42 FA. These data demonstrate a remarkable diversity of NZGLM FA.

Recent advances in gas chromatography combustion-isotope ratio mass spectrometry (GCC-IRMS) has made compound-specific isotope analysis routine, but reports on position-specific isotopic analysis are still scarce. On-line GC-pyrolysis (Py) coupled to GCC-IRMS is reported here for isolation and isotopic characterization of alaninol and phenethylamine, analogues of alanine and phenylalanine, respectively. Ideally, pyrolytic fragments will originate from unique sites within the parent molecule, and isotope ratios for each position within the parent can either be measured directly or calculated from fragment isotope ratios without substantially degrading the analytical precision. Alaninol pyrolysis yielded several fragments, of which CO and CH4 were used for isotope ratio calculations. Isotope labeling experiments showed that CO derived entirely from the C(1) position, while all three positions of alaninol contributed to CH4 (29.0 +/- 0.3% from C(1), 3.6 +/- 0.2% from C(2), and 66.9 +/- 1.1% from C(3)). We demonstrate iterative use of mass balance to calculate isotope ratios from all positions despite the nonideal positional fidelity of CH4. Pyrolysis of phenethylamine generated benzene and toluene fragments. Benzene derived entirely from C(ring), and toluene was proportionately formed from C(3) and C(ring). Relative intramolecular isotope ratios (Deltadelta13C) were calculated directly from delta13C of fragments or indirectly by mass balance. Though the C(3) isotope ratio was calculated from the benzene and toluene fragments, propagation of errors showed that the final precision of the determination was degraded due to the small contribution that C(3) makes to toluene. Samples of each amino acid from four different vendors showed natural variability between sources, especially at the C(1) position of alaninol (range of Deltadelta13C approximately 50 per thousand). The average precision was SD(Deltadelta13C) < 0.20 per thousand for directly measured positions of alaninol and phenethylamine. The precision of indirectly measured positions was poorer (SD(Deltadelta13C) = 0.94 per thousand for alaninol, 6.54 per thousand for phenethylamine) due to propagation of errors. These data demonstrate that GC-Py-GCC-IRMS data can be used to extract high-precision isotope ratios from amino acids despite nonideal positional fidelity in fragments and that natural intramolecular variability in delta13C can be used to distinguish different sources of amino acids.

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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.

Presented here is the first experimental evidence that natural, intramolecular, isotope ratios are sensitive to physiological status, based on observations of intramolecular delta(15)N of lysine in the mitochondrial mimic Paracoccus denitrificans. Paracoccus denitrificans, a versatile, gram-negative bacterium, was grown either aerobically or anaerobically on isotopically-characterized ammonium as sole cell-nitrogen source. Nitrogen isotope composition of the biomass with respect to source ammonium was Delta(15)N(cell - NH4) = delta(15) - delta(15)N(NH4) = -6.2 +/- 1.2 per thousand for whole cells under aerobic respiration, whereas cells grown anaerobically produced no net fractionation (Delta(15)N(cell - NH4) = -0.3 +/- 0.23 per thousand). Fractionation of (15)N between protein nitrogen and total cell nitrogen increased during anaerobic respiration and suggests that residual nitrogen-containing compounds in bacterial cell membranes are isotopically lighter under anaerobic respiration. In aerobic cells, the lysine intramolecular difference between peptide and sidechain nitrogen is negligible, but in anaerobic cells was a remarkable Delta(15)N(p - s) = delta(15)N(peptide) - delta(15)N(sidechain) = +11.0 per thousand, driven predominantly by enrichment at the peptide N. Consideration of known lysine pathways suggests this to be likely due to enhanced synthesis of peptidoglycans in the anaerobic state. These data indicate that distinct pathway branching ratios associated with microbial respiration can be detected by natural intramolecular Deltadelta(15)N measurements, and are the first in vivo observations of position-specific measurements of nitrogen isotope fractionation.