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Carbon-13 isotopomics of triacylglycerol (TAG) fatty acids or free fatty acids in biological matrices holds considerable potential in food authentication, forensic investigations, metabolic studies, and medical research. However, challenges arise in the isotopic analysis of short- and medium-chain (C4 to C10) fatty acid methyl esters (SMCFAMEs) through gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The high volatility of these esters results in losses during their preparation, leading to isotopic fractionation. Moreover, the methoxy group added to acyl chains requires the correction of δ13C values, thereby increasing the uncertainty of the final results. Analyzing free fatty acids (FFAs) addresses both issues encountered with SMCFAMEs. To achieve this objective, we have developed a new protocol enabling the isotopomics of individual fatty acids (FAs) by GC-C-IRMS. The same experiment also provides the FA profile, i.e., the relative percentage of each FA in the TAG hydrolysate or its concentration in the studied matrix. The method exhibited high precision, as evidenced by the repeatability and within-lab reproducibility of results when tested on TAGs from both animal and vegetal origins. Compared to the analysis of FAMEs by GC-C-IRMS, the current procedure also brings several improvements in alignment with the principles of green analytical chemistry and green sample preparation. Thus, we present a two-in-one method for 13C-isotopomic and metabolomic biomarker quantitation within quasi-universal TAG compounds, encompassing the short- and medium-acyl chains.

Tramadol, previously only known as a synthetic analgesic, has now been found in the bark and wood of roots of the African medicinal tree Nauclea latifolia . At present, no direct evidence is available as to the biosynthetic pathway of its unusual skeleton. To provide guidance as to possible biosynthetic precursors, we have adopted a novel approach of retro-biosynthesis based on the position-specific distribution of isotopes in the extracted compound. Relatively recent developments in isotope ratio monitoring by ¹³C NMR spectrometry make possible the measurement of the nonstatistical position-specific natural abundance distribution of ¹³C (δ ¹³C ᵢ) within the molecule with better than 1‰ precision. Very substantial variation in the ¹³C positional distribution is found: between δ ¹³C ᵢ = −11 and −53‰. Distribution is not random and it is argued that the pattern observed can substantially be interpreted in relation to known causes of isotope fractionation in natural products. Thus, a plausible biosynthetic scheme based on sound biosynthetic principals of precursor–substrate relationships can be proposed. In addition, data obtained from the ¹⁸O/ ¹⁶O ratios in the oxygen atoms of the compound add support to the deductions made from the carbon isotope analysis. This paper shows how the use of ¹³C NMR at natural abundance can help with proposing a biosynthetic route to compounds newly found in nature or those difficult to tackle by conventional means.

Cholesterol, the principal zoosterol, is a key metabolite linked to several health complications. Studies have shown its potential as a metabolic biomarker for predicting various diseases and determining food origin. However, the existing INEPT (insensitive nuclei enhanced by polarization transfer) 13C position-specific isotope analysis method of cholesterol by NMR was not suitable for very precise analysis of small quantities due to its long acquisition time and therefore is restricted to products rich in cholesterol. In this work, a symmetric and adiabatic heteronuclear single quantum coherence (HSQC) 2D NMR sequence was developed for the high-precision (few permil) analysis of small quantities of cholesterol. Adiabatic pulses were incremented for improving precision and sensitivity. Moreover, several strategies such as the use of non-uniform sampling, linear prediction, and variable recycling time were optimized to reduce the acquisition time. The number of increments and spectral range were also adjusted. The method was developed on a system with a cryogenically cooled probe and was not tested on a room-temperature system. Our new approach allowed analyzing as low as 5 mg of cholesterol in 31 min with a long-term repeatability lower than 2‰ on the 24 non-quaternary carbon atoms of the molecule comparing to 16.2 h for the same quantity using the existing INEPT method. This result makes conceivable the isotope analysis of matrices low in cholesterol.

The application of isotope ratio methods in authenticity and traceability relies on the accuracy and robustness of the methodology employed. An unexpected source of error has now been identified, which can introduce major and variable inaccuracies into the determination of site-specific isotope ratio measurement by quantitative ¹³C NMR spectrometry if not correctly controlled. This is the isotope chemical shift effect, which comes into play when hydrogen atoms in the target molecule enter into exchange with deuterated water present at trace levels in the deuterated solvent used as the frequency lock. Even at a level of contamination as low as 0.02%, an error of 5‰ can be introduced, fivefold the required accuracy of 1‰. How to avoid this source of error is discussed.

The application of isotope ratio methods in authenticity and traceability relies on the accuracy and robustness of the methodology employed. An unexpected source of error has now been identified, which can introduce major and variable inaccuracies into the determination of site-specific isotope ratio measurement by quantitative [.sup.13]C NMR spectrometry if not correctly controlled. This is the isotope chemical shift effect, which comes into play when hydrogen atoms in the target molecule enter into exchange with deuterated water present at trace levels in the deuterated solvent used as the frequency lock. Even at a level of contamination as low as 0.02%, an error of 5‰ can be introduced, fivefold the required accuracy of 1‰. How to avoid this source of error is discussed.

The stable carbon isotope ¹³C is used as a universal tracer in plant eco-physiology and studies of carbon exchange between vegetation and atmosphere. Photosynthesis fractionates against ¹³CO ₂ so that source sugars (photosynthates) are on average ¹³C depleted by 20‰ compared with atmospheric CO ₂. The carbon isotope distribution within sugars has been shown to be heterogeneous, with relatively ¹³C-enriched and ¹³C-depleted C-atom positions. The ¹³C pattern within sugars is the cornerstone of ¹³C distribution in plants, because all metabolites inherit the ¹³C abundance in their specific precursor C-atom positions. However, the intramolecular isotope pattern in source leaf glucose and the isotope fractionation associated with key enzymes involved in sugar interconversions are currently unknown. To gain insight into these, we have analyzed the intramolecular isotope composition in source leaf transient starch, grain storage starch, and root storage sucrose and measured the site-specific isotope fractionation associated with the invertase (EC 3.2.1.26) and glucose isomerase (EC 5.3.1.5) reactions. When these data are integrated into a simple steady-state model of plant isotopic fluxes, the enzyme-dependent fractionations satisfactorily predict the observed intramolecular patterns. These results demonstrate that glucose and sucrose metabolism is the primary determinant of the ¹³C abundance in source and sink tissue and is, therefore, of fundamental importance to the interpretation of plant isotopic signals.

Secondary organic aerosol (SOA) plays a central role in air pollution and climate. However, the description of the sources and mechanisms leading to SOA is elusive despite decades of research. While stable isotope analysis is increasingly used to constrain sources of ambient aerosol, in many cases it is difficult to apply because neither the isotopic composition of aerosol precursors nor the fractionation of aerosol forming processes is well characterised. In this paper, SOA formation from ozonolysis of α-pinene – an important precursor and perhaps the best-known model system used in laboratory studies – was investigated using position-dependent and average determinations of 13C in α-pinene and advanced analysis of reaction products using thermal-desorption proton-transfer-reaction mass spectrometry (PTR-MS). The total carbon (TC) isotopic composition δ13C of the initial α-pinene was measured, and the δ13C of the specific carbon atom sites was determined using position-specific isotope analysis (PSIA). The PSIA analysis showed variations at individual positions from −6.9 to +10. 5 ‰ relative to the bulk composition. SOA was formed from α-pinene and ozone in a constant-flow chamber under dark, dry, and low-NOx conditions, with OH scavengers and in the absence of seed particles. The excess of ozone and long residence time in the flow chamber ensured that virtually all α-pinene had reacted. Product SOA was collected on two sequential quartz filters. The filters were analysed offline by heating them stepwise from 100 to 400 °C to desorb organic compounds that were (i) detected using PTR-MS for chemical analysis and to determine the O : C ratio, and (ii) converted to CO2 for 13C analysis. More than 400 ions in the mass range 39–800 Da were detected from the desorbed material and quantified using a PTR-MS. The largest amount desorbed at 150 °C. The O : C ratio of material from the front filter increased from 0.18 to 0.25 as the desorption temperature was raised from 100 to 250 °C. At temperatures above 250 °C, the O : C ratio of thermally desorbed material, presumably from oligomeric precursors, was constant. The observation of a number of components that occurred across the full range of desorption temperatures suggests that they are generated by thermal decomposition of oligomers. The isotopic composition of SOA was more or less independent of desorption temperature above 100 °C. TC analysis showed that SOA was enriched in 13C by 0.6–1.2 ‰ relative to the initial α-pinene. According to mass balance, gas-phase products will be depleted relative to the initial α-pinene. Accordingly, organic material on the back filters, which contain adsorbed gas-phase compounds, is depleted in 13C in TC by 0.7 ‰ relative to the initial α-pinene, and by 1.3 ‰ compared to SOA collected on the front filter. The observed difference in 13C between the gas and particle phases may arise from isotope-dependent changes in the branching ratios in the α-pinene + O3 reaction. Alternatively, some gas-phase products involve carbon atoms from highly enriched and depleted sites, as shown in the PSIA analysis, giving a non-kinetic origin to the observed fractionations. In either case, the present study suggests that the site-specific distribution of 13C in the source material itself governs the abundance of 13C in SOA.

Recent developments in ¹³C NMR spectrometry have allowed the determination of intramolecular ¹³C/¹²C ratios with high precision. However, the analysis of carbohydrates requires their derivatization to constrain the anomeric carbon. Fructose has proved to be particularly problematic because of a byproduct occurring during derivatization and the complexity of the NMR spectrum of the derivative. Here, we describe a method to determine the intramolecular ¹³C/¹²C ratios in fructose by ¹³C NMR analysis of the acetyl-isopropylidene derivative. We have applied this method to measure the intramolecular ¹³C/¹²C distribution in the fructosyl moiety of sucrose and have compared this with that in the glucosyl moiety. Three prominent features stand out. First, in sucrose from both C₃ and C₄ plants, the C-1 and C-2 positions of the glucosyl and fructosyl moieties are markedly different. Second, these positions in C₃ and C₄ plants show a similar profile. Third, the glucosyl and fructosyl moieties of sucrose from Crassulacean acid metabolism (CAM) metabolism have a different profile. These contrasting values can be interpreted as a result of the isotopic selectivity of enzymes that break or make covalent bonds in glucose metabolism, whereas the distinctive ¹³C pattern in CAM sucrose probably indicates a substantial contribution of gluconeogenesis to glucose synthesis.

The potential of wine .sup.13C isotope composition (δ.sup.13C) is presented to assess vine water status during grape ripening. Measurements of δ.sup.13C have been performed on a set of 32 authentic wines and their ethanol recovered after distillation. The data, obtained by isotope ratio monitoring by mass spectrometry coupled to an elemental analyser (irm-EA/MS), show a high correlation between δ.sup.13C of the bulk wine and its ethanol, indicating that the distillation step is not necessary when the wine has not been submitted to any oenological treatment. Therefore, the ethanol/wine δ.sup.13C correlation can be used as an indicator of possible enrichment of the grape must or the wine with exogenous organic compounds. Wine ethanol δ.sup.13C is correlated to predawn leaf water potential (R.sup.2 = 0.69), indicating that this parameter can be used as an indicator of vine water status. Position-specific .sup.13C analysis (PSIA) of ethanol extracted from wine, performed by isotope ratio monitoring by nuclear magnetic resonance (irm-.sup.13C NMR), confirmed the non-homogenous repartition of .sup.13C on ethanol skeleton. It is the δ.sup.13C of the methylene group of ethanol, compared to the methyl moiety, which is the most correlated to predawn leaf water potential, indicating that a phase of photorespiration of the vine during water stress period is most probably occurring due to stomata closure. However, position-specific .sup.13C analysis by irm-.sup.13C NMR does not offer a greater precision in the assessment of vine water status compared to direct measurement of δ.sup.13C on bulk wine by irm-EA/MS.

The potential of wine ¹³C isotope composition (δ¹³C) is presented to assess vine water status during grape ripening. Measurements of δ¹³C have been performed on a set of 32 authentic wines and their ethanol recovered after distillation. The data, obtained by isotope ratio monitoring by mass spectrometry coupled to an elemental analyser (irm-EA/MS), show a high correlation between δ¹³C of the bulk wine and its ethanol, indicating that the distillation step is not necessary when the wine has not been submitted to any oenological treatment. Therefore, the ethanol/wine δ¹³C correlation can be used as an indicator of possible enrichment of the grape must or the wine with exogenous organic compounds. Wine ethanol δ¹³C is correlated to predawn leaf water potential (R ² = 0.69), indicating that this parameter can be used as an indicator of vine water status. Position-specific ¹³C analysis (PSIA) of ethanol extracted from wine, performed by isotope ratio monitoring by nuclear magnetic resonance (irm-¹³C NMR), confirmed the non-homogenous repartition of ¹³C on ethanol skeleton. It is the δ¹³C of the methylene group of ethanol, compared to the methyl moiety, which is the most correlated to predawn leaf water potential, indicating that a phase of photorespiration of the vine during water stress period is most probably occurring due to stomata closure. However, position-specific ¹³C analysis by irm-¹³C NMR does not offer a greater precision in the assessment of vine water status compared to direct measurement of δ¹³C on bulk wine by irm-EA/MS.