Liquid Chromatography Isotope Ratio Mass Research Articles

C/H/O stable isotope of rape honey and its components combined with machine learning algorithmsto identify geographical origin.

The distribution of rape honey is among the largest and most diverse of all honeys available to humankind with respect to the geographical origin. Accurate isotopic reference values for rape honey are therefore important for precise verification of honey origin and its traceability. New combined rape honey δ13 C, δ2 H, and δ18 O values in combination with values on its compounds (protein and saccharides) were used to complement existing databases to better identify the geographical origin of Chinese rape honey. Traceability methods based on elemental analyzer isotope ratio mass spectrometry and liquid chromatography isotope ratio mass spectrometry were established for geographical origin of rape honey. Rape honey harvested in the high-altitude region (QH; Qinghai) had significantly higher values (1.4 to 5.3‰ for δ13 C, 7.9 to 12.9‰ for δ2 Hprotein ) for the δ13 C of whole honey (-23.8‰), its protein (-24.4‰), fructose (-23.5‰), glucose (-23.6‰), and disaccharide (-24.7‰), and also δ2 H of the protein (103.5‰) than those in low-altitude regions (HB; Hubei, SC; Sicuan, and JS; Jingsu). The δ18 Orape honey was a useful index to differentiate whether rape honey from coastal (JS) or non-coastal (HB, SC, and QH) regions. The δ13 C, δ2 H, and δ18 O values in rape honey are affected by geographical factors, such as temperature and altitude. The δ13 Cprotein and δ13 Crape honey values were better to identify the geographical origin of rape honey than δ13 Csaccharides . The δ18 O and δ2 H values of rape honey protein were more suitable for traceability than those of rape honey. The combination of the δ13 C, δ2 H, and δ18 O values of rape honey and its extracted protein and saccharides improved the precision of three models (linear discriminant analysis, SVM, and random forest) used to discriminate rape honey from different regions in China. The SVM model obtained the best accuracy (93.2%). Stable isotopes could be significant predictors in determining the geographical origin of rape honey.

Results of an Interlaboratory Comparison of a Liquid Chromatography-Isotope Ratio Mass Spectrometry Method for the Determination of 13C/12C Ratios of Saccharides in Honey.

BackgroundStable carbon isotope analysis of sugars in honey by LC–isotope ratio mass spectrometry (IRMS) is a useful tool for detecting adulteration of honey with extraneous sugar. Purity criteria based on 13C/12C ratios of saccharides in honey, determined by LC–IRMS of a large number of authentic honey samples, have been elaborated. However, no interlaboratory comparison (ILC) has yet been performed to estimate the precision of the method under reproducibility conditions.ObjectiveTo address this knowledge gap an ILC involving 14 laboratories and using six honey samples was conducted.MethodsThe participants were allowed to use their LC–IRMS-based method of choice for sample preparation and compound separation.ResultsThe precision figures were estimated according to ISO 5725:1994. The repeatability relative standard deviation (RSDr) for the determination of δ13C values of fructose and glucose varied between 0.3 and 0.5%, with 0.3 and 1.0% for disaccharides, and 0.7 and 2.8% for trisaccharides. The RSDR varied between 0.8 and 1.8% for the monosaccharides, 1.0 and 1.5% for disaccharides, and 1.4 and 2.8% for trisaccharides.ConclusionBased on the obtained precision data the LC–IRMS method for the determination of 13C/12C ratios of saccharides in honey was considered fit for the conformity assessment of honey with established purity criteria.HighlightsPrecision estimates for a LC–IRMS method to determine 13C/12C ratios of saccharides in honey were obtained through an ILC. The data created can form the basis for the standardization of the method by interested standards-developing organizations for use in official control.

Geographical Origin Classification of Chinese Wines Based on Carbon and Oxygen Stable Isotopes and Elemental Profiles

Wines from different regions have different qualities due to the impact of geographical location and climate. The sale of inferior wines seriously violates the fair-trade rights of consumers. This article provides an elemental analysis classification method for verifying the geographical origin of wines in the People's Republic of China. Inductively coupled plasma mass spectrometry, liquid chromatography isotope ratio mass spectrometry, and an isotope ratio mass spectrometer were used to analyze 142 wine samples collected from Helan Mountain, Xinjiang, Yunchuanzang, the Yanhuai Valley, and the Hexi Corridor regions. The data included elemental profiles, carbon isotope ratios (δ13C), and oxygen isotope ratios (δ18O). The results of multivariate analysis revealed that the geographical origin of wine is closely related to variations in elemental profiles and isotope ratios. Introducing δ18O and the elements Li, Mn, Ag, In, Th, Ta, and Re into the discriminant model yielded correct classification rates of the linear discriminant model of 90.8% for the training set and 87.3% for the test set.

New Concepts for the Determination of Oxidation Efficiencies in Liquid Chromatography-Isotope Ratio Mass Spectrometry.

In liquid chromatography coupled to isotope ratio mass spectrometry (LC-IRMS), analytes are separated on an LC system and consecutively oxidized to CO2, which is required for the determination of compound-specific carbon isotope ratios. Oxidation is performed in an online reactor by sulfate radicals. Reaction conditions in the interface depend on the flow conditions determined by the LC method and the flow rates and concentrations of oxidation agent and phosphoric acid added in the interface. To determine accurate isotope ratios, a quantitative conversion of the carbon contained in the analyte to the CO2 measurement gas is a prerequisite. Oxidation efficiencies are not commonly evaluated during method development, although certain analytes are known to be difficult to be oxidized by sulfate radicals. For the assessment of the oxidation efficiency of the LC-IRMS system, three different approaches were evaluated. (1) Residual organic carbon in the eluent stream of the interface was determined to calculate oxidation yields depending on the initial analyte concentration. (2) The IRMS response was calibrated to an inorganic carbon reference material to determine oxidation efficiencies with the help of the IRMS as a detector. (3) The oxidation temperature was deliberately reduced while monitoring the δ13C and signal intensity. The common assumption that a linear relation of IRMS signal to analyte concentration is an indicator for complete oxidation in LC-IRMS could be disproved. All three approaches can be applied for future method development in LC-IRMS, monitoring of existing flow injection applications, as well as for verification of complete oxidation in established LC-IRMS methods.

Linking molecular size, composition and carbon turnover of extractable soil microbial compounds

Microbial contribution to the maintenance and turnover of soil organic matter is significant. Yet, we do not have a thorough understanding of how biochemical composition of soil microbial biomass is related to carbon turnover and persistence of different microbial components. Using a suite of state-of-the-art analytical techniques, we investigated the molecular characteristics of extractable microbial biomass and linked it to its carbon turnover time. A 13CO2 plant pulse labelling experiment was used to trace plant carbon into rhizosphere soil microbial biomass, which was obtained by chloroform fumigation extraction (CFE). 13C content in molecular size classes of extracted microbial compounds was analysed using size exclusion chromatography (SEC) coupled online to high performance liquid chromatography–isotope ratio mass spectrometry (SEC-HPLC-IRMS). Molecular characterization of microbial compounds was performed using complementary approaches, namely SEC-HPLC coupled to Fourier transform infrared spectroscopy (SEC-HPLC-FTIR) and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS). SEC-HPLC-FTIR suggests that mid to high molecular weight (MW) microbial compounds were richer in aliphatic CH bonds, carbohydrate-like compounds and possibly PO derivatives from phospholipids. On the contrary, the lower size range was characterized by more oxidised compounds with hydroxyl, carbonyl, ether and/or carboxyl groups. ESI-FT-ICR-MS suggests that microbial compounds were largely aliphatic and richer in N than the background detrital material. Both molecular characterization tools suggest that CFE derived microbial biomass was largely lipid, carbohydrate and protein derived. SEC-HPLC-IRMS analysis revealed that 13C enrichment decreased with increasing MW of microbial compounds and the turnover time was deduced as 12.8 ± 0.6, 18.5 ± 0.6 and 22.9 ± 0.7 days for low, mid and high MW size classes, respectively. We conclude that low MW compounds represent the rapidly turned-over metabolite fraction of extractable soil microbial biomass consisting of organic acids, alcohols, amino acids and sugars; whereas, larger structural compounds are part of the cell envelope (likely membrane lipids, proteins or polysaccharides) with a much lower renewal rate. This relation of microbial carbon turnover to its molecular size, structure and composition thus highlights the significance of cellular biochemistry in determining the microbial contribution to soil carbon cycling and specifically soil organic matter formation.

Soil microbial carbon turnover decreases with increasing molecular size

It is well established that soil microorganisms play an important role in respiration of newly fixed plant carbon. Recent results show that they also contribute significantly to soil organic matter (SOM) formation. We hypothesized that different molecular size classes of compounds in soil microbial biomass (SMB) have variable turnover time and in consequence influence SOM formation differentially. Here we used natural differences in carbon stable isotope signatures (δ13C values) after C3–C4 vegetation change to track newly fixed C4 plant carbon into SMB molecular size classes. SMB was obtained by chloroform fumigation extraction (SFE) and δ13C values of its size classes were measured using size exclusion chromatography coupled online to liquid chromatography‒isotope ratio mass spectrometry (SEC–LC–IRMS). Resolved SMB was assigned to 5 size classes of 1800–9800, 800–1800, 380–800, 180–380 and 50–180 Da respectively. The contribution of recent C4 plant carbon to size classes of SMB decreased with increasing molecular weight (MW). It ranged from 77 ± 19% in the lowest MW size class size class to 41 ± 14% in the highest MW size class in a sandy soil and from 59 ± 18% in the lowest MW size class to 8 ± 15% in the highest MW size class in a clayey soil. A decreasing carbon turnover of compounds in SMB extracts along a continuum of molecular size from small to large implies that low molecular weight microbial compounds are rapidly metabolized products that link to fast respiratory carbon fluxes, whereas high molecular weight ones could be products of microbial synthesis like structural compounds that have slower turnover rates and link to slower SOM formation. Our methods help avoid contamination of CFE extracts and the results help explain why SMB turnover is faster in CFE extracts when compared to calculations using membrane lipids (e.g. PLFA-based).

δ13C analysis of bulk organic matter in speleothems using liquid chromatography–isotope ratio mass spectrometry

The determination of δ13C values in speleothems is of considerable importance in palaeoenvironmental research, but has focussed solely on analysis of the carbonate. Here we demonstrate a new method for analysing the δ13C values of organic matter (OM) trapped in speleothems, utilising flow injection liquid chromatography–isotope ratio mass spectrometry (LC–IRMS). Developmental analysis using a homogenised speleothem powder showed that the method is robust, with repeated digests and analyses having an average standard deviation of 0.1‰. Dilution tests with samples of 4–23μg total organic carbon (TOC) show relatively small linearity effects, with the overall standard deviation across a peak response range of 1700–9000mV being 0.2‰.

Kinetics of amino sugar formation from organic residues of different quality

Amino sugars are key compounds of microbial cell walls, which have been widely used as biomarker of microbial residues to investigate soil microbial communities and organic residue cycling processes. However, the formation dynamics of amino sugar is not well understood. In this study, two agricultural Luvisols under distinct tillage managements were amended with uniformly 13C-labeled wheat residues of different quality (grain, leaf and root). The isotopic composition of individual amino sugars and CO2 emission were measured over a 21-day incubation period using liquid chromatography–isotope ratio mass spectrometry (LC–IRMS) and trace gas IRMS. Results showed that, the amount of residue derived amino sugars increased exponentially and reached a maximum within days after residue addition. Glucosamine and galactosamine followed different formation kinetics. The maxima of residue derived amino sugars formation ranged from 14 nmol g−1 dry soil for galactosamine (0.8% of the original concentration) to 319 nmol g−1 dry soil for glucosamine (11% of the original concentration). Mean production times of residue derived amino sugars ranged from 2.1 to 9.3 days for glucosamine and galactosamine, respectively. In general, larger amounts of amino sugars were formed at a higher rate with increasing plant residue quality. The microbial community of the no-till soil was better adapted to assimilate low quality plant residues (i.e. leaf and root). All together, the formation dynamics of microbial cell wall components was component-specific and determined by residue quality and soil microbial community.

Determination of Suitable Column Geometries by Means of van Deemter and Kinetic Plots for Isothermal and Isocratic Method Development in High-Temperature Liquid Chromatography Isotope Ratio Mass Spectrometry

The method of high-temperature liquid chromatography isotope ratio mass spectrometry (HTLC-IRMS) is used to determine the origin or authenticity of compounds. Currently, the drawback of this hyphenation is the interface which causes pronounced band broadening due to a large extra-column volume. Therefore, the aim of this study is to determine suitable column geometries and particle sizes at different temperature and to study the effect of extra-column band broadening. The tools to assess the efficiency of columns are van Deemter and kinetic plots. By comparison of different column geometries and particle sizes, it could be shown that 3.0 mm ID columns achieve a higher performance than 2.1 mm ID columns and a particle size of 1.7 μm is advantageous over 3.5 and 5.0 μm particles when the injection volume is adjusted to 2 μL and the temperature is higher than 60 °C. Because water was the mobile phase, the retention factor could not be kept constant at different column temperatures. The lower retention factor at elevated temperatures leads to a decrease of the plate number, because of the relatively larger contribution to extra-column band broadening at lower retention factors. This is the reason why 3.0 mm ID columns should be preferred for the HTLC-IRMS hyphenation when the separation is carried out under isothermal and isocratic conditions.

Comparison of liquid chromatography–isotope ratio mass spectrometry (LC/IRMS) and gas chromatography–combustion–isotope ratio mass spectrometry (GC/C/IRMS) for the determination of collagen amino acid δ13C values for palaeodietary and palaeoecological reconstruction

Results are presented of a comparison of the amino acid (AA) δ(13)C values obtained by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) and liquid chromatography-isotope ratio mass spectrometry (LC/IRMS). Although the primary focus was the compound-specific stable carbon isotope analysis of bone collagen AAs, because of its growing application for palaeodietary and palaeoecological reconstruction, the results are relevant to any field where AA δ(13)C values are required. We compare LC/IRMS with the most up-to-date GC/C/IRMS method using N-acetyl methyl ester (NACME) AA derivatives. This comparison involves the analysis of standard AAs and hydrolysates of archaeological human bone collagen, which have been previously investigated as N-trifluoroacetyl isopropyl esters (TFA/IP). It was observed that, although GC/C/IRMS analyses required less sample, LC/IRMS permitted the analysis of a wider range of AAs, particularly those not amenable to GC analysis (e.g. arginine). Accordingly, reconstructed bulk δ(13)C values based on LC/IRMS-derived δ(13)C values were closer to the EA/IRMS-derived δ(13)C values than those based on GC/C/IRMS values. The analytical errors for LC/IRMS AA δ(13)C values were lower than GC/C/IRMS determinations. Inconsistencies in the δ(13)C values of the TFA/IP derivatives compared with the NACME- and LC/IRMS-derived δ(13)C values suggest inherent problems with the use of TFA/IP derivatives, resulting from: (i) inefficient sample combustion, and/or (ii) differences in the intra-molecular distribution of δ(13)C values between AAs, which are manifested by incomplete combustion. Close similarities between the NACME AA δ(13)C values and the LC/IRMS-derived δ(13)C values suggest that the TFA/IP derivatives should be abandoned for the natural abundance determinations of AA δ(13)C values.

Investigation of amino acid δ 13C signatures in bone collagen to reconstruct human palaeodiets using liquid chromatography–isotope ratio mass spectrometry

This research presents the individual amino acid δ 13C values in bone collagen of humans ( n = 9) and animals ( n = 27) from two prehistoric shell midden sites in Korea. We obtained complete baseline separation of 16 of the 18 amino acids found in bone collagen by using liquid chromatography–isotope ratio mass spectrometry (LC–IRMS). The isotopic results reveal that the humans and animals in the two sites had similar patterns in essential amino acids (EAAs) and non-essential amino acids (NEAAs). The EAA and NEAA δ 13C values in humans are intermediate between those in marine and terrestrial animals. However, the threonine δ 13C values in humans and animals measured in this study are more highly enriched than those of other amino acids. At both sites, all amino acids in marine animals are 13C-enriched relative to those of the terrestrial animals. The isotopic evidence suggests that the Tongsamdong human had EAAs and NEAAs from marine food resources, while the Nukdo humans mainly had EAAs from terrestrial food resources but obtained NEAAs from both terrestrial and marine resources. The δ 13C isotopic differences in amino acids between marine and terrestrial animals were the largest for glycine (NEAA) and histidine (EAA) and the smallest for tyrosine (NEAA) and phenylalanine (EAA). In addition, threonine among the EAAs also had a large difference (∼8‰) in δ 13C values between marine and terrestrial animals, and has the potential to be used as an isotopic marker in palaeodietary studies. Threonine δ 13C values were used in conjunction with the established Δ 13C Glycine–phenylalanine values and produced three distinct dietary groups (terrestrial, omnivorous, and marine). In addition, threonine δ 13C values and Δ 13C Serine–phenylalanine values were discovered to separate between two dietary groups (terrestrial vs. marine), and these δ 13C values may provide a potential new indicator for investigating the distinction between marine and terrestrial protein sources in human diets.

A three-phase liquid chromatographic method for δ 13C analysis of amino acids from biological protein hydrolysates using liquid chromatography–isotope ratio mass spectrometry

We report a three-phase chromatographic method for the separation and analysis of δ 13C values of underivatized amino acids from biological proteins (keratin, collagen, and casein) using liquid chromatography–isotope ratio mass spectrometry (LC–IRMS). Both precision and accuracy of δ 13C values for standard amino acid mixtures over the range of approximately 8 to 1320 ng of carbon per amino acid on the column were assessed. The precision of δ 13C values of amino acids was found to be better at higher concentrations, whereas accuracy improved at lower concentrations. The optimal performance for this method was achieved with between 80 and 660 ng of carbon of each amino acid on the column. At amino acid amounts lower than 20 ng of carbon on the column, precision and accuracy may become compromised. The application of this new three-phase chromatographic technique will allow the analysis of δ 13C of amino acids to be carried out as a routine method and benefit fields of research such as biomedicine, forensics, ecology, nutrition, and palaeodiet reconstruction in archaeology.

Online stable carbon isotope ratio measurement in formic acid, acetic acid, methanol and ethanol in water by high performance liquid chromatography–isotope ratio mass spectrometry

A suitable analysis condition was determined for high performance liquid chromatography–isotope ratio mass spectrometry (HPLC–IRMS) while making sequential measurements of stable carbon isotope ratios of δ 13C in formic acid, acetic acid, methanol and ethanol dissolved in water. For this online column separation method, organic reagents are not applicable due to carbon contamination; thus, water and KH 2PO 4 at low concentrations were tested as mobile phase in combination with a HyPURITY AQUASTAR™ column. Formic acid, acetic acid, methanol and ethanol were separated when 2 mM KH 2PO 4 aqueous solution was used. Under the determined analysis condition for HPLC–IRMS, carbon concentrations could be measured quantitatively as well as carbon isotope ratio when carbon concentration was higher than 0.4 mM L for each chemical.