Liquid Chromatography/isotope Ratio Mass Spectrometry Research Articles

Purity Assessment of Honey Based on Compound Specific Stable Carbon Isotope Ratios Obtained by LC-IRMS.

The use of stable carbon isotope ratios (δ13C) of sugar fractions of honey is a powerful tool to detect adulteration with sugar syrups. This is accomplished by calculating differences of the δ13C values between individual honey saccharides and comparing them to published purity criteria. A liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) method for the determination of δ13C values of sugars in honey was previously validated by an interlaboratory comparison but no further guidance was given how to include the obtained precision figures of the compound specific δ13C values in the purity assessment of honey. To use existing data to estimate the standard deviation of the repeatability (sr) and reproducibility (sR) of differences (Δ δ13C) between the δ13C values of individual honey saccharides. Previously published δ13C values were used to calculate differences (Δ δ13C values) between δ13C fructose-δ13C glucose, δ13C glucose-δ13C disaccharides, etc in a honey sample; sr and sR of Δ δ13C values were calculated according to ISO 5725-2:2019. The Δ δ13C sr and sR values were essentially of the same magnitude as the sr and sR values of δ13C values of the sugar fractions. The precision of the Δ δ13C values was used to estimate the critical difference for comparing a test result with a reference value according to ISO 5725-6:1994. This varied between 0.02 ‰ and 0.40 ‰. The estimated critical differences can be used to determine whether a honey test result complies with published Δ δ13C purity criteria. The proposed procedure will increase the confidence in decisions based on compound specific δ13C values regarding the conformity of honey with published purity criteria.

Using Liquid Chromatography-Isotope Ratio Mass Spectrometry for Detection of Economically Motivated Adulteration of Maple Syrup.

Maple syrup is a sought-after commodity, and used as a condiment and a sweetener. Also, it is an active target of economically motivated adulteration (EMA), similar to other foods such as lemon juice and honey. This study is aimed to detect low cost sugar adulteration in maple syrup via an internal standard method using malic acid through solid-phase extraction (SPE) and LC with isotope ratio mass spectrometric detection (LC-IRMS). In this work, an optimized SPE sample preparation procedure was used for the isolation of organic acids from maple syrup. Using LC-IRMS, malic acid was separated from other organic acids and the δ13C value of malic acid was determined. Eleven maple syrup samples, domestic or imported from Canada, were evaluated for 13C/12C ratios (δ13C values) using combustion module-cavity ring down spectrometry (CM-CRDS) and compared to the δ13C values obtained from well-established elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) methods. The δ13C values of isolated malic acid analyzed by SPE-LC-IRMS were used as internal standards and compared to the δ13C values of bulk maple syrup; difference (δ13Csugars - δ13Cmalic acid) values greater than 3.6‰ are indicative of low-cost sugar adulteration. Overall, the results obtained from SPE-LC-IRMS provided a faster, novel analysis approach for determining low-cost sugar adulteration in maple syrup for regulatory purposes. This method also provided lower detectable limits of adulteration versus current literature reports using bulk analysis and comparable detection limits to Tremblay and co-workers who utilized an internal standard method. SPE-LC-IRMS is a robust method that can be used for detecting adulteration in maple syrup samples for regulatory purposes. SPE-LC-IRMS is a faster, novel analysis approach for determining C4 adulteration in maple syrup with lower detection limits.

How to Couple LC-IRMS with HRMS─A Proof-of-Concept Study.

Compound-specific stable isotope analysis (CSIA) is a unique analytical technique for determining small variations in isotope ratios of light isotopes in analytes from complex mixtures. A problem of CSIA using gas chromatography (GC) and liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) is that any structural information of the analytes is lost due to the processes involved in determining the isotope ratio. To obtain the isotopic composition of, for example, carbon from organic compounds, all carbon in each analyte is quantitatively converted to CO2. For GC-IRMS, open split GC-IRMS-MS couplings have been described that allow additional acquisition of structural information of analytes and interferences. Structural analysis using LC-IRMS is more difficult and requires additional technical and instrumental efforts. In this study, LC was combined for the first time with simultaneous analysis by IRMS and high-resolution mass spectrometry (HRMS), enabling the direct identification of unknown or coeluting species. We have thoroughly investigated and optimized the coupling and showed how technical problems, arising from instrumental conditions, can be overcome. To this end, it was successfully demonstrated that a consistent split ratio between IRMS and HRMS could be obtained using a variable postcolumn flow splitter. This coupling provided reproducible results in terms of resulting peak areas, isotope values, and retention time differences for the two mass spectrometer systems. To demonstrate the applicability of the coupling, we chose to address an important question regarding the purity of international isotope standards. In this context, we were able to confirm that the USGS41 reference material indeed contains substantial amounts of pyroglutamic acid as suggested previously in the literature. Moreover, the replacement material, USGS41a, still has significant amounts of pyroglutamic acid as impurity, rendering some caution necessary when using this material for isotopic calibration.

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.

Carbon isotope ratio of organic acids in sake and wine by solid-phase extraction combined with LC/IRMS.

We developed an analytical procedure for determining the δ13C values of organic acids in sake and wine using solid-phase extraction combined with liquid chromatography/isotope ratio mass spectrometry (LC/IRMS). First, the solid-phase extraction (SPE) procedure was performed and various tests were conducted to extract organic acids from alcoholic beverages using the simulated sake sample. Under the optimal SPE procedure, high recovery rates (96-118%) and good accuracies (≤ 0.7‰) were thus achieved for the simulated sake and wine samples. Next, we determined the δ13C of organic acid (tartaric acid, malic acid, lactic acid, succinic acid) in 9 sake and 11 wine samples. Finally, the δ13C values of lactic acid in nine sake samples suggested that lactic acid had been added during the brewing process. The high correlation between the δ13C values of tartaric acid and malic acid in 11 wine samples was consistent with their common source, grapes. This analytical method may help to identify when organic acids have been added to sake and wine and to elucidate the process of organic acid production therein. Graphical abstract.

Compound-Specific Isotope Analysis of Human Hair: Predicting Behaviors and Biometrics beyond Dietary Factors.

This study describes the use of amino acid quantitation and amino-acid-specific isotope ratio analysis of scalp hair of American individuals to predict soft biometric traits about the donors. The scalp hair of each donor was washed, dried, homogenized and acid hydrolyzed before analysis using liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). A variety of statistical tests examined the relationships between the amino acid variables and biometric questionnaire responses, the latter of which could be assessed as continuous variables-in the case of age or body mass index (BMI)-or as categorical variables in the case of sex and alcohol consumption. Correlations between biometric factors and amino acid δ13C values were more significant after controlling for the extent of 13C in the subjects' diets. Multivariate analysis revealed that the sex of a donor could be correctly predicted with cross-validated accuracies of 80% using the isotope ratios and 89% using amino acid quantities. Using amino acid δ13C values or quantities, the age of a subject could only be predicted with an accuracy of ±27 years (95% CI). Hair treatments, such as chemical straitening and dying, did not have any measurable effect on the isotope ratios or quantities of amino acids in the hair. Unexpectedly, the δ13C values of sulfur-containing amino acids were significantly different between teetotalers and subjects who consumed alcohol daily. Further refinement of this study, including larger cohorts with controlled behavioral states or genetic factors, could provide helpful investigative leads in forensic casework.

Stable carbon isotope analysis of polyphosphonate complexing agents by anion chromatography coupled to isotope ratio mass spectrometry: method development and application.

Compound-specific carbon isotope analysis (carbon CSIA) by liquid chromatography/isotope ratio mass spectrometry (LC-IRMS) is a novel and promising tool to elucidate the environmental fate of polar organic compounds such as polyphosphonates, strong complexing agents for di- and trivalent cations with growing commercial importance over the last decades. Here, we present a LC-IRMS method for the three widely used polyphosphonates 1-hydroxyethane 1,1-diphosphonate (HEDP), amino tris(methylenephosphonate) (ATMP), and ethylenediamine tetra(methylenephosphonate) (EDTMP). Separation of the analytes, as well as ATMP and its degradation products, was carried out on an anion exchange column under acidic conditions. Quantitative wet chemical oxidation inside the LC-IRMS interface to CO2 was achieved for all three investigated polyphosphonates at a comparatively low sodium persulfate concentration despite the described resilience of HEDP towards oxidative breakdown. The developed method has proven to be suitable for the determination of carbon isotope fractionation of ATMP transformation due to manganese-catalyzed reaction with molecular oxygen, as well as for equilibrium sorption of ATMP to goethite. A kinetic isotope effect was associated with the investigated reaction pathway, whereas no detectable isotope fractionation could be observed during sorption. Thus, CSIA is an appropriate technique to distinguish between sorption and degradation processes that contribute to a concentration decrease of ATMP in laboratory batch experiments. Our study highlights the potential of carbon CSIA by LC-IRMS to gain a process-based understanding of the fate of polyphosphonate complexing agents in environmental as well as technical systems.

Heart-cutting two-dimensional liquid chromatography combined with isotope ratio mass spectrometry for the determination of stable carbon isotope ratios of gluconic acid in honey

Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) is used to analyze various types of samples, including foodstuffs, to determine their authenticity and trace their origin on the basis of their stable carbon isotope ratios (δ13C). However, multicomponent samples are difficult to analyze. For example, determining the δ13C values of the organic acids in honey is complicated by the presence of large amounts of carbohydrates. Herein, we present a heart-cutting two-dimensional LC/IRMS method for analysis of honey samples. In this method, the organic acids in the samples were first separated from the carbohydrates by a size-exclusion column, and then the organic acids were separated from each other by a reverse-phase column connected to the first column via a switching valve. By means of this method, the δ13C values for three organic acids in high-carbohydrate-content simulated honey samples could be determined with high accuracy and precision (≤0.3‰ and ≤0.1‰, respectively). In addition, the gluconic acid δ13C values for 25 honey samples were determined with high precision and found to range from −31.7 to −28.5‰ (mean: −30.0 ± 0.7‰). These values shed some light on the mechanism of gluconic acid production. Taken together, our results suggest that this two-dimensional LC method has the potential to be more effective than one-dimensional LC for use in isotopic research.

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.

13C- and 15N-Isotope Analysis of Desphenylchloridazon by Liquid Chromatography-Isotope-Ratio Mass Spectrometry and Derivatization Gas Chromatography-Isotope-Ratio Mass Spectrometry.

The widespread application of herbicides impacts surface water and groundwater. Metabolites (e.g., desphenylchloridazon from chloridazon) may be persistent and even more polar than the parent herbicide, which increases the risk of groundwater contamination. When parent herbicides are still applied, metabolites are constantly formed and may also be degraded. Evaluating their degradation on the basis of concentration measurements is, therefore, difficult. This study presents compound-specific stable-isotope analysis (CSIA) of nitrogen- and carbon-isotope ratios at natural abundances as an alternative analytical approach to track the origin, formation, and degradation of desphenylchloridazon (DPC), the major degradation product of the herbicide chloridazon. Methods were developed and validated for carbon- and nitrogen-isotope analysis (δ13C and δ15N) of DPC by liquid chromatography-isotope-ratio mass spectrometry (LC-IRMS) and derivatization gas chromatography-IRMS (GC-IRMS), respectively. Injecting standards directly onto an Atlantis LC-column resulted in reproducible δ13C-isotope analysis (standard deviation <0.5‰) by LC-IRMS with a limit of precise analysis of 996 ng of DPC on-column. Accurate and reproducible δ15N analysis with a standard deviation of <0.4‰ was achieved by GC-IRMS after derivatization of >100 ng of DPC with 160-fold excess of (trimethylsilyl)diazomethane. Application of the method to environmental-seepage water indicated that newly formed DPC could be distinguished from "old" DPC by the different isotopic signatures of the two DPC sources.

Determination of carbon isotope ratios for honey samples by means of a liquid chromatography/isotope ratio mass spectrometry system coupled with a post-column pump.

Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) has been used to authenticate and trace products such as honey, wine, and lemon juice, and compounds such as caffeine and pesticides. However, LC/IRMS has several disadvantages, including the high cost of the CO2 membrane and blocking by solidified sodium persulfate. Here, we developed an improved system for determining carbon isotope ratios using LC/IRMS. The main improvement was the use of a post-column pump. Using the improved system, we determined δ13 C values for glucose with high accuracy and precision (0.1‰ and 0.1‰, respectively; n=3). The glucose, fructose, disaccharide, trisaccharide, and organic acid constituents of honey samples were analyzed using LC/IRMS. The δ13 C values for glucose, fructose, disaccharides, trisaccharides, and organic acids ranged from -27.0 to -24.2‰, -26.8 to -24.0‰, -28.8 to -24.0‰, -27.8 to -22.8‰, and-30.6 to -27.4‰, respectively. The analysis time was a third to a half of that required for analysis by previously reported methods. The column flow rate could be arbitrarily adjusted with the post-column pump. We applied the improved method to 26 commercial honey samples. Our results can be expected to be useful for other researchers who use LC/IRMS.

MoDIE: Moderate dissolved inorganic carbon (DI13C) isotope enrichment for improved evaluation of DIC photochemical production in natural waters

Photochemical reactions in natural waters are a sink for dissolved organic carbon (DOC) and a source of dissolved inorganic carbon (DIC). Although considered significant to the carbon budget of the oceans, DIC photoproduction rates remain poorly constrained due to the analytical challenge involved in accurately measuring very low production rates (likely sub-μM h− 1 for blue ocean waters) relative to high background DIC concentrations (~ 2 mM). In an attempt to overcome this analytical limitation, almost all previous DIC photoproduction studies in marine systems have relied on the stripping of background DIC prior to irradiation, with unknown consequences for the integrity of the DOC pool and its photoreactivity, and none have resulted in a satisfactory determination of photoproduced DIC in open ocean waters. Here, we use small additions of NaH13CO3 (< 10% of background DIC concentrations) to achieve moderate DI13C isotope enrichment (MoDIE) of river, estuarine, and seawater samples. We then quantify the shift in δ13C of DIC in irradiated samples via liquid chromatography - isotope ratio mass spectrometry (LC-IRMS) and use these shifts to calculate photoproduced DIC. MoDIE provides a new method to more precisely (± 56 to ± 340 nM DIC) and more rapidly quantify DIC photoproduction compared to previous methods. MoDIE was evaluated by determining initial DIC photoproduction rates, along with associated broadband photochemical efficiency data (ranging from 144 to 280 μmol DIC per mol photons absorbed), in riverine to offshore waters, and produced the earliest time point and most precise measurement of DIC photoproduction in unmodified, low-CDOM, blue water (ag(325) = 0.30 m− 1) reported to date. In addition to measuring small DIC changes from photochemistry, the use of MoDIE could provide a major advance for other ocean carbon cycling studies, including examination of oceanic respiration, carbonate mineral precipitation/dissolution, and assessment of ocean acidification.

High-resolution palaeodietary reconstruction: Amino acid δ13C analysis of keratin from single hairs of mummified human individuals

Abstract Stable isotope analysis of mummified human hair allows the reconstruction of the recent life histories of individuals that died thousands of years ago. The objective of this study is to improve the resolution of hair palaeodietary reconstruction by sequentially analyzing 0.5 cm segments of a single hair using liquid chromatography-isotope ratio mass spectrometry (LC/IRMS). The subsistence strategies of seven individuals, spanning different cultures/periods (Chinchorro, Formative, Inca) and covering different geographic areas (coasts and hinterland) of the Atacama Desert, northern Chile, were reconstructed by analyzing δ 13 C amino acid compositions using an improved methodology that requires only 0.5 cm segments of a single hair. The amino acid δ 13 C values were supplemented with bulk carbon, nitrogen and sulfur isotope analysis performed on multiple hairs. Our results show that the archaic hunter-gatherers strongly relied on aquatic resources, as did the first transitional Formative individuals living on the coasts. Conversely, the Formative inhabitants of the nearby valley exhibit consumption of terrestrial resources in a seasonal pattern. A broader dietary spectrum made of mixed terrestrial and aquatic foods is identified in the Inca individual. The sequential analysis of 0.5 cm increments from a single hair has permitted the high-resolution reconstruction (approximately fortnightly) of the recent life history of these pre-Columbian individuals, discerning short-term nutritional changes related to agricultural cycle, multiple dietary intakes or mobility. Although bulk methods can detect changes in diet and track seasonal shifts, the variations in the carbon isotope signal can be highly attenuated with respect to the dietary intake because of the use of multiple hairs.

Using liquid chromatography‐isotope ratio mass spectrometry to measure the δ13C of dissolved inorganic carbon photochemically produced from dissolved organic carbon

AbstractDissolved inorganic carbon (DIC) has long been recognized as the main identifiable product of dissolved organic carbon (DOC) photochemical remineralization. However, quantification of DIC photoproduction in natural waters has been hampered by low photoproduction rates (nM to μM h−1) relative to high DIC background concentrations (μM to mM). Here, we describe a novel method to determine the δ13C of photoproduced DIC in three southeastern US river water samples (Savannah, Altamaha, St. Marys) using liquid chromatography‐isotope ratio mass spectrometry (LC‐IRMS). Initial (&lt; 4 h) DIC photoproduction rates were similar to those quantified using published methods that rely on extensive sample manipulation, but were 3.3 times higher than measured via the previous methods for prolonged exposures (&gt; 14 h). Keeling plots indicated that the average δ13C value for photoproduced DIC (−33.3 ± 1.4‰) was depleted in 13C relative to bulk riverine DOC, consistent with the preferential photomineralization of lignin. This average was used with a simple isotope mass balance approach to calculate the concentration of photoproduced DIC. The LC‐IRMS measures δ13C‐DIC shifts (typically ± 0.01 to 0.1‰) more precisely than DIC concentration changes in the early stages of irradiation. Therefore, the combination of LC‐IRMS analysis and our simple mass balance approach provides a new, higher precision method to quantify initial rates of DIC photoproduction and associated photochemical efficiency (i.e., apparent quantum yield) spectra in aquatic systems without any sample manipulation. A full description, sensitivity analysis and recommendations for the method are presented.

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.

Performance of the wet oxidation unit of the HPLC isotope ratio mass spectrometry system for halogenated compounds.

The performance of liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) for polar halogenated compounds was evaluated. Oxidation capacity of the system was tested with halogenated acetic acids and halogenated aromatic compounds. Acetic acid (AA) was selected as a reference compound for complete oxidation and compared on the molar basis to the oxidation of other analytes. The isotope values were proofed with calibrated δ(13)C values obtained with an elemental analyzer (EA). Correct isotope values were obtained for mono- and dichlorinated, fluorinated, and tribrominated acetic acids and also for aniline, phenol, benzene, bromobenzene, chlorobenzene, 1,2-dichlorobenzene, 2,4,6-trichlorophenol, pentafluorophenol, and nitrobenzene. Incomplete oxidation of trichloroacetic acid (TCA) and trifluoroacetic acid (TFA) resulted in lower recovery compared to AA (37% and 24%, respectively) and in isotopic shift compared to values obtained with EA (TCA Δδ(13)C(EA/LC-IRMS) = 8.8‰, TFA Δδ(13)C(EA/LC-IRMS) = 6.0‰). Improvement of oxidation by longer reaction time in the reactor and increase in the concentration of sulfate radicals did not lead to complete combustion of TCA and TFA needed for δ(13)C analysis. To the best of our knowledge, this is the first time such highly chlorinated compounds were studied with the LC-IRMS system. This work provides information for method development of LC-IRMS methods for halogenated contaminants that are known as potential threats to public health and the environment.

Carbon Isotope Ratio Analysis of Steroids by High-Temperature Liquid Chromatography-Isotope Ratio Mass Spectrometry

Generally, compound-specific isotope analysis of steroids is carried out by gas chromatography combined with isotope ratio mass spectrometry. Thus, a derivatization of the steroids prior to the measurement is compulsory, and a correction of the isotopic data is often necessary. To overcome this limitation, we present a new approach of high-temperature liquid chromatography coupled with photodiode array detection and isotope ratio mass spectrometry (HT-LC/PDA/IRMS) for the carbon isotope ratio analysis of unconjugated steroids. A steroid mixture containing 19-norandrosterone, testosterone, epitestosterone, androsterone, and 5β-pregnane-3α,17α,20α-triol was fully separated on a C4 column under high-temperature elution with water as the sole eluent. The accuracy for isotope analysis (±0.5 ‰) was around 20 μg g(-1) for testosterone, epitestosterone (79 ng steroid absolute on column), and 30 μg g(-1) for 19-norandrosterone, androsterone, and 5β-pregnane-3α,17α,20α-triol (119 ng steroid absolute on column). The applicability of the method was tested by measuring a pharmaceutical gel containing testosterone. With this work, the scope of LC/IRMS applications has been extended to nonpolar compounds.

Determination of exogenous γ-amylase residue in honey

A novel method for the determination of exogenous gamma-amylase residue in honey using liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) was established. After pre-separation by gel column chromatography, the gamma-amylase in honey samples was separated from the sugars. The gamma-amylase was then used to catalyze maltose into glucose. This enzymatic reaction was under the conditions of 55 degrees C and 0.03 mol/L phosphate buffer solution (pH 4.5) for 48 h. The maltose and glucose in the above enzymatic reaction solution were separated using liquid chromatography. By measuring the content of glucose with isotope ratio mass spectrometry, the gamma-amylase in honey can be determined. The linear range of gamma-amylase was 5 - 200 U/kg with the quantification limit of 5 U/kg. The recoveries were between 89.6% and 108.2% with the relative standard deviations from 3.3% to 4.9%. This method was used to analyze 38 honey and rice syrup samples, and the detection rate of gamma-amylase was 76.3%. To further verify the detection capability of this method, an authentic honey was adulterated with 15% (mass fraction) rice syrup. The gamma-amylase content in this sample was 10.2 U/kg. This method can effectively identify honey adulteration with rice syrups from the perspective of enzymology.

Determination of exogenous γ-amylase residue in honey

A novel method for the determination of exogenous γ-amylase residue in honey using liquid chromatography-isotope ratio mass spectrometry(LC-IRMS) was established.After pre-separation by gel column chromatography,the γ-amylase in honey samples was separated from the sugars.The γ-amylase was then used to catalyze maltose into glucose.This enzymatic reaction was under the conditions of 55 ℃ and 0.03 mol/L phosphate buffer solution(pH 4.5) for 48 h.The maltose and glucose in the above enzymatic reaction solution were separated using liquid chromatography.By measuring the content of glucose with isotope ratio mass spectrometry,the γ-amylase in honey can be determined.The linear range of γ-amylase was 5-200 U/kg with the quantification limit of 5 U/kg.The recoveries were between 89.6% and 108.2% with the relative standard deviations from 3.3% to 4.9%.This method was used to analyze 38 honey and rice syrup samples,and the detection rate of γ-amylase was 76.3%.To further verify the detection capability of this method,an authentic honey was adulterated with 15%(mass fraction) rice syrup.The γ-amylase content in this sample was 10.2 U/kg.This method can effectively identify honey adulteration with rice syrups from the perspective of enzymology.

Importance of microbial soil organic matter processing in dissolved organic carbon production

Soil dissolved organic carbon (DOC) sources and its seasonal dynamics are poorly known. We aimed to determine the contribution of plant and soil organic matter (SOM) to size classes of DOC in a field experiment with C3 to C4 vegetation change on two soil types through different seasons. Stable isotope ratios of DOC size classes were measured using size exclusion chromatography (SEC) coupled online to liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). SEC resolved DOC into three size classes: very high molecular weight/vHMW (>10kDa), high molecular weight/HMW (0.4-10kDa), and low molecular weight/LMW (<0.4kDa). HMW DOC was most abundant in all seasons, soil types, and depths. In contrast, vHMW DOC was only seen postsnowmelt in upper 20cm and was mainly (87±9%) plant-derived. Through all seasons, HMW and LMW DOC had less than 30% recent plant contribution. Similar size range and source of DOC size classes and soil chloroform fumigation extracts suggest microbial origin of DOC. Thus, microbial SOM recycling is an important process in DOC production. We suggest that DOC molecules get partitioned manifold between soil solution and the mineral matrix (chromatography), thereby getting constantly decomposed, altered, or produced anew by soil microorganisms (reactive transport).