A new concept for isotope ratio monitoring liquid chromatography/mass spectrometry

A new interface for the on-line coupling of a liquid chromatograph to a stable isotope ratio mass spectrometer has been developed and tested. The interface is usable for (13)C/(12)C determination of organic compounds, allowing measurement of small changes in (13)C abundance in individual analyte species. All of the carbon in each analyte is quantitatively converted into CO(2) while the analyte is still dissolved in the aqueous liquid phase. This is accomplished by an oxidizing agent such as ammonium peroxodisulfate. The CO(2) is separated from the liquid phase and transferred to the mass spectrometer. It is shown that the whole integrated process does not introduce isotope fractionation. The measured carbon isotope ratios are accurate and reproducible. The sensitivity of the complete system allows isotope ratio determination down to 400 ng of compound on-column. By-passing the high-performance liquid chromatography (HPLC) separation allows bulk isotopic analysis with substantially lower sample amounts than those required by conventional elemental analyzers. The results of the first applications to amino acids, carbohydrates, and drugs, eluted from various types of HPLC columns, are presented. The wide range of chromatographic methods enables the analysis of compounds never before amenable to isotope ratio mass spectrometry techniques and may lead to the development of many new assays.     

Improved isotope ratio measurement performance in liquid chromatography/isotope ratio mass spectrometry by removing excess oxygen

A low dead volume oxygen scrubbing system was introduced in a commercially available liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) interface to enhance the analytical capability of the system. In the LC/IRMS interface carbon from organic samples is converted into CO(2) inside the mobile phase by wet chemical oxidation using peroxodisulfate (Na(2)S(2)O(8)). After passing the hot reaction zone, surplus oxygen (O(2)) remains dissolved in the liquid phase. Both CO(2) and O(2) diffuse through a transfer membrane into the helium carrier and are transferred to the mass spectrometer. The presence of O(2) in the ion source may have detrimental effects on measurement accuracy and precision as well as on filament lifetime. As a remedy, a new on-line O(2)-removing device has been incorporated into the system.The new O(2) scrubber consists of two parallel hot copper reduction reactors (0.8 mm i.d., active length 120 mm) and a switch-over valve between them. One reactor is regenerated using He/H(2) while the other is actively scavenging O(2) from the gas stream. The capacity of each reduction reactor, expressed as usage time, is between 40 and 50 min. This is sufficient for a single LC run for sugars and organic acids. A further increase of the reduction capacity is accompanied by a peak broadening of about 100%. After switching to a freshly reduced reactor the oxygen background and the delta(13)C values of the reference gas need up to 500 s to stabilize. For repeated injections the delta(13)C values of sucrose remain constant (+/-0.1 per thousand) for about 3000 s. The long-term stability for measurements of sucrose was 0.11 per thousand without the reduction oven and improved slightly to 0.08 per thousand with the reduction oven. The filament lifetime improved by more than 600%, thereby improving the long-term system stability and analytical efficiency. In addition the costs per analysis were reduced considerably.     

Direct analysis of carbon isotope variability in albumins by liquid flow-injection isotope ratio mass spectrometry

We demonstrate the high precision C isotopic analysis of a series of purified albumins by liquid chromatography-combustion isotope ratio mass spectrometry (IRMS) by using direct aqueous liquid injection. Albumins from 18 species and albumens from chicken and turkey egg were obtained from a commercial source and shown to be of > 98% purity by capillary zone electrophoresis and high-performance liquid chromatography. One microliter of an aqueous protein solution with a total of < 40-pmol protein (2. 5 µg), which contained about 150-nmol C, was injected directly into a flowing stream of high-performance liquid chromatography grade water. The solution passed through a pneumatic nebulizer, was sprayed onto a moving wire, passed through a drying oven, and was combusted in a furnace. After the water of combustion was removed, the resulting CO₂ gas was directed to a high precision IRMS instrument operated in continuous flow mode. The average precision across the 20 samples analyzed was SD(δ ¹³C)=0.45%., and the average accuracy was δ¹³C < 0.4%. compared to aliquots analyzed by conventional preparation by using combustion tubes and dual inlet analysis. The observed isotope ratio range was about ?22.5%. < δ ¹³C_PDB < ?16%. as expected for modern materials from a natural source. These results demonstrate rapid, high precision, and accurate C isotopic analysis of untreated macromolecules in an aqueous stream by liquid source IRMS.     

Position‐specific carbon isotope analysis of trichloroacetic acid by gas chromatography/isotope ratio mass spectrometry

Trichloroacetic acid (TCAA) is an important environmental contaminant present in soils, water and plants. A method for determining the carbon isotope signature of the trichloromethyl position in TCAA using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) was developed and tested with TCAA from different origins. Position‐specific isotope analysis (PSIA) can provide direct information on the kinetic isotope effect for isotope substitution at a specific position in the molecule and/or help to distinguish different sources of a compound. The method is based on the degradation of TCAA into chloroform (CF) and CO₂ by thermal decarboxylation. Since thermal decarboxylation is associated with strong carbon isotope fractionation (ε = ?34.6 ± 0.2‰) the reaction conditions were optimized to ensure full conversion. The combined isotope ratio of CF and CO₂ at the end of the reaction corresponded well to the isotope ratio of TCAA, confirming the reliability of the method. A method quantification limit (MQL) for TCAA of 18.6 µg/L was determined. Samples of TCAA produced by enzymatic and non‐enzymatic chlorination of natural organic matter (NOM) and some industrially produced TCAA were used as exemplary sources. Significant different PSIA isotope ratios were observed between industrial TCAA and TCAA samples produced by chlorination of NOM. This highlights the potential of the method to study the origin and the fate of TCAA in the environment. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantification of intracellular homocysteine by stable isotope dilution liquid chromatography/tandem mass spectrometry

A precise and accurate stable isotope dilution liquid chromatography/tandem mass spectrometry method for the analysis of intracellular homocysteine has been developed. An internal standard, [(2)H(8)]-homocystine, was added to cell pellets from EA.hy 926 cells grown in culture under low and high folate concentrations. D,L-dithiothreitol was used to reduce cellular homocystine to homocysteine. Cellular proteins were precipitated by the addition of formic acid in acetonitrile. After centrifugation, a portion of the supernatant was analyzed by liquid chromatography/tandem mass spectrometry. Using a Supelcosil cyano column with an Applied Biosystems API 4000 triple quadrupole mass spectrometer, the SRM transitions for homocysteine (m/z 136 to m/z 90) and [(2)H(4)]-homocysteine (m/z 140 to m/z 94) were monitored. The method was validated by conducting five replicate analyses on three different days at four different concentrations (concentrations at the lower limit of quantitation and expected lower quartile, mid-range and upper quartile). The limit of detection was 2 ng/10(6) EA.hy 926 cells. Using this method, the intracellular homocysteine concentration in EA.hy 926 cells ranged from 10 to 36 ng/10(6) cells.     

Determination of phenylalanine isotope ratio enrichment by liquid chromatography/time- of-flight mass spectrometry

The application of time-of-flight mass spectrometry to isotope ratio measurements has been limited by the relatively low dynamic range of the time-to-digital converter detectors available on commercial LC/ToF-MS systems. Here we report the measurement of phenylalanine isotope ratio enrichment by using a new LC/ToF-MS system with wide dynamic range. Underivatized phenylalanine was injected onto a C18 column directly with 0.1% formic acid/acetonitrile as the mobile phase. The optimal instrument parameters for the time-of-flight mass spectrometer were determined by tuning the instrument with a phenylalanine standard. The accuracy of the isotope enrichment measurement was determined by the injection of standard solutions with known isotope ratios ranging from 0.02% to 9.2%. A plot of the results against the theoretical values gave a linear curve with R2 of 0.9999. The coefficient of variation for the isotope ratio measurement was below 2%. The method is simple, rapid, and accurate and presents an attractive alternative to traditional GC/MS applications.     

Gas chromatography/combustion/isotope ratio mass spectrometric analysis of the stable carbon composition of tetrols

The stable carbon isotope compositions of tetrols, erythritol and threitol were determined by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Using four tetrols with various δ¹³C values derivatized by methylboronic acid, the carbon isotope analysis method achieved excellent reproducibility and high accuracy. There was no carbon isotopic fractionation during the derivatization processes. The differences in the carbon isotopic compositions of methylboronates between the measured and calculated ranged from ?0.20 to 0.12‰, within the specification of the GC/C/IRMS system. It was demonstrated that δ¹³C values of tetrols could be calculated by a simple mass balance equation between tetrols, methylboronic acid, and methylboronates. The analogous 2‐methyltetrols, marker compounds of photooxidation products of atmospheric isoprene, should have similar behavior using the same derivatization reagent. This method may provide insight on sources and sinks of atmospheric isoprene. Copyright © 2009 John Wiley & Sons, Ltd.     

High-temperature reversed-phase liquid chromatography coupled to isotope ratio mass spectrometry

Compound‐specific isotope analysis (CSIA) by liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) has until now been based on ion‐exchange separation. In this work, high‐temperature reversed‐phase liquid chromatography was coupled to, and for the first time carefully evaluated for, isotope ratio mass spectrometry (HT‐LC/IRMS) with four different stationary phases. Under isothermal and temperature gradient conditions, the column bleed of XBridge C₁₈ (up to 180 °C), Acquity C₁₈ (up to 200 °C), Triart C₁₈ (up to 150 °C), and Zirchrom PBD (up to 150 °C) had no influence on the precision and accuracy of δ¹³C measurements, demonstrating the suitability of these columns for HT‐LC/IRMS analysis. Increasing the temperature during the LC/IRMS analysis of caffeine on two C₁₈ columns was observed to result in shortened analysis time. The detection limit of HT‐RPLC/IRMS obtained for caffeine was 30 mg L^–1 (corresponding to 12.4 nmol carbon on‐column). Temperature‐programmed LC/IRMS (i) accomplished complete separation of a mixture of caffeine derivatives and a mixture of phenols and (ii) did not affect the precision and accuracy of δ¹³C measurements compared with flow injection analysis without a column. With temperature‐programmed LC/IRMS, some compounds that coelute at room temperature could be baseline resolved and analyzed for their individual δ¹³C values, leading to an important extension of the application range of CSIA. Copyright © 2011 John Wiley & Sons, Ltd.     

Carbon isotope ratio measurements of glyphosate and AMPA by liquid chromatography coupled to isotope ratio mass spectrometry

The interest in compound-specific isotope analysis for product authenticity control and source differentiation in environmental sciences has grown rapidly during the last decade. However, the isotopic analysis of very polar analytes is a challenging task due to the lack of suitable chromatographic separation techniques which can be used coupled to isotope ratio mass spectrometry. In this work, we present the first method to measure carbon isotope compositions of the widely applied herbicide glyphosate and its metabolite aminomethylphosphonic acid (AMPA) by liquid chromatography coupled to isotope ratio mass spectrometry. We demonstrate that this analysis can be carried out either in cation exchange or in reversed-phase separation modes. The reversed-phase separation yields a better performance in terms of resolution compared with the cation exchange method. The measurement of commercial glyphosate herbicide samples show its principal applicability and reveals a wide range of δ¹³C values between ?24 and ?34 ‰ for different manufacturers. The absolute minimum amounts required to perform a precise and accurate determination of carbon isotope compositions of glyphosate and AMPA were in the sub-microgram range. The method proposed is sensitive enough to further perform the experiments that are necessary to better understand the carbon isotope fractionation associated to the natural degradation of glyphosate into AMPA. Furthermore, it can be used for contaminant source allocation and product authenticity as well.     

Congener-specific analysis of hexabromocyclododecane by high-performance liquid chromatography/electrospray tandem mass spectrometry

A congener-specific method based on high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS) in the negative ion mode was developed for the analysis of hexabromocyclododecane (HBCDD). On a C(18) analytical column, with a methanol/water mobile phase, the alpha-isomer was completely resolved from the beta- and gamma-isomers while the beta- and gamma-isomers were sufficiently resolved at half their peak heights. The ES spray voltage strongly influenced the intensity of the ion signal. For MS, a source temperature of 500 degrees C and a collision energy of 50 eV were found to be optimum for the [M-H](-) to Br(-) transition. Run-to-run and day-to-day (n = 3) variability was minimal, with relative standard deviations of 2.6-4.1 and 2.4-4.4%, respectively. The limit of detection was 4-6 pg on-column. When applied to tissue samples from Lake Winnipeg fish both alpha- and gamma-isomers of HBCDD were found in low-ng/g (lipid corrected) concentrations.     

Site-specific carbon isotope analysis of aromatic carboxylic acids by elemental analysis/pyrolysis/isotope ratio mass spectrometry

Site-specific carbon isotope composition of organic compounds can provide useful information on their origin and history in natural environments. Site-specific isotope analyses of small amounts of organic compounds (sub-nanomolar level), such as short-chain carboxylic acids and amino acid analogues, have been performed using gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/pyrolysis/IRMS). These analyses were previously limited to volatile compounds. In this study, site-specific carbon isotope analysis has been developed for non-volatile aromatic carboxylic acids at sub-micromolar level by decarboxylation using a continuous flow elemental analysis (EA)/pyrolysis/IRMS technique. Benzoic acid, 2-naphthylacetic acid and 1-pyrenecarboxylic acid were pyrolyzed at 500-1000 degrees C by EA/pyrolysis/IRMS to produce CO2 for delta13C measurement of the carboxyl group. These three aromatic acids were most efficiently pyrolyzed at 750 degrees C. Conventional sealed-tube pyrolysis was also conducted for comparison. The delta13C values of CO2 generated by the continuous flow technique were within 1.0 per thousand of those performed by the conventional technique, indicating that the new continuous flow technique can accurately analyze the carbon isotopic composition of the carboxyl group in aromatic carboxylic acids. The new continuous flow technique is simple, rapid and uses small sample sizes, so this technique will be useful for characterizing the isotopic signature of carboxyl groups in organic compounds.     

Solid-phase microextraction method for carbon isotopic analysis of volatile carboxylic acids in human plasma by gas chromatography/combustion/isotope ratio mass spectrometry

A new analytical method is described for the determination of the physiological concentration and low-level enrichment of (13)C-short-chain volatile organic acids (SCVAs) (e.g. (13)C-acetate and (13)C-butyrate) in human plasma. This two-step method involves solid-phase microextraction (SPME) coupled to gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) without any organic solvents or derivatizing agents. Two SCVA extraction methods were compared using a carboxen/polydimethylsiloxane fiber: headspace sampling (HS) and liquid sampling (LS) SPME. The influences of extraction temperature and time were tested to optimize the adsorption of SCVAs onto the fiber. The comparison of the peak area responses of the acids in the two adsorption methods showed better sensitivity in the human physiological concentration range in the LS mode than in the HS mode.The accuracy of isotopic enrichment measurement was determined using plasma spiked with (13)C-acetate and (13)C-butyrate solution from 0 to 1 mol percent excess (MPE). The linearity and repeatability (RSD < 5%) were measured in LS mode. Plasma SCVA concentrations were also determined relative to 3-methylvalerate (internal standard). Linearity and repeatability were observed from 0 to 400 microM for acetate, from 0 to 20 microM for propionate, and from 0 to 10 microM for butyrate. This method was also used to determine plasma acetate production obtained from lactulose (an undigestible disaccharide) fermentation in one healthy volunteer over 3 h. The acetate concentration increased twofold, 2 h after oral lactulose intake. These results are in agreement with the data obtained by GC/MS in healthy volunteers and obese adults following a lactulose intake by using higher amounts of labelled tracers.SPME coupled with GC/C/IRMS can be used to analyze (13)C-SCVAs at low enrichment (<0.5 MPE) within the physiological concentration measured in human plasma.     

Determination of dithiocarbamate fungicide residues by liquid chromatography/mass spectrometry and stable isotope dilution assay

A rapid and very sensitive high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method for the simultaneous determination of dithiocarbamate (DTC) fungicide residues in fruits and vegetables was developed. The surface extraction of samples used an alkaline buffer consisting of sodium hydrogen carbonate and DL-penicillamine. The three DTC subclasses, i.e. dimethyldithiocarbamates (DMDs), ethylenebis(dithiocarbamates) (EBDs), and propylenebis(dithiocarbamates) (PBDs), were separated on a Sequant ZIC-pHILIC column using an acetonitrile/10 mM ammonia gradient. Because of the instability of DTC residues extracted from plant samples, a stable isotope dilution assay was applied. For each DTC subclass, the limits of detection and quantification were approximately 0.03 mg kg(-1) and 0.05 mg kg(-1), respectively. Recoveries from grapes, cucumbers, tomatoes, and rucola, spiked in the range of 0.01-0.9 mg kg(-1), averaged between 90 and 100%.