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 (13)C)=0.45%., and the average accuracy was δ(13)C < 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 (13)CPDB < -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.

Quantitative evaluation of carbon isotopic fractionation during reversed-phase high-performance liquid chromatography.

The fractionation of 13C during low-performance preparative LC and high-performance LC is reported quantitatively for methyl palmitate and using high-precision isotope ratio mass spectrometry (IR-MS). For both preparative and high-performance analytical columns, 13C enrichment is about 7% greater than the parent starting material, drops sharply in the first section of the peak and then settles to a value about 1% below that of the starting material. Recycling over a single HPLC column did not induce greater fractionation. These results emphasize the importance of quantitative peak collection for high-precision IR-MS studies, particularly the first part of the peak where the isotope ratio changes rapidly.

High-precision liquid chromatography-combustion isotope ratio mass spectrometry.

Online liquid chromatography-combustion high-precision carbon isotope ratio mass spectrometry (LCC-IRMS) is demonstrated for the first time with a direct interface to the liquid source. The interface is based on a continuously coated moving wire which facilitates reproducible solvent removal and analyte combustion, followed by drying of the CO2 and admission to the mass spectrometer. Routine precision and accuracy for compounds analyzed with the interface in the flow injection mode is about delta 13CPDB = 0.5/1000 over the range -28 < delta 13CPDB < 83. Precision in the liquid chromatography mode is routinely approximately 1/1000 and is limited by sample size. This system expands the range of online compound-specific isotope ratio analysis (CSIA) to thermally labile and nonvolatile compounds.

Condensed-phase carbon isotopic standards for compound-specific isotope analysis.

A protocol has been developed for the preparation of isotopically characterized internal standards for compound-specific isotope analysis. Fatty acids and their corresponding methyl esters are loaded into flame seal vials by a protocol carefully designed to minimize isotopic fractionation and contamination. Carbon isotope analysis of randomly selected vials yields isotopic precision with an average standard deviation of 0.09/1000 (delta 13CPDB). Absolute values for carbon isotope ratios are determined against an international standard (NIST RM8541:USGS24). The procedure is general and may be used for all compounds soluble in volatile solvents. The fatty acid and fatty acid methyl esters standards developed here will be useful for day-to-day quality control and assessment of derivative carbon addition due to methylation of fatty acids.

High-precision continuous-flow isotope ratio mass spectrometry.

Although high-precision isotope determinations are routine in many areas of natural science, the instrument principles for their measurements have remained remarkably unchanged for four decades. The introduction of continuous-flow techniques to isotope ratio mass spectrometry (IRMS) instrumentation has precipitated a rapid expansion in capabilities for high-precision measurement of C, N, O, S, and H isotopes in the 1990s. Elemental analyzers, based on the flash combustion of solid organic samples, are interfaced to IRMS to facilitate routine C and N isotopic analysis of unprocessed samples. Gas/liquid equilibrators have automated O and H isotopic analysis of water in untreated aqueous fluids as complex as urine. Automated cryogenic concentrators permit analysis at part-per-million concentrations in environmental samples. Capillary gas chromatography interfaced to IRMS via on-line microchemistry facilitates compound-specific isotope analysis (CSIA) for purified organic analytes of 1 nmol of C, N, or O. GC-based CSIA for hydrogen and liquid chromatography-based interfaces to IRMS have both been demonstrated, and continuing progress promises to bring these advances to routine use. Automated position-specific isotope analysis (PSIA) using noncatalytic pyrolysis has been shown to produce fragments without appreciable carbon scrambling or major isotopic fractionation, and shows great promise for intramolecular isotope ratio analysis. Finally, IRMS notation and useful elementary isotopic relationships derived from the fundamental mass balance equation are presented.