Lawrence C. Thomas

Quantitative measurements via co-elution and dual-isotope detection by gas chromatography-mass spectrometry

Dual-isotope measurements by gas chromatography-mass spectrometry (GC-MS) which mimic isotope dilution may suffer from irreproducibilities or unduly large uncertainties because of variations in ionization efficacies for the respective forms in the MS source. Such variations are sometimes avoided via extensive pretreatments and high-resolution GC separations. However, in some circumstances, an alternative approach is feasible which instead exploits the advantages of decreasing GC resolution. By forcing both forms of each analyte to co-elute, their ionization efficacies in the MS source will be virtually identical, thereby allowing for highly reproducible relative response ratios to be attained despite dramatically lowered GC resolution. The co-elution results described here are nearly as precise as results from moderate-resolution separations in the absence of interferents. Thus, dual-isotope GC-MS measurements with co-elution of the target analytes and their respective isotopically labeled internal standards offer a powerful alternative to the conventional approach of requiring expensive and labor-intensive additional pretreatments and separations; however, the effects of interferences may be exacerbated by the forced co-elution and must also be considered.

Measurements using gas chromatography with coelution and dual-isotope atomic emission detection.

Dual-isotope measurements by gas chromatography (GC)-atomic emission detection (AED) may enhance results for quantitative analyses. Adding a known amount of an isotopically labelled form of target analytes in each sample can compensate for irreproducibilities or uncertainties associated with sample pretreatments and sample loading. Similarly, fluctuations in AED temperatures, flows and interferants can be compensated via the added labelled forms if each target analyte and its isotopically labelled form coelute. Under these conditions they are subject to identical excitation environments and are measured from the same viewed volumes. Consequently, improved quantitative results may be attained by coelution in GC-AED methods which mimic isotope dilution.

Quantitative comparisons of reaction products using liquid chromatography with dual-label radioactivity measurements

Abstract Dual-label methods for radiometric measurements with liquid chromatography are evaluated. The procedures show marked improvements in data quality for comparisons of product formations and relative product abundances in multiple-pathway reaction systems. The results from these procedures are shown to be relatively unaffected by large variations in uncertainties in pretreatment steps and volume measurements. Moreover, these methods yield much better quantitative results than do commonly used corresponding conventional comparisons. These quantitative comparisons use reference substances generated by a reference reaction system as internal standards. A suite of reaction products is compared via the internal standards for components common to both reference and sample reaction systems. The dual-label methods are especially suitable for toxicologic metabolism comparisons, but could easily be adapted to other reaction systems such as syntheses or degradations. Moreover, the dual-label procedures should be amenable to non-radioactive measurements with isotope-selective methods such as mass fragmentometry, emission spectrometry or resonance methods.

Comparison of Integration Vs. Fixed-Time Methods For Kinetic Analyses

Abstract Results for an integration method of enzymatic analysis are directly compared to concurrently-achieved fixed-time rate data. Two to fiftyfold enhancement of signal-to-noise ratios are obtained for the integration method vs. the fixed-time method for theoretically-calculated response vs. time data. the signal-to-noise ratio improves substantially as the number of increments increases for estimating the integral of changes in product concentration over time. Also, advantages of the integration method are enchanced as enzyme activities increase and as instrument noise decreases.

Quantitative comparisons using liquid chromatography with dual-label radioactivity measurements for evaluation of isotope and impurity effects

Dual-label methods with radiometric measurements with liquid chromatography are evaluated for assessing effects of impurities and isotopic composition in reaction experiments. The procedure is suitably sensitive to detect interferences caused by effects of impurities in reactants, isotope exchange during reaction and effects of kinetic differences due to differing masses of the differently labeled forms. The methods show marked improvements in data quality for comparisons of product formations and relative product abundances in multiple-pathway reaction systems. The results from computer simulations for the procedures are shown to be relatively unaffected by large variations in uncertainties in pretreatment steps and volume measurements. Moreover, these methods yield much better quantitative results than do commonly used corresponding conventional comparisons, and the benefits are corroborated by results from experiments with microsomal reactions.

Comparisons of integration vs. rate measurements for oxidase enzymatic analyses

Results obtained by integration of net indicator-substance signals during the progress of a reaction are compared to those concurrently achieved by rate estimation with the time vs. signal data, for glucose oxidase assays and enzymatic glucose determinations. Computer-simulated integration results typically exhibit data quality superior to that of rate measurements, for the entire very wide ranges of oxidase activities and concentrations studied. Only at extreme instrument-noise levels and low analyte values did any of the rate procedures (the variable-time method) show some advantage over integration. The data quality, expressed as relative uncertainties, of the simulated results obtained by use of the integration method was typically up to 120 times better than that obtained by rate methods, with greater enhancement at higher enzyme activities or substrate concentrations. Experimental results spanning physiological concentrations showed similar trends, but the advantage was not as great as that shown in the simulations.

Laser-excited fluorescence detection of gas-phase chromatography eluates

A high-spectral-resolution laser-excited molecular fluorescence gas chromatographic (GC) detection system is evaluated. It utilizes a pulsed supersonic jet expansion to yield very-narrow-bandwidth (e.g. less than or equal to 0.1 nm) fluorescence excitation spectra that may be rapidly scanned via a tunable dye laser. A microcomputer synchronizes the entire system, collects data, performs calculations, and creates visual displays of results. The laser-excited fluorescence detection system was interfaced to a gas chromatograph to exploit both the selectivity of the chromatography and the excellent spectral selectivity of the detector. Fluorescence excitation chromatograms were acquired by monitoring fluorescence emission from selected transition wavelengths characteristic of the GC eluates. The excitation wavelengths were also programmed to change at appropriate retention times to provide greatest selectivities for individual analytes as they eluted, and to allow multiple analytes to be determined in a single elution. Response factors for the system varied appreciably from run to run, which precluded the use of external standard quantitation procedures. However, excellent stabilities for within-run relative response factors were sufficient to allow for good quantitative measurements using internal standard techniques.

Quantitative comparisons using gas chromatography-mass spectrometry and dual-isotope techniques for detection of isotope and impurity interferences

Abstract Dual-isotope methods for special quantitative comparisons using GC-MS are evaluated for assessing interferences in reaction experiments. The procedure is suitable for detecting interferences from impurities in reactants, errors due to isotope exchange and effects of kinetic differences between labeled and non-labeled forms. Combined reaction of both isotopic forms of test compounds in the same reaction system, followed by concurrent pretreatment and GC-MS measurements, allow for special ratios to be tested statistically to determine if the interferences are significant. The comparisons may be relatively unaffected by large uncertainties in pretreatment and measurement steps and thus may yield greatly improved results relative to corresponding conventional assessments. Moreover, concentrations of the individual eluates need not be known and eluate identifications may not be required for proper use of this approach.

Relative response ratios for dual-isotope measurements via coelution and GC/MS

Dual-isotope internal standard measurements by GC/MS which mimic isotope dilution may suffer from non-linear response relations, irreproducibilities or unduly large uncertainties because of variations in ionization efficiences for the respective isotopic forms in the MS source. Such variations may sometimes be avoided via extensive pretreatments, high resolution GC separations and careful control of instrumental parameters. However, an alternative approach is feasible which instead exploits advantages of decreasing GC resolution. By forcing both forms of each analyte to coelute, their relative ionization efficiencies in the MS source should be nearly constant, thereby effectively allowing for constant relative sensitivities over several orders of magnitude in concentration. Thus, constant relative response ratios, required for internal standard calculations, may be attained as a consequence of dramatically lowered GC resolution. Coelution results described herein show linear relative sensitivity relations over much broader ranges than observed for corresponding conventional calibrations with separated components. Coelution methods for dual-isotope GC/MS determinations are compatible with internal standard calculations and thereby offer a powerful alternative to the conventional approach of requiring expensive and labor-intensive additional pretreatments and separations to assure resolution of measured eluates.

Quantitative comparisons of reaction products using gas chromatography-mass spectrometry and dual-isotope techniques

Abstract Special dual-isotope methods for GC-MS measurements show response necessary for quantitative comparisons of reaction product abundances, even for multiple-pathway reaction systems. The quantitative comparisons are compatible with the use of isotopically labeled reference substances generated by reference reactions, for which suites of reaction products may be compared via compounds common to both reference and sample reaction systems. The results of comparisons studied are fairly insensitive to large variations in relative concentrations and to high uncertainties in individual analyte measurements. Moreover, the approach may, for some circumstances, be used for analytes of unknown identities or with reference compounds of unknown concentrations. The dual-isotope GC-MS methods described are suitable for photolysis reactions and should be especially helpful in toxicologic metabolism comparisons, environmental degradation studies or other kinetic systems.