Stable Isotope Reference Research Articles

Nicotine, acetanilide and urea multi‐level 2H‐, 13C‐ and 15N‐abundance reference materials for continuous‐flow isotope ratio mass spectrometry

Accurate determinations of stable isotope ratios require a calibration using at least two reference materials with different isotopic compositions to anchor the isotopic scale and compensate for differences in machine slope. Ideally, the delta values of these reference materials should bracket the isotopic range of samples with unknown delta values. While the practice of analyzing two isotopically distinct reference materials is common for water (VSMOW-SLAP) and carbonates (NBS 19 and L-SVEC), the lack of widely available organic reference materials with distinct isotopic composition has hindered the practice when analyzing organic materials by elemental analysis/isotope ratio mass spectrometry (EA-IRMS). At present only L-glutamic acids USGS40 and USGS41 satisfy these requirements for delta13C and delta15N, with the limitation that L-glutamic acid is not suitable for analysis by gas chromatography (GC). We describe the development and quality testing of (i) four nicotine laboratory reference materials for on-line (i.e. continuous flow) hydrogen reductive gas chromatography-isotope ratio mass-spectrometry (GC-IRMS), (ii) five nicotines for oxidative C, N gas chromatography-combustion-isotope ratio mass-spectrometry (GC-C-IRMS, or GC-IRMS), and (iii) also three acetanilide and three urea reference materials for on-line oxidative EA-IRMS for C and N. Isotopic off-line calibration against international stable isotope measurement standards at Indiana University adhered to the 'principle of identical treatment'. The new reference materials cover the following isotopic ranges: delta2H(nicotine) -162 to -45 per thousand, delta13C(nicotine) -30.05 to +7.72 per thousand, delta15N(nicotine) -6.03 to +33.62 per thousand; delta15N(acetanilide) +1.18 to +40.57 per thousand; delta13C(urea) -34.13 to +11.71 per thousand, delta15N(urea) +0.26 to +40.61 per thousand (recommended delta values refer to calibration with NBS 19, L-SVEC, IAEA-N-1, and IAEA-N-2). Nicotines fill a gap as the first organic nitrogen stable isotope reference materials for GC-IRMS that are available with different delta15N values. Comparative delta13C and delta15N on-line EA-IRMS data from 14 volunteering laboratories document the usefulness and reliability of acetanilides and ureas as EA-IRMS reference materials.

Vitamin A equivalence of spirulina β-carotene in Chinese adults as assessed by using a stable-isotope reference method

BackgroundSpirulina is a high-protein food supplement that contains carotenoids. ObjectiveThe objective of the study was to determine the vitamin A equivalence of spirulina β-carotene in humans. DesignSpirulina was grown in a 23 atom% 2H2O cultural solution. Spirulina β-carotene showed the greatest enrichment as [2H10]trans β-carotene. Ten healthy Chinese men with a mean (±SD) serum retinol concentration of 1.7 ± 0.3 μmol/L and a body mass index (in kg/m2) of 23 ± 3 consumed 5.8 μmol [13C10]retinyl acetate in oil as a reference dose with a breakfast containing 13 g fat. One week later, each subject consumed 7.9 μmol trans β-carotene in spirulina with a breakfast containing 22 g fat. All subjects followed diets low in carotenoid and vitamin A. Forty blood samples were collected from each subject over a span of 56 d. Concentrations and enrichments of retinol and β-carotene in serum samples were determined by using HPLC and a mass spectrometer. ResultsCompared with the serum response to [13C10]retinyl acetate dose, the mean conversion factor of spirulina β-carotene to retinol was 4.5 ± 1.6 (range: 2.3–6.9) by weight. It was estimated that 80% of the conversion occurred within the first 24 h after spirulina administration. ConclusionIn a group of well-nourished, normal-weight Chinese men following low-vitamin A diets, 4.5 mg spirulina β-carotene consumed with 22 g fat has the same vitamin A activity as does 1 mg retinyl acetate.

Normalization of measured stable isotopic compositions to isotope reference scales – a review

In stable isotope ratio mass spectrometry (IRMS), the stable isotopic composition of samples is measured relative to the isotopic composition of a working gas. This measured isotopic composition must be converted and reported on the respective international stable isotope reference scale for the accurate interlaboratory comparison of results. This data conversion procedure, commonly called normalization, is the first set of calculations done by the users. In this paper, we present a discussion and mathematical formulation of several existing routinely used normalization procedures. These conversion procedures include: single-point anchoring (versus working gas and certified reference standard), modified single-point normalization, linear shift between the measured and the true isotopic composition of two certified reference standards, two-point and multi-point linear normalization methods. Mathematically, the modified single-point, two-point, and multi-point normalization methods are essentially the same. By utilizing laboratory analytical data, the accuracy of the various normalization methods (given by the difference between the true and the normalized isotopic composition) has been compared. Our computations suggest that single-point anchoring produces normalization errors that exceed the maximum total uncertainties (e.g. 0.1 per thousand for delta(13)C) often reported in the literature, and, therefore, that it must not be used for routinely anchoring stable isotope measurement results to the appropriate international scales. However, any normalization method using two or more certified reference standards produces a smaller normalization error provided that the isotopic composition of the standards brackets the isotopic composition of unknown samples.

Inter‐laboratory comparison of elemental analysis and gas chromatography combustion isotope ratio mass spectrometry (GC‐C‐IRMS). Part I: δ13C measurements of selected compounds for the development of an isotopic Grob‐test

This study was directed towards investigating suitable compounds to be used as stable isotope reference materials for gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) calibration. Several compounds were selected from those used in the 'Grob-test' mixture. Oxygen- and nitrogen-containing substances were added to these compounds to allow the mixture to be used as a possible multi-isotopic calibration tool for 2H/1H, 13C/12C, 15N/14N and 18O/16O ratio determinations. In this paper we present the results of delta13C measurements performed by the consortium of the five laboratories taking part in this inter-calibration exercise. All the compounds were individually assessed for homogeneity, short-term stability and long-term stability by means of EA-IRMS, as required by the bureau communitaire de reference (BCR) Guide for Production of Certified Reference Materials. The results were compared then with the GC-C-IRMS measurements using both polar and non-polar columns, and the final mixture of selected compounds underwent a further certification exercise assessing limits of accuracy and reproducibility under specified GC-C-IRMS conditions.

Plasma appearance of labeled β-carotene, lutein, and retinol in humans after consumption of isotopically labeled kale

The bioavailability of carotenoids from kale was investigated by labeling nutrients in kale with 13C, feeding the kale to seven adult volunteers, and analyzing serial plasma samples for labeled lutein, beta-carotene, and retinol. Ingested doses of labeled carotenoids were 34 micromol for beta-carotene and 33 micromol for lutein. Peak plasma concentrations, areas under the plasma concentration-time curves (AUCs), and percentages of dose recovered at peak plasma concentrations were calculated. Average peak plasma concentrations were 0.38, 0.068, and 0.079 microM for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. Average AUC values (over 28 days) were 42.8, 13.6, 13.2 microM h for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. Percentages of dose recovered at peak plasma concentrations were 3.6, 0.7, and 0.7% for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. A positive relationship was observed between baseline plasma retinol levels and [13C]retinol plasma response. It is possible that this relationship was mediated either through some aspect of beta-carotene absorption or via the common pathways of metabolism for postdose and endogenous retinoid.

A re-evaluation of the carbon isotopic composition of organic reference materials

In 1983, Schoell et al. [Schoell, M., Faber, E., Coleman, M.L., 1983. Carbon and hydrogen isotopic compositions of the NBS 22 and NBS 21 stable isotope reference materials: an inter-laboratory comparison. Organic Geochemistry 5, 3–6] carried out an inter-laboratory comparison of the International Atomic Energy Agency (IAEA) reference materials NBS 21 (a graphite) and NBS 22 (an oil). At that time they found that NBS 22 in particular to be more depleted in 13C than the values published by the IAEA at that time (i.e., δ 13C of −29.81‰ PDB ±0.06 as opposed to δ 13C of −29.4‰ PDB). Even so, to this day, the IAEA has subsequently used a value of δ 13C of −29.7‰ ± 0.2 VPDB. Following the advent of the new IAEA VPDB scale, defined by NBS 19 (a carbonate), this laboratory at the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Petroleum and Exploration and Mining Divisions in North Ryde, NSW, Australia found that a number of the reference materials depleted in 13C (especially “organic” type reference materials) to be more depleted in 13C on the VPDB scale than expected. In particular, NBS 22 appears to be 0.25‰ more depleted in 13C than generally accepted values compared with a suite of carbonate standards run together. We suggest that the NBS 22 and other “organic” reference materials have probably all been calibrated to NBS 21 or 22 and should now be redefined on the VPDB scale.

Calcite‐CO2 mixed into CO2‐free air: a new CO2‐in‐air stable isotope reference material for the VPDB scale

AbstractIn order to generate a reliable and long‐lasting stable isotope ratio standard for CO2 in samples of clean air, CO2 is liberated from well‐characterized carbonate material and mixed with CO2‐free air. For this purpose a dedicated acid reaction and air mixing system (ARAMIS) was designed. In the system, CO2 is generated by a conventional acid digestion of powdered carbonate. Evolved CO2 gas is mixed and equilibrated with a prefabricated gas comprised of N2, O2, Ar, and N2O at close to ambient air concentrations. Distribution into glass flasks is made stepwise in a highly controlled fashion. The isotopic composition, established on automated extraction/measurement systems, varied within very small margins of error appropriate for high‐precision air‐CO2 work (about ±0.015‰ for δ13C and ±0.025‰ for δ18O). To establish a valid δ18O relation to the VPDB scale, the temperature dependence of the reaction between 25 and 47°C has been determined with a high level of precision. Using identical procedures, CO2‐in‐air mixtures were generated from a selection of reference materials; (1) the material defining the VPDB isotope scale (NBS 19, δ13C = +1.95‰ and δ18O = −2.2‰ exactly); (2) a local calcite similar in isotopic composition to NBS 19 (‘MAR‐J1’, δ13C = +1.97‰ and δ18O = −2.02‰), and (3) a natural calcite with isotopic compositions closer to atmospheric values (‘OMC‐J1’, δ13C = −4.24‰ and δ18O = −8.71‰). To quantitatively control the extent of isotope‐scale contraction in the system during mass spectrometric measurement other available international and local carbonate reference materials (L‐SVEC, IAEA‐CO‐1, IAEA‐CO‐8, CAL‐1 and CAL‐2) were also processed. As a further control pure CO2 reference gases (Narcis I and II, NIST‐RM 8563, GS19 and GS20) were mixed with CO2‐free synthetic air. Independently, the pure CO2 gases were measured on the dual inlet systems of the same mass spectrometers. The isotopic record of a large number of independent batches prepared over the course of several months is presented. In addition, the relationship with other implementations of the VPDB‐scale for CO2‐in‐air (e.g. CG‐99, based on calibration of pure CO2 gas) has been carefully established. The systematic high‐precision comparison of secondary carbonate and CO2 reference materials covering a wide range in isotopic composition revealed that assigned δ‐values may be (slightly) in error. Measurements in this work deviate systematically from assigned values, roughly scaling with isotopic distance from NBS 19. This finding indicates that a scale contraction effect could have biased the consensus results. The observation also underlines the importance of cross‐contamination errors for high‐precision isotope ratio measurements.As a result of the experiments, a new standard reference material (SRM), which consists of two 5‐L glass flasks containing air at 1.6 bar and the CO2 evolved from two different carbonate materials, is available for distribution. These ‘J‐RAS’ SRM flasks (‘Jena‐Reference Air Set’) are designed to serve as a high‐precision link to VPDB for improving inter‐laboratory comparability.a Copyright © 2005 John Wiley & Sons, Ltd.

Short-term (intestinal) and long-term (postintestinal) conversion of β-carotene to retinol in adults as assessed by a stable-isotope reference method

Quantitative information on the conversion of beta-carotene to vitamin A in humans is limited. We determined the short- and long-term conversion of labeled beta-carotene to vitamin A by using a stable-isotope reference method. [(2)H(8)]beta-Carotene (11,011 nmol, or 6 mg) in oil was given with a liquid diet (25% of energy from fat) to 22 adult volunteers (10 men, 12 women). Three days after the [(2)H(8)]beta-carotene dose, the volunteers each took a dose of [(2)H(8)]retinyl acetate (8915 nmol, or 3 mg) in oil with the same liquid diet. Blood samples were collected over 56 d. The 53-d area under the serum [(2)H(4)]retinol response curve (from the [(2)H(8)]beta-carotene dose) was 569 +/- 385 nmol. d, and the 53-d area under the serum [(2)H(8)]retinol response curve (from the [(2)H(8)]retinyl acetate dose) was 1798 +/- 1139 nmol. d. With the use of [(2)H(8)]retinyl acetate as the vitamin A reference, the [(2)H(4)]retinol formed from [(2)H(8)]beta-carotene (11,011 nmol) was calculated to be equivalent to 3413.9 +/- 2298.4 nmol retinol. The conversion factor of beta-carotene to retinol varied from 2.4 to 20.2, and the average conversion factor was 9.1 to 1 by wt or 4.8 to 1 by mol. This conversion factor was positively correlated with body mass index (r = 0.57, P = 0.006). The postabsorption conversion of beta-carotene was estimated as 7.8%, 13.6%, 16.4%, and 19.0% of the total converted retinol at 6, 14, 21, or 53 d after the [(2)H(8)]beta-carotene dose, respectively. The quantitative determination of the conversion of beta-carotene to vitamin A in humans can be accomplished by using a stable-isotope reference method. This approach provides in vivo metabolic information after a physiologic dose of beta-carotene.

Bioavailability and vitamin A value of carotenes from red palm oil assessed by an extrinsic isotope reference method.

Red palm oil (RPO) contains high concentrations of beta- and alpha-carotene, and is presumed to possess a higher vitamin A value than other foods. The objective was to determine the metabolic vitamin A and carotene values of refined red palm oil in healthy adult subjects, using a stable isotope reference method. Twelve healthy subjects were administered a small standardised meal containing 10 g RPO (2.4 mg beta-carotene and 1.8 mg alpha-carotene) in a blended juice-based drink also containing 2 mg tetradeuterated retinyl acetate (d4-RA) as a metabolic reference. At baseline and at several times after the test meal, the concentrations of carotenes and of d4- and d0-(unlabelled) retinyl esters, in the plasma chylomicron-rich (d < 1.006) fraction were determined by high high-performance liquid chromatography and gas chromatography mass spectrometry, respectively. The masses of palm oil-derived vitamin A and carotenes absorbed ('yield') were calculated assuming 80% absorption of the d4-RA reference dose. The mean yield of retinol from the RPO was 0.41 mg, ranging from 0.17 mg to 0.86 mg. The mean yields of beta- and alpha-carotene were 0.29 mg and 0.25 mg, respectively, suggesting that beta-carotene was more extensively metabolised than alpha-carotene. Subjects assimilated an average of 23% of the dose of carotenes, as the sum of retinol and unmetabolised carotenes. The vitamin A values of red palm oil obtained under these conditions, a mean of 0.17 mg retinol absorbed per mg beta-carotene consumed (beta-carotene : retinol equivalency of 5.7:1) is higher than that of all other vegetable sources we have evaluated to date.

α- and β-Carotene from a Commercial Carrot Puree Are More Bioavailable to Humans than from Boiled-Mashed Carrots, as Determined Using an Extrinsic Stable Isotope Reference Method

The extent to which processing affects the carotene or vitamin A value of foods is poorly understood. An extrinsic reference method was used to estimate the mass of carotenes and vitamin A derived from various preparations made from the same lot of carrots. Using a repeated-measures design, nine healthy adult subjects consumed test meals of either carrot puree (commercial baby food) or boiled-mashed carrots on separate days; six of the subjects also consumed a test meal of raw-grated carrot. Test meals supplied 34.7 micromol (18.6 mg) carrot beta-carotene (beta C), plus 6 micromol deuterium-labeled retinyl acetate (d(4)-RA) in oil solution. Baseline-adjusted carotene and retinyl ester (R-ester) area-under-curve (AUC) responses in the triacylglycerol-rich lipoprotein (TRL) fraction (0-8.5 h) were determined using HPLC and gas chromatography-mass spectrometry. The masses of absorbed beta C, alpha-carotene (alpha C) and R-ester were estimated by comparing their AUC values with that of deuterium-labeled retinyl ester (d(4)-R-ester), assuming the latter represented 80% of the d(4)-RA reference dose. Absorption of beta C and alpha C was approximately twofold greater from carrot puree than from boiled-mashed carrots, whereas the retinol yield was only marginally (P = 0.11) influenced by treatment. Carotene and R-ester absorption from raw-grated carrot was intermediate to, and did not differ significantly from the cooked preparations. The vitamin A yield (puree, 0.53 mg; boiled-mashed, 0.44 mg) of cooked carrot containing 18.6 mg beta C was substantially less than that predicted by current convention and limited primarily by intestinal carotene uptake. Processing can therefore significantly improve bioavailability of carrot carotenes, and in some cases influence the carotene value more than the intrinsic vitamin A value.

Vitamin A equivalence of β-carotene in a Woman as determined by a stable isotope reference method

Quantitative information on conversion of beta-carotene to vitamin A in humans is limited. Our laboratory has developed a stable isotope method for studying the conversion of beta-carotene (beta-C) to vitamin A. Two dosage levels (a pharmacological dose, 126.0 mg beta-C-d8, and a physiological dose, 6.0 mg beta-C-d8) were used 2.5 y apart in an adult female volunteer to study dose effects on the conversion of beta-C to vitamin A. Blood samples were collected over 21 d. beta-C and retinol were extracted from serum and isolated by high performance liquid chromatography. The retinol fraction was derivatized to a trimethylsilyl ether which was analyzed by gas chromatograph/mass spectrometry with electron capture negative chemical ionization. The retinol-d4 response in the circulation peaked at 24 hours after the beta-C-d8 dose, with a higher percent enrichment after the pharmacological dose than after the physiological dose. By using retinyl acetate-d8 as the vitamin A reference, the retinol-d4 formed from 6 mg of beta-C-d8 (11.2 mumol) was calculated to be equivalent to 1.6 mg of retinol (i.e., 3.8 mg of beta-C was equivalent to 1 mg of retinol). However, the retinol-d4 formed from 126 mg of beta-C-d8 (235 mumol) was equivalent to 2.3 mg of retinol (i.e., 55 mg beta-C was equivalent to 1 mg retinol). These results provide evidence that it is feasible to use stable isotope reference method to study retinol equivalence of beta-C and that there may be a dose-dependence on bioconversion of beta-carotene to retinol.

Ratios for light‐element isotopes standardized for better interlaboratory comparison

Stable isotope‐ratio measurements of the light elements have been used in increasingly important ways to understand processes in geochemistry, hydrology, oceanography, atmospheric sciences, environmental studies, and many other fields. Progress in these fields requires better precision and reproducibility of stable isotope‐ratio measurements. Some laboratories are claiming accuracies of 0.02‰; or better for δ13C and δ180. However, δ13C and δ180 analyses of the same sample by different laboratories can differ by more than 0.3‰. Recognizing that isotopic analyses of the same homogeneous material reported from different credible laboratories should yield the same isotopic composition within the uncertainty of the measurements, the International Atomic Energy Agency (IAEA) held an Advisory Group Meeting on stable isotope reference and intercomparison materials for light elements in Vienna from December 11 to 14, 1995.

Avilability of enriched stable isotope reference materials for medical and biological studies

Avilability of enriched stable isotope reference materials for medical and biological studies

I.A.E.A. advisory group meeting on stable isotope reference samples for geochemical and hydrological investigations: Vienna, Austria, September 19–21, 1983

I.A.E.A. advisory group meeting on stable isotope reference samples for geochemical and hydrological investigations: Vienna, Austria, September 19–21, 1983

I.A.E.A. advisory group meeting on stable isotope reference samples for geochemical and hydrological investigations: Vienna, Austria, September 19–21, 1983

Unconventional hydrocarbon resources such as shale oil/gas and coal-bed methane have become an increasingly important source of energy over the past decade. The Vaca Muerta Shale (Neuquén Basin, Argentina) contains the second largest technically recoverable quantity of shale gas in the world. Exploitation of the play has been complicated by elevated concentrations of CO2 in several fields, the origin of which is currently poorly understood. Elevated CO2 levels are consistently encountered when deep-rooted faults in the Auquilco Evaporite Formation, present below the Vaca Muerta Shale, overlap with shallower faults that propagate from the top of evaporites into the shale, indicating a sub-evaporate origin of the CO2. Here we report new isotopic analysis of CO2-rich gases from two producing fields. CO2 concentrations increase with C1/(C2 + C3) values (4.8–33.5) and fractionation of δ13CCO2 (−0.9 to −7.7‰), suggest that CH4 have been displaced by CO2 which entered the shale after hydrocarbon maturation. The noble gas composition (3He/4He of 3.43–3.95 RA, where RA is the atmospheric ratio of 1.399 × 10−6, 21Ne/22Ne of 0.0310–0.0455, 20Ne/22Ne of 9.89–10.52, 40Ar/36Ar of 2432–3674 and CO2/3He 6.8–20.2 × 107) of the gases is consistent with mixing of magmatic CO2 with crustal hydrocarbon-rich gases and provides evidence for the loss of significant CO2. Using inverse modelling techniques, we determine that the magmatic gas has a 3He/4He of 3.95–4.08 RA, CO2/3He of 8.8–16 × 108 and 20Ne/22Ne of 12.13−0.10+0.08, 21Ne/22Ne of 0.074−0.003+0.004. Based on the radiogenic He and Ne this is consistent with a depleted asthenosphere mantle source, which has been trapped in the crust since 6.0–22.8 Ma. This is significantly younger than Late Cretaceous maturation of the hydrocarbon source rocks. Mantle melting as a result of asthenosphere upwelling induced by the collision of the South Chile Ridge and the Chile Trench at ~14 Ma is the most likely source of the CO2. Gases from below the shale contain two air saturated water-derived noble gas components, distinguished on the basis of 20Ne†/36Ar, 84Kr/36Ar, 132Xe/36Ar ratios. These are consistent with early and late stage open system Rayleigh fractionation of groundwater-derived noble gases. We find evidence that these mix with the magmatic component prior to entering the Vaca Muerta and mixing with an adsorption derived gas retained in the source kerogen.

Comparison of stable isotope reference samples

Use of light stable isotope ratio measurements has proliferated in the past decade. The need for procuring additional stable isotope reference materials was recognized at an International Atomic Energy Agency (IAEA) consultants' meeting convened in 19761. This group recommended acquisition of two carbonates, two carbon dioxide samples, a biotite, a sulphate, and other reference materials. We report here on the mass-spectrometric analysis of these and other reference samples in a single laboratory to minimize interlaboratory calibration errors. A result of this work is an improved equation for relating the PDB isotope scale (belemnite from the Peedee Formation of South Carolina adopted in the 1950s as a reference in palaeotemperature studies2) to the V-SMOW (Vienna-Standard Mean Ocean Water) scale1.

Carbon and hydrogen isotopic compositions of the NBS 22 and NBS 21 stable isotope reference materials: An inter-laboratory comparison

Measurement of 17 laboratories on the stable isotope reference materials, NBS 22 lubricating oil and NBS 21 graphite, resulted in a revised calibration on the PDB scale. NBS 22 13C/ 12C ratios of all laboratories did not demonstrate a gausssian distribution. Therefore, values which resulted from direct comparison with NBS 20 carbonate standards were selected the mean value of which is −29.81 ± 0.06ℵ; this value is recommended as new value for NBS 22. The reported variations in δ 13C for NBS 21 were consistent within the accuracies for single meaurements; the mean value is −28.16 ± 0.01. The variations of D/H ratios of NBS 22 also were consistent with the accuracy quoted, with a mean value of −119ℵ.

Preparation and stable isotope determination of NBS-16 and NBS-17 carbon dioxide reference samples

There was a need for stable isotope reference samples other than water for interlaboratory calibration discussion. Two carbon dioxide reference samples, NBS-16 and NBS-17, were prepared for oxygen and carbon stable isotope calibration of mass spectrometers. NBS-16 was prepared from carbon dioxide taken directly from a tank of Air Products liquified carbon dioxide, produced as an industrial byproduct. NBS-17 was prepared from carbon dioxide taken directly from a tank of Chemetron Corp. gaseous carbon dioxide, obtained from a carbon dioxide well in the southwestern United States. This selection ensured that there would be a large difference in carbon and oxygen isotope ratios between the two NBS reference gases. The preparation of several hundred isotopically homogeneous carbon dioxide reference samples proved to be a nontrivial exercise, plus this method can be used to prepare gaseous laboratory reference samples for internal use to keep an accurate check on drift of the working standard. The preparation technique was described in detail. 1 figure, 2 tables.