Isotope Dilution Gas Chromatography Research Articles

Simultaneous supercritical fluid extraction and chemical derivatization for the gas chromatographic–isotope dilution mass spectrometric determination of amphetamine and methamphetamine in urine

An in-situ supercritical fluid extraction (SFE) and chemical derivatization (ChD) procedure followed by gas chromatography–isotope dilution mass spectrometry (GC–MS) for the determination of amphetamines in urine is described and evaluated. While using celite as the SFE wet-support, the one-pot sample pretreatment procedure also employs ammonium water to alkalize the urine matrix that contains protonated amphetamine (AP) and methamphetamine (MA). The mean recoveries achieved by simultaneous SFE–ChD, i.e., 95% (RSD=3.8%) for AP and 89% (RSD=4.0%) for MA, are significantly better than the corresponding overall recoveries obtained upon stepwise SFE–ChD, suggesting the unreacted trifluoroacetic anhydride (TFA) in the former procedure has strengthened the extracting power of CO 2 fluid as has been evidenced by a control test. As to GC–MS analysis, the optimal qualitative ions and quantitative ions of the respective analytes were determined via a rigorous evaluation process. Thus, the regression calibration curves for AP and MA in urine are linear within 100∼50 000 ng/ml, with correlation coefficients typically exceeding 0.999. The limits of detection determined by two methods for AP and MA vary from 19 to 50 ng/ml, and limits of quantitation from 21 to 100 ng/ml. Precisions calculated for the triplicate analyses of AP and MA in a 500-ng/ml spiked control, two real-case samples and two quasi real-case samples, respectively, using regression calibration are typically below 10%. The method is simple and reliable. It may serve as an alternative to the existing confirmatory protocol for forensic urine drug testing.

Selenomethionine content of candidate reference materials.

Selenium has been identified as an antioxidant of importance in the diet. Accurate determination of its chemical forms depends on the availability of suitable reference materials (RMs). Two candidate reference materials for determination of selenomethionine (Semet) in food-related materials, a standard wheat gluten sample (NIST RM 8418 Wheat Gluten) and a commercial selenium enriched yeast, have been examined by use of a gas chromatography-isotope dilution mass spectrometry (IDMS) procedure, after treatment of the matrix with 0.1 mol L(-1) hydrochloric acid containing stannous chloride, addition of CNBr, and extraction with chloroform. This procedure results in cleavage of the CH3Se group to form volatile CH3SeCN. Addition of isotopically enriched 74Semet to an analytical sample enables estimation of the naturally occurring protein-bound 80Semet by IDMS without a protein-digestion process. We found that the Wheat Gluten RM contains a significant amount of Semet as a portion of its assigned value of 2.58 microg Se(total g(-1). Commercial selenium yeast tablets are labeled as containing an elevated level of "organic selenium", usually as Semet. The sample we investigated contained 210 microg Se(total) g(-1) sample as determined separately by IDMS, measuring elemental selenium after digestion. 73% of this total (153 +/- 21 microg Se(semet) g(-1); n = 23) was present as Semet. Thus, these two materials contain significant amounts of their total selenium content as Semet and would be good candidates for further study and characterization as reference materials for determining this important food component. The CNBr reaction used will also enable the determination of Se-(methyl)selenocysteine, the biological role of which is of recent interest. In addition to matrix RMs for Semet, it is important to have standard materials of the pure substance. We have examined a sample of a candidate standard material of selenomethionine being prepared by the USP. It was confirmed that this material is pure selenomethionine.

Determination of styrene and styrene-7,8-oxide in human blood by gas chromatography–mass spectrometry

Methods of isotope-dilution gas chromatography–mass spectrometry (GC–MS) are described for the determination of styrene and styrene-7,8-oxide (SO) in blood. Styrene and SO were directly measured in pentane extracts of blood from 35 reinforced plastics workers exposed to 4.7–97 ppm styrene. Using positive ion chemical ionization, styrene could be detected at levels greater than 2.5 μg/l blood and SO at levels greater than 0.05 μg/l blood. An alternative method for measurement of SO employed reaction with valine followed by derivatization with pentafluorophenyl isothiocyanate and analysis via negative ion chemical ionization GC–MS–MS (SO detection limit=0.025 μg/l blood). The detection limits for SO by these two methods were 10–20-fold lower than gas chromatographic assays reported earlier, based upon either electron impact MS or flame ionization detection. Excellent agreement between the two SO methods was observed for standard calibration curves while moderate to good agreement was observed among selected reinforced plastics workers ( n=10). Levels of styrene in blood were found to be proportional to the corresponding air exposures to styrene, in line with other published relationships. Although levels of SO in blood, measured by the direct method, were significantly correlated with air levels of either styrene or SO among the reinforced plastics workers, blood concentrations were much lower than previously reported at a given exposure to styrene. The two assays for SO in blood appear to be unbiased and to have sufficient sensitivity and specificity for applications involving workers exposed to styrene and SO during the manufacture of reinforced plastics.

Preparation and value assignment of Standard Reference Material 968c Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum

Standard Reference Material 968c Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for retinal, δ-, γ-, and α-tocopherol, trans- and total β-carotene, and cholesterol in human serum. Values are also reported for 16 additional compounds including lutein, zeaxanthin, α- and β-cryptoxanthin, lycopene, α-carotene, retinyl palmitate, and 25-hydroxyvitamin D. The certified values for the fat-soluble vitamins and carotenoids in SRM 968c were based on the agreement of results from the means of at least two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the medians from an interlaboratory comparison study among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol in the SRM are the means of results obtained using the NIST definitive method, gas chromatography–isotope dilution mass spectrometry.

Regional Water-Quality Analysis of 2,4-D and Dicamba in River Water Using Gas Chromatography-Isotope Dilution Mass Spectrometry

Gas chromatography with isotope dilution mass spectrometry (GC-MS) and enzyme-linked immunosorbent assay (ELISA) were used in regional National Water Quality Assessment studies of the herbicides, 2,4-D and dicamba, in river water across the United States. The GC-MS method involved solid-phase extraction, derivatized with deuterated 2,4-D, and analysis by selected ion monitoring. The ELISA method was applied after preconcentration with solid-phase extraction. The ELISA method was unreliable because of interference from humic substances that were also isolated by solid-phase extraction. Therefore, GC-MS was used to analyzed 80 samples from river water from 14 basins. The frequency of detection of dicamba (28%) was higher than that for 2,4-D (16%). Concentrations were higher for dicamba than for 2,4-D, ranging from less than the detection limit (7lt; 0.05 μg/L) to 3.77μg/L, in spite of 5 times more annual use of 2,4-D as compared to dicamba. These results suggest that 2,4-D degrades more rapidly in the environment than dicamba.

Preparation of 4,4′-(1-[ 2H 6]methylethylidene)bis-[2,3,5,6- 2H 4]phenol and its application to the measurement of bisphenol A in beverages by stable isotope dilution mass spectrometry

The preparation of 4,4′-(1-[ 2H 6]methylethylidene)bis-[2,3,5,6- 2H 4]phenol, bisphenol A-d 14, was achieved in excellent yield by reaction of [ 2H 6]acetone with [ 2H 6]phenol in the presence of deuterium chloride. The product thus obtained was shown by mass spectroscopy to be a mixture of the 2H 14, 2H 13 and 2H 12 isotopomers in the relative proportions of 82.3:16.2:1.5, respectively. An isotope dilution gas chromatography–mass spectrometric method using bisphenol A-d 14 as an internal standard was developed to measure the level of bisphenol A in beverages. The procedure involves extracting bisphenol A into dichloromethane, and then purifying the analyte by back extraction into dilute aqueous sodium hydroxide. Conversion of bisphenol A and its internal standard, bisphenol A-d 14, to their corresponding O-bis(trifluoroacetyl) derivatives by treatment with trifluoroacetic anhydride gave compounds with good chromatographic properties and whose mass fragmentomerty is such that loss of M−CH 3 and M−C 2H 3 are the base peaks in the mass spectra of the analyte and internal standard, respectively. Quantification of bisphenol A was achieved by comparing the area of the M−15 ion to that of the corresponding ion of bisphenol A-d 14. The characteristics of our assay are: an analyte recovery of better than 95%, a root mean square signal-to-noise ratio of 79:1 for 1.7 pg on column and an inter-assay RSD of better than 4% ( n=5).

Trace-level determination of 1,4-dioxane in water by isotopic dilution GC and GC-MS.

The volatile and polar solvent 1,4-dioxane has recently been reported as a contaminant of ground and surface waters, establishing the need to determine this substance in drinking water. This investigation established that 1,4-dioxane can be determined in water by various techniques including direct aqueous injection (DAI) gas chromatography (GC) and purge and trap GC-mass spectrometry (MS). Purge and trap GC-MS is limited by 1,4-dioxane's poor purge efficiency, resulting in detection limits up to 100 times greater than the efficiently purged volatile organic compounds. To attain the sensitivity required for drinking water monitoring, a method based on continuous liquid-liquid extraction with dichloromethane was developed. Isotope dilution was more accurate and reproducible than quantification with external standards, and the improvement in precision led to a lower method detection limit, 0.2 microgram L-1, using a quadrupole ion trap instrument in the electron ionization mode. Isotope dilution accuracy approached 100% in ppb determinations. Isotopic dilution quantification was also possible using a non-selective GC detector owing to the high efficiency of capillary GC columns that resolve the deuterium-labeled solvent from the natural isotopes.

Quantitative analysis of urinary acylglycines for the diagnosis of β-oxidation defects using GC-NCI-MS

The analysis of acylglycines is an important biochemical tool for the diagnosis of inherited disorders of mitochondrial fatty acid β-oxidation. A stable isotope dilution gas chromatography negative chemical ionisation mass spectrometry method for the quantitative analysis of short- and medium-chain acylglycines as their bis(trifluoromethyl)benzyl (BTFMB) ester derivatives is described. The diagnostic usefulness of the method was demonstrated in nine patients with medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) deficiency, and seven patients with multiple acyl-CoA dehydrogenation defect (MAD). The urinary acylglycine profiles in these patients were compared to those in controls ( n=19), children on a medium-chain triglyceride (MCT) supplemented diet ( n=4), and patients with various other diseases ( n=5).

Hydroxyl radical generation during exercise increases mitochondrial protein oxidation and levels of urinary dityrosine

Isolated mitochondria are well-established sources of oxidants in vitro. There is little direct evidence that mitochondria promote oxidative stress in vivo, however . Model system studies demonstrate that ortho-tyrosine, meta-tyrosine, and o,o′-dityrosine increase in proteins oxidized by hydroxyl radical. To determine whether mitochondria generate oxidants in vivo, we used isotope dilution gas chromatography mass spectrometry to quantify levels of these markers in the heart muscle of control and exercised rats. Exercise led to a 50% increase in ortho-tyrosine, meta-tyrosine, and o,o′-dityrosine in the mitochondrial proteins but not cytosolic proteins of heart muscle. This increase was transient, and levels returned to normal when exercised animals were allowed to rest. There also was a transient increase in the level of o,o′-dityrosine in the urine of exercised rats. This relationship between mitochondrial and urine levels of o,o′-dityrosine suggests that urine assays of this oxidized amino acid may serve as noninvasive measures of oxidative stress . These observations also provide direct evidence that heart muscle mitochondria produce an intermediate resembling the hydroxyl radical that promotes protein oxidation in vivo.

Quantitative acylcarnitine profiling in fibroblasts using [U- 13C] palmitic acid: an improved tool for the diagnosis of fatty acid oxidation defects

A method was developed for the investigation of mitochondrial fatty acid β-oxidation in cultured fibroblasts. Monolayer cultures were incubated without foetal calf serum with commercially available [U- 13C] palmitic acid and l-carnitine for 96 h. The acylcarnitines produced by the cells were extracted from the cell suspension and analysed either by quantitative stable isotope dilution gas chromatography chemical ionization mass spectrometry, or by fast atom bombardment mass spectrometry. Characteristic acylcarnitine profiles were obtained for all the different enzyme deficiencies investigated, with the exception of carnitine palmitoyltransferase II deficiency and carnitine/acylcarnitine carrier deficiency which showed similar patterns. Comparison between this method and the 3H-myristate and 3H-palmitate tritium release assays revealed that the method described here is superior, allowing unequivocal identification of patients.

Quantitative determination of trimethylamine in urine by solid-phase microextraction and gas chromatography–mass spectrometry

Trimethylaminuria (fish odour syndrome) is diagnosed from an increase in urinary excretion of trimethylamine with decreased trimethylamine oxide. We report a new quantitative stable isotope dilution gas chromatography–mass spectrometry procedure for the analysis of these metabolites using solid-phase microextraction (SPME). Both polydimethylsiloxane and mixed Carboxen–polydimethylsiloxane SPME fibres were found to be suitable for the headspace extraction of TMA. This new sampling technique could have wide application for the analysis of volatile and semi-volatile compounds by metabolic screening laboratories.

Analysis of pristanic acid β-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry

In this paper we report the development of highly sensitive, selective, and accurate stable isotope dilution gas chromatography negative chemical ionization mass spectrometry (GC-NCI-MS) methods for quantification of peroxisomal β-oxidation intermediates of pristanic acid in human plasma: 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid. The carboxylic groups of the intermediates were converted into pentafluorobenzyl esters, whereas hydroxyl groups were acetylated and ketogroups were methoximized. Hereafter, the samples were subjected to clean-up by high performance liquid chromatography. Analyses were performed by selected monitoring of the carboxylate anions of the derivatives. Control values of all three metabolites were established (2,3-pristenic acid: 2–48 nm, 3-hydroxypristanic acid: 0.02–0.81 nm, 3-ketopristanic acid: 0.07–1.45 nm). A correlation between the concentrations of pristanic acid and its intermediates in plasma was found. The diagnostic value of the methods is illustrated by measurements of the intermediates in plasma from patients with peroxisomal disorders. It is shown that in generalized peroxisomal disorders, the absolute concentrations of 2,3-pristenic acid, 3-hydroxypristanic acid, and 3-ketopristanic acid were comparable to those in the controls, whereas relative to the pristanic acid concentrations these intermediates were significantly decreased. In bifunctional protein deficiency, elevated levels of 2,3-pristenic acid and 3-hydroxypristanic acid were found. 3-Ketopristanic acid, although within the normal range, was relatively low when compared to the high pristanic acid levels in these patients.—Verhoeven, N. M., D. S. M. Schor, E. A. Struys, E. E. W. Jansen, H. J. ten Brink, R. J. A. Wanders, and C. Jakobs. Analysis of pristanic acid β-oxidation intermediates in plasma from healthy controls and patients affected with peroxisomal disorders by stable isotope dilution gas chromatography mass spectrometry. J. Lipid Res. 1999. 40: 260–266.

Influence of soybean processing, habitual diet, and soy dose on urinary isoflavonoid excretion

In an attempt to explain the wide individual variation seen in urinary isoflavonoid phytoestrogen excretion, we conducted a series of 3 human feeding studies: a large crosssectional study of equol production in humans with a soy challenge, a comparison of phytoestrogen metabolism when subjects consumed fermented and unfermented soy products, and a dose-response study of urinary isoflavonoid excretion at the low end of soy consumption. All studies were conducted in young, healthy humans. Urinary isoflavonoids were measured by isotope-dilution gas chromatography–mass spectrometry. Similar to results from other studies, 35% of screened subjects (30 men and 30 women) excreted equol (> 2000 nmol/d). In women, equol excretion was associated with higher intake of dietary fiber and carbohydrate. Fermentation of soy decreased the isoflavone content of the product fed but increased the urinary isoflavonoid recovery, suggesting that fermentation increases availability of isoflavones in soy. When soy-protein powder was fed at 0, 5, 10, and 20 g/d (0–36 mg isoflavones), there was a linear dose response of urinary isoflavonoid excretion to soy consumption that did not differ between subjects with high and low equol excretion. These results suggest that equol excretion may be related to the fermentable carbohydrate content of the diet; additional study is needed. Processing of soy affects isoflavone metabolism and must be considered in recommending exposure to isoflavones from soyfoods. Although optimal isoflavone exposure for disease protection has not been determined, urinary isoflavonoid excretion appears linear at low-to-moderate soy consumption.

Urinary excretion of phenol, catechol, hydroquinone, and muconic acid by workers occupationally exposed to benzene.

OBJECTIVES: Animal inhalation studies and theoretical models suggest that the pattern of formation of benzene metabolites changes as exposure to benzene increases. To determine if this occurs in humans, benzene...

Isotope Dilution Mass Spectrometric Quantification of 3-Nitrotyrosine in Proteins and Tissues Is Facilitated by Reduction to 3-Aminotyrosine

Oxidative damage by reactive nitrogen species has been implicated in the pathogenesis of atherosclerosis and other inflammatory diseases. The mechanisms of tissue damage are poorly understood, however, because the toxic intermediates are short-lived. Previousin vitrostudies have suggested that 3-nitrotyrosine represents a specific marker of protein oxidation by reactive nitrogen species. The detection of this nitrated aromatic amino acid may thus serve as an indicator of tissue injury by nitrogen speciesin vivo.Here we describe a highly sensitive and specific analytical method for quantifying free and protein-bound 3-nitrotyrosine. The assay involves acid hydrolysis of proteins, isolation of 3-nitrotyrosine by ion exchange chromatography, and reduction of 3-nitrotyrosine to 3-aminotyrosine with dithionite. The reduced amino acid is then converted to itsn-propyl, per-heptafluorobutyryl derivative and quantified by isotope dilution gas chromatography negative-ion chemical ionization mass spectrometry. Attomole levels of 3-nitrotyrosine can be reproducibly measured in this manner. Quantifying 3-nitrotyrosine levels of tissues by stable isotope dilution gas chromatography/mass spectrometry should provide a powerful tool for exploring the impact of reactive nitrogen species on oxidative reactionsin vivo.

Chronic Administration of Methylmalonic Acid (MMA) to Rats Causes Proteinuria and Renal Tubular Injury • 1815

Children with methylmalonic acidemia frequently develop chronic renal insufficiency, for reasons unknown. We administered MMA chronicaally to rats to test the hypothesis that MMA is nephrotoxic. Methods: 3-wk-old Sprague-Dawley rats received MMA (pH 7.2-7.4), 20mg/100g BW daily by SQ injection X 6 wks (N=6), then in drinking water (40mg/100g BW/day) for 6 more wks. Two rats received MMA in drinking water from the outset (20mg/100g BW/day X 4 wks, 40mg/100g BW/day X 8 wks). Five rats received saline injections twice daily X 6 wks. Urine and blood were collected every 2 wks. Renal biopsy was performed at 6 wks and necropsy at 12 wks. Urinary MMA was measured by stable isotope dilution gas chromatography- mass spectometry. Results: MMA-treated rats and controls were similar in food and water intake, weight gain, serum creatinine and BUN. SQ or oral MMA produced high urinary MMA levels (e.g., 1646 μM/mM creatinine at 6 wks (mean), vs. 8.26 in controls). Six of 8 MMA-treated rats had proteinuria, that was first detected at 2 wks and thereafter increased (Table). After 12 wks of MMA, glomeruli appeared normal but tubules of proteinuric rats were variably dilated (especially in the renal medulla) with proteinaceous casts and flattened epithelium. By EM, tubular epithelium showed variable mitochondrial swelling and disorganization of cristae, dilated smooth endoplasmic reticulum, dilatation of basolateral intercellular spaces, and more secondary lysosomes than controls. Conclusions: Chronic administration of MMA rats causes renal injury, which histologically appears to predominantly affect tubular epithelial cells. These findings suggest that chronic renal insufficiency associated with methylmalonic acidemia results from nephrotoxicity of MMA.

Subnanomolar quantification of caffeine's in vitro metabolites by stable isotope dilution gas chromatography–mass spectrometry

A method for the quantification of subnanomolar levels of in vitro metabolites of caffeine by an isotope dilution gas chromatographic–mass spectrometric (GC–MS) assay has been developed and applied. Trideuteromethylated analogs of each primary metabolite were synthesized and added after incubations of caffeine with human liver microsomes high in cytochrome P4501A2. HPLC separation of the metabolites prior to GC–MS quantification allowed the isolation of theobromine and paraxanthine which coeluted by GC and enabled quantification over a larger dynamic range. Quantitative analysis was performed on the n-propylated derivatives by selected-ion monitoring of either the M +⋅ ions for the dimethylxanthines or [M−C 3H 6] +⋅ ions for 1,3,7-trimethyluric acid. For the least abundant metabolite (1,3,7-trimethyluric acid), the detection level on column was 200 pg. Replicate analyses exhibited intra- and inter-day variability of 4.2 and 7.9%, respectively. This assay has been successfully used in the quantification of caffeine's primary metabolites in more than 180 incubations, at varying substrate concentrations and with multiple enzyme sources.

Pristanic acid β-oxidation in peroxisomal disorders: studies in cultured human fibroblasts

To investigate the individual steps of peroxisomal β-oxidation, human fibroblasts from controls and patients affected by different peroxisomal disorders were incubated for 96 h with pristanic acid. Hereafter, 2,3-pristenic acid and 3-hydroxypristanic acid in the incubation medium were quantified by stable isotope dilution gas chromatography mass spectrometry (GC-MS). In control fibroblasts, both intermediates were formed and excreted into the medium in significant amounts. In cells from patients affected with different types of generalized peroxisomal disorders, the formation of both intermediates was absent or low, depending on the clinical severity of the disorder. In fibroblasts from patients affected with bifunctional protein deficiency, the concentrations of 2,3-pristenic acid and 3-hydroxypristanic acid in the medium were higher than in control cell lines.

Comparison of Oxidative Base Damage in Mitochondrial and Nuclear DNA

The levels of endogenous pig liver cells mitochondrial DNA oxidative base damage have been investigated using isotope dilution gas chromatography mass spectrometry (GC/MS). Higher levels of five measured bases were found in mtDNA in relation to nuclear DNA. We have also detected large differences in the modified base ratios of mitochondrial versus nuclear DNA. These ratios for the bases with promutagenic properties as 8OHGua and 5OHCyt are much lower then for other bases (5OHHyd, 5OHMeHyd, 5OHMeUra).

Amino Acid Analysis of Peptides and Proteins on the Femtomole Scale by Gas Chromatography/Mass Spectrometry

Amino acid analysis (AAA) is a useful aid in protein chemistry, but its routine application is limited by a modest limit of detection. Typically, 10 pmol of material is required, but even at this level the reproducibility can be poor. We have employed isotope dilution gas chromatography electron capture negative ionization mass spectrometry (GC/ECNI/MS) to provide accurate and reliable data on less than 100 fmol of material. Precision and accuracy are good, and all 20 non-hydrolyzed amino acids can be determined in this manner. The protein is hydrolyzed (HCl), and then a cocktail, composed of all 20 amino acids as stable isotope-labeled forms (i.e., (13)C and (2)H), is added. The mixture of protein-derived and stable isotope-labeled amino acids is then converted to volatile electron-capturing derivatives with a multistep approach employing heptafluorobutyric anhydride (HFBA), pentafluorobenzyl bromide (PFBBr), and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). These derivatives are then injected directly into the GC/MS system. Groups of selected ions, characteristic of each derivatized amino acid, are thereafter monitored at appropriate time intervals. The ratios of the ion current for the selected ions for the native amino acid and its labeled form are determined and converted to absolute amounts of the native amino acids in the protein hydrolyzate by reference to standard samples prepared at the time of the analysis.