Stable Isotope Dilution Gas Chromatography-mass Research Articles

Determination of lead in urine and whole blood by stable isotope dilution gas chromatography-mass spectrometry

A stable isotope dilution gas chromatography-mass spectrometry (GC-MS) method is described for the determination of lead (Pb) in urine and whole blood. The use of lithium bis(trifluoroethyl)dithiocarbamate Pb(FDEDTC) as a chelating agent showed strong memory effect, restricting the range of Pb isotope ratios that can be measured in unknown samples. To overcome this carryover problem, we further derivatized the Pb(FDEDTC)2 chelate with 4-fluorophenyl magnesium bromide to form Pb(FC6H4)4. The sequential analyses of solutions of natural Pb and enriched 204Pb with Pb(FC6H4)4 chelate by GC-MS demonstrated no observable memory effect. Precision and accuracy of Pb isotope ratio measurements with Pb(FC6H4)4 were established, and the isotope dilution GC-MS method was validated by determining Pb concentrations in urine standards from the National Institute of Standards and Technology, urine and blood reference materials from the New York State Department of Health, and blood Pb survey samples from the College of American Pathologists.

Measurement of Excitatory Sulfur Amino Acids, Cysteine Sulfinic Acid, Cysteic Acid, Homocysteine Sulfinic Acid, and Homocysteic Acid in Serum by Stable Isotope Dilution Gas Chromatography-Mass Spectrometry and Selected Ion Monitoring

Oxidized sulfur-containing amino acids are recognized as agonists of excitatory amino acid receptors in the mammalian nervous system. Homologues of glutamic acid (homocysteine sulfinic acid and homocysteic acid) and aspartic acid (cysteine sulfinic acid and cysteic acid) have been shown to be agonistic to N-methyl-D-aspartate receptors in animal brain and have been demonstrated in brain tissue. Considerable evidence exists for the role of homocysteic acid and cysteine sulfinic acid as endogenous ligands for excitatory amino acid receptors. We report, for the first time, the quantitation of these compounds in normal human serum, by a newly developed gas chromatography-mass spectrometry method that employs stable isotope-dilution selected ion monitoring using internal standards prepared in our laboratory. We also report new methods of synthesis of stable isotope-labeled internal standards used in measuring cysteine sulfinic acid, cysteic acid, homocysteine sulfinic acid, and homocysteic acid.

Catechol estrogen production in rat microsomes after treatment with indole-3-carbinol, ascorbigen, or β-naphthaflavone: A comparison of stable isotope dilution gas chromatography-mass spectrometry and radiometric methods

Compounds like indole-3-carbinol (I3C) have been shown to increase catechol estrogen formation and reduce mammary tumor incidence in mice. These compounds may exert a protective effect for breast cancer development by decreasing the overall estrogen pool available for the formation of 16α-hydroxyestrone (16α-OHE 1), a metabolite that retains significant estrogenic activity, may be mutagenic and could represent a potential carcinogenic intermediate of estradiol degradation. I3C and ascorbigen originate from the breakdown of glucobrassicin. We have compared the inductive effects of I3C with ascorbigen and β-naphthaflavone (Bnf) in microsomes from rats pretreated with these compounds using isotope dilution GC-MS and a radiometric method. Incubated microsomes from rats pretreated with I3C and ascorbigen yielded high levels of 2-hydroxyestradiol (2-OHE 2) that were comparable to levels induced by Bnf and were significantly above control group levels ( p < 0.005). Absolute values determined by the radiometric method were approximately 40% lower than 2-OHE 2 concentrations determined by GC-MS, although the relative changes in each group were the same. These differences may be attributed to the radiolabel becoming trapped in microsomal intermediates in the sequence leading to tritium entering the aqueous compartment. Both ascorbigen- and Bnf-treated animals exhibited significant increases in 2-hydroxyestrone (2-OHE 1) ( p < 0.05). The ability of ascorbigen to induce estradiol C-2 hydroxylation has not been previously reported. Based on these data, we speculate that ascorbigen will act as an anticarcinogenic agent and will inhibit or reduce the incidence of mammary tumor formation.

Mercury determination in blood by gas chromatography-mass spectrometry.

A stable isotope dilution gas chromatography-mass spectrometry method using 196Hg as an internal standard is described for determining Hg in blood. In this method, the blood samples are not subjected to any digestion to avoid the loss of Hg. A solution of 0.6M HCl is used to free Hg present in blood from proteins. The pH of the solution is adjusted to 9 using borate buffer and Hg chelated using lithium bis(trifluoroethyl)dithiocarbamate. All isotope ratio measurements are made using an organic mass spectrometer. Overall precision values for the five major Hg isotopes relative to 202Hg are 1.6-2.3% when 10 ng samples of chelated Hg are analyzed. No appreciable memory or carryover effect is observed when two synthetic mixtures differing in 196Hg/202Hg ratios by a factor of 30 are sequentially analyzed. The method is validated by determining Hg in blood samples using isotope dilution GC-MS.

New developments in the diagnosis and investigation of mitochondrial fatty acid oxidation disorders.

Since the discovery of muscle carnitine palmitoyltransferase deficiency in 1973, a dozen separate defects of mitochondrial fatty acid beta-oxidation in man have been identified. With the exception of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, which occurs with a frequency approaching 1:10,000 among Caucasians of Northern European origin, the other defects are quite rare. Collectively, however, they are common causes of disease resembling Reye syndrome in early life, and some have a later and more chronic presentation with cardiomyopathy and skeletal muscle weakness. They also represent a small, but significant, proportion of cases of sudden and unexplained death within the first 2 years of life. Diagnosis of these disorders has become increasingly sophisticated, with the advent of new analytical technologies and an increased awareness of the appropriate clinical and laboratory investigations needed in order to evaluate potential defects of this pathway. The combination of provocative testing (e.g., carnitine loading, phenylpropionic acid loading, long-chain fat loading) and advanced analytical techniques for the measurement of blood and urinary metabolites (e.g., tandem fast atom bombardment-mass spectrometry, stable isotope dilution gas chromatography-mass spectrometry) permits a specific diagnosis in the case of several, although not all, of the disorders of this pathway. Methods for the measurement of all of the enzymes of beta-oxidation are now available to enhance this diagnostic capability. There remain, however, many patients in whom clinical and laboratory signs point to a defect in beta-oxidation, but in whom no specific diagnosis has yet been made.(ABSTRACT TRUNCATED AT 250 WORDS)

High Prevalence of Cobalamin Deficiency in Elderly Outpatients

To measure the prevalence of cobalamin (vitamin B12) deficiency in geriatric outpatients as documented by both low serum cobalamin levels and elevations of serum methylmalonic acid and homocysteine and to determine the response to cobalamin treatment. Prospective study screening elderly subjects for cobalamin deficiency using radiodilution cobalamin assays as well as stable isotope dilution gas chromatography-mass spectrometry methylmalonic acid and homocysteine assays. In patients with serum cobalamin levels < or = 300 pg/mL, the response to cobalamin treatment in the group with levels of methylmalonic acid and/or homocysteine > 3 standard deviations (SD) above the mean for normals was compared with that of those without such elevations. Outpatient geriatric clinics at the VA Medical Center and University Health Sciences Center, Denver, CO. One-hundred and fifty-two consecutive outpatients, ages 65 to 99, were screened. Twenty-nine subjects with serum cobalamin levels < or = 300 pg/mL were prospectively evaluated and treated with cobalamin. Cobalamin, methylmalonic acid, homocysteine, complete blood counts, neurologic examination, and neuropsychological testing. The prevalence of cobalamin deficiency as defined by a serum cobalamin level < or = 300 pg/mL and levels of serum methylmalonic acid and/or homocysteine elevated to > 3 SD was 14.5% of the screened outpatients. A similar proportion of patients with low normal serum cobalamin levels (between 201 and 300 pg/mL) demonstrated elevated metabolites > 3 SD (56%) compared with patients with low serum cobalamin levels (< or = 200 pg/mL) (62%). Cobalamin therapy caused a marked fall or complete correction of the elevated methylmalonic acid and homocysteine levels in each patient who was treated prospectively. Results for complete blood count, lactate dehydrogenase, bilirubin, baseline neurologic score, and baseline neuropsychologic scores did not differ in the group of patients with elevated metabolites compared with those with normal metabolites. The mean red cell volume fell significantly in the patients with elevated metabolites after 6 months of cobalamin treatment. One patient with elevated metabolites had marked improvement in his neurologic abnormalities after 6 months of cobalamin treatment. There was a high (14.5%) prevalence of cobalamin deficiency as demonstrated by elevations in serum methylmalonic acid and homocysteine in addition to low or low normal serum cobalamin levels in elderly outpatients. The serum cobalamin level was insensitive for screening since similar numbers of patients with low normal serum cobalamin levels of 201-300 pg/mL compared with patients with low cobalamin levels (< or = 200 pg/mL) had markedly elevated metabolites which fell with cobalamin treatment. Additional studies will be required to define the full clinical benefit from treatment with Cbl in elderly subjects.

Determination of copper in urine and serum by gas chromatography-mass spectrometry

A stable isotope dilution gas chromatography-mass spectrometry method using enriched 65Cu as an internal standard is described for the determination of Cu in urine and serum. Chelating agents N,N′-ethylenebis-(trifluoroacetylacetoneimine) [H 2(enTFA 2)] and lithium bis(trifluoroethyl)dithiocarbamate [Li(FDEDTC)] were used and evaluated for memory effect. H 2(enTFA 2) did not show any appreciable memory effect, whereas Li(FDEDTC) was found to have a strong memory effect. Overall precision of 1.6% was obtained for determining Cu isotope ratios at a 10-ng level using H 2(enTFA 2). Cu concentrations in the National Institute of Standards and Technology (NIST) reference materials, freeze-dried urine SRM 2670, and human serum SRM 909 determined using the H 2(enTFA 2) chelating agent were in good agreement with the NIST-certified values. Isotope ratios determined by gas chromatography-mass spectrometry on samples with altered isotopic composition were in good agreement with the inductively coupled plasma-mass spectrometry data.

A sensitive method for the quantification of the mass of inositol phosphates using gas chromatography-mass spectrometry

A sensitive method to directly measure the mass of inositol phosphates from biologic samples is described. The procedure uses ammonium sulfate gradient elution anion exchange column chromatography to isolate inositol monophosphate, bisphosphate, trisphosphate, and tetrakisphosphate. The isolated fractions are dephosphorylated and subsequently desalted by a novel approach using solid barium hydroxide in a 1:1 stoichiometric ratio to the amount of ammonium sulfate present in the dephosphorylated sample. The myo-inositol derived from each inositol phosphate species was quantified by stable isotope dilution gas chromatography-mass spectrometry of the hexakis(trimethylsilyl) derivative using hexadeutero-myo-inositol as the internal standard. The applicability and sensitivity of this method are illustrated by measuring the mass of individual inositol phosphates in isolated adult canine cardiac myocytes.

Prenatal Diagnosis of Propionic and Methylmalonic Acidaemia by Stable Isotope Dilution Analysis of Amniotic Fluid

Stable isotope dilution gas chromatography—mass spectrometry (GC/MS) analysis of methylcitric acid in amniotic fluid for the rapid and accurate prenatal diagnosis of propionic acidaemia (McKusick 23200) and methylmalonic acidaemia (McKusick 25100) was first described in 1980 (Naylor et al., 1980). This was soon followed by similar methods for the determination of methylmalonic acid in amniotic fluid for prenatal diagnosis of methylmalonic acidaemia (Trefz et al.,1981; Zinn et al., 1982; Sweetman et al.,1982). We describe here our results for the prenatal diagnosis of 102 pregnancies at risk for either propionic acidaemia or methylmalonic acidaemia.

Isotope dilution gas chromatography—mass spectrometry for the study of eicosanoid metabolism in human blood platelets

Stable isotope dilution gas chromatography—mass spectrometry provides one of the most important techniques for the quantitative measurement of eicosanoids. This technique was aplied to the quantitation of hydroxyeicosatetraenoic acids, hydroxyheptadecatrienoic acid, thromboxane B 2 and prostaglandin F 2α formed during platelet aggregation after stimulation of gel-filtered platelets with thrombin (0.25 U/ml) or collagen (2 μg/ml). Similar amounts of hydroxyheptadecatrienoic acid and thromboxane B 2 were found after platelet activation. The ratio of formation of 12-hydroxyeicosatetraenoic acid to thromboxane B 2 varied from donor to donor. Only small amounts of prostaglandin F 2α (up to 200 pg per 2.0·10 8 platelets) and basic values of 15-hydroxyeicosatetraenoic acid (up to 100 pg per 2.0·10 8 platelets) were measured using gas chromatography with negative ion chemical ionization mass spectrometry. In addition, different stable isotope dilutions were prepared and are discussed in detail.

Methylcitrate in maternal urine during a pregnancy with a fetus affected with propionic acidaemia

Patients homozygous for propionic acidaemia have high levels of methylcitric acid in urine while heterozygotes have normal levels. Methylcitric acid was determined by stable isotope dilution gas chromatography-mass spectrometry in maternal urine during a pregnancy with a fetus affected with propionic acidaemia. Although the levels increased linearly between 23 and 39 weeks of gestation, the levels were not significantly elevated above normal until after 30 weeks of gestation. Thus, determination of methylcitric acid in maternal urine is not of value for early prenatal diagnosis of propionic acidaemia.

Analysis of the plasticiser acetyltributyl citrate in foods by stable isotope dilution gas chromatography-mass spectrometry

Description d'une technique de dosage de cet adjuvant des matieres plastiques d'emballage des produits alimentaires en vue de mesurer la migration de ce compose, apres marquage au deuterium

Analysis of di-(2-ethylhexyl)adipate plasticiser in foods by stable isotope dilution gas chromatography—mass spectrometry

Realisation d'une technique de dosage de ce compose present dans les emballages alimentaires et qui peut contaminer les produits par migration (essai sur du fromage, des gâteaux)

Vascular sensitivity to prostaglandin I2 and urinary excretion of 6-keto-prostaglandin F1 alpha in conscious dogs.

A method is described for measuring the urinary excretion of 6-keto-prostaglandin F1 alpha, the stable hydrolysis product of prostaglandin I2, by stable isotope dilution gas chromatography-mass spectrometry. Three different doses of prostaglandin I2 were infused intravenously into conscious dogs and the effects on systemic and renal haemodynamics and urinary sodium excretion were observed. The two highest infusion rates of prostaglandin I2 (15 and 30 ng min-1 kg-1 body weight) induced significant decreases in systematic blood pressure and dose-related increases in sodium excretion, but no change in renal haemodynamics. There was a linear relationship between urinary excretion of 6-keto-prostaglandin F1 alpha and the rate of infusion of prostaglandin I2. The calculated basal rate of entry of prostaglandin I2 into the systematic circulation in conscious dogs is 4 ng min-1 kg-1 body weight, which is substantially higher than that previously reported in man.

Qualitative and quantitative measurement of hydroxy fatty acids, thromboxanes and prostaglandins using stable isotope dilutions and detection by gas chromatography—mass spectrometry

Methods for measurement of the metabolites of arachidonic acid (AA), namely prostaglandins (PGs), thromboxanes (TXs) and hydroxy fatty acids, using stable isotope dilution gas chromatography—mass spectrometry are described. With a few exceptions, labelled species of the various AA metabolites are not commercially available and were therefore synthesized in our laboratory. [ 2H 8] AA, produced by deuteration of eicosatetraynoic acid, was used for comparing the metabolism of exogenously added and endogenously present AA in fibroblast cultures. After derivatization and catalytic hydrogenation, structure elucidation and quantification of the different hydroxy fatty acids was carried out by determination of the fragment ions resulting form α-cleavage at the site of the hydroxy function. During catalytic hydrogenation a significant hydrogen—deuterium exchange was observed. To eliminate this problem, 18O-labelled standards were prepared by exchanging the oxygen of the carboxylic acid group. The preparation and the use of hydroxy fatty acids, PGs and TXs labelled with 18O is described.

Measurement of plasma histamine by stable isotope dilution gas chromatography-mass spectrometry: Methodology and normal values

A new method for the determination of histamine by stable isotope dilution mass fragmentography is described. The method is specific, sensitive, and accurate, resulting in a within-day coefficient of variation of 4.1% and a day-to-day variation of 7.9%. It was shown that the first blood sample after a venipuncture can contain an artificially elevated plasma histamine concentration. Platelets contain about 7 pmol histamine/10 9 cells. Serum histamine was elevated about four times in comparison with plasma histamine. This phenomenon was mainly ascribed to degranulation of basophilic leukocytes by complement activation during blood clotting. Normal values for plasma histamine were ( n = 25) 2.07 ± 0.75 nmol/liter (mean ± 1 SD), which is one of the lowest values reported up to now.

Analysis of modified bases in DNA by stable isotope dilution gas chromatography-mass spectrometry: 5-Methylcytosine

A sensitive assay for 5-methylcytosine in DNA has been developed based on stable isotope dilution gas chromatography-mass spectrometry with selected ion monitoring. 5-([ 2H 3]-Methyl)cytosine and [ methyl- 2H 3]thymine have been synthesized as internal standards for analysis of DNA following acid digestion, conversion of pyrimidines to volatile t-butyldimethylsilyl derivatives, and separation in 3 min by gas chromatography. Submicrogram amounts of DNA have been analyzed for 5-methylcytosine content in the range 0.02–1.5 mol%. The estimated limit of quantitative measurement is 0.3 pmol of methylated base in a DNA hydrolysate. The method is compared with other techniques for quantitative measurement of methylated bases in DNA, and 5-methylcytosine levels and precision of analysis for calf thymus, pBR322, and ΦX-174 DNAs are reported and compared with literature values. The method can readily be adapted to the accurate high-sensitivity analysis of other methylated bases in DNA.

Determination of selenium in biological materials by stable isotope dilution gas chromatography-mass spectrometry.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDetermination of selenium in biological materials by stable isotope dilution gas chromatography-mass spectrometryDonald C. Reamer and Claude. VeillonCite this: Anal. Chem. 1981, 53, 14, 2166–2169Publication Date (Print):December 1, 1981Publication History Published online1 May 2002Published inissue 1 December 1981https://doi.org/10.1021/ac00237a004RIGHTS & PERMISSIONSArticle Views137Altmetric-Citations74LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (514 KB) Get e-Alerts Get e-Alerts

Quantitative analysis of two dinor urinary metabolites of prostaglandin I 2

The synthesis of deuterium- and tritium-labeled analogs of 2,3-dinor-6-keto-prostaglandin F 1α and of 6,15-diketo-13,14-dihydro-2,3-dinor-prostaglandin F 1α is described. These analogs were used as internal standards in the assay of the corresponding unlabeled metabolites in human urine by stable isotope dilution and combined gas chromatography-mass spectrometry. In male subjects the 24-h urinary excretion of the two metabolites was found to be 719 ± 264 and 314 ± 115 ng, respectively. The method offers a noninvasive approach to the study of prostaglandin I 2 synthesis in man.

Quantitative determination of the major metabolite of prostaglandins F 1α and F 2α in human urine by stable isotope dilution and combined gas chromatography-mass spectrometry

An improved gc-ms method has been developed for the measurement of 5α,7α- dihydroxy - 11 - ketotetranorprosta - 1,16 - dioic acid (PGF-M), the major urinary metabolite of PGF 1α and PGF 2α in man. The assay is based on gas chromatography and selected ion monitoring mass spectrometry, employing a specifically deuterated analog of the metabolite, labeled in the cyclopentane ring, as internal standard. The analytical procedure involves extraction from urine, methylation and selective hydrolysis at pH10 to afford the PGF-M 16-monomethyl ester. Dehydration of this compound to the corresponding δ-lactone methyl ester and subsequent solvent extraction effects a substantial purification of the urinary extract. Following conversion to its t-BDMS ether methyl ester derivative, the metabolite is purified by thin-layer chromatography and analyzed by gas chromatography-mass spectrometry. Measurements of urinary PGF-M in the normal range of 5–40 ng/ml may be made with 2% precision from a 10 ml sample while the lower limit of detection is in the order of 1 ng/ml.