Stable Isotope Dilution Mass Spectrometry Research Articles

Effect of SRI 63-675 on hemodynamics and blood PAF levels during porcine endotoxemia

We evaluated the effect of SRI 63-675, a specific platelet-activating factor (PAF) receptor antagonist, on hemodynamics and PAF biosynthesis during 4 h of porcine endotoxemia. Hexadecyl PAF was extracted from blood, purified by normal-phase and reverse-phase high-performance liquid chromatography (RP-HPLC), and quantitated by stable isotope dilution and thermospray mass spectrometry. Infusion of either saline or SRI 63-675 alone caused no change in hexadecyl PAF concentrations. In contrast, endotoxin increased blood hexadecyl PAF concentrations from 1.5 +/- 0.1 ng/ml at 0 h (baseline) to a peak value of 8.3 +/- 1.9 ng/ml at 0.5 h of endotoxemia (P less than 0.05). Blood PAF levels gradually declined toward the baseline value after 0.5 h of endotoxemia. Because endotoxin did not modify plasma acetylhydrolase activity ex vivo, the increased hexadecyl PAF levels were probably secondary to increased PAF biosynthesis and not decreased biodegradation. Bioassay of RP-HPLC fractions that were derived from endotoxemic blood and that eluted at a retention time consistent with [3H]alkyl PAF caused aggregation of washed rabbit platelets that was inhibited by SRI 63-675. The PAF receptor antagonist blocked the 0.5 h endotoxin-induced increase in blood hexadecyl PAF concentration concomitant with blockade of thrombocytopenia. The endotoxin-induced pulmonary hypertension, decreased cardiac index, and increases in pulmonary vascular resistance and alveolar-arterial O2 gradient were attenuated by SRI 63-675. The data suggest that PAF-stimulated PAF biosynthesis may substantially contribute to blood hexadecyl PAF levels and cardiopulmonary dysfunction during the initial phase of endotoxemia.

Diurnal changes in plasma levels of 2-pyrrolidinone determined by isotope dilution mass spectrometry

A new capillary gas chromatographic method with mass spectrometric detection for the determination of 2-pyrrolidinone was developed. Using quantification based on stable isotope dilution mass spectrometry by monitoring selected ions in the ammonia chemical ionization mode diurnal changes of 2-pyrrolidinone levels in plasma of three healthy adults were established. Although substantial fluctuations, ranging from 5 to 30 μg/l, occurred during a 24-h daytime period, no consistent picture related to clock time or food intake was obtained. Endogenous oscillations should be taken into consideration, for example when evaluating transdermal penetration of 2-pyrrolidinone, used as enhancer in topical drug formulation.

Serum theophylline analysis by isotope dilution mass spectrometry

Serum theophylline analysis was attempted using stable isotope dilution mass spectrometry. Theophylline 15N-labelled in position 7 was used as internal standard. The analysis was performed at resolution 3000 by selected ion monitoring with temperature programming. The sample was introduced in underivatized form by means of the direct inlet probe. Good linearity was obtained in the concentration range 5-40 micrograms ml-1 with relative standard deviations less than 6%. The method is simple and permits six samples to be run per hour. Sensitivity permits analysis at 0.5 micrograms ml-1 at a signal to noise ratio 4:1.

Analysis of abnormal urinary metabolites in the newborn period in medium‐chain acyl‐CoA dehydrogenase deficiency

In order to determine which are useful early diagnostic markers for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, we have analysed urine from an asymptomatic neonate. Profiling of urinary organic acids followed by peak confirmation by electron impact mass spectrometry revealed a high suberate/adipate ratio (greater than 1.0) and the presence of n-hexanoylglycine (HG). Acylcarnitine analysis by fast atom bombardment mass spectrometry (FAB-MS) was inconclusive, but FAB-MS/MS (tandem mass spectrometry) revealed diagnostic amounts of octanoylcarnitine and hexanoylcarnitine. Quantitative analysis of acylglycines by stable isotope dilution and chemical ionization mass spectrometry revealed a 30-fold increase in HG and increased suberylglycine, but no increase in 3-phenylpropionylglycine.

Determination of the new aromatic retinoic acid Ro 13-7410 in plasma by two-dimensional gas chromatography negative ion chemical ionization mass spectrometry with selected-ion monitoring

A highly sensitive and specific method for the determination of the aromatic retinoic acid Ro 13-7410 in plasma at the picogram per millilitre level is described. The method involves extraction of plasma using Extrelut-1 columns, purification of the extract with Bond Elut-NH2 cartridges, derivatization with pentafluorobenzyl bromide, and subsequent analysis by two-dimensional capillary gas chromatography using the zone-cutting technique, stable isotope dilution and selective negative ion monitoring chemical ionization mass spectrometry. A tetradeuterated analogue is used as internal standard. Quantification is possible down to 25 pg/ml using 1 ml of plasma. The coefficients of variation of the method as calculated from quality control samples are 8.5 and 4.2% at the 100 and 400 pg/ml levels. The method has been applied to the analysis of plasma of volunteers following an oral dose of 40 micrograms Ro 13-7410 and plasma of dogs following an intravenous and oral dose of 25 and 50 micrograms, respectively.

Quantitative stereochemical analysis of subnanogram amounts of 12-hydroxy-(5,8,10,14)-eicosatetraenoic acid by sequential chiral phase liquid chromatography and stable isotope dilution mass spectrometry

The two enantiomers of 12-hydroxy-(5,8,10,14)-eicosatetraenoic acid (12-HETE) are products of different biosynthetic pathways and have distinct biologic actions. Conventional methods of stereochemical analysis of 12-HETE require multimicrogram amounts of material and cannot be applied to systems where the availability of tissue is limited and only trace quantities of 12-HETE are generated. We have developed a method capable of measuring subnanogram amounts of 12-HETE enantiomers which involves addition of racemic, 18O 2-labeled 12-HETE as an internal standard, chiral phase HPLC of the pentafluorobenzyl ester derivative of 12-HETE, and stable isotope dilution gas chromatographic-negative ion chemical ionization mass spectrometric quantitation of the resolved stereoisomers. This method has been employed to determine the stereochemical composition of 12-HETE produced by isolated pancreatic islets.

Stable isotope dilution mass spectrometry for the diagnostic study of histidinaemia.

Stable isotope dilution mass spectrometry for a diagnosis to detect the heterozygote state of histidinaemia has been developed. We have synthesized L-[3-15N, 5,beta,beta-2H3]histidine (histidine-[M + 4]) for use as a biological internal standard and DL-[1,3-15N2, 5,alpha,beta,beta-2H4]histidine (histidine-[M + 6]) as an analytical internal standard. For the gas chromatographic/mass spectrometric assay of stable isotopically substituted histidine in human plasma, histidine was derivatized to alpha N-trifluoroacetyl-imN-ethoxycarbonylhistidine n-butyl ester. Quantification was carried out by selected ion monitoring on the molecular ions (m/z 379, 383 and 385) of the respective gas chromatographic derivatives of non-labelled histidine, histidine-[M + 4] and histidine-[M + 6]. The calibration curve was linear over the range of 5-2000 ng ml-1 plasma (r = 0.9999). The intra- and inter-assay coefficients of variation were in the range 1.1-3.9%. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for application to a pharmacokinetic study of histidine after administration of a trace amount of stable isotopically substituted histidine in man.

Determination of cortisol in human plasma by stable isotope dilution mass spectrometry.

Cortisol selectively labelled with 2H at the 19-methyl and the C-1 position (cortisol-d5) was synthesized and used as an internal standard in stable isotope dilution mass spectrometry in order to determine cortisol in human plasma. A capillary gas chromatographic/mass spectrometric method provided a sensitive and reliable technique with good accuracy, precision and reproducibility without complex corrections for contributions by using cortisol-d5 as an internal standard. For calculation of plasma cortisol, peak areas were measured by selected ion monitoring on the characteristic fragment ions of the dimethoxime tri(trimethylsilyl) derivatives of cortisol and cortisol-d5 (m/z 605 and 610, respectively). The sensitivity of the gas chromatographic/mass spectrometric assay was 1.02 ng per injection, with a signal-to-noise ratio of about 6. The inter- and intra-assay coefficients of variation for plasma sample were 3.07% and 1.77%, respectively.

Average mass approach to stable isotope dilution mass spectrometry

AbstractThe application of the fundamental equation for isotopic dilution mass spectrometry to real samples often requires using approximations which can lead to significant errors. A new approach relating the composition of isotopic mixtures to the average mass of the mass spectral pattern is presented. The fundamental equations are completely general; they should be applicable to a wide range of problems and may provide improved accuracy and precision in some cases.

Preparation of biological tissue for determination of arsenic and selenium by graphite furnace atomic absorption spectrometry.

A great deal of interest has been generated in the measurement of selenium in biological tissue. Consequently, a method was needed for the digestion of large numbers of tissue samples (mainly eggs and liver of avian species) for arsenic and selenium analysis. Several wet digestion methods are described for the solubilizing of biological tissues and perchloric acid is often used in these procedures. However, this acid can be dangerous and particularly so in the presence of organic material. To avoid the use of perchloric acid, the authors have used an open flask digestion method which involves the careful evaporation of a 20-mL nitric acid/tissue solution down to 3 mL, directly on a hot plate. The authors have overcome many of the shortcomings of this method through a modification of the nitric acid digestion method in which hydrogen peroxide is added during the digestion process to enhance the solubilizing of the biological tissue. Samples solubilized in this manner were then quantified directly for arsenic and/or selenium by using graphite furnace atomic absorption spectrometry (GFAAS) with Zeeman background correction. The authors were able to have the method for selenium independently verified by stable isotope dilution mass spectrometry (SIDMS). The lower limit ofmore » detection, based on a 0.5-g subsample (wet weight) and a 25-mL final volume dilution, was 0.10 pp for both arsenic and selenium. The lower limit of detection, based on a 0.25-g subsample (dry weight) and a 50-mL final volume dilution, was 0.40 ppm for the two elements.« less

Determination of glycolic acid level in higher plants during photorespiration by stable isotope dilution mass spectrometry with double-labeling experiments

Determination of glycolic acid by stable isotope dilution was applied to the measurement of the glycolic acid pool size in tomato and maize leaves during photorespiration. Detached leaves were maintained in the presence of 18O 2; [ 13C]glycolate was added to the foliar extract as an internal standard and the mixture of biological glycolate and [ 13C]glycolate was analyzed by combined gas chromatography-mass spectrometry. The level of foliar glycolate pool was measured via the 13C label, and 18O incorporation was determined.

Quantitative Determination of Dexamethasone in Human Plasma by Stable Isotope Dilution Mass Spectrometry

An analytical method for the quantitation of nanogram to subnanogram amounts of dexamethasone is described. Dexamethasone was isolated from human plasma using a C18-bonded reverse-phase cartridge, purified by subsequent normal-phase HPLC, and the corresponding trimethylsilyl derivative analyzed by gas chromatography-mass spectrometry (GC-MS). The quantitation by isotope-dilution MS was carried out by selected-ion monitoring on the (M + 1)+ ion of the trimethylsilyl derivative of dexamethasone and its stable isotopically labeled diluent, [13C6,2H3]dexamethasone (681 and 690m/z, respectively). Methane was used as the GC carrier gas and as the chemical-ionization reagent gas. The sensitivity of the method, judged from the lower limit of detection of the mass spectrometer, was at ~ 100pg. The inter-and intraassay coefficients of variation (CV) determined at two different concentrations were 3.83 and 3.78% for 2 ng/mL and 2.64 and 1.29% for 5 ng/mL, respectively. Plasma concentration profiles for dexamethasone following a single 1-mg iv and a 2-mg oral dose of dexamethasone administered 24h apart to two healthy volunteers are presented. The mass fragmentographic method described here is useful for bioavailability and pharmacokinetic studies of the synthetic glucocorticoid.

Cytoarchitectonic distribution of zinc in the hippocampus of man and the rat

Zinc was measured in whole hippocampus and in hippocampal sub-regions by stable-isotope dilution mass spectrometry. In both man and the rat, the most zinc (102–145 ppm, dry weight) was found in the hilar region, the least (27–37) in the fimbria. The amount of zinc directly associated with mossy-fiber axons was estimated to be approximately 8% of the total zinc in the hippocampus, and the concentration of mossy-fiber zinc was estimated at 220–300 μM. Methodological and theoretical implications of the quantitative findings were discussed.

Accurate assay of lithium by isotope dilution mass spectrometry.

A need for accurate assay of lithium exists in a number of different fields, e.g. in the nuclear field and in clinical chemistry. Stable isotope dilution mass spectrometry is a well established technique that can be used successfully for this purpose. The technique has an inherent accuracy which is constant over a wide dynamic concentration range. This work reports on the assay of lithium in LiF deposits of amounts of the order of mg and in human serum with lithium concentration in the ppm range. In both cases the assay has been achieved to an accuracy of 0.5 percent.

Stable-isotope dilution measurement of zinc and lead in rat hippocampus and spinal cord

Zinc and lead concentrations in hippocampus and spinal cord of rats were measured using the highly-accurate method of stable isotope dilution mass-spectrometry. In hippocampus, average zinc concentration was 72.7 ppm (dry weight), average lead, 0.053 ppm; in spinal cord, zinc averaged 26.1 ppm, lead, 0.018 ppm. Possible explanations for apparent overestimations of rat CNS metal content in previously publisjed work were discussed.

State-of-the-art contamination control techniques for ultratrace elemental analysis

The detection limits of many nonnuclear methods for ultratrace (≤0.1 μg/g) elemental determinations lie well below the level at which precise and accurate practical analysis can be executed routinely. Advances in analytical methodologies which are rapidly eroding such disparities are reviewed. The applications of X-ray fluorescence, atomic absorption, stable isotope dilution mass spectrometry, and laser intracavity absorption spectrophotometry to ultratrace analyses not amenable to solution by nuclear methods are discussed.

Accurate quantification of nonesterified and total cholesterol in plasma and urine from crude lipid extracts by stable isotope dilution and field desorption mass spectrometry.

Levels of free and total cholesterol were quantified in control sera and human urine using [2H7]cholesterol or [13C2]cholesterol as internal standard and field desorption mass spectrometry for the quantification. These analyses are performed using a crude chloroform extract without chromatographic separation of the constituents. Due to the extremely soft ionization conditions in field desorption, the constituents of the crude lipid extract predominantly form molecular ion species and only a very low amount of fragment ions of low relative abundance thus minimizing the possibility of ion overlap in the mass spectrum. Mass spectrometric quantification of cholesterol in two control sera resulted in data within the range of assigned concentrations. The standard deviations of the mass spectrometric data were around +/- 2%. Generally, 50 microliters of serum or plasma or 500 microliters of urine are extracted and the mass spectrometric quantification performed using an aliquot of 2-10%. A characteristic pattern of intact cholesteryl esters is also obtained.

Determination of uracil and thymine and their nucleosides and nucleotides in picomole amounts by gas chromatography mass spectrometry selected ion monitoring.

A stable isotope dilution method is presented by which uracil (Ura) and thymine (Thy) can be determined with high precision and sensitivity at the picomole level utilizing stable isotope dilution and gas chromatography electron impact mass spectrometry in the selected ion monitoring mode. [15N2]Ura and [2H3]Thy served as internal standards. The molecular ions as well as the [M-CH3]+ ion fragments of silylated Ura and Thy (Ura-TMS and Thy-TMS) were suitable for the assay which provides evidence of specificity, if identical results are obtained at both ions. Nucleosides and nucleotides of Ura and Thy were determined following quantitative hydrolysis in 6 N HCl at 180 degrees C for two hours. Other hydrolysis procedures did not give satisfactory results. Levels of free Ura and Thy were measured in human and rat plasma after solvent extraction with a sensitivity of 20-40 pm ml-1 demonstrating ready applicability of the assay method to biological samples. The potential physiological role of circulating Ura and Thy is discussed.

Determination of lithium traces in microliters of water, wine and high purity solvents by stable isotope dilution and field desorption mass spectrometry

In this work, the applicability of field desorption mass spectrometry for the determination of lithium in mineral and tap water, wine and high-purity solvents has been examined. Because of the outstanding sensitivity of field desorption mass spectrometry for alkali cations and the high specificity of mass spectrometry, it was possible to determine lithium from microlitre samples by using the method of stable isotope dilution. Lithium, at levels of 10−7–10−4 mg/ ml, was determined in the samples without any pretreatment. The time required for one analysis was about 20–30 min. The results from quantitative analyses of mineral water were compared with others obtained by atomic-absorption spectrophotometry and were in good agreement. The very small concentrations of lithium in wine and high-purity solvents, which are too low to be measured by conventional atomic-absorption techniques, can be determined accurately without difficulty by field desorption mass spectrometry.

Determination of choline and acetylcholine in distinct rat brain regions by stable isotope dilution and field desorption mass spectrometry

The determination of choline and acetylcholine by field desorption mass spectrometry from rat brain tissue samples has been demonstrated. Essential points of the assay are the use of stable isotope labelled internal standards, a simple ion pair extraction procedure, direct analysis of the quaternary ammonium ions without derivatization, and accumulation of the electrically recorded field desorption ion signals with a multi-channel analyser. The analysis of sample amounts in the picogram range gave quantitative data of good precision (0.6--10% standard deviation).