Stable isotope dilution method for the determination of serum glucose using discharge-assisted thermospray liquid chromatography/mass spectrometry

A discharge-assisted thermospray liquid chromatography/mass spectrometric method for the determination of serum glucose was studied. Isotope dilution technique was used with uniformly labelled (13C6) glucose as an internal standard. Successful liquid chromatography/mass spectrometry was achieved by post-column addition of aqueous ammonium acetate to the mobile phase. Quantification was performed by measuring the peak intensity ratios of the unlabelled and labelled [M + NH4]+ ions. Analytical results using the National Institute of Standards and Technology standard reference material serum showed satisfactory agreement with the certified value, and a relative standard deviation of about 1% was obtained.     

Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry

Trimethylamine-N-oxide (TMAO) levels in blood predict future risk for major adverse cardiac events including myocardial infarction, stroke, and death. Thus, the rapid determination of circulating TMAO concentration is of clinical interest. Here we report a method to measure TMAO in biological matrices by stable isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with lower and upper limits of quantification of 0.05 and >200 μM, respectively. Spike and recovery studies demonstrate an accuracy at low (0.5 μM), mid (5 μM), and high (100 μM) levels of 98.2, 97.3, and 101.6%, respectively. Additional assay performance metrics include intraday and interday coefficients of variance of <6.4 and <9.9%, respectively, across the range of TMAO levels. Stability studies reveal that TMAO in plasma is stable both during storage at −80 °C for 5 years and to multiple freeze thaw cycles. Fasting plasma normal range studies among apparently healthy subjects (n = 349) show a range of 0.73–126 μM, median (interquartile range) levels of 3.45 (2.25–5.79) μM, and increasing values with age. The LC/MS/MS-based assay reported should be of value for further studies evaluating TMAO as a risk marker and for examining the effect of dietary, pharmacologic, and environmental factors on TMAO levels.     

Determination of aldosterone in human serum by isotope dilution gas chromatography/mass spectrometry using a new heptafluorobutyryl derivative.

The formation of a new derivative of aldosterone with heptafluorobutyric anhydride for gas chromatography/mass spectrometry (GC/MS) is presented. The highest and also the most prominent ion of this derivative is observed at m/z 734. The new derivative is, under the described conditions, reproducibly formed, is stable and has good gas chromatographic properties. These characteristics make the new derivative extremely suitable for selective, sensitive, precise and accurate analysis of aldosterone in serum by GC/MS in association with isotope dilution. This is proven by the agreement between the measurement results obtained by two laboratories for samples of three batches of lyophilized control serum. The sample pretreatment procedures used in each laboratory are described.     

Stable isotope dilution analysis of salicylic acid and hydroquinone in human skin samples by gas chromatography with mass spectrometric detection

A sensitive and accurate gas chromatographic-mass spectrometric (GC-MS) method has been developed for the quantitative determination of salicylic acid (SA) and hydroquinone (HQ) from human skin samples and cosmetic emulsions. Deuterium labeled SA-d(6) and HQ-d(6) were used as internal standards (IS). The samples were extracted with methanol, dried under nitrogen and derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+1% trimethylchlorosilane (TMCS). Quantification was performed in SIM mode with a limit of quantification (LOQ) of 50 ng ml(-1) for SA and 10 ng ml(-1) for HQ. The inter-day variation (R.S.D.) was less than 5% and the accuracy was better than 13.3% for both compounds. The recoveries from the different matrices ranged between 93.1 and 103.3% for SA, and 97.3 and 100.8% for HQ.     

Quantitation of tetramethylene disulfotetramine in human urine using isotope dilution gas chromatography mass spectrometry (GC/MS and GC/MS/MS)

Tetramethylene disulfotetramine (tetramine) is a rodenticide associated with numerous poisonings was extracted and quantified in human urine using both gas chromatography/mass spectrometry (GC/MS) and GC/tandem mass spectrometry (MS/MS). 1200 μL samples were prepared using a ¹³C₄-labeled internal standard, a 96-well format, and a polydivinyl-benzene solid phase extraction sorbent bed. Relative extraction recovery was greater than 80% at 100 ng/mL. Following extraction, samples were preconcentrated by evaporation at 60 °C, and reconstituted in 50 μL acetonitrile. One-microliter was injected in a splitless mode on both instruments similarly equipped with 30 m × 0.25 mm × 25 μm, 5% phenyl-methylpolysiloxane gas chromatography columns. A quantification ion and a confirmation ion (GC/MS) or analogous selected reaction monitoring transitions (GC/MS/MS) were integrated for all reported results. The method was characterized for precision (5.92–13.4%) and accuracy (96.4–111%) using tetramine-enriched human urine pools between 5 and 250 ng/mL. The method limit of detection was calculated to be 2.34 and 3.87 ng/mL for GC/MS and GC/MS/MS, respectively. A reference range of 100 unexposed human urine samples was analyzed for potential endogenous interferences on both instruments—none were detected. Based on previous literature values for tetramine poisonings, this urinary method should be suitable for measuring low, moderate, and severe tetramine exposures.     

Measurement of enterolactone and enterodiol, the first mammalian lignans, using stable isotope dilution and gas chromatography mass spectrometry

Methods are described to extract and measure 2,3-bis(3-hydroxybenzyl)-gamma-butyrolactone (enterolactone) and 2,3-bis(3-hydroxybenzyl)butane-1,4-diol (enterodiol) from physiological fluids. They are based on the use of stable labelled analogues of each compound as internal standards with end point assay being by gas chromatography mass spectrometry. The methods were developed to quantify enterolactone and enterodiol in experiments designed to investigate the significance and source of these compounds in man and animals. Examples of studies on human ovarian blood and investigations of their excretion in urine following dietary manipulation are described.     

Stable isotope dilution quantification of mutagens in cooked foods by combined liquid chromatography-thermospray mass spectrometry

A method of general applicability for the detection and quantification of mutagens in cooked foods at the ppb level is presented. A minimal sample prefractionation is employed and [Me-²H₃-labeled analogs of the compounds of interest are added for identification and quantification of mutagens by accurate measurement of chromatographic retention (K′) in reverse-phase high-performance liquid chromatography (HPLC), and by measurement of the ratio of response of the protonated molecular ions of analyte and internal standard by directly coupled liquid chromatography-mass spectrometry (LC/MS). Initial application is demonstrated in the analysis of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quonoline (MeIQ) in broiled salmon. Measured levels of IQ and MeIQ in broiled salmon flesh were 0.3–1.8 ppb and 0.6–2.8 ppb, respectively, and for the skin of broiled salmon 1.1–1.7 ppb and 1.5–3.1 ppb, respectively. Results on cooked beef and sardine are also reported.     

Assessment of threonine metabolism in vivo by gas chromatography/mass spectrometry and stable isotope infusion

The fractional contributions (FC) of threonine to glycine and 2-ketobutyrate (KB) fluxes in fed pigs have been assessed by the constant infusion of L-[1-13C]-threonine. The analysis of the enantiomeric purity of labeled threonine by gas chromatography/mass spectrometric (GC/MS) analysis is reported as the N-TFA isopropyl ester derivative. The commercially available [1-13C]threonine comprised 98.7% of the L-enantiomer, enriched at 99 atom percentage excess (APE), and 1.3% of L-allo-threonine contaminant, also enriched at 99 APE. The enantiomeric purity of threonine in plasma of pigs infused for 10 h with [1-13C]threonine showed that the L-allo contaminant did not accumulate. The t-butyl dimethylsilyl derivatives of threonine, glycine, and 2-aminobutyrate (ABA) were used to measure the enrichment of these compounds in plasma and liver samples by GC/MS/selected ion monitoring analysis. Analyses were performed on between 1 and 5 nmol of each amino acid extracted from biological fluids and a 1:10 split injection. GC/MS parameters were assessed with standards at similar quantities and found to be satisfactory; e.g., injection of 1-10 nmol of glycine did not significantly alter the slope and the precision of the standard curve. The coefficient of variation of enrichment determination was less than 10% for standards enriched at 0.4 APE or more and biological samples enriched at 0.6 APE or greater. Within-animal coefficients of variation for four plasma samples obtained at equal intervals between 8 and 10 h of [1-13C]threonine infusion were 4, 21, and 24% for threonine, ABA, and glycine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification of monofluoroacetate and monochloroacetate in human urine by isotope dilution liquid chromatography tandem mass spectrometry

The rodenticide monofluoroacetate (MFA) and monochloroacetate (MCA), a chemical intermediate from several chemical syntheses, have been identified as potential agents of chemical terrorism due to their high toxicity. In preparation for response to poisonings and mass exposures, we have developed a quantification method using isotopic dilution to determine MFA and MCA in urine from 50 to 5000 ng/mL. Both analytes were extracted from urine using solid-phase extraction; extraction recoveries were 62% (MFA) and 76% (MCA). The extracts were then separated with isocratic high-performance liquid chromatography and identified using electrospray ionization tandem mass spectrometry, with detection limits of 0.9 and 7.0 ng/mL for MFA and MCA, respectively. Selectivity was established for both analytes with unique chromatographic retention times which were correlated with isotopically labeled internal standards and the use of two mass spectral transitions for each compound. The intra-day variability was less than 5% for both analytes and the inter-day variability was 7% for MFA and 6% for MCA.     

Stable isotope mass spectrometry in childhood lead poisoning

In order to better understand the relative importance of various sources of lead in childhood lead poisoning, high-precision, isotoperatio, solid-source-mass spectrometry of microgram-sized lead samples was applied to three hospitalized cases in Boston, ranging in age from 1.5 to 14 yr, that had blood-lead levels of 0.7–1.2 μg/g. The lead isotopes in the ambient Boston environment (air, soil, and dust) were also measured. In each case, the isotopic composition (IC) of the child's blood lead was identical with the IC of lead paint taken from the child's residence at a site accessible to the child. Fecal lead samples were also identical to that particular paint. Soil lead IC did not always match the IC of local paints. Paint samples vary widely in their IC's (206/204=17.5–19.4, about 200 times analytical reliability). Dust in homes that never had lead paint contained lead that resembled lead in urban soils. Dust lead IC did not necessarily have the same IC as current automobile lead emissions, but appeared to reflect the long-term accumulation of several sources of urban lead fallout. Limitations and implications of this data are discussed.     

Measurement of pyrethroid,organophosphorus, and carbamate insecticides in human plasma using isotope dilution gas chromatography–high resolution mass spectrometry

We have developed a gas chromatography–high resolution mass spectrometry method for measuring pyrethroid, organophosphorus, carbamate and fipronil pesticides and the synergist piperonyl butoxide in human plasma. Plasma samples were extracted using solid phase extraction and were then concentrated for injection and analysis using isotope dilution gas chromatography–high resolution mass spectrometry. The limits of detection ranged from 10 to 158 pg/mL with relative recoveries at concentrations near the LODs (e.g., 25 or 250 pg/mL) ranging from 87% to 156% (9 of the 16 compounds were within ±15% of 100%). The extraction recoveries ranged from 20% to 98% and the overall method relative standard deviations were typically less than 20% with some exceptions. Analytical characteristics were determined at 25, 250, and 1000 pg/mL.     

Sensitive isotope dilution liquid chromatography/tandem mass spectrometry method for quantitative analysis of bumetanide in serum and brain tissue

We have developed and validated a simple and sensitive stable isotope dilution liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for the quantification of bumetanide in human serum. Samples were prepared with a simple acetonitrile based protein precipitation. The supernatant was then analyzed directly using LC-MS/MS. Chromatographic separation was achieved on a C18 reversed phase column using a methanol and water gradient. The detection was performed in selected reaction monitoring (SRM) mode via a positive electrospray ionization (ESI) interface. The method had a lower limit of quantification (LLOQ) of 1 ng/mL, linearity up to 1250 ng/mL, intra- and inter-day precision less than 10%, and accuracy within ±10%. This method was also demonstrated to be suitable for the analysis of bumetanide in rat serum and brain tissue. Bumetanide concentrations in rat serum and brain were determined for samples collected at several intervals following intraperitoneal (i.p.) injection of bumetanide, and were used to calculate bumetanide permeability through the blood-brain barrier.     

Urinary biomarkers of 1,3-butadiene in environmental settings using liquid chromatography isotope dilution tandem mass spectrometry

Although, 1,3-butadiene is a known human carcinogen emitted from mobile sources, little is known about traffic-related human exposure to this toxicant. This pilot study was designed to characterize traffic-related environmental exposure to 1,3-butadiene and evaluate its urinary mercapturic acids as biomarkers of exposure in these settings. Personal air samples and multiple urine samples were collected on two separate occasions from three groups of individuals that differed by spatial proximity as well as intensity of traffic: (i) toll collectors, (ii) urban-weekday and (iii) suburban-weekend group. Air samples were analyzed using thermal desorption followed by GC/MS and urine samples were analyzed using isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) for two mercapturic acids of 1,3-butadiene: monohydroxy-3-butenyl mercapturic acid (MHBMA) and 1,2-dihydroxybutyl mercapturic acid (DHBMA). Exposure differed between groups (p<0.05) with median values of 2.38, 1.62 and 0.88 microg/m(3) for toll collectors, the urban-weekday group and the suburban-weekend group, respectively. A refined ID-LC-MS/MS method enabled detection of MHBMA, previously detected only in occupational settings, with high frequency. MHBMA and DHBMA were detected in 95 and 100% of urine samples at levels (mean+/-S.D.) of 9.7+/-9.5, 6.0+/-4.3 and 6.8+/-2.6 ng/mL for MHBMA and 378+/-196, 258+/-133 and 306+/-242 ng/mL for DHBMA for the three different groups, respectively. Mean biomarker levels were higher among the toll collectors compared to the other two groups, however, the differences were not statistically significant (p>0.05). This study is the first to evaluate 1,3-butadiene biomarkers for subtle differences in environmental exposures. However, additional research will be required to ascertain whether the lack of statistical association observed here is real or attributable to unexpectedly small differences in exposure between groups (<1 microg/m(3)), non-specificity of the biomarker at low exposure, and/or small sample size.     

Stable isotope dilution mass spectrometric assay for PRPP using enzymatic procedures

5-Phosphoribosyl-1-pyrophosphate (PRPP) is an important regulator of de novo purine synthesis. A method for the measurement of PRPP in erythrocytes was designed, which is based on the determination of [(13)C(5)]glutamate derived from [(13)C(5)]glutamine following the utilization of PRPP by the action of amidophosphoribosyltransferase. The present study describes a gas chromatographic-mass spectrometric method for determination of [(13)C(5)]glutamate using [(13)C(2)]glutamate as an internal standard. The methods involved purification by anion-exchange chromatography using a BondElut SAX and derivatization with isobutyl chlorocarbonate in water-methanol-pyridine. Quantitation was performed by selected ion monitoring of the protonated molecular ions in the chemical ionization mode. The intra-day reproducibility in the amounts of [(13)C(5)]glutamate determined was in good agreement with the actual amounts added in erythrocytes. A linear relationship was found between the amount of PRPP added and the amount of [(13)C(5)]glutamate formed from [(13)C(5)]glutamine using amidophosphoribosyltransferase.     

Stable isotope gas chromatography-tandem mass spectrometry determination of aminoethylcysteine ketimine decarboxylated dimer in biological samples

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD; systematic name: 1,2-3,4-5,6-7,8-octahydro-1,8a-diaza-4,6-dithiafluoren-9(8aH)-one) is a previously described metabolite of cysteamine that has been reported to be present in mammalian brain, urine, plasma, and cells in culture and vegetables and to possess potent antioxidative properties. Here, we describe a stable isotope gas chromatography-tandem mass spectrometry (GC-MS/MS) method for specific and sensitive determination of AECK-DD in biological samples. (13)C(2)-labeled AECK-DD was synthesized and used as the internal standard. Derivatization was carried out by N-pentafluorobenzylation with pentafluorobenzyl bromide in acetonitrile. Quantification was performed by selected reaction monitoring of the mass transitions m/z 328 to 268 for AECK-DD and m/z 330 to 270 for [(13)C(2)]AECK-DD in the electron capture negative ion chemical ionization mode. The procedure was systematically validated for human plasma and urine samples. AECK-DD was not detectable in human plasma above approximately 4nM but was present in urine samples of healthy humans at a maximal concentration of 46nM. AECK-DD was detectable in rat brain at very low levels of approximately 8pmol/g wet weight. Higher levels of AECK-DD were detected in mouse brain (～1nmol/g wet weight). Among nine dietary vegetables evaluated, only shallots were found to contain trace amounts of AECK-DD (～6.8pmol/g fresh tissue).     

Estrogens, androgens, and progestins in follicular fluid from preovulatory follicles: identification and quantification by gas chromatography/mass spectrometry associated with stable isotope dilution

The synthesis and use of stable isotope-labeled analogs of various steroids have made it possible to undertake a study of follicular fluid (FF) aspirated from mature and preovulatory follicles. Our previous results have been brought together here in order to review quantitative work done by gas chromatography-mass spectrometry. A positive gradient between peripheral plasma and FF concentrations of a steroid suggests the possibility of ovarian biosynthesis. This is particularly relevant to the catecholestrogens, 19-norsteroids, and some corticosteroids.