Isotope Dilution Gas Chromatography-mass Spectrometry Research Articles

A gas chromatography–mass spectrometry method for the quantitation of N-nitrosoproline and N-acetyl- S-allylcysteine in human urine: Application to a study of the effects of garlic consumption on nitrosation

Biomarkers in urine can provide useful information about the bioactivation of chemical carcinogens and can be used to investigate the chemoprotective properties of dietary nutrients. N-Nitrosoproline (NPRO) excretion has been used as an index for endogenous nitrosation. In vitro and animal studies have reported that compounds in garlic may suppress nitrosation and inhibit carcinogenesis. We present a new method for extraction and sensitive detection of both NPRO and N-acetyl- S-allylcysteine from urine. The latter is a metabolite of S-allylcysteine, which is found in garlic. Urine was acidified and the organic acids were extracted by reversed-phase extraction (RP–SPE) and use of a polymeric weak anion exchange (WAX–SPE) resin. NPRO was quantified by isotope dilution gas chromatography–mass spectrometry (GC–MS) using [ 13C 5]NPRO and N-nitrosopipecolinic acid (NPIC) as internal standards. This method was used to analyze urine samples from a study that was designed to test whether garlic supplementation inhibits NPRO synthesis. Using this method, 2.4 to 46.0 ng NPRO/ml urine was detected. The method is straightforward and reliable, and it can be performed with readily available GC–MS instruments. N-Acetyl- S-allylcysteine was quantified in the same fraction and detectable at levels of 4.1 to 176.4 ng/ml urine. The results suggest that 3 to 5 g of garlic supplements inhibited NPRO synthesis to an extent similar to a 0.5-g dose of ascorbic acid or a commercial supplement of aged garlic extract. Urinary NPRO concentration was inversely associated with the N-acetyl- S-allylcysteine concentration. It is possible that allyl sulfur compounds found in garlic may inhibit nitrosation in humans.

Impact of rosiglitazone and glyburide on nitrosative stress and myocardial blood flow regulation in type 2 diabetes mellitus

Cardiovascular disease, the leading cause of death in patients with type 2 diabetes mellitus (T2DM), is usually preceded by endothelial dysfunction and altered myocardial blood flow (MBF) regulation. Hyperglycemia, oxidative-nitrosative stress, systemic inflammation, and insulin resistance are implicated in the pathogenesis of abnormal MBF regulation, myocardial ischemia, and apoptosis. However, the impact of oral antihyperglycemic therapy on myocardial perfusion is controversial. Our objective was to explore the effect of rosiglitazone and glyburide on nitrosative stress and MBF regulation in subjects with T2DM. [ 13N]ammonia positron emission tomography and cold pressor testing were used in 27 diabetic subjects (mean age, 49 ± 11 years; glycohemoglobin, 7% ± 1.5%) randomized to either rosiglitazone 8 mg/d or glyburide 10 mg/d for 6 months. Isotope dilution gas chromatography–mass spectrometry was used to quantify plasma 3-nitrotyrosine, a stable marker of reactive nitrogen species. At 6 months, there were no significant differences between groups in the mean glycohemoglobin, blood pressure, or plasma lipids. Rosiglitazone significantly reduced plasma nitrotyrosine, high-sensitivity C-reactive protein, and von Willebrand antigen ( P < .03 for all) and significantly increased plasma adiponectin ( P < .05). No significant changes in these parameters were observed with glyburide. Treatment with glyburide, but not rosiglitazone, resulted in a significant deterioration in both resting and stress MBF. Rosiglitazone, but not glyburide, ameliorated markers of nitrosative stress and inflammation in subjects with T2DM without impairing myocardial perfusion.

The HDL proteome: a marker–and perhaps mediator–of coronary artery disease

One important cardioprotective function of HDL is to remove cholesterol from lipid-laden macrophages in the artery wall. HDL also exerts anti-inflammatory effects that might inhibit atherogenesis. However, HDL has been proposed to be dysfunctional in humans with established coronary artery disease (CAD), though the underlying mechanisms are unclear. Therefore, we used mass spectrometry to investigate the roles of HDL proteins in inflammation and cardiovascular disease. Shotgun proteomic analysis identified multiple complement regulatory proteins, protease inhibitors, and acute-phase response proteins in HDL, strongly implicating the lipoprotein in inflammation and the innate immune system. Moreover, mass spectrometry and biochemical analyses demonstrated that HDL3 from subjects with clinically significant CAD was selectively enriched in apolipoprotein E, suggesting that it carries a distinctive protein cargo in humans with atherosclerosis. HDL from CAD subjects also contained markedly elevated levels of chlorotyrosine and nitrotyrosine, two characteristic products of myeloperoxidase, indicating that oxidative damage might generate dysfunctional HDL. Aggressive lipid therapy with a statin and niacin remodeled the HDL proteome to resemble that of apparently healthy subjects. Collectively, our observations indicate that quantifying the HDL proteome by mass spectrometry should help identify novel anti-inflammatory and cardioprotective actions of HDL and provide insights into lipid therapy.

Gas chromatography–mass spectrometry assay for determination of Nτ-methylhistamine concentration in canine urine specimens and fecal extracts

To develop and validate a gas chromatography-mass spectrometry (GC-MS) method for determination of Ntau-methylhistamine (NMH) concentration in canine urine and fecal extracts and to assess urinary NMH concentrations in dogs with mast cell neoplasia and fecal NMH concentrations in dogs with protein-losing enteropathy. Urine specimens were collected from 6 healthy dogs and 7 dogs with mast cell neoplasia. Fecal extracts were obtained from fecal specimens of 28 dogs with various severities of protein-losing enteropathy, as indicated by fecal concentration of alpha1-proteinase inhibitor. NMH was extracted directly from urine, and fecal specimens were first extracted into 5 volumes of PBSS containing 1% newborn calf serum. Ntau-methylhistamine in specimens was quantified via stable isotope dilution GC-MS. The assay was validated via determination of percentage recovery of known amounts of NMH and interassay coefficients of variation. Urinary excretion of NMH was evaluated by means of NMH-to-creatinine concentration ratios. Recovery of NMH in urine and fecal extracts averaged 104.6% and 104.5%, respectively. Interassay coefficients of variation ranged from 5.4% to 11.7% in urine and 12.6% to 18.1% in fecal extracts. Urinary NMH excretion was significantly increased in dogs with mast cell neoplasia, compared with that in healthy dogs. No correlation was detected between severity of protein-losing enteropathy and fecal NMH concentration. This method provided a sensitive, reproducible means of measuring NMH in canine urine and fecal extracts. High urinary NMH-to-creatinine concentration ratios in dogs with mast cell neoplasia are consistent with increased histamine release in this disease.

4β‐Hydroxycholesterol as an endogenous marker for CYP3A4/5 activity. Stability and half‐life of elimination after induction with rifampicin

The oxysterol 4beta-hydroxycholesterol has been suggested as a marker for CYP3A4/5 activity. We have previously shown that plasma 4beta-hydroxycholesterol continues to increase for several weeks after maximal induction of CYP3A4/5 by carbamazepine at the dose given. In the present study we aimed to determine the time course of the decrease in plasma 4beta-hydroxycholesterol after termination of induction of CYP3A4/5 by rifampicin. An additional aim was to determine the variation in plasma level of 4beta-hydroxycholesterol with time in 12 untreated healthy volunteers. Twenty-four healthy subjects were allocated into three study groups of equal sizes. The volunteers were treated with rifampicin (either 20 mg day(-1), 100 mg day(-1) or 500 mg day(-1)) for 2 weeks. Blood samples were taken before, during and after rifampicin treatment. In another group of 12 untreated volunteers blood samples were collected at different time points in order to determine the intraindividual variations in plasma 4beta-hydroxycholesterol concentrations. Plasma levels of 4beta-hydroxycholesterol were determined by isotope-dilution gas chromatography-mass spectrometry. Rifampicin treatment increased plasma 4beta-hydroxycholesterol levels. After termination of rifampicin treatment plasma levels of 4beta-hydroxycholesterol decreased slowly with an apparent half-life of 17 days. The intraindividual variation in plasma levels of 4beta-hydroxycholesterol in untreated subjects was low, with coefficients of variation of between 4.8 and 13.2% over a period of 3 months. After termination of induction of CYP3A4/5, plasma 4beta-hydroxycholesterol levels decreased slowly during 8 weeks. The half-life of elimination (17 days) resembled that of cholesterol rather than other oxysterols. The long half-life results in stable plasma concentrations with time.

Quantification of 13 priority polycyclic aromatic hydrocarbons in human urine by headspace solid-phase microextraction gas chromatography–isotope dilution mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants in both living and working environments. The aim of this study was the development of a headspace solid-phase microextraction gas chromatography–isotope dilution mass spectrometry (HS-SPME/GC–IDMS) method for the simultaneous quantification of 13 PAHs in urine samples. Different parameters affecting PAHs extraction by HS-SPME were considered and optimized: type/thickness of fiber coatings, extraction temperature/time, desorption temperature/time, ionic strength and sample agitation. The stability of spiked PAHs solutions and of real urine samples stored up to 90 days in containers of different materials was evaluated. In the optimized method, analytes were absorbed for 60 min at 80 °C in the sample headspace with a 100 μm polydimethylsiloxane fiber. The method is very specific, with linear range from the limit of quantification to 8.67 × 10 3 ng L −1, a within-run precision of <20% and a between-run precision of <20% for 2-, 3- and 4-ring compounds and of <30% for 5-ring compounds, trueness within 20% of the spiked concentration, and limit of quantification in the 2.28–2.28 × 10 1 ng L −1 range. An application of the proposed method using 15 urine samples from subjects exposed to PAHs at different environmental levels is shown.

Occurrence of ethanol and other drugs in blood and urine specimens from female victims of alleged sexual assault

Results of toxicological analysis of blood and urine specimens from 1806 female victims of alleged non-consensual sexual activity are reported. After making contact with the police authorities, the victims were examined by a physician for injuries and biological specimens were taken for forensic toxicology and other purposes (e.g. DNA). Urine if available or otherwise on an aliquot of blood after protein precipitation was screened for the presence of drugs by enzyme immunoassay methods (EMIT/CEDIA). All positive results from screening were verified by more specific methods, involving isotope dilution gas chromatography–mass spectrometry (GC–MS) for illicit drugs. A large number of prescription drugs were analyzed in blood by capillary column gas chromatography with a nitrogen–phosphorous (N–P) detector. Ethanol was determined in blood and urine by headspace gas chromatography and concentrations less than 0.1 g/L were reported as negative. The number of reported cases of alleged sexual assault was highest during the warmer summer months and the mean age of victims was 24 years (median 20 years), with ∼60% being between 15 and 25 years. In 559 cases (31%) ethanol and drugs were negative. In 772 cases (43% of total) ethanol was the only drug identified in blood or urine. In 215 cases (12%) ethanol occurred together with at least one other drug. The mean, median and highest concentrations of ethanol in blood ( N = 806) were 1.24 g/L, 1.19 g/L and 3.7 g/L, respectively. The age of victims and their blood-alcohol concentration (BAC) were positively correlated ( r = 0.365, p < 0.001). Because BAC decreases at a rate of 0.10–0.25 g/(L h), owing to metabolism the concentration in blood at time of sampling is often appreciably less than when the crime was committed several hours earlier. Licit or illicit drugs were identified in blood or urine in N = 262 cases (15%). Amphetamine and tetrahydrocannabinol were the most common illicit drugs at mean (median) concentrations in blood of 0.22 mg/L (0.1 mg/L) and 0.0012 mg/L (0.0006 mg/L), respectively. Among prescription drugs, sedative-hypnotics such as diazepam and zopiclone were common findings along with SSRI antidepressants and various opiate analgesics. Interpreting the analytical results in terms of voluntary vs. surreptitious administration of drugs and the degree of incapacitation in the victim as well as ability to give informed consent for sexual activity is fraught with difficulties.

Preparation of reference material for organochlorine pesticides in a herbal matrix

The development of reference material for four organochlorine pesticides, namely hexachlorobenzene and three isomers of hexachlorocyclohexane (alpha-hexachlorocyclohexane, beta-hexachlorocyclohexane and gamma-hexachlorocyclohexane), in a ginseng root sample is presented. Raw materials (Panax ginseng) were purchased from a local market and confirmed to contain certain levels of incurred organochlorine pesticide residues by a validated gas chromatography-mass selective detection method. A total of more than 300 bottles each containing 25 g of samples were prepared after the materials had been freeze-dried, milled and thoroughly mixed. The homogeneity and stability of samples from randomly selected bottles were verified and the reference values were characterized using a highly precise isotope dilution gas chromatography-mass spectrometry (ID-GCMS) method that was recently developed by our laboratory. The purity of standard organochlorine chemicals was determined against certified reference materials to establish the accuracy of the ID-GCMS analysis. The concentrations (+/- expanded uncertainty) of hexachlorobenzene, alpha-hexachlorocyclohexane, beta-hexachlorocyclohexane and gamma-hexachlorocyclohexane in the reference material were 0.198 +/- 0.015, 0.450 +/- 0.022, 0.213 +/- 0.011 and 0.370 +/- 0.032 mg kg(-1), respectively. A portion (70 bottles) of the samples was also used in a proficiency testing (PT) scheme for assessing the testing capabilities of field laboratories. The consensus mean values of the PT obtained from the 70 participants were on the same order but deviated by -2.7 to -14.1% from those of the assigned reference values. Because of the wide spread of participants' data (relative standard deviation ranging from 44 to 56%), the PT results were not included in the calculation of the assigned values of the reference materials. The materials served as suitable reference materials to ascertain the quality control and validation processes for the determination of organochlorine pesticides in herbal matrices.

Method validation for measurement of hair nicotine level in nonsmokers

The development of strategies to address the growing worldwide burden of exposure to secondhand smoke (SHS) would be facilitated by sensitive and accurate methods for assessing SHS exposure. Hair provides a readily available matrix for assessing biomarkers of typical SHS exposure. We developed and applied an optimized analytical method using an isotope dilution gas chromatography-mass spectrometry (GC/MS) for hair nicotine measurement. The utility of this optimized method is illustrated by presenting data on SHS exposure of women and children from 31 countries. Using this isotope dilution method with spiked samples (3.3 ng/mg), we found that the greatest hair nicotine extraction efficiency was obtained with a 60 min shaking time. In the field study (n = 2400), a positive association was evident between hair nicotine concentrations from nonsmokers and higher numbers of cigarettes smoked per day in a household.

Measurement of nicotine in household dust

An analytical method of measuring nicotine in house dust was optimized and associations among three secondhand smoking exposure markers were evaluated, i.e., nicotine concentrations of both house dust and indoor air, and the self-reported number of cigarettes smoked daily in a household. We obtained seven house dust samples from self-reported nonsmoking homes and 30 samples from smoking homes along with the information on indoor air nicotine concentrations and the number of cigarettes smoked daily from an asthma cohort study conducted by the Johns Hopkins Center for Childhood Asthma in the Urban Environment. House dust nicotine was analyzed by isotope dilution gas chromatography–mass spectrometry (GC/MS). Using our optimized method, the median concentration of nicotine in the dust of self-reported nonsmoking homes was 11.7 ng/mg while that of smoking homes was 43.4 ng/mg. We found a substantially positive association ( r=0.67, P<0.0001) between house dust nicotine concentrations and the numbers of cigarettes smoked daily. Optimized analytical methods showed a feasibility to detect nicotine in house dust. Our results indicated that the measurement of nicotine in house dust can be used potentially as a marker of longer term SHS exposure.

Snail bioaccumulation of triclocarban, triclosan, and methyltriclosan in a north texas, usa, stream affected by wastewater treatment plant runoff

Grazing by freshwater snails promotes nutrient turnover in algal communities. Grazed algal compartments may include antimicrobial agents and metabolites, such as triclocarban (TCC), triclosan (TCS), and methyltriclosan (MTCS), which are incompletely removed by wastewater treatment plant (WWTP) processing. The present study quantifies snail bioaccumulation factors (BAFs) for TCC, TCS, and MTCS at the outfall of Pecan Creek (TX, USA), the receiving stream for the city of Denton (TX, USA) WWTP. Helisoma trivolvis (Say) is ubiquitous and thrives under standard laboratory conditions, leading to its choice for this bioaccumulation study in conjunction with Cladophora spp. Along with providing substrate for epiphytic growth, Cladophora spp. provide a source of food and shelter for H. trivolvis. After being caged for two weeks, algae and snails were collected from the WWTP outfall, along with water-column samples, and analyzed by isotope dilution gas chromatography-mass spectrometry for TCS and MTCS and by liquid chromatography-mass spectrometry for TCC. Algal and snail samples were analyzed before exposure and found to be below practical quantitation limits for all antimicrobial agents. Triclocarban, TCS, and MTCS in water samples were at low-ppt concentrations (40-200 ng/L). Triclocarban, TCS, and MTCS were elevated to low-ppb concentrations (50-300 ng/g fresh wt) in caged snail samples and elevated to low-ppb concentrations (50-400 ng/g fresh wt) in caged algal samples. Resulting snail and algal BAFs were approximately three orders of magnitude, which supports rapid bioaccumulation among algae and adult caged snails at this receiving stream outfall. The results further support TCC, TCS, and MTCS as good candidate marker compounds for evaluation of environmental distribution of trace WWTP contaminants.

Migration of Bisphenol A from Polycarbonate Baby and Water Bottles into Water under Severe Conditions

The isotope dilution headspace solid-phase microextraction and gas chromatography-mass spectrometry method for bisphenol A (BPA) developed previously was used successfully in a BPA migration study at 70 degrees C of polycarbonate baby and reusable water bottles recently sold in Canada by using the whole bottles instead of pieces cut from the bottles. Migration of BPA from the PC bottles heated at 70 degrees C was found to increase over the time in the quadratic equations. Migration levels of BPA in water varied from 228 to 521 microg L (-1) or from 0.26 to 0.90 microg cm (-2) after being heated at 70 degrees C for 6 days. The average migration rates of BPA from the PC bottles into water at 70 degrees C ranged from 1.84 to 4.83 ng cm (-2) h (-1).

Quantification of polycyclic aromatic hydrocarbons based on comprehensive two-dimensional gas chromatography–isotope dilution mass spectrometry

Comprehensive two-dimensional gas chromatography (GC × GC) offers favourable resolution and sensitivity compared with conventional one-dimensional gas chromatography (1D-GC), as reported in many studies. These characteristics are of major interest when analytes are in trace concentration, and are present in complex mixtures, as is the case of polycyclic aromatic hydrocarbons (PAHs) in atmospheric particulate samples. Whilst GC × GC has been widely applied to identification of different types of analytes in several matrices, less seldom has it been used for quantification of these analytes. Although several quantitative methods have been proposed, they may be tedious and/or require considerable user development. Whereas quantification in 1D-GC is a routine and well-established procedure, in GC × GC, it is not so straightforward, especially where novel or untested procedures have yet to be incorporated into software packages. In the present study, it is proposed that a subset of the modulated peaks generated for each solute may be summed, based on the specific target ion mass of each compound present in a certified standard reference material (SRM) 1649a (urban dust). The ratio between a PAH and its corresponding deuterated (PAH-d) form showed that there is no statistical loss of sensitivity when this ratio is calculated based on whether the total sum of modulated peaks, or if only the two or the three most intense modulated peaks, are employed. Manual integration may be required, and here was found to give more acceptable values than automatic integration. Automated integration has been shown here to underestimate the modulated peak responses when low concentrations of PAHs were analyzed. Although for most PAHs good agreement with the certified values were observed, the analytical method needs to be further optimized for some of the other PAH, as can be see with those PAH with high variability in the range of urban dust analyzed.

Gas Chromatographic−Mass Spectrometric Analysis of Biomarkers Related to Folate and Cobalamin Status in Human Serum after Dimercaptopropanesulfonate Reduction and Heptafluorobutyl Chloroformate Derivatization

Methylmalonic acid and total homocysteine belong to useful clinical indicators of cobalamin and folate status and are commonly measured separately. A sensitive and rapid method has been developed for simultaneous determination of both biomarkers and related metabolites in serum or plasma by isotope dilution gas chromatography-mass spectrometry (GC/MS). Thiols bound in disulfide bonds were released with 2,3-dimercapto-1-propanesulfonic acid (DMPS), and after deproteinization, they were phase-transfer derivatized with heptafluorobutyl chloroformate (HFBCF) in a single step. The reducing capability of the DMPS agent was comparable to that of the dithiothreitol, but exceeded the latter in much cleaner extracts obtained. The new method enabled GC/MS screening of amino acidic metabolites, including cystathionine and thiol-containing dipeptides, as their N(S,O)-heptafluorobutoxycarbonyl heptafluorobutyl ester derivatives in serum or plasma. Accurate quantitation of seven biomarkers was accomplished by using deuterated internal standards; the detection limits ranged from 7 to 20 nmol/L, the between-day precision from 1.5 to 8.8%, and the recoveries were between 83 and 103%. The results suggest that the new combined procedure of DMPS reduction and HFBCF derivatization make the method efficient for diagnostics of folate and cobalamin status as well as for screening of amino acidic metabolites in body fluids.

Reference measurement procedure for Δ9-tetrahydrocannabinol in serum

An isotope dilution gas chromatography/mass spectrometry (ID-GC/MS) reference measurement procedure for Delta9-tetrahydrocannabinol (THC) in serum was developed and validated. The method complies with the concept of a ratio primary reference measurement procedure. The uncertainty was determined for two concentrations of THC in serum (1 ng/mL and 2.4 ng/mL). The calculation procedure is based on the Guide to the Expression of Uncertainty in Measurement (GUM). The relative expanded uncertainty was found to be less than 2% for both concentration levels, corresponding to a 95% confidence interval. For the reference method, it was shown that the measurement of THC within the concentration range covered by the current threshold values is very accurate. The method has the potential to provide traceability for the methods used in practical forensics.

Modifications to the Organophosphorus Nerve Agent-Protein Adduct Refluoridation Method for Retrospective Analysis of Nerve Agent Exposures

Organophosphorus nerve agents (OPNAs) continue to pose a threat to military personnel and the general public because of their toxicity and their potential use as weapons of mass destruction. An effective method for the detection of human exposure to OPNAs involves the refluoridation of nerve agents adducted to the serum protein butyrylcholinesterase. The regenerated agents are then enriched by solid-phase extraction and quantified by isotope-dilution gas chromatography-mass spectrometry. We have previously reported improvements that resulted in a 10-fold increase in sensitivity. We have now made further changes to the method that include the addition of confirmation ions, the addition of soman (GD) to the assay, the expansion of the linear range, and the elimination of high-volume injection to decrease background noise and run time while improving sensitivity. This report includes the standard operating procedures for this method for tabun, sarin, soman, cyclohexylsarin, and VX and validation studies. The method's limits of detection ranged from 5.5 to 16.5 pg/mL for the G analogue of VX and GD, respectively. Characterization of quality control (QC) materials resulted in an average coefficient of variation of 15.1% for the five analytes in low QC pools and 11.7% in high QC pools.

Determination of aldosterone in serum by liquid chromatography–tandem mass spectrometry

Measurement of serum aldosterone is clinically important in the diagnosis of hypertension. While isotope dilution gas chromatography–mass spectrometry (ID-GC–MS) provides reliable results, it requires derivatization and is lengthy and time-consuming. Detection by liquid chromatography–mass spectrometry (LC–MS) is a potentially superior method. The analysis utilizes 0.5 mL of serum. The samples were extracted with dichloromethane–ether. The extract was evaporated to dryness and aldosterone was analyzed by LC–MS/MS operating in the negative mode ESI after separation on a reversed-phase column. Aldosterone was also measured by RIA. The calibration curves for analysis of serum aldosterone exhibited consistent linearity and reproducibility in the range of 60–3000 pmol/L. Interassay CVs were 4.3–7.5% at aldosterone concentrations of 97–993 pmol/L. The lower limit of quantitation (LOQ) was 30 pmol/L (signal to noise ratio = 10). The mean recovery of the analyte added to serum ranged from 95 to 102%. The regression equation by LC–MS/MS ( x) and RIA ( y) method was: y = 1.33 x + 185 ( r = 0.95; n = 124). Sensitivity and specificity of the LC–MS/MS method for serum aldosterone offer advantages over GC–MS by eliminating derivatization. The novel method is rapid, reliable and simple to perform with a routine LC–MS/MS spectrometer. The sensitivity is adequate for patient samples. Aldosterone concentrations reported by nonextraction RIA were consistently higher than those produced by LC–MS/MS.

Quantification of fenitrothion and its main metabolites in poplar leaves by isotope dilution gas chromatography–mass spectrometry coupled with solid-phase microextraction

A sensitive and specific method for determining fenitrothion and its main metabolites, 3-methyl-4-nitrophenol and fenitrooxon, in poplar leaves using deuterated isotopes as the internal standard is described. The analytes and the labeled isotopes were extracted from leaves by solid-phase microextraction and subsequently analysed by gas chromatography coupled with mass spectrometry. The method had a chromatographic run time of 17.0 min and good linearity over the range 0.01–10 mg kg −1. The detection limits ranged between 2.5 and 0.6 μg kg −1. The isotopic dilution technique allowed improving significantly the repetitivity even using different fibers with the same coating (RSD < 5.1%). The method was applied successfully to study the persistence of fenitrothion in forestal matrices in a poplar forest after cannon spray application of the insecticide.

Intrahepatic cholestasis of pregnancy: Amelioration of pruritus by UDCA is associated with decreased progesterone disulphates in urine

Intrahepatic cholestasis of pregnancy (ICP) is characterized by pruritus, elevated bile acids, and, specifically, elevated disulphated progesterone metabolites. We aimed to study changes in these parameters during treatment with dexamethasone or ursodeoxycholic acid (UDCA) in 40 out of 130 women included in the Swedish ICP intervention trial (26 randomized to placebo or UDCA, 14 randomized to dexamethasone). Serum bile acid profiles and urinary steroid hormone metabolites were analyzed using isotope-dilution gas chromatography-mass spectrometry and electrospray-mass spectrometry. We found that all patients displayed ICP-typical serum bile acid profiles with >50% cholic acid at baseline but almost 80% UDCA upon treatment with this bile acid. In UDCA-treated patients, relative amounts of disulphated progesterone metabolites in urine decreased by 34%, 48% (P < 0.05), and 55% (P < 0.05) after 1, 2, and 3 weeks of treatment, respectively, which was significantly correlated to improvements of pruritus scores but not to serum bile acid levels. In contrast, in patients randomized to dexamethasone or placebo, no changes in steroid metabolites or pruritus scores were observed. UDCA treatment in ICP decreased urinary excretion of disulphated progesterone metabolites, suggesting that amelioration of pruritus is connected to stimulation of hepatobiliary excretion of progesterone disulphates.

Mass spectrometric and physiological validation of a sensitive, automated, direct immunoassay for serum estradiol using the Architect®

Background Measurement of estradiol (E 2) plays a critical role in the diagnosis and clinical management of reproductive disorders. The challenge for all currently available direct methods for measuring E 2 is to provide accuracy and precision across a wide dynamic range. Methods We describe the development and multi-site performance evaluation of a direct E 2 assay on the Architect i2000®. Assay performance and method comparisons were performed by testing specimens from men, healthy women with regular menstrual cycles, and post-menopausal women using the Architect assay and isotope dilution, gas chromatography-mass spectrometry (ID/GC-MS). Reference intervals were established by testing prospectively collected daily blood draws from 42 healthy women, 72 postmenopausal women and 101 males. Results No unexpected cross-reactivity or interference was observed for over 40 compounds tested. Recovery was 100 ± 10% in the presence of estrone and estriol. Functional sensitivity (%CV < 20%) was < 15 pg/ml. 1 1 To convert conventional pg/ml [ng/l] to SI units [pmol/l] multiply by 3.671. The imprecision of the assay was < 7.1% (total CV), < 2.5%, and < 2.3% for control sera containing 45, 190, and 600 pg/ml estradiol, respectively. The assay had a correlation of y = 1.033 x + 0.3156, r 2 = 0.99, n = 131 compared to ID/GC-MS. Reference intervals for the current Architect Estradiol assay are reported. Conclusions Format changes resulted in dramatic improvement in the performance and accuracy of this direct, fully automated assay. The assay is standardized by ID/GC-MS. The assay is clinically useful for serum concentrations from 15 to > 4000 pg/ml.