Gas Chromatographic-mass Spectrometric Assay Research Articles

Nutrient Composition, Antioxidant Capacity and Natural Products in Livingstone Potato (Plectranthus esculentus )

The nutrients, natural products and antioxidant capacity of flour from Livingstone potato (Plectranthus esculentus) tubers were investigated. The flour contained 8.26 ± 0.20% moisture, 2.58 ± 0.18% ash, 2.60 ± 0.07% dietary fiber, 5.30 ± 0.17% lipid, 7.87 ± 0.04% protein and 76.00 ± 2.33% carbohydrate, but contained low amount of thiamine, riboflavin and niacin. The total flavonoid content of flour was lower (P < 0.05) than its total phenolic content. The antioxidant assay of flour (determined using 2,2-diphenyl-1-picrylhydrazyl radical and reducing power assays) indicated its strong antioxidant activity, although lower than that of quercetin. Gas chromatographic mass spectrometric assay of methanol/chloroform extract of flour revealed the presence of 13 compounds, whereas the major compounds in the extract with identified biological properties were 9,12-octadecadienoic (55.31%), 12-methyl-E,E-2,13-octadecadien-1-ol (21.68%), 10-octadecenoic acid methyl ester (2.74%) and 3,5-di-t-butyl phenol (2.34%). Livingstone potato contains strong antioxidant activity and some bioactive compounds that support its biological properties. Practical Applications This paper reveals the bioactive principles/natural products in Livingstone potato that justify its biological properties as used in folklore medicine. By isolating and identifying some compounds with biological activities in this plant, new drugs could be developed from this plant in the future to treat various diseases.

An Attempt to Degradation of Anticancer Drug and Odor in the Medical Environment by Photocatalyst

Currently, there is a need to reduce the occupational exposure of health care workers to anticancer drugs. Environmental contamination by anticancer drugs and subsequent exposure of health care workers are associated with vaporization of anticancer drugs. Furthermore, carcinomatous unpleasant odor is an additional problem to vaporized anticancer drugs in the field of clinical cancer therapy. We attempted to degrade vaporized anticancer drug and unpleasant odor using a photocatalyst. Cyclophosphamide or unpleasant odors (ammonia, formaldehyde, isovaleric acid, and butyric acid) were vaporized by heating in a closed chamber. Plates of photocatalyst coated with titanium dioxide were placed into the chamber and irradiated by light source. Vaporized cyclophosphamide in the chamber was recovered by bubbling the internal air through acetone and derivatized by trifluoroacetic anhydride for analysis by gas chromatographic-mass spectrometric assay. Vaporized odors were determined using a gas-detector tube, which changed color depending on the concentration. Following activation of the photocatalyst by a light source, the residual amounts of anticancer drug and unpleasant odor components were significantly decreased compared with when the photocatalyst was not activated without a light source. These results indicate that the photocatalysts can accelerate the degradation of vaporized anticancer drugs and odor components. Air-cleaning equipment using a photocatalyst is expected to be useful in improving the QOL of cancer patients experiencing carcinomatous unpleasant odor, and in reducing occupational exposure of health care workers to anticancer drugs.

Determination of MTBE, TBA and BTEX in Soil by Headspace Gas Chromatography-Mass Spectrometry

A headspace gas chromatographic mass spectrometric (GC-MS) assay method was developed for the simultaneous determination of methyl tertiary butyl ether (MTBE), -butyl alcohol (TBA) and benzene, toluene, ethyl benzene and xylene (BTEX) in soil contaminated with gasoline. 2 g of soil sample were placed in a 10 mL headspace vial filled with 5 mL of phosphoric acid solution (pH 3) saturated with NaCl, and the solution was spiked with fluorobenzene as an internal standard and sealed with a cap. The vial was heated in a heating block for 40 min at . The detection limits of the assay were 0.08-0.12 /kg for the analytes. For five independent determinations at 10 and 50 /kg, the relative standard deviations were less than 10%. The method was used to analyze fifty six soil samples collected from various regions contaminated with gasoline in Korea. The developed method may be valuable for the monitoring of the analytes in soil.

Rapid determination of natural steroidal hormones in saliva for the clinical diagnoses

BackgroundSaliva samples are easily collectable and non-invasive, and the monitoring of natural steroidal hormones, such as estrone (E1), 17β-estradiol (E2), estriol (E3), progesterone (P), and testosterone (T), in saliva has attracted much attention due to its numerous potential clinical and health-related applications. Because E1, E2, E3, P and T are useful indicators in numerous clinical and health-related diagnoses, there is a need for simultaneous determination.ResultsA gas chromatography-mass spectrometric assay was developed for rapid simultaneous determination of E1, E2, E3, P and T in saliva for clinical diagnoses. Extraction was achieved with a liquid extraction using 3.0 mL of pentane. The extract was dried and silylated with N-methyl-N-(trimethylsilyl) trifluoroacetamide/NH4I (100:2) under a catalysis of 1.5% dithioerythritol for 10 min at 90°C. The accuracy of the analytes was in the range of 96% to 112% at concentrations of 0.05 and 0.10 μg/L (5.0 and 10.0 μg/L for E3), respectively, with relative standard deviations of less than 11%. The lowest quantification limits were from 0.002 to 0.6 μg/L for 1.0 mL of saliva.ConclusionNatural steroidal hormones were detected in the concentration ranges of nd to 0.2 μg/L in human saliva. The salivary testosterone values in the patients with prostatic carcinoma were significantly lower than in normal males. The method may useful in numerous clinical and health-related diagnoses.

Determination of 1,4-Dioxane in Water by Isotopic Dilution Headspace GC–MS

A headspace gas chromatographic mass spectrometric assay method was developed for determining 1,4-dioxane in water. A 5-mL water sample was placed in a 10-mL headspace vial and saturated with NaCl, and the solution was spiked with 1,4-dioxane-d 8 as an internal standard and sealed with a cap. The vial was heated in a headspace for 30 min at 90 °C. The method detection limit of the assay was 0.02 μg L−1 for 1,4-dioxane, and the relative standard deviations were less than 3%. 1,4-Dioxane was detected in the concentration range of 0.05–2.4 μg L−1 in 20 raw water. The developed method may be valuable for monitoring 1,4-dioxane in water.

A Non-lethal Water-based Removal-reapplication Technique for Behavioral Analysis of Cuticular Compounds of Ants

Interspecific relationships among insects are often mediated by chemical cues, including non-volatile cuticular compounds. Most of these compounds are hydrocarbons that necessitate the use of solvents for their extraction, identification, and manipulation during behavioral assays. The toxicity of these solvents often precludes the removal and reapplication of hydrocarbons from and to live insects. As a consequence, dummies often are used in behavioral assays, but their passivity can bias the behavior of the responding insects. To overcome these limitations, we propose a method where cuticular compounds are extracted from live ants by placing them into glass vials half-filled with tepid water (ca. 34 degrees C) and vigorously shaking the vials to form an emulsion whose supernatant can be analyzed and/or reapplied to other ants. We demonstrate that cuticular compounds can be extracted from workers of the red fire ant, Solenopsis saevissima, and reapplied to the cuticle of workers from a sympatric species, Camponotus blandus (both Hymenoptera: Formicidae), while keeping the ants alive. Gas chromatographic-mass spectrometric analysis and behavioral assays were used to confirm the successful transfer of the behaviorally active compounds.

Pharmacokinetics of Phenytoin: Reminders and Discoveries

Phenytoin Half-Life and Clearance during Maintenance Therapy in Adults and Elderly Patients with Epilepsy. Ahn JE, Cloyd JC, Brundage RC, Marino SE, Conway JM, Ramsay RE, White JR, Musib LC, Rarick JO, Birnbaum AK, Leppik IE. Neurology 2008;71(1):38–43. BACKGROUND: Phenytoin (PHT) is widely used to treat epilepsy in elderly patients, but information on its pharmacokinetics in this population is limited. OBJECTIVE: The purpose of this study was to investigate the effects of age and sex on PHT pharmacokinetics using stable-labeled (SL) isotopes of PHT or fosphenytoin (FOS) administered IV or IM while patients remained on their oral maintenance regimen. METHODS: Subjects were patients 18 years or older with epilepsy, but otherwise healthy, on a maintenance regimen of PHT who were not taking interacting medications. Subjects were given a single injection of a 100 mg dose of SL-PHT or SL-FOS followed by their usual morning PHT dose less 100 mg. Serial blood samples were collected up to 196 hours after the SL dose. Plasma PHT and SL-PHT concentrations were measured by a gas chromatographic-mass spectrometric assay. PHT pharmacokinetics were characterized using a population-based, nonlinear, mixed-effects model. RESULTS: Sixty-three subjects completed the study, 45 of whom were 65 years or older. There was no difference between adult and elderly or men and women in PHT clearance, distribution volume, and elimination half-life. The mean elimination half-life was 40 hours. CONCLUSIONS: Healthy elderly adults appear to have the same phenytoin (PHT) pharmacokinetics as younger adults. Reduced PHT dosage requirements may be due to age-related changes in patients’ sensitivity to the therapeutic and toxic effects of the drug. The prolonged elimination half-life suggests that most patients can take PHT once daily and the time to reach steady-state may extend to 2–3 weeks.

Validation of a sensitive gas chromatographic–mass spectrometric method for the simultaneous determination of β-elemene and β-elemenal in human plasma

A sensitive gas chromatographic–mass spectrometric assay was described for determination of β-elemene and β-elemenal in human plasma, which has been successfully applied in clinical trial. After liquid–liquid extraction and gas chromatographic separation, the analytes were identified and quantitated. Calibration curves were linear in range from 31.25 to 8000 ng mL −1 and the limit of quantification for both was 31.25 ng mL −1. Intra- and inter-day precision at three concentrations were 2.3–8.3% with accuracy of −8.5 to 6.1% for elemene and 3.0–9.9% with accuracy of −2.3 to 5.9% for elemenal. The method was validated to be suitable for further pharmacokinetic study.

Key issues in F2-isoprostane analysis

A large body of evidence indicates that measurement of F2-isoprostanes, specific prostaglandin F2-like compounds derived from the non-enzymatic peroxidation of arachidonic acid, is a reliable biomarker of oxidant stress in the human body. Since the discovery of F2-isoprostanes in the early 1990s, a variety of analytical approaches has been introduced for the quantification of these novel compounds. The aim of the present review is to shed light on the available gas chromatographic-mass spectrometric assays for the measurement of plasma or urinary F2-isoprostanes and to highlight a number of issues which need to be addressed in order to implement F2-isoprostane measurement as a gold-standard biomarker of oxidative stress in biological samples.

Phenytoin half-life and clearance during maintenance therapy in adults and elderly patients with epilepsy

Phenytoin (PHT) is widely used to treat epilepsy in elderly patients, but information on its pharmacokinetics in this population is limited. The purpose of this study was to investigate the effects of age and sex on PHT pharmacokinetics using stable-labeled (SL) isotopes of PHT or fosphenytoin (FOS) administered IV or IM while patients remained on their oral maintenance regimen. Subjects were patients 18 years or older with epilepsy, but otherwise healthy, on a maintenance regimen of PHT who were not taking interacting medications. Subjects were given a single injection of a 100 mg dose of SL-PHT or SL-FOS followed by their usual morning PHT dose less 100 mg. Serial blood samples were collected up to 196 hours after the SL dose. Plasma PHT and SL-PHT concentrations were measured by a gas chromatographic-mass spectrometric assay. PHT pharmacokinetics were characterized using a population-based, nonlinear, mixed-effects model. Sixty-three subjects completed the study, 45 of whom were 65 years or older. There was no difference between adult and elderly or men and women in PHT clearance, distribution volume, and elimination half-life. The mean elimination half-life was 40 hours. Healthy elderly adults appear to have the same phenytoin (PHT) pharmacokinetics as younger adults. Reduced PHT dosage requirements may be due to age-related changes in patients' sensitivity to the therapeutic and toxic effects of the drug. The prolonged elimination half-life suggests that most patients can take PHT once daily and the time to reach steady-state may extend to 2-3 weeks.

Prevalence of gestational exposure to cannabis in a Mediterranean city by meconium analysis

To investigate the prevalence of in utero exposure to cannabis in a cohort of 974 mother-infants dyads from Barcelona (Spain). A validated gas chromatographic-mass spectrometric assay was used for meconium analysis. Prenatal exposure to cannabis was detected in 5.3% newborns; however, only 1.7% of the participating mothers disclosed gestational drug use. Ethnicity was not associated with cannabis use, nor were the maternal features or newborns somatometry. A significantly higher percentage of cannabis using mothers had a managerial professional job versus nonusers. Meconium analysis is especially important to identify neonates who have been exposed to cannabis in utero, which appear normal at birth and therefore may not be recognized. Timely detection of these newborns at risk provides the basis for appropriate treatment and adequate medical and social follow-up.

Comparison of electron and chemical ionization modes by validation of a quantitative gas chromatographic–mass spectrometric assay of new generation antidepressants and their active metabolites in plasma

A gas chromatographic–mass spectrometric method (GC–MS) for the simultaneous determination of the ‘new’ antidepressants (mirtazapine, viloxazine, venlafaxine, trazodone, citalopram, mianserin, reboxetine, fluoxetine, fluvoxamine, sertraline, maprotiline, melitracen, paroxetine) and their active metabolites (desmethylmirtazapine, O-desmethylvenlafaxine, m-chlorophenylpiperazine, desmethylcitalopram, didesmethylcitalopram, desmethylmianserin, desmethylfluoxetine, desmethylsertraline, desmethylmaprotiline) in plasma using different ionization modes was developed and validated. Sample preparation consisted of a strong cation exchange mechanism and derivatisation with heptafluorobutyrylimidazole. The GC separation was performed in 24.8 min. Identification and quantification were based on selected ion monitoring in electron (EI) and chemical ionization (CI) modes. Calibration by linear and quadratic regression for electron and chemical ionization, respectively, utilized deuterated internal standards and a weighing factor 1/ x 2. Limits of quantitation were established between 5 and 12.5 ng/ml in EI and positive ionization CI (PICI), and 1 and 6.25 ng/ml in negative ionization CI (NICI). During validation stability, sensitivity, precision, accuracy, recovery, and selectivity were evaluated for each ionization mode and were demonstrated to be acceptable for most compounds. While it is clear that not all compounds can be quantitated either due to chromatographic (trazodone) or derivatisation problems ( O-desmethylvenlafaxine), this method can quantitate most new antidepressants (ADs) in the therapeutic range using EI. PICI and NICI lead to higher selectivity. Moreover, NICI is of interest for small sample volumes and high sensitivity requirements. This paper draws the attention to the pros and cons of the different ionization modes in the GC–MS analysis of these antidepressants in plasma.

Quantification of Δ9-tetrahydrocannabinol and its Major Metabolites in Meconium by Gas Chromatographic-mass Spectrometric Assay: Assay Validation and Preliminary Results of the “Meconium Project”

A rapid and simple procedure based on gas chromatography-mass spectrometry (GC-MS) is described for determination of Delta-tetrahydrocannabinol (THC), 11-hydroxy-Delta-tetrahydrocannabinol (THC-OH) and 11-nor-Delta-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in meconium using Delta-tetrahydrocannabinol (Delta-THC) and deuterated THC-COOH as internal standards. The biological matrix was subjected to liquid-liquid extraction after enzyme hydrolysis for conjugated analytes.Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The method was validated in the range 20 to 500 microg/g using 1g of meconium per assay. The method was applied to the analysis of meconium in a cohort of newborns to assess eventual fetal exposure to cannabis. Within positive samples, THC-COOH and THC-OH (range: 33.7 to 182.1 and 20.7 to 493.3 microg/g, respectively) were both present in the majority of cases with only 1 specimen with THC-OH as the most abundant metabolite and 2 with THC only.

Hyperoxaluria, Hypocitraturia, Hypomagnesiuria, and Lack of Intestinal Colonization byOxalobacter formigenesin a Cervical Spinal Cord Injury Patient with Suprapubic Cystostomy, Short Bowel, and Nephrolithiasis

Although urolithiasis is common in spinal cord injury patients, it is presumed that the predisposing factors for urinary stones in spinal cord injury patients are immobilization-induced hypercalciuria in the initial period after spinal injury and, in later stages, urine infection by urease-producing micro-organisms, e.g., Proteus sp., which cause struvite stones. We describe a patient who sustained C-7 complete tetraplegia in a road traffic accident in 1970, when he was 16 years old. Left ureterolithotomy was performed in 1971 followed by left nephrectomy in 1972. Probably due to adhesions, this patient developed volvulus of the intestine in 1974. As he had complete tetraplegia, he did not feel pain in the abdomen and there was a delay in the diagnosis of volvulus, which led to ischemia of a large segment of the small bowel. All but 1 ft of jejunum and 1 ft of ileum were resected leaving the large bowel intact. In 1998, suprapubic cystostomy was performed. In 2004, this patient developed calculus in the solitary right kidney. Complete stone clearance was achieved by extracorporeal shock wave lithotripsy. Stone analysis: calcium oxalate 60% and calcium phosphate 40%. Metabolic evaluation revealed hyperoxaluria, hypocitraturia, and hypomagnesiuria. Since this patient had hyperoxaluria, the stool was tested for Oxalobacter formigenes, a specific oxalate-degrading, anerobic bacterium inhabiting the gastrointestinal tracts of humans; absence of this bacterium appears to be a risk factor for development of hyperoxaluria and, subsequently, calcium oxalate kidney stone disease. DNA from the stool was extracted using the QIAamp DNA stool Mini Kit (Qiagen, Chatsworth, CA). The genomic DNA was amplified by polymerase chain reaction using specific primers for oxc gene (developed by Sidhu and associates). The stool sample tested negative for O. formigenes. The patient was prescribed potassium citrate mixture; he was advised to avoid oxalate-rich food, maintain recommended levels of calcium in his diet, and take live bio-yogurt. Two months later, 24-h urinary oxalate decreased from 0.618 to 0.411 mmol/day; 24-h urine citrate increased from 0.58 to 1.10 mmol/day. Six months later, an oxalate absorption test was performed. The patient swallowed a capsule, soluble in gastric juice, containing 50 mg (0.37 mmol) sodium [13C2]oxalate corresponding to 33.8 mg of [13C2]oxalic acid. The amount of labeled oxalate, excreted in urine, was measured by a gas chromatographic-mass spectrometric assay. Oxalate absorption, expressed as the percentage of the labeled dose recovered in the 24-h urine after dosing, was 8.3% (reference range: 2.3–17.5%). In addition to other conventional measures, oral administration of O. formigenes or lactic acid bacteria mixture to promote bacterial degradation of oxalate in the gut, and thus combat hyperoxaluria, may play a role in prevention of calcium oxalate kidney stones.

Drug Testing in Blood: Validated Negative-Ion Chemical Ionization Gas Chromatographic–Mass Spectrometric Assay for Enantioselective Measurement of the Designer Drugs MDEA, MDMA, and MDA and Its Application to Samples from a Controlled Study with MDMA

The enantiomers of the designer drugs 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) differ in their pharmacologic and toxicologic potency. The aim of this study was to develop an assay for measuring these enantiomers in small plasma volumes and to analyze samples from a controlled study with MDMA. The analytes were extracted from < or = 0.2 mL of plasma by mixed-mode solid-phase extraction. After derivatization with S-(-)-heptafluorobutyrylprolyl chloride, the resulting diastereomers were separated by gas chromatography (HP-5MS) within 17 min and detected by mass spectrometry in the negative-ion chemical ionization mode. The method was fully validated and applied to samples from a controlled study in which a single dose of racemic MDMA (75 mg) was administered. The derivatized enantiomers were well separated and detected with good sensitivity. The assay was linear (per enantiomer) at 1-50 microg/L for MDA and 5-250 microg/L for MDMA and MDEA. Analytical recovery, accuracy, repeatability, and intermediate precision data were within required limits. Extraction yields were 82.1%-95.3%. In the study samples, concentrations of R-(-)-MDMA significantly exceeded those of S-(+)-MDMA. Their ratios (R vs S) were always >1.0 and increased over time. Concentrations of S-(+)-MDA exceeded those of R-(-)-MDA, their ratios (R vs S) also increasing over time but remaining <1.0. This assay enables sensitive, reliable, and fast enantioselective measurement of MDA, MDMA, and MDEA in small volumes of plasma. The controlled study data confirm previous findings of MDMA and MDA enantiomer ratios (R vs S) increasing over time after ingestion of racemic MDMA.

Fast, Simple, and Validated Gas Chromatographic-Mass Spectrometric Assay for Quantification of Drugs Relevant to Diagnosis of Brain Death in Human Blood Plasma Samples

In addition to total anamnesis, one of the important aspects in diagnosis of brain death is the exclusion of effective plasma concentrations of drugs that might mimic brain death. A minimum consensus for toxicological analysis in this context includes relevant analytes (thiopental, pentobarbital, methohexital, phenobarbital, diazepam, nordazepam, and midazolam) and proposes limits of quantification. Propofol is another relevant drug. After liquid-liquid extraction of 200 microL of plasma using 50 microL of a solution of deuterated internal standards in butyl acetate and 50 microL of butyl acetate, 2 microL of the organic phase was analyzed by gas chromatography-mass spectrometry using selected-ion monitoring mode. Validation included the parameters selectivity, calibration model, precision and accuracy, and extraction efficiency. Accuracy and precision data obtained using 6-point and 1-point calibration were compared. The abovementioned analytes were separated within 10 minutes and sensitively detected. No interfering peaks were observed in blank samples from 10 different sources. The linearity ranges were 0.5-6 mg/L for propofol, 0.25-10 mg/L for pentobarbital and thiopental, 0.125-10 mg/L for methohexital, 2.5-50 mg/L for phenobarbital, 0.05-2.5 mg/L for diazepam and nordazepam, and 0.01-0.5 mg/L for midazolam. Extraction efficiency ranged from 85% to 111%. The acceptance criterion for accuracy and precision (99% confidence interval of measured mean within +/-50% of target value) was fulfilled for all analytes, even with 1-point calibration using a calibrator close to the center of the linearity range. The assay was applied to analysis of real brain death cases. In conclusion, the described assay allowed fast and reliable determination of analytes relevant to diagnosis of brain death, and 1-point calibration kept the workload low.

Determination of Phenolic Antioxidants in Spilled Aviation Fuels by Gas Chromatography-Mass Spectrometry

A gas chromatography-mass spectrometric assay method was developed for the simultaneous determination of 2,6-di-tert-butylphenol (DTBP) and 2,4-dimethyl-6-tert-butylphenol (DMTBP) in aviation fuel. Extraction and purification were achieved with a solid phase extraction procedure using silica gel. Elution was performed with 30 mL of methylene chloride: pentane (2:3) following washing with 10 mL of n-pentane. The extract was concentrated to about 100 μL and analyzed by GC-MS (SIM). The peaks had good chromatographic properties by using a semi-polar column (Innowax) and the extraction of these compounds from samples gave recoveries of about 87% for DTBP and about 75% for DMTBP with small variations. Method detection limits were 5.0 ng mL−1 for DBMP and 7.0 ng mL−1 for DMTBP. The method may be useful for spilled fuel type differentiation between kerosene and JP-8.

Serum levels of total homocysteine, homocysteine metabolites and of advanced glycation end-products (AGEs) in patients after renal transplantation.

Hyperhomocysteinemia has been described as an independent risk factor for cardiovascular diseases (CVD) influencing patient outcome. Advanced glycation end-products (AGEs) are involved in the pathogenesis of vascular damage in uremia. This study was undertaken to assess whether high serum levels of total homocysteine (tHcy) with its metabolites methylmalonic acid (MMA), methylcitric acid (MCA) and cystathionine (CYSTA) as well as elevated serum concentrations of the AGEs pentosidine and Nepsilon-carboxymethyllysine (CML) are independent risk factors for CVD, left ventricular hypertrophy (LVH) or hypertension as well as kidney dysfunction in renal transplant recipients (RTR). Serum levels of tHCy, MMA, MCA and CYSTA were measured by a gas chromatographic-mass spectrometric assay, pentosidine by HPLC and CML using an ELISA assay. All measured parameters were significantly higher in RTRs than in healthy subjects (p < 0.0001). The levels of pentosidine and CML as well as of tHcy and its metabolites correlated significantly with each other, but not with those ofMMA and CYSTA. Significant correlations were also found between pentosidine and tHcy, MMA or MCA as well as between CML, MMA and MCA, respectively. Acute or chronic rejection did not influence these values. No significant differences were observed between patients with or without CVD or with hypertension. In RTRs with LVH, only the tHcy levels were significantly higher than in those RTRs without LVH (p = 0.006). Logistic regression analysis revealed an independent influence of tHcy on the presence of LVH. These results may indicate an association between high tHcy values and LVH. Further investigation is needed to determine whether a reduction of tHcy and Hcy metabolites and/or AGE serum concentrations would significantly improve patient outcome after undergoing renal transplantation.

New sensitive determination method of benzidine–hemoglobin adducts by gas chromatography–electron impact mass spectrometry

A gas chromatographic–mass spectrometric assay was developed for the determination of benzidine (BZ)–hemoglobin adducts. Adducts were released from hemoglobin by alkaline hydrolysis and extraction at pH 8 with ethyl ether. The dried extract was completely derivatized with N-methyl- N-( tert.-butyldimethylsilyl) trifluoroacetamide (MTBDMSTFA)–NH 4I (1000:3) under catalysis of dithioerythritol. The recovery of BZ, acetylbenzidine (ABZ) and diacetylbenzidine (DABZ) in the extraction procedure was 76–98%. The detection limits of the assay were 0.1 ng/g for both BZ and ABZ, and 0.5 ng/g for DABZ based upon assayed hemoglobin of 0.1 g. The method was applied to the determination of BZ–hemoglobin adducts formed in young female Sprague–Dawley rats after treatment for 1, 2 and 3 weeks with 0.008% BZ via the drinking water. Two adducts were detected by proposed procedure. The structure of these adducts could be assigned to BZ and ABZ. After 1 week, the total mean amount of adducts determined was 2.8 ng/g hemoglobin. The adduct levels increased up to about 7.5 ng/g after a week and, thereafter, remained essentially constant. The relative contribution of BZ and ABZ to the total hemoglobin adduct level was strongly treatment time-dependent. After 1 week, the BZ and ABZ adducts were formed at similar levels, whereas after 3 weeks the ABZ adducts was predominant. Treatment of rats for 3 weeks in the dose range 12.2–36.8 mg of BZ in drinking water resulted in a dose-proportional increase in the total amount of hemoglobin adducts formed.

Quantitative gas chromatographic-mass spectrometric assay of 4-hydroxynonenal bound to thiol proteins in ischemic/reperfused rat hearts.

Increasing evidence indicates that protein-aldehyde adducts involving mostly 4-hydroxynonenal could be causally involved in both pathophysiological and adaptive events following an oxidative stress insult such as ischemia/reperfusion. The goal of this study was to assess if isotope dilution chromatography-mass spectrometry can be used to quantitate changes in the cardiac levels of 4-hydroxynonenal and 1,4-dihydroxynonene, one of its major metabolites, bound to thiol proteins during ischemia/reperfusion. For this purpose, we modified a previously published method to include treatment with Raney Nickel, which specifically cleaves thioether linkages. Our study model was the isolated Langendorff-perfused rat heart subjected to various ischemia/reperfusion protocols. Hearts perfused under normoxia contained small amounts of protein-bound 4-hydroxynonenal and 1,4-dihydroxynonene (1.38 +/- 0.29 and 2.69 +/- 0.17 nmol/g wet weight, respectively). The accumulation of these adducts after global ischemia depended on the severity of the ischemic insult up to a plateau and was not exacerbated by reperfusion. In conclusion, our method allows the quantification of time-dependent changes in 4-hydroxynonenal and 1,4-dihydroxynonene bound to proteins via thioether linkage in ischemic/reperfused heart tissues. The presence of protein-bound 1,4-dihydroxynonene in heart tissues suggests that this organ can detoxify protein-bound 4-hydroxynonenal.