Microdialysis for the determination of acetaldehyde and ethanol concentrations in the striatum of freely moving rats

The subject of the present paper is the simultaneous determination of ethanol (EtOH) and acetaldehyde (AcH) concentrations in the striatum of freely moving rats using an in vivo microdialysis followed by head-space gas chromatography (GC). Major operation conditions of GC were as follows: column, injector and detector temperatures 90, 110 and 200 degrees C, respectively; Supelcowax wide bore capillary column (60 m length, 0.53 mm i.d., 2 microm film thickness); carrier gas, nitrogen; flow rate, 20 ml/min. The recovery of EtOH and AcH at a concentration 40 mM and 250 microM, respectively, by microdialysis showed a maximum of 83.8+/-12.2 and 51.2+/-6.5%, respectively, at a flow rate of 0.8 microl/min. A good linear calibration curve in the concentration range of 5-50 mM for EtOH (r=0.998), and 10-250 microM for AcH (r=0.988) was observed. Microdialysates were collected for 10 min each after insertion of probe into the striatum. Rats were treated with cyanamide (100mg/kg, a potent aldehyde dehydrogenase inhibitor) and 60 min later with EtOH (1g/kg) intraperitoneally. A 10 min sample was about 8 microl. This volume was mixed with 40 microl of 0.002% t-butanol as an internal standard in 0.6N perchloric acid, and then analyzed by head-space GC method. The peak EtOH and AcH concentrations in the striatal dialysates reached maximum at 30 min, and then gradually decreased. This method represents a reasonable tool to quantify in vivo both AcH and EtOH levels simultaneously in rat brain.     

The effect of indomethacin and adenosine 5'-triphosphate on the excitatory innervation of the rate urinary bladder

Adenosine 5'-triphosphate (ATP) (20-400 microM) contracted 48% of isolated rat urinary bladder preparations but induced no response in the remainder. The response to ATP never exceeded 25% of the response to electrical stimulation in the presence of indomethacin (50 microM) plus hyoscine (25 microM) and usually developed more slowly than that to electrical stimulation. Autoinhibition could be produced to ATP by incubating the tissue with ATP (200 microM) for 20 min. Incubation of the tissue with ATP (200 microM) for 60 min in the presence of indomethacin (50 microM) and either hyoscine (25 microM) or hemicholinium-3 (500 microM) reduced but failed to abolish responses to electrical stimulation. Responses to acetylcholine were not affected by ATP (200 microM) in the presence of indomethacin and the output of acetylcholine induced by neuronal stimulation at 10 Hz was not inhibited by ATP (200 microM) or by indomethacin (50 microM). The results suggest a possible modulatory role for ATP in the excitatory innervation of the rat urinary bladder.     

Trifluoroacetic anhydride-catalyzed nitration of toluene as an approach to the specific analysis of nitrate by gas chromatography-mass spectrometry.

The nitration of aromatic compounds by electrophilic substitution is often utilized in analyses of nitrate concentrations in physiological samples by gas chromatographic methods. Problems associated with the use of concentrated sulfuric acid, which is normally used to catalyze this reaction, led us to investigate an alternative method. We describe here a facile GC/MS assay for nitrate in plasma or urine samples which takes advantage of the ability of trifluoroacetic anhydride (TFAA) to catalyze the nitration of aromatics. Toluene, utilized as both reaction solvent and electrophile, was shown to react with nitrate in the presence of TFAA to quantitatively produce the three nitrotoluene isomers (ratio o-:m:p-, approx 57:3:40). Following the incorporation of 15N-labeled nitrate as internal standard, nitrotoluene was quantified using GC/MS by analysis of the selected the ion pairs m/z 120 and 121 (M+ -OH) for the o-isomer or m/z 137 and 138 (molecular ion, M+) for the p-isomer. The limit of detection for nitrate after TFAA-catalyzed conversion to nitrotoluene was less than 100 fmol on column (s/n; 40:1). The TFAA-based GC/MS assay was compared with that utilizing the usual catalyst, concentrated sulfuric acid. With the exception of samples containing nitroarginine analogues, good correlation was found for urine or plasma samples analyzed using either a standard sulfuric acid-catalyzed method or the TFAA-catalyzed procedure. Nitroarginine analogues, which can be present in samples following their use as nitric oxide synthase inhibitors, did not decompose under the conditions of the TFAA-catalyzed assay and, hence, do not give rise to significant interference with nitrate analysis in this procedure. In contrast, catalytic sulfuric acid caused nitroarginine analogues to decompose (essentially quantitatively) and cause spuriously high nitrate levels in samples. The use of TFAA as a catalyst for the nitration of toluene enables a facile and sensitive GC/MS analysis for nitrate which offers improved safety and sample integrity.     

Assay of coenzyme Q(10) in plasma by a single dilution step

A new method is described for determining coenzyme Q(10) (CoQ(10)) in plasma. The method is based on oxidation of CoQ(10) in the sample by treating it with para-benzoquinone followed by extraction with 1-propanol and direct injection into the HPLC apparatus. This method achieves a linear detector response for peak area measurements over the concentration range of 0.05-3.47 microM. Diode array analysis of the peak was consistent with CoQ(10) spectrum. Supplementation of the samples with known amounts of CoQ(10) yielded a quantitative recovery of 96-98.5%; the method showed a level of quantitation of 1.23 nmol per HPLC injection (200 microl of propanol extract containing 33.3 microl of plasma). A correlation of r = 0.99 (P < 0.0001) was found with a reference electrochemical detection method. Within run precision showed a CV% of 1.6 for samples approaching normal values (1.02 microM). Day-to-day precision was also close to 2%.     

Negative chemical ionization gas chromatography/mass spectrometry to quantify urinary 3-methylhistidine: application to burn injury

A rapid method for measuring 3-methylhistidine (3MH) in rat and human urine with higher sensitivity and precision than any previously reported method is described using internal standard [1-(13)C]3MH (M+1) and negative chemical ionization (NCI) gas chromatography/mass spectrometry (GC/MS). Internal standard [1-(13)C]3MH (M+1) was added to rat and human urine samples, hydrolyzed, and absorbed onto cation exchange columns. The column eluent was dried and derivatized for GC/MS analysis. Quantification of 3MH levels was accomplished by monitoring the m/z 204 fragment. The m/z 204 fragment was chosen due to the fragment's abundance and stability as determined by analysis of [methyl-(2)H(3), (18)O(2)]3MH (M+7) and [methyl-(13)C]3MH (M+1) fragmentation patterns under NCI conditions. This method shows excellent linearity (0.9989) over the range studied (0-0.5 mol), high recovery (95.9%), and low coefficient of variation (4.7%). The described method is sensitive enough to detect 6.8 pmol amount of urinary 3MH with a precision of 9.1%. The in vivo utility of this method to quantify urinary 3MH was tested in a burn injury rat model and on urine specimens from pediatric burn patients. Data obtained from the urine of burn-injured rats and pediatric burn patients match previously reported trends and validate the in vivo utility of this method.     

Capillary electrophoretic assay for nitrate levels in the vitreous of proliferative diabetic retinopathy

The determination of nitric oxide (NO) in human vitreous samples is complicated by the relatively short half-life of the analyte and the viscous, high salt and protein biological matrix. In this work, we developed a fast (<5min) and useful CE method to determine the stable metabolite, nitrate, from vitreous samples. This proposed method has been successfully applied to determine the nitrate levels from the vitreous humor of patients undergoing vitrectomy for a variety of conditions. A statistically significant increase (P=0.000001) of the mean level of nitrate was observed in vitreous humor of patients with proliferative diabetic retinopathy (41.17+/-4.09microM, n=27) versus controls (15.22+/-0.86microM, n=35). The elevated levels of nitrate in the vitreous of patients known to have diabetic retinopathy suggests that NO is involved with the pathology of this disease.     

Indirect determination of nitric oxide production by reduction of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli MC1061

Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described. To obtain the crude bacterial lysate containing nitrate reductase activity, E. coli MC1061 was subjected to 16-20 freeze-thawing cycles, from -70 to 60 degrees C, until nitrite reductase activity was < or = 25%. Nitrate reductase activity was detected mainly in the crude preparation. To validate the nitrate reduction procedure, standard nitrate solutions (1.6-100 microM) were incubated with the nitrate reductase preparation for 3 h at 37 degrees C, and nitrite was estimated by the Griess reaction in a microassay. Nitrate solutions were reduced to nitrite in a range of 60-70%. Importantly, no cofactors were necessary to perform nitrate reduction. The biological samples were first reduced with the nitrate reductase preparation. After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified. The utility of the nitrate reductase preparation was assessed by nitrate+nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E. coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages. Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate+nitrite in biological fluids by spectrophotometric methods.     

S100B and brain natriuretic peptide predict functional neurological outcome after intracerebral haemorrhage

Colorimetric test strip assays are a convenient and inexpensive means for the determination of cotinine in human urine because they can be performed in a nonlaboratory environment using a trained technician. Four hundred human urine samples were separated into four categories: (1) heavy smokers (>20 cigarettes smoked per day), (2) light smokers (<20 cigarettes smoked per day), (3) non-smokers, and (4) vegetarian non-smokers. Samples were evaluated by a gas chromatography/mass selective detector (GC/MSD) method as a reference and using NicCheck I? (DynaGen, Inc.). Colour intensity can range from 0 (no colour) to 14 (deep pink). Qualitative values were assigned as negative (0), low (1-6) and high (7-14). Comparison of the test strip and GC/MSD results showed: (1) 43 (10.75%) false negatives using the criterion of a GC/MSD cotinine level above 200 ng ml^-1 and test strip reading of 0, (2) 31 (7.75%) false positives using the criterion of a GC/MSD cotinine level below 1 ng ml^-1 and a test strip reading of 1 or greater, and (3) no correlation between the test strip and GC/MSD results (r = 0.597, p < 0.05). The fact that the colorimetric reaction is sensitive to many nicotine metabolites and/or heterocyclic amine structures whereas the GC/MSD method measures nicotine and cotinine selectively might explain the false positive results. False negative results were likely to be due to a lack of sensitivity of the test strip.     

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.     

Gas chromatography mass spectrometry analysis of carboxyethyl-hydroxychroman metabolites of alpha- and gamma-tocopherol in human plasma

Alpha- and gamma-tocopherol (alpha- and gamma-T, respectively) metabolite analysis is of key relevance in the study of vitamin E metabolism. Whilst there is information on urinary excretion of the two major metabolites of these vitamin E homologues, namely the 2,5,7,8-tetramethyl-2-(beta-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), their concentration and response to supplements in plasma remains poorly investigated. In this study we describe a gas chromatography-mass spectrometry (GC/MS)-based assay to measure both alpha- and gamma-T and their corresponding CEHC metabolites in human plasma. As an example of the application of this method we report data obtained following the supplemention of two healthy volunteers with 100 mg of deuterium-labeled gamma-T acetate (d(2)-gamma-TAC). Under routine analytical conditions a good linearity in the range 0.0025--1 microM was observed for both the alpha- and gamma-CEHC deuterated standards. In plasma samples, the detection limit for alpha- and gamma-CEHC was 2.5 and 5 nmol/l, respectively. The minimum amount of plasma required for the assay was 500 microl. The plasma concentrations of alpha-CEHC and gamma-CEHC in unsupplemented healthy subjects were 12.6 +/-7.5 and 160.7 +/- 44.9 nmol/l, respectively. In the two volunteers supplemented with 100 mg of d(2)-gamma-TAC, plasma d(2)-gamma-T concentrations increased 250 to 450-fold 6 h postsupplementation. Plasma and urinary d(2)-gamma-CEHC concentrations increased 20 to 40-fold 9--12 h postsupplementation. Interestingly, the acute increase in d(2) gamma-T did not significantly affect the baseline plasma concentrations of d(0)-gamma-T and only slight lowered alpha-T concentrations. Likewise, plasma alpha-CEHC levels were not influenced and urinary excretion of alpha-CEHC were unaltered. This GC/MS method provides a versatile and accurate mean for assessing carboxyethyl-hydroxychroman metabolites of vitamin E in plasma.     

Determination of 3-nitrotyrosine in human urine at the basal state by gas chromatography-tandem mass spectrometry and evaluation of the excretion after oral intake

3-Nitrotyrosine (NO(2)Tyr) is a potential biomarker of reactive-nitrogen species (RNS) including peroxynitrite. 3-Nitrotyrosine occurs in human plasma in its free and protein-associated forms and is excreted in the urine. Measurement of 3-nitrotyrosine in human plasma is invasive and associated with numerous methodological problems. Recently, we have described an accurate method based on gas chromatography (GC)-tandem mass spectrometry (MS) for circulating 3-nitrotyrosine. The present article describes the extension of this method to urinary 3-nitrotyrosine. The method involves separation of urinary 3-nitrotyrosine from nitrite, nitrate and l-tyrosine by HPLC, preparation of the n-propyl-pentafluoropropionyltrimethylsilyl ether derivatives of endogenous 3-nitrotyrosine and the internal standard 3-nitro-l-[(2)H(3)]tyrosine, and GC-tandem MS quantification in the selected-reaction monitoring mode under negative-ion chemical ionization conditions. In urine of ten apparently healthy volunteers (years of age, 36.5+/-7.2) 3-nitrotyrosine levels were determined to be 8.4+/-10.4 nM (range, 1.6-33.2 nM) or 0.46+/-0.49 nmol/mmol creatinine (range, 0.05-1.30 nmol/mmol creatinine). The present GC-tandem MS method provides accurate values of 3-nitrotyrosine in human urine at the basal state. After oral intake of 3-nitro-l-tyrosine by a healthy volunteer (27.6 microg/kg body weight) 3-nitro-l-tyrosine appeared rapidly in the urine and was excreted following a biphasic pharmacokinetic profile. Approximately one third of administered 3-nitro-l-tyrosine was excreted within the first 8 h. The suitability of the non-invasive measurement of urinary 3-nitrotyrosine as a method of assessment of oxidative stress in humans remains to be established.     

Determination of plasma aminothiols by high performance liquid chromatography after precolumn derivatization with N-(2-acridonyl)maleimide

Design, synthesis and properties of new derivatization reagent N-(2-acridonyl)-maleimide (MIAC) for thiol groups is presented. The reaction of MIAC with aminothiols is specific, very fast and yield highly fluorescent products. The HPLC method for determination of homocysteine, cysteine and glutathione based on utilization of MIAC is developed. A baseline separation of derivatives is achieved by isocratic elution on reverse phase column within 6 min. The method is linear in the range of 0.5-25 microM for homocysteine and glutathione, and in the range of 0.5-200 microM for cysteine. The limits of detection for homocysteine, cysteine and glutathione are 1.2, 1.4 and 2.0 pmol, respectively, per 20 microl injection. Within and between-run precision expressed as relative standard deviations are in the range of 1.35-4.38% and 0.89-4.13%, respectively.     

Rapid and sensitive static headspace gas chromatography-mass spectrometry method for the analysis of ethanol and abused inhalants in blood

A sensitive and specific method using static headspace gas chromatography coupled with mass spectrometry (GC/MS) has been developed for the quantitative determination of ethanol in biological fluids using n-propanol as internal standard. Gas chromatography was performed in isothermal mode with a GC run time of 2.6 min. The quantification was performed using scan mode abstracting a quantitative ion and a qualifier ion for ethanol and for the internal standard. The method was linear (r(2), 0.999, in the concentration range of 5-200 mg/dl), specific (no interference from methanol acetaldehyde, acetone or from endogenous materials), sensitive (limit of quantification and limit of detection of 0.2 and 0.02 mg/dl, respectively) and robust (less than 5% inter- and intra-assay coefficient of variation). A slightly modified method was also developed for the quantification of five commonly abused inhalants (dichloromethane, ethyl acetate, benzene, toluene and xylene) in blood. The method used a gradient GC program with a run time of 8 min. The quantification was performed using scan mode and integrating the area under the peak using trichloroethane as an internal standard. Without optimization, the method was linear (from 5 to 100 mg/l) and sensitive.     

Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

High-resolution microanalysis of nitrite and nitrate in neuronal tissues by capillary electrophoresis with conductivity detection

Nitrites and nitrates are widely used reporters of endogenous activity of nitric oxide synthases (NOS), an important group of enzymes producing the gaseous signal molecule nitric oxide (NO). However, due to the great chemical heterogeneity of neuronal tissues, standard analytical protocols for evaluation of neuronal nitrite/nitrate concentrations are inefficient. We optimized a high-performance capillary zone electrophoresis (CZE) technique to analyze nitrite/nitrate concentrations in submicroliter samples from mammalian neuronal tissues. The measurements were made using a PrinCE 476 computerized capillary electrophoresis system with a Crystal 1000 contact conductivity detector. Isotachophoretic stacking injection of 1000- to 10000-fold diluted samples, which had been pretreated with a custom-designed solid-phase microextraction (SPME) cartridge, was employed to assay micromolar and nanomolar nitrite and nitrate levels in the presence of the high millimolar chloride concentrations characteristic of many biological samples. In the presented technique, a 10-microl volume of diluted ganglionic sample was used for chloride removal and sample cleanup. The method yields high analytical performance, including good reproducibility, resolution, and accuracy. The limits of detection relative to undiluted sample matrix were 8.9 nM (0.41 ppb) and 3.54 nM (0.22 ppb) for nitrite and nitrate, respectively. In addition, this technique resolves other anions that are present in neuronal tissues at sub-nanomolar concentrations and can be broadly applied for high-throughput anionic profiling. In rat dorsal root ganglia, endogenous levels of nitrate (231+/-29 microM; n=6) and nitrite (24-96 microM) were found. These concentrations exceeded those previously found in neuronal tissue homogenates using different techniques.     

Colorimetric determination of chloride in biological samples by using mercuric nitrate and diphenylcarbazone

A colorimetic method is outlined for the determination of the chloride ion in biological samples (blood serum, plasma, and urine). The present method is based on the quantitative reduction of free mercuric ions by chloride ions. Chloride ions form an indissociable complex with mercuric ions. The remaining free mercuric ions form a purple complex with diphenylcarbazone with an absorption maximum at 550 nm. The reduction of color intensity at 550 nm is directly proportional to chloride concentration in the sample. The linear concentration range in the final reaction mixture was 0–100 μM with a correlation coefficient of −0.9997. The coefficient of variation for the 50 μM chloride ion in the final reaction mixture was 0.9% (n=6). The analyzed value of chloride concentration in the human control serum Accutrol™ Normal (Sigma) was 101±4 mM (mean±SD, n=12). The certified value of chloride in Accutrol Normal by Sigma is 102 mM, with a mean in the range 91–113 mM. This method was applied to the measurement of urinary chloride excretion in experimental rats. During 16-h urine collection, no food was given and rats had free access to purified water. The urinary excretion rate of chloride was 23.6±9.3 μmol/h (mean±SD, n=8) and 126.2±28.0 μmol/h (n=8) for rats fed a normal diet (2.6 g NaCl/kg diet) and a high-salt diet (82.6 g NaCl/kg diet) for 70 d prior to urine collection, respectively. This method is appropriate for low concentrations of chloride in samples or when sample volume is limiting, as in many animal studies such as metabolic urine collection from rats. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of the products that may also be suitable.     

Lisinopril quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of Lisinopril in human plasma using Enalaprilat as internal standard. The analyte and internal standard were extracted from the plasma samples by solid-phase extraction using Waters HLB Oasis SPE cartridges and chromatographed on a C8 analytical column. The mobile phase consisted of acetonitrile/water (60:40, v/v) + 20 mM acetic acid + 4.3 mM of triethylamine. The method had a chromatographic total run-time of 6.5 min and was linear within the range 2.00-200 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM). The precision (CV%) and accuracy, calculated from limit of quantification (LOQ) samples (n = 8), were 8.9 and 98.9%, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of Lisinopril 20mg.     

A turbidimetric assay for quantitating functional fibrin(ogen) using polystyrene-divinylbenzene microparticles.

A sensitive assay has been developed to quantitate fibrinogen in plasma or in other aqueous solutions. Microscopic latex particles, modified with a mixed monomolecular film of lecithin and fibrinogen, are used as a solid-phase reagent. These lecithin/fibrinogen-coated beads aggregate when stirred in the presence of thrombin and, when solution-phase fibrinogen is added, the increased rate of aggregation is proportional to the concentration of soluble fibrinogen. Using a sample volume of 200 microl, as little as 15 nM ( approximately 5 microg ml-1) fibrinogen can be measured. Fibrinogen determinations using the bead assay compared favorably with those derived from a standard clinical assay, with a correlation coefficient (r2) of 0.9710 over a range of 2.5 to 28.0 microM. Analytic precision was comparable to available assays, with typical coefficients of variation of 12.7 and 7.1% for fibrinogen concentrations of 30 nM and 15.0 microM, respectively. The method has a dynamic range of 15 nM to over 3.0 microM and offers the advantage of being sensitive to 20-fold lower concentrations of fibrinogen compared to routine clot-based methods. Unlike immunological assays, e.g., ELISA, it measures only the functional protein. This bead method should prove to be of greatest use to investigators measuring low levels of functional fibrin(ogen).     

Determination of phenolic flame-retardants in human plasma using solid-phase extraction and gas chromatography–electron-capture mass spectrometry

A method for determination of phenolic flame-retardants in human plasma utilizing solid-phase extraction (SPE) and gas chromatography with electron-capture mass spectrometric detection (GC–ECMS), has been developed. The plasma lipids were decomposed by application of concentrated sulphuric acid directly on the polystyrene–divinylbenzene SPE column. The method has been validated for 2,4,6-tribromophenol (TriBP), pentabromophenol (PeBP), tetrachlorobisphenol-A (TCBP-A) and tetrabromobisphenol-A (TBBP-A) in the concentration range 1.2–25, 0.4–40, 4–200 and 4–200 pg g⁻¹ plasma, respectively. The average absolute recovery of the analytes ranged from 51 to 85%. Tetrabromo-o-cresol and chlorotribromobisphenol-A were found suitable as internal standards, and the average recovery of the analytes relative to the internal standards was in the range 93–107%. The repeatability of the method was in the range 4–30% relative standard deviation. The estimated detection limits of TriBP, PeBP, TCBP-A and TBBP-A were 0.3, 0.4, 3.0 and 0.8 pg g⁻¹ plasma, respectively. The method has been used for analysis of plasma samples from potentially occupationally exposed human individuals.     

An amperometric inhibitor biosensor for the determination of reduced glutathione (GSH) without any derivatization in some plants

In this study, an amperometric biosensor based on cucumber tissue homogenate was developed for the determination of glutathione. Cucumber (Cucumis sativus L.) tissue homogenate was used as the biological material. The cucumber tissue homogenate was cross-linked with gelatine using glutaraldehyde and fixed on a pretreated teflon membrane. The principle of the measurements was based on the determination of the decrease in the differentiation of oxygen level which had been caused by the inhibition of ascorbate oxidase in the biological material by glutathione. Determinations were carried out by standard curves which were obtained by the measurement of the decrease in the consumed oxygen level related to glutathione concentration. Optimization and characterization studies of the biosensor were carried out and a linearity in the gamma-L-glutamyl-L-cysteinyl-glycine (GSH) concentration range 0.1-2 microM was obtained when 600 microM ascorbic acid was used as a substrate. The repeatability experiments (n = 7) revealed that for 1.5 microM GSH, the average value (x), standard deviation (S.D.) and variation coefficient (C.V.) were 1.517 microM, 4.72 x 10(-5) 3.11%, respectively. The biosensor useful lifetime was at least 2 months. The results of some plant samples analyzed with the presented biosensor agreed well with the spectrophotometric method (Ellman's reagent) used as a reference.