Determination of butorphanol in horse race urine by immunoassay and gas chromatography-mass spectrometry

An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC-MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC-MS system. The ELISA test (20 microl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC-MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC-MS method in the SIM mode using nalbuphine as internal standard included linearity studies (10-250 ng/ml), recovery (+/-100%), intra-assay (4.1-14.9%) and inter-assay (9.3-45.1%) precision, stability (10 days), limit of detection (10 ng/ml) and limit of quantitation (20 ng/ml).     

Development of a fluorescence polarization immunoassay for the routine detection of N-desmethylzopiclone in urine samples

A polarization fluoroimmunoassay was developed for the detected of N-desmethylzopiclone in urine and the reagents were adapted for use on the Vitalab Eclair analyser. Therefore, N-fluoresceinthiocarbamyldesmethylzopiclone was synthesized as a fluorescent tracer and used in combination with an existing pool of antibodies, raised against the hemisuccinyl derivative of N-desmethylzopiclone. The optimization of the assay was performed on a semi-quantitative basis, relative to a cut-off value of 300 ng ml-1. No significant interference was observed from a selection of existing drugs and endogenous compounds. The minimum detectable dose of the immunoassay was calculated to be 30 ng ml-1 (pooled-variance t-distribution, p = 0.01, degrees of freedom = 10). Intra- and inter-assay relative standard deviations were < 10 and < 12%, respectively. For the confirmation of positive samples, an established reverse-phase HPLC technique, in combination with fluorescence detection, was used. The combined screening/confirmation procedure was applied to cumulative excretion samples, after ingestion of one tablet of Imovane (zopiclone, 7.5 mg).     

Speciation of ionic alkyllead compounds in human urine by gas chromatography-mass spectrometry after butylation through a Grignard reaction

The interaction of ethanol and neurotrophin-mediated cell survival was examined in primary cultures of cortical neurons. Cells were obtained from rat fetuses on gestational day 16 and maintained in a medium supplemented with either 10% or 1.0% fetal calf serum (FCS). Exogenous nerve growth factor (NGF; 20 ng/ml), brain-derived neurotrophic factor (BDNF; 20 ng/ml) or neurotrophin 3 (NT-3; 20 ng/ml) was added to the cultures alone, or in combination with ethanol (400 mg/dl). The number of viable neurons was determined after a 48 h treatment with a growth factor and/or ethanol. The effects of ethanol on the expression of high affinity neurotrophin receptors (trkA, trkB, and trkC) and the low-affinity receptor (p75), were analyzed using Western immunoblots. In untreated cultures, 22.7% and 26.3% of the cells raised in a medium containing 10% and 1.0% FCS, respectively, were lost. Only NGF prevented the death of the cultured cortical neurons. Ethanol was toxic; it caused a 23.5% and 16.7% loss of cells (for cells grown in a medium containing 10% and 1.0% FCS, respectively) beyond that occurring 'naturally' in an untreated culture. Ethanol completely blocked the NGF-mediated cell survival. In general, BDNF and NT-3 did not offset the toxic effect of ethanol. Immunoblotting studies showed that the expression of p75 was significantly (p < 0.05) lower (40%) in ethanol-treated cultures, but ethanol did not affect trk expression. Thus, ethanol has specific effects upon NGF-mediated cell survival and the effects on the low affinity receptor imply that p75 specifically plays an important role in NGF signaling.     

Determination of loratadine and pheniramine from human serum by gas chromatography-mass spectrometry

PURPOSE: To report initial experiences with stent implantation in the treatment of native and recurrent aortic coarctation in adults. METHODS: Two adult patients were diagnosed with aortic coarctation: in one, the native aorta was involved, and in the other, the stenosis involved a prior coarctation repair. Both patients were offered and selected angioplasty with possible stent implantation as an alternative to surgery. RESULTS: In the patient with recurrent narrowing, thrombolysis and balloon dilation preceded the successful deployment of three Palmaz stents along the grafted segment. In the case of native disease, one Palmaz stent was implanted primarily at the site of a critical, focal stenosis. No complications were encountered, and recovery was uneventful. Follow-up at 12 and 6 months, respectively, showed sustained clinical improvement with resolution of symptoms and excellent hemodynamic values. CONCLUSIONS: The positive outcome in these early cases supports further evaluation of the efficacy of adjunctive or primary stenting for treatment of native or recurrent aortic coarctation in adults.     

Application of solid-phase microextraction and gas chromatography-mass spectrometry for the determination of chlorophenols in urine

Previously, we have demonstrated that chronic exposure to immobilization (IMO) did not modify the influence of catecholamines on active behaviour of rats in the holeboard, but clearly increased the role of these amines in the forced swimming test (FST). In the present experiment, it was studied whether or not chronic IMO altered the role of dopamine in the two tests. Adult male Sprague-Dawley rats were left either undisturbed or subjected daily to 2 h of IMO stress for 12 days. On the following day, half of the rats were administered saline and the others the dopamine antagonist haloperidol (0.5 mg/kg). Then the rats remained undisturbed in the animal room (controls) or were subjected to acute IMO for 2 h. Finally, all animals were exposed consecutively to the holeboard (4 min) and the FST (5 min). In non-chronically stressed rats, acute IMO depressed behaviour in the holeboard but not in the FST. In chronic IMO rats the inhibitory effect of acute IMO on holeboard activity was slightly reduced as compared to controls. Acute IMO increased struggling in rats previously exposed to chronic IMO but did not alter struggling in non-chronically stressed rats. Whereas the inhibition caused by haloperidol treatment in the active behaviour of rats in the holeboard was not altered by chronic IMO, the inhibitory effect of haloperidol in the active behaviour of rats in the FST was greater after chronic IMO, particularly in rats also subjected to acute IMO. These data suggest that chronic IMO stress potentiates the role of dopamine in a specific behavioural task such as the FST and adds support to the previously published data demonstrating enhanced behavioural and neurochemical responses to dopamine-related drugs after chronic stress.     

Determination of thevetin B in serum by fluorescence polarization immunoassay

Thevetin B, a cardiac glycoside of Thevetia neriifolia Juss. seeds, was determined in serum by fluorescence polarization immunoassay. Anti-digitoxin antibody was used, thevetin B genin being structurally identical to digitoxigenin. Cross-reactivity of 94% was found by this method, for concentrations from 5 to 80 ng ml-1.     

Determination of acrolein in human urine by headspace gas chromatography and mass spectrometry

The aim of the present study is to examine whether noradrenergic neurons of the locus coeruleus (LC) of the rat contain monoamine oxidase (MAO) activity. Sections were processed initially for MAO enzyme histochemistry using tyramine as a substrate, followed by fluorescence immunohistochemistry for tyrosine hydroxylase (TH). In the LC, virtually all TH-immunoreactive neurons (i.e., noradrenergic neurons) were also positive for MAO. No MAO activity was found in any TH-negative neurons. Neurons in the LC have previously been shown to form dopamine during noradrenaline biosynthesis and to produce serotonin from exogenously administered l-5-hydroxytryptophan. Moreover, dopamine- and serotonin-degrading MAO activity has also been found in LC neurons. Therefore, our results indicate that MAO activity is localized within noradrenergic neurons in the LC and is likely involved in the degradation of dopamine that is endogenously synthesized, and also in the elimination of serotonin that is produced from exogenous precursors.     

Determination of gacyclidine enantiomers in human plasma by gas chromatography-mass spectrometry using selected-ion monitoring

A sensitive gas chromatographic assay using mass selective-detection has been developed for the simultaneous quantitation of the enantiomers of (+/-)-gacyclidine (a non competitive N-methyl-D-aspartate antagonist) in human plasma. Gacyclidine enantiomers and phencyclidine (PCP), the internal standard, were extracted using a single-step liquid-liquid extraction with hexane at pH 8.0. Each enantiomer was separated on a chiral gas chromatography capillary column and specifically detected by mass spectrometry (MS) in selected-ion monitoring (SIM) mode. Gacyclidine enantiomers and PCP were monitored using the fragment ions at m/z 206 and 200, respectively. No interference was observed from endogenous components. The limit of quantitation (LOQ) for each enantiomer of gacyclidine was 300 pg/ml by using plasma samples of 500 microl. The calibration curves were linear (r2=0.998) over a range of 0.3125 to 20 ng/ml. The extraction efficiency was higher than 95% for both enantiomers. Intra- and inter-day bias were less than 10% at every standard curve concentration. Intra-day precision was less than 19% for (-)-gacyclidine and 15% for (+)-gacyclidine. Inter-day precision was below 15% for both enantiomers. The assay was validated for an enantioselective pharmacokinetic study in healthy male volunteers.     

Analysis of beta-agonists in urine and tissues by capillary gas chromatography-mass spectrometry: in vivo study of salbutamol disposition in calves

The fate of salbutamol sulphate given orally has been investigated in calves. The urinary excretion rate and the tissue distribution of this beta-agonistic drug were studied by capillary gas chromatography coupled to low resolution mass spectrometry (GC-LRMS) under electron impact (EI) ionization mode, using an hexadeuterated salbutamol analogue as the internal standard. The parent drug and metabolites were extracted via solid phase extraction (SPE) mixed-phase-containing disposable columns and analysed as their trimethylsilyl derivatives. A more efficient clean-up had to be carried out for tissue samples. An acidic precipitation followed by a liquid-liquid extraction were therefore performed before the SPE. Moreover, the problem of tissue digestion was elucidated by means of an ultrasonic probe. Samples were also analysed before and after enzymic hydrolysis using purified beta-glucuronidase and a mixture of beta-glucuronidase and arylsulphatase, to obtain evidence of phase II conjugation mechanisms. Both free salbutamol and conjugated metabolites were detected in urine and tissue samples. Except for liver or kidney, salbutamol was rapidly cleared from most tissues after a withdrawal period. The possible excretion of some phase I metabolites was also investigated, using further analyses under positive chemical ionization LRMS and high resolution mass spectrometry (HRMS).     

Determination of norfloxacin in human plasma and urine by high-performance liquid chromatography and fluorescence detection

A method for the determination of norfloxacin in human plasma and urine is described. Plasma samples were deproteinized using acetonitrile. The supernatant was analysed by C18 HPLC. Fluorescence detection at an excitation wavelength of 300 nm and an emission wavelength of 450 nm was utilized. The assay was validated in the concentration range of 31 to 2507 ng/ml when 0.5-ml aliquots of plasma were handled. The intra-day precision of the spiked quality control samples ranged from +/- 0.37 to +/- 4.14% in plasma (concentration range: 70.3-2109.2 ng/ml) and from +/- 0.51 to +/- 1.56% in urine (concentration range: 7.5-299.4 micrograms/ml). The intra-day accuracy obtained for norfloxacin in the quality control samples ranged from -5.18% to -9.47% in plasma and from -10.56% to - 5.91% in urine. The assay has been used to support human pharmacokinetic studies.     

Parent cyclosporine in whole blood by monoclonal fluorescence polarization immunoassay for axsym and monoclonal enzyme-multiplied immunoassay for cobas-fara

Secondary tumors of the thyroid are very rare, being the kidney the most frequent place of the primary tumor. The majority of these metastases appear months or years after the primary renal tumor. We report the case of an asymptomatic renal carcinoma discovered after the histological analysis of a thyroidectomy piece. The treatment of the primary renal tumor was radical nephrectomy. Three years after diagnosis and treatment the patient is free of relapse.     

Multiresidue determination of pesticides in apples and pears by gas chromatography-mass spectrometry

This paper describes a rapid, specific and sensitive multiresidue method for the routine analysis of several classes of pesticides used for the treatment of apples and pears, involving a rapid extraction procedure at pH 4.5 with a mixture of acetone-dichloromethane-hexane (50:20:30, v/v/v) and gas chromatography coupled to mass-selective detection, in order to achieve quantitative analysis down to their respective maximum residue limit. Extraction recoveries were between 55 and 98%. Limits of detection and limits of quantitation ranged respectively, from 0.01 to 0.05 mg/kg and from 0.02 to 0.1 mg/kg. Intra-assay relative standard deviation was less than 19% for all compounds. An excellent linearity was observed from these LOQs up to 500 mg/kg. Intermediate (inter-assay) precision and accuracy were satisfactory. The method has been applied to many fruit samples intended for commercialisation.     

Screening of chlorpropamide in horse plasma by high-performance liquid chromatography with ultraviolet absorbance detection, and confirmation by gas chromatography-mass spectrometry

A chromatographic method was developed to detect and confirm the presence of chlorpropamide (I) in horse plasma samples, for antidoping control. The plasma sample (1 ml) was extracted with dichloromethane and screened by high-performance liquid chromatography, and confirmation of the drug's presence was accomplished by using gas chromatography-mass spectrometry (GC-MS). The limit of detection was found to be 3.5 ng/ml at a signal-to-noise ratio of three. Derivatization of I with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane allowed for highly stable, accurate and sensitive GC-MS analysis. Plasma samples collected after the administration of diabinese were positive for I (one-five days) in all samples analysed.     

A sensitive assay of 5-fluorouracil in plasma by gas chromatography-mass spectrometry

1 A gas chromatographic-mass spectrometric method was developed for determining 5-fluorouracil in plasma, using methylated thymine as an internal standard. 2 5-fluorouracil was extracted from plasma by a novel procedure which removed plasma components interferring with the sensitivity of the assay. The method included heating the plasma, washing with ether and extracting the drug under optimum conditions. 3 The sensitivity of the assay was 10 ng/ml plasma, sufficient to determine the low concentrations of 5-fluorouracil found in plasma during continuous infusion of the drug in patients receiving chemotherapy for cancer.     

Separation and detection of phencyclidine in urine by gas chromatography

A rapid, sensitive and selective analytical procedure for phencyclidine and one of its metabolites in urine has bee developed. Three techniques have been studied for extraction of the drug from the biological matrix: (a) reversed-phase XAD resin, (b) charcoal absorption, and (c) solvent extraction using chloroform. Temperature-programmed gas chromatography was used to quantitate the illicit drug. Solvent extraction appears to offer the most efficient separation of the drug and its metabolite, as the recovery was 94% and the technique required only 7-8 min per sample. Reversedphase column extraction is also quite useful; although more time-consuming for an indivisual sample, it would be useful for screening purposes.     

Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of omega- and (omega-1)-hydroxylated metabolites of elaidic and oleic acids in human and rat liver microsomes

A peptide that inhibits the human cholesteryl ester transfer protein (CETP) was isolated from hog plasma by ultracentrifugation, two sequential column chromatographies and electroelution from gels. Molecular weight of the peptide was determined to be approximately 3 kDa on the SDS-PAGE. The peptide contained 28 amino acids with an identical sequence to the amino terminus of hog apolipoprotein-CIII except two amino acid residues: -Pro-Glu- at the fifth and sixth amino acids from the amino terminus in the isolated peptide, in contrast to -Leu-Leu- in hog apo-CIII. A peptide synthesized chemically according to the amino acid sequence of the peptide (designated P28) showed approximately the same degree of CETP inhibitory activity as the isolated peptide. Synthetic peptides with different number of amino acids were also tested for CETP inhibition. Among the peptides, the one with 20 amino acid residues (P20) from the amino terminus showed the highest inhibitory activity against the CETP. The peptide appeared to be associated with the hog high-density lipoproteins (HDL), as determined by immunoblot analysis using antibody against P28. The CETP-inhibitory activity of the peptide was examined in vivo using diet-induced hypercholesterolemic rabbits. When the peptide was injected into the rabbits (7-9 mg/kg body weight), approximately 75% CETP activity disappeared from the plasma in 1 h after the injection and the effect lasted up to 30 h. The inhibition of CETP in vivo led to a concomitant decrease in total plasma cholesterol level up to 30% and an increase in the level of HDL-cholesterol up to 32%. The cholesterol concentrations in the rabbit plasma gradually recovered to the initial level after 48 h.     

Direct analysis of several Fusarium mycotoxins in cereals by capillary gas chromatography-mass spectrometry

This article describes three experiments relating to the legibility of TV menus. Special emphasis is placed on the influence of a relatively new feature in TVs: the possibility to blend graphics and video. Three experiments are presented: one concentrating on the influences of blending level and video content; one concentrating on the influences of content and of colour combinations; and one concentrating on the influences of various font characteristics. The results are interpreted in terms of guidelines for blended TV menus.