Gas chromatographic-mass spectrometric identification and quantification of aniline after extraction from serum and derivatization with 2,2,2-trichloroethyl chloroformate, a novel derivative

The effect of the neurosteroid progesterone on the kainate receptor-mediated response was studied in cultured chick spinal cord neurons using the whole-cell voltage-clamp recording technique. Progesterone rapidly and reversibly potentiates the kainate-induced current in a dose-dependent manner, with an EC50 of 35 microM and a maximal potentiation of 30%. Potentiation of the kainate response by extracellularly applied progesterone is not significantly affected by inclusion of a saturating concentration of progesterone in the electrode buffer, indicating that progesterone acts at the extracellular surface of the membrane. Furthermore, progesterone enhances the kainate maximal response with little effect on the kainate EC50.     

A novel derivatization of phenol after extraction from human serum using perfluorooctanoyl chloride for gas chromatography-mass spectrometric confirmation and quantification

Phenol (carbolic acid) is widely used as a disinfectant as well as in the chemical industry as an intermediate in the synthesis of a variety of chemicals. Phenol is also the major metabolite of benzene which is used in many commercial solvents. Phenol is toxic and caustic and may cause death even from dermal absorption. Therefore, measurement of phenol in postmortem blood is essential. The concentration of phenol in blood can be measured by gas chromatography with flame ionization or mass spectrometry. Phenol can also be analyzed by high performance liquid chromatography. However, in forensic toxicology, unambiguous confirmation of phenol by mass spectrometry is as important as quantification in blood. Here we describe a novel derivatization of phenol after extraction with chloroform from human serum using perfluorooctanoyl chloride. The perfluorooctanoyl derivative of phenol showed a strong molecular ion at m/z 490 (relative abundance: 23%) whereas the base peak was observed at m/z 77. The derivative of the internal standard 3,4-dimethylphenol showed a very strong molecular ion at m/z 518 (relative abundance: 56%) and the base peak was observed as m/z 121. The derivative of p-cresol, a chemically related phenolic compound, showed a strong molecular ion at 504 m/z (relative abundance: 54%) and a base peak at m/z 107. We observed baseline separation between derivatized phenol (retention time: 6.1 min), p-cresol (retention time: 7.8 min), and the internal standard (retention time: 9.4 min). We observed no interferences in our assay from grossly hemolyzed serum. Within and between run precision was studied using a serum standard containing 25 mg/L of phenol. The within run precision was 6.6% (mean = 24.3, SD = 1.6 mg/L, n = 8) whereas the between run precision was 8.6% (mean = 25.5, SD = 2.2 mg/L, n = 8). The assay was linear for serum phenol concentrations of 10-200 mg/L. The detection limit was 1 mg/L of serum phenol concentration. The average recoveries were 92.1% to 94.0% for various serum phenol concentrations.     

Gas chromatographic-mass spectrometric identification of isosaccharino-1,4-lactone in human serum and urine

We have identified alpha-isosaccharino-1,4-lactone and beta-isosaccharino-1,4-lactone in the ethyl acetate extract of an ultrafiltrate of blood and of normal human urine, using high-resolution gas chromatography-mass spectrometry. The ultrafiltrate of blood was obtained from three patients on hemodialysis, one with psoriasis vulgaris, one with uremia, and one with amyotrophic lateral sclerosis. The concentration of beta-isosaccharino-1,4-lactone in the ultrafiltrate was two- to threefold that of alpha-isosaccharino-1,4-lactone. Mass fragmentography showed that isosaccharino-1,4-lactone is normally present in human serum in a concentration of 1.7 (SD 1.5) mg/L. In physiological fluid, isosaccharino-1,4-lactone apparently is present in its hydrated form, isosaccharinate.     

Gas chromatography-electron ionization and chemical ionization mass spectrometric analysis of urinary phenmetrazine after derivatization with 4-carbethoxyhexafluorobutyryl chloride--a new derivative

Phenmetrazine is a central nervous system stimulant currently used as an anorectic agent. The drug is abused and is reported to cause death from overdose. We describe a new derivatization method for phenmetrazine using 4-carbethoxyhexafluorobutyryl chloride. Quantitation of urinary phenmetrazine can be easily achieved by using N-ethyl amphetamine as an internal standard. The electron ionization mass spectrum of 4-carbethoxyhexafluorobutyryl derivative of phenmetrazine showed a molecular ion at m/z 427 and a base peak at m/z 70. In the methane chemical ionization mass spectrum, the base peak was observed at m/z 428 (protonated molecular ion). In the electron ionization mass spectrum of 4-carbethoxyhexafluorobutyryl derivative of the internal standard, N-ethyl amphetamine we did not observe a molecular ion. However, in the chemical ionization mass spectrum, the protonated molecular ion at m/z 414 was the base peak. The retention time of derivatized phenmetrazine (8.4 min) was substantially longer than the retention time of the underivatized molecule. Moreover, underivatized phenmetrazine showed poor peak shape (substantial tailing) while derivatized phenmetrazine had excellent chromatographic properties. The within-run and between-run precisions of the assay were 2.6% and 3.1% respectively at a urinary phenmetrazine concentration of 10 micrograms/mL. The assay was linear for urinary phenmetrazine concentration of 1 to 100 micrograms/mL with a detection limit of 0.2 microgram/mL.     

Gas chromatographic-mass spectrometric determination of benzo[a]pyrene and chrysene diol epoxide globin adducts in humans

The efficacy of a newly developed gas chromatography-negative ion chemical ionization-mass spectrometry-selected ion monitoring (GC-NICI-MS-SIM) assay for measuring globin adducts of benzo[a]pyrene (B[a]P) and chrysene diol epoxides in human was evaluated. In this pilot study, smokers and nonsmokers were selected as exposed and nonexposed groups. Using [2H12]r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyren e ([2H12]trans,anti-B[a]P-tetraol) as an internal standard, B[a]P-tetraols released from globin after hydrolysis and derivatization were quantified by GC-NICI-MS-SIM. Levels of trans-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene (chrysene-DE)-globin adducts were estimated by assuming that the recovery and the MS response of the perdeuterated B[a]P-tetraol internal standard reflected the recovery and MS response of chrysene tetraols. The assay was found to be reproducible and sensitive enough to detect both analytes in all samples. The mean levels of B[a]P-tetraols released from the corresponding benzo[a]pyrene diol epoxide (BPDE) globin adducts in smokers were significantly higher than those in nonsmokers, i.e., 2.6 +/- 0.6 SE fmol/mg globin (ranging from 1.2 to 7.8 fmol/mg globin) in smokers and 0.97 +/- 0.05 SE fmol/mg globin (ranging from 0.7 to 1.3 fmol/mg globin) in nonsmokers (P < 0.01). Interestingly, estimated levels of chrysene-DE-globin adducts in the same subjects were about two orders of magnitude higher than those of the globin adducts of BPDE. The mean of the chrysene-DE adducts in smokers was estimated to be 310 +/- 30 SE fmol/mg globin (ranging from 190 to 460 fmol/mg globin) and that in nonsmokers was 250 +/- 25 SE fmol/mg globin (ranging from 110 to 380 fmol/mg). Although the estimated mean of chrysene-DE adducts with globin in smokers appeared to be about 25% higher than in nonsmokers, the difference was not significant (P = 0.06). The results of this study demonstrate the feasibility of the GC-NICI-MS-SIM method for measurement of BPDE globin adducts in humans.     

Gas chromatographic-mass spectrometric investigations on the formation of phenolic and aromatic hydrocarbons in food (author''s transl)

Experience from arthroscopy in 224 patients with knee complaints is reported. The common method for arthroscopy is compared with a new modification developed during the study. The new method includes introduction of a 5 mm arthroscope through the patellar tendon at the level of the joint line and the use of hooks to test the menisci and the ligaments. The experience from this method shows that it gives an expanded field of vision and that technical failures are less frequent. No complications occurred. It is stated that arthroscopy is of great value in the diagnosis of knee injuries. However, a high diagnostic accuracy can only be reached by an experienced operator using a strictly standardized method.     

Gas chromatographic quantitation of dextropropoxyphene and norpropoxyphene in urine after solid-phase extraction

A gas chromatographic technique with flame ionization detection, which is based on a solid-phase extraction (SPE) procedure using mixed-mode SPE columns, for the simultaneous quantitation of dextropropoxyphene and norpropoxyphene in urine is presented. Urine is treated with sodium hydroxide in order to rearrange, by base catalysis, norpropoxyphene to norpropoxyphene amide, which is then extracted with these columns and chromatographed. The method is specific, linear over the range 0-2000 ng/mL, sensitive, and reproducible. The extracts are cleaner than those obtained with traditional liquid-liquid extraction procedure, which is an important feature in view of further mass spectrometric confirmation of narcotics and other drugs.     

First identification of microcystins in Irish lakes aided by a new derivatisation procedure for electrospray mass spectrometric analysis

Recent animal and bird deaths at several lakes in Ireland were indicative of possible cyanobacterial poisoning. Using protein phosphatase inhibition assays, microcystins (MCs) were identified in extracts of cyanobacteria from several lakes at concentrations ranging from 1.6 to 168 micrograms/g. This is the first report of MCs in Irish freshwaters. The protein phosphatase inhibition assay was used to screen fractions during HPLC purification of the MCs in cyanobacteria (Anabaena and Oscillatoria) and water samples from Corbally and Caragh Lakes. MC-LR, MC-HtyR, MC-FR, and MC-YR and 3 unidentified MCs of m/z values 1028.5, 981, 1042.7 were isolated from the Corbally sample; while the Caragh Lake sample contained largely MC-LR and MC-YR. A new microanalytical technique was developed for the confirmation of MCs which involved the derivatisation of the methyldehydroalanine group of MCs with 2-aminoethanethiol. Electrospray mass spectrometry of these products showed characteristic double-charged ions, and this novel technique was useful for differentiating MCs from co-eluting impurities in HPLC fractions of cyanobacterial extracts.     

Development of a gas chromatographic-mass spectrometric drug screening method for the N-dealkylated metabolites of fentanyl, sufentanil, and alfentanil

A sensitive, specific urinary assay for fentanyl, sufentanil, and alfentanil based on their N-dealkylated metabolites is described. Norfentanyl, norsufentanil-noralfentanil, and 2H5-norfentanyl are synthesized and characterized by standard analytical techniques. Derivatization of these secondary amines to yield the pentafluorobenzamides produces stable products with good gas chromatographic properties and unique, high-mass fragments in their mass spectra. These properties are utilized to develop a drug screening procedure based on gas chromatography-mass spectrometry to detect these major metabolites in human urine. The metabolites are isolated from urine samples by a liquid-liquid extraction procedure. The method allows for detection of metabolite concentrations as low as 0.3 ng/mL.     

Meperidine metabolites: identification of N-hydroxynormeperidine and a hydroxy-methoxy derivative of meperidine in biological fluids

Gas chromatographic and gas chromatographic-mass spectrometric techniques were used to identify non-acidic metabolites of meperidine (N-methyl-4-phenyl-4-carbethoxypiperidine) excreted in human, rat, and guinea pig urine. Following enzymic hydrolysis N-hydroxynormeperidine was identified in the urine of all three species in addition to the expected metabolites normeperidine and meperidine N-oxide. In rat urine the p-hydroxyphenyl metabolite of meperidine was present in appreciable amounts. Also present in small quantity was a new phenolic metabolite of meperidine containing both hydroxyl and O-methoxyl substituents in the phenyl ring of the parent drug. The latter two metabolites were excreted as conjugates in the rat.     

Percutaneous treatment of a congenital splenic cyst with alcohol: a new therapeutic approach

Percutaneous treatment of a huge congenital splenic cyst in a 23-year-old man is presented. The cyst had been catheterized and drained two times within a 3-month period without injecting any sclerosing agent into the cavity. On the third attempt, catheter drainage and injection of alcohol into the cyst cavity were performed because of insufficient response to drainage alone. He was discharged symptom-free after the procedure. The cyst diminished in size considerably 9 months after the treatment with alcohol. The volume of the cyst was reduced from 5200 to 8 ml. Although percutaneous treatment of a congenital splenic cyst with tetracyclin has been reported, to our knowledge this is the first case of a congenital splenic cyst treated with alcohol as a sclerosing agent. Percutaneous treatment of splenic cyst can obviate the need for partial or total splenectomy and may be an alternative to surgical treatment.     

Derivatization procedures for gas chromatographic-mass spectrometric determination of xenobiotics in biological samples, with special attention to drugs of abuse and doping agents

The development of low cost MS detectors in recent years has promoted an important increase in the applicability of GC-MS system to analyze for the presence of foreign substances in the human body. Drugs and toxic agents are in vivo metabolized in such a way that more polar compounds are usually formed. Derivatization of these metabolites is often an unavoidable requirement for gas chromatographic analysis. Application of derivatization methods in recent years has been relevant, especially for silylation, acylation, alkylation and the formation of cyclic or diastereomeric derivatives. Given the relevance of drug of abuse testing in modern toxicology, main derivatization procedures for opiates, cocaine, cannabis, amphetamines, benzodiazepines and LSD have been reviewed. Papers describing the analyses of drugs of abuse in matrixes other than blood, such as hair or sweat, have received special attention. Advances in derivatization for sports drug testing have been particularly relevant for anabolic steroids, diuretics and corticosteroids. Among the several methodologies applied, the formation of trimethylsilyl, perfluoroacyl or methylated derivatives have proved to be both versatile and extensively used. Further advances in derivatization for GC-MS applications in clinical and forensic toxicology will depend on the one hand on the degree of further use of GC-MS for routine applications and, on the other hand, on the alternative progress made for developments in LC-MS or CE-MS. Last but not least, the appearance of comprehensive libraries in which reference spectra for different derivatives of many drugs and their metabolites are collected will have an important impact on the expansion of derivatization in GC-MS for toxicological applications.     

Biotransformation of 17-alkylsteroids in the equine: gas chromatographic-mass spectral identification of ten intermediate metabolites of methyltestosterone

Parenterally administered domoic acid, a structural analog of the excitatory amino acids glutamic acid and kainic acid, has specific effects on brain histology in rats, as measured using different anatomic markers. Domoic acid-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex, and different anatomic markers can detect these neurotoxic effects to varying degrees. Here we report effects of domoic acid administration on quantitative indicators of brain metabolism and gliosis. Domoic acid, 2.25 mg/kg i.p., caused stereotyped behavior and convulsions in approximately 60% of rats which received it. Six to eight days after domoic acid or vehicle administration, the animals were processed to measure regional brain incorporation of the long-chain fatty acids [1-(14)C]arachidonic acid ([14C]AA) and [9,10-(3)H]palmitic acid ([3H]PA), or regional cerebral glucose utilization (rCMRglc) using 2-[1-(14)C]deoxy-D-glucose, by quantitative autoradiography. Others rats were processed to measure brain glial fibrillary acidic protein (GFAP) by enzyme-linked immunosorbent assay. Domoic acid increased GFAP in the anterior portion of cerebral cortex, the caudate putamen and thalamus compared with vehicle. However, in rats that convulsed after domoic acid GFAP was significantly increased throughout the cerebral cortex, as well as in the hippocampus, septum, caudate putamen, and thalamus. Domoic acid, in the absence of convulsions, decreased relative [14C]AA incorporation in the claustrum and pyramidal cell layer of the hippocampus compared with vehicle-injected controls. In the presence of convulsions, relative [14C]AA incorporation was decreased in hippocampus regions CA1 and CA2. Uptake of [3H]PA into brain was unaffected. Relative rCMRglc decreased in entorhinal cortex following domoic acid administration with or without convulsions. These results suggest that acute domoic acid exposure affects discrete brain circuits by inducing convulsions, and that domoic acid-induced convulsions cause chronic effects on brain function that are reflected in altered fatty acid metabolism and gliosis.     

Relationship between alcohol intake and immunoglobulin a immunoreactivity with acetaldehyde-modified bovine serum albumin

Acetaldehyde, the main metabolite of ethanol, is a highly reactive species that reacts with macromolecules to produce unstable and stable adducts. Acetaldehyde-modified proteins are immunogenic and have been detected in the liver and blood of alcoholics. Furthermore, antibodies reactive with acetaldehyde-modified proteins have been detected in the plasma of social drinkers and alcoholics. However, the class distribution of immunoglobulins reactive with modified proteins was different in the two groups, being predominantly immunoglobulin (Ig)M in social drinkers, but IgM and IgA in alcoholics. In this study, we demonstrate that heavy drinkers (alcohol intake > 130 g/week for females and 150 g/week for males) also exhibit IgA reactivity with acetaldehyde-modified proteins. The IgA adduct-specific reactivity (IgA reactivity with acetaldehyde-modified bovine serum albumin-reactivity with native bovine serum albumin) showed a moderate correlation with self-reported alcohol intake, but did not correlate with markers such as plasma transaminase, gamma-glutamyltransferase activity, or mean corpuscular volume. IgA adduct-specific reactivity had similar specificity to the conventional tests of alcohol abuse, but had higher sensitivity than the other tests, especially with heavy drinkers. Data presented herein demonstrate that elevated IgA reactivity with acetaldehyde-modified epitopes is associated with heavy drinking and is a potential marker for high alcohol intake.     

Determination of 5-hydroxytryptophol in urine by high-performance liquid chromatography: application of a new post-column derivatization method with fluorometric detection

The aim of the present study was to develop a high-performance liquid chromatographic (HPLC) method for determination of the serotonin metabolite 5-hydroxytryptophol (5HTOL) in human urine. 5HTOL was liberated from its conjugated form by enzymatic hydrolysis and isolated by a sample clean-up procedure on a small Sephadex G-10 column. The eluate was injected onto an isocratically eluted C18 reversed-phase column and 5HTOL was converted into a fluorescent oxazole derivative by on-line post-column reaction with benzylamine in the presence of potassium hexacyanoferrate(III). The limit of detection was about 10 nM and the intra-assay coefficients of variation were below 4% with urine samples and standard solutions. The results indicate that the method can be used as a screening method to discriminate between normal and elevated levels of total (free + conjugated) 5HTOL in urine.     

Swelling-activated chloride currents in a drug-sensitive cell line and a P glycoprotein-expressing derivative are underlied by channels with the same pharmacological properties

It has been proposed that P glycoprotein (Pgp) expression is associated with swelling-activated Cl- currents in multidrug-resistant cells. The Pgp substrate vinblastine and the modulator verapamil produced a reversible concentration-dependent block of swelling-activated Cl- currents in both a drug-sensitive cell line (MCF-7) and a Pgp-expressing derivative (BC19/3). The similarity of the results obtained in both cell lines suggests that the mechanism of block is not related to Pgp expression and supports the hypothesis that Pgp expression is not necessary for the swelling activation of Cl- currents. In contrast to the results obtained with vinblastine, two other cytoskeleton-disrupting agents, colchicine and cytochalasin D, were not able to affect the swelling-activated Cl- currents in either cell line. The data provided no evidence for the involvement of the cytoskeleton in the swelling activation of Cl- channels in these cell lines. The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid and 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, each produced a similar reversible concentration-dependent block in the swelling-activated currents in both the Pgp-expressing and nonexpressing cells. This strongly suggests that the Cl- channel(s) responsible for the swelling-dependent current in both cell lines are the same and, since MCF-7 cells do not express Pgp, that Pgp is not the channel responsible for the volume-activated Cl- currents in these cells.     

Polymorphism and identification of metallothionein isoforms by reversed-phase HPLC with on-line ion-spray mass spectrometric detection

Microbore reversed-phase HPLC with on-line ion-spray mass spectrometric detection is proposed for a study of polymorphism of rabbit liver metallothionein (MTRL) and its major isoforms MT-1 and MT-2. Separation conditions had been optimized until each chromatographic peak could be attributed to a single metalloprotein species, of which the molecular mass could be determined by ionspray MS. At the optimized conditions (elution with the gradient 5-8% of acetonitrile within 50 min using a 5 mM acetate buffer at pH 6), the chromatogram of MTRL showed five peaks, whereas those of MT-1 and MT-2 showed nine and seven different peaks, respectively. The on-line determination of the molecular masses (+/- 1 Da) of the compounds eluted permits the unambiguous cataloguing and further referencing of putative and true MT subisoforms. The results obtained are compared with those obtained by direct ion-spray MS of the MT preparations at different pHs, with a goal to identify possible chromatographic artifacts. Metals (Cd, Cu) in the eluted complexes were studied by HPLC with on-line ICP MS detection.     

Families caring for children with fetal alcohol syndrome: the nurse''s role in early identification and intervention

Mislabeled umbilical cord blood specimens were identified as an important and difficult problem to solve. Quality improvement principles were employed after education-based interventions failed to achieve measurable improvement. A small interdisciplinary working group of key stakeholders investigated, designed, and evaluated interventions for a solution. This article describes a system-based change where substantial qualitative and quantitative improvements were measured. The success of the change is attributed to the involvement and commitment by key stakeholders and use of systems reengineering principles.