Quantitative measurement of N-acetyl-L-aspartic acid in urine by gas chromatography with negative-ion chemical ionization mass spectrometry.

A highly specific and sensitive assay for N-acetyl-L-aspartic acid has been developed. The trideuterated compound was synthesized and used as an internal standard for gas chromatography with negative-ion chemical ionization mass spectrometry. Urine samples were acidified and extracted with ethyl acetate, and the compounds converted into their pentafluorobenzyl ester derivatives. Under these conditions, sub-picogram amounts of the pure derivatives could be detected. Thus, only microliter volumes of urine samples have to be processed to achieve reliable quantification of "basal" levels of N-acetyl-L-aspartic acid.


Determination of urinary beta-phenylethylamine as its N-benzenesulphonamide derivative by gas chromatography with flame photometric detection.

A selective and sensitive method for the determination of urinary beta-phenylethylamine (PEA) by gas chromatography (GC) has been developed. After extraction of the urine sample with n-pentane, PEA was converted into its N-benzenesulphonamide derivative and then determined by GC with flame photometric detection using a DB-1301 capillary column. By using this method, nanogram amounts of PEA in urine could be accurately and precisely determined without any influence from coexisting substances. Analytical results for the determination of PEA in urine samples from normal subjects are presented.


Quantitation of phenacyl esters of retinal fatty acids by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the separation and quantitation of retinal fatty acids containing long-chain polyunsaturated fatty acids is described. Fatty acids from frog retinal lipids were converted to the corresponding phenacyl derivatives which were separated on a C18 reversed-phase column and detected at 242 nm. Molar absorptivities (peak area units/nmol) of up to seventeen fatty acid phenacyl derivatives were determined and used for quantitation of fatty acids separated by HPLC. Compared with gas chromatography, the HPLC method gave a similar molar percent distribution of the fatty acids and was twenty to fifty times more sensitive. This HPLC method provides a useful means for the study of chemistry and metabolism of long-chain polyunsaturated fatty acids in retina and other tissues where amounts of material may be limited or recovery of individual components desirable.


Rapid method to isolate serum amyloid P component from human plasma. Characterization of the isolated protein.

A rapid and reproducible method to isolate serum amyloid P component from healthy human plasma has been developed. It uses affinity chromatography on an agarose column followed by anion-exchange chromatography. It was found that the isolated compound has a significantly different isoelectric point (pI 5.7) from that reported previously (pI 4.1). The new data are in good agreement with calculated values determined from the amino acid composition of the protein.


Automated high-performance liquid chromatographic assay for the determination of 7-ethoxycoumarin and umbelliferone.

An improved high-performance liquid chromatographic assay is presented for the determination of 7-ethoxycoumarin O-deethylase activity. Following a 30-min microsomal incubation, 7-ethoxycoumarin, 4-methylumbelliferone (internal standard), and the metabolite umbelliferone were extracted with chloroform. Separation was achieved with an isocratic mobile phase using a microBondapak phenyl (300 mm x 3.9 mm I.D.) analytical column. The effluent was monitored by fluorescence detection with an excitation wavelength of 360 nm and an emission wavelength of 470 nm. The intra- and inter-assay coefficients of variation were 10 and 6%, respectively. A detection limit of 0.07 micrograms/ml was achieved, making this method suitable for characterizing P-450 activity of human livers.


Separation and determination of aminohalogenbenzophenones by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection has been developed for the determination of three aminohalogenbenzophenones: 2-amino-2',5-dichlorobenzophenone, 2-amino-5-chlorobenzophenone and 2-amino-5-bromo-2'-fluorobenzophenone, metabolites of benzodiazepinooxazoles and other psychotropic drugs. A mobile phase of methanol-water (65:35), containing 5 mM KH2PO4 appeared to be the optimal when a 4-microns, 60-A Nova-Pak C18 column and a flow-rate of 0.75 ml/min (130 bar) were used. The temperature was optimized at 30 degrees C. The amperometric detector, equipped with glassy carbon electrode, was operated at 1.3 V versus Ag/AgCl in the DC mode. The method was applied to the determination of these compounds at two concentration levels: ppm and ppb (ng/cm3) using 2-amino-5-chlorobenzophenone as internal standard. The limit of determination was 750 pg/ml of biological fluid for each compound, and recoveries greater than 97% were obtained for spiked samples of urine and serum, using C18 Sep-Pak cartridges in the sample clean-up procedure.


Determination of AJ-3941, a possible agent for the treatment of cerebrovascular disorders, in plasma and brain by means of high-performance liquid chromatography with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic method with fluorescence detection is described for the determination of AJ-3941 (I), a possible agent for the treatment of cerebrovascular disorders, in plasma and brain tissue. A simple hexane extraction was used for plasma, and for brain homogenate the hexane extract was further purified by solid-phase extraction. The determination limit was ca. 3 ng/ml for both plasma (0.5 ml) and 10% (w/v) brain homogenate (1 ml). The method was applied to the determination of I in plasma and brain samples of experimental animals.


Determination of the catecholamines and serotonin, their precursors tyrosine and tryptophan, and their main metabolites in rat brain using reversed-phase high-performance liquid chromatography with fluorimetric and oxidative amperometric detection in series.

A high-performance liquid chromatographic method for the determination of catecholamines and serotonin, their precursors and their main metabolites was developed applied to rat cerebellum, hypothalamus, striatum and cortex. A fluorimetric and an oxidative amperometric detector were used in series. For both detectors, detection limits (25-520 pg) were useful for this application, linearity of standards was excellent (average r greater than 0.9997), between-run precision for sample analytes was generally acceptable (coefficient of variation less than 10% with appreciable concentrations present) and average recoveries of standard additions to sample analytes were better than 90%. Particular attention was paid to peak identification, including both a thorough treatment of retention time agreement of peaks in standards and sample analytes, and a comparison of results for the seven compounds amenable to quantitation by both detectors. Considerable attention was also given to determining the stability of standards and sample analytes under a wide variety of conditions, and practical recommendations were made.


Simultaneous determination of myocardial nucleotides, nucleosides, purine bases and creatine phosphate by ion-pair high-performance liquid chromatography.

An ion-pair reversed-phase high-performance liquid chromatographic method is described for the separation and quantification of myocardial nucleotides, nucleosides, their metabolites and creatine phosphate-related compounds in a single run. Separation of a standard mixture containing 21 compounds was achieved on a 5-microns Hypersil ODS column with a 5-min isocratic elution (buffer: 0.1 M NaH2PO4, pH 5.5, containing 5.9 mM tetrabutylammonium hydrogen-sulphate) followed by a slow linear gradient to 17% acetonitrile. The method was applied to extracts of freeze-clamped rat heart tissue samples as well as to extracts of neonatal rat heart cardiomyocytes, and it provided good resolution of high-energy phosphates, including creatine phosphate, as well as of their degradation products.


Determination of vitamin B6 vitamers and pyridoxic acid in biological samples.

For the determination of vitamin B6 vitamers (pyridoxal phosphate, pyridoxamine phosphate, pyridoxal, pyridoxine, pyridoxamine) and 4-pyridoxic acid in biological samples such as plasma, cerebrospinal fluid and rat brain regions, a sensitive micromethod using high-performance liquid chromatography (HPLC) with fluorescence detection in combination with post-column derivatization is described. Metaphosphoric acid tissue extracts with deoxypyridoxine as an internal standard were injected into the HPLC system with a binary gradient elution at a flow-rate of 1.2 ml/min. The excitation wavelength of the fluorescence detector was set at 328 nm and the emission wavelength at 393 nm with a 15-nm slit width for the photocell. This method allows the assay of vitamin B6 vitamers within 30 min in one chromatographic run. The present method has been applied extensively for the measurement of vitamin B6 vitamer levels in discrete brain regions of small animals, cells in culture and biopsy samples.


Silver staining of collagen type I after sodium dodecylsulphate polyacrylamide gel electrophoresis: effect of Maillard reaction.

Differences in the acidic silver staining, after sodium dodecylsulphate polyacrylamide gel electrophoresis, between the alpha 1 and alpha 2 collagen chains, as well as between rat-tail tendon and calf-skin collagen type I, were observed. The staining conditions at which the staining differences are both most expressed and reproducible were characterized. Age differences between staining of the alpha 1 CB6 fragment from young rats (2 and 12 months) and old rats (29 months) indicated that different susceptibilities of collagen species to the silver staining can be the result of different extents of some age-dependent post-translational modification, such as glycation. In vitro incubation of acid-soluble rat-tail tendon collagen with various sugars led to an increase in silver staining compared with samples incubated in the absence of sugar. This effect was inhibited by sodium cyanoborohydride, diethylenetriamine pentaacetic acid and aminoguanidine, i.e. compounds inhibiting the Maillard reaction at various stages. It can be concluded that the enhanced silver susceptibility of glycated collagen is related to advanced-phase Maillard reaction products attached to collagen.


High-performance liquid chromatographic determination of sulphobromophthalein and its conjugates.

A simple, sensitive and selective high-performance liquid chromatographic method for the determination of sulphobromophthalein and its mercaptide conjugates in rat bile was developed. These pigments, which have an absorption maximum at 580 nm in alkaline solution, were separated isocratically on an alkali-resistant ODS column by paired-ion chromatography. Analysis of bile samples obtained after intravenous administration of sulphobromophthalein to rats showed the presence of at least twenty peaks of metabolites, of which thirteen were identified and seven quantified.


Determination of cefcanel in plasma and urine by high-performance liquid chromatography using coupled columns, after administration of the new cephalosporin prodrug cefcanel daloxate hydrochloride.

A rapid and sensitive high-performance liquid chromatographic method has been developed for the determination in plasma and urine of the new cephalosporin cefcanel. The method involves a simple deproteinizing step followed by separation on a coupled-column chromatographic system with ultraviolet detection. Limits of quantification were 0.2 microM for plasma samples and 2 microM for urine samples. The method has been used for the determination of cefcanel in various clinical studies.


Automated-extraction, high-performance liquid chromatographic method and pharmacokinetics in rats of a highly A2-selective adenosine agonist, CGS 21680.

CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine, I) is a highly A2-selective (A2/A1 = 140), high-affinity adenosine agonist. A method has been devised to extract the compound from biological matrices with automated solid-phase extraction using C18 bonded silica columns. This is followed by reversed-phase, paired-ion chromatography on a Supelco LC-18-S column with fluorescence detection. The limit of quantitation is 5 ng/ml, but 1 ng/ml (five times the signal-to-noise ratio) can readily be detected. Tritium-labeled compound was used to study the pharmacokinetics in rats. After an intravenous dose of 0.3 mg/kg, biphasic elimination kinetics were observed for parent I, characterized by half-lives of 1.8 min (distribution) and 15 min (elimination). The volume of distribution in the terminal phase (V beta) was low (0.27 l/kg) and plasma clearance was moderate (0.83 l/kg/h). Although the compound was rapidly absorbed (mean Tmax = 13 min), low concentrations (mean Cmax = 94 ng/ml) were observed after an oral dose of 3.0 mg/kg, and bioavailability was only approximately 1.4%. Radioactivity persisted in plasma longer than parent compound after either dose, but levels were too low for isolation and structure identification of drug-derived compounds.


Determination of 2-naphthylamine in urine by a novel reversed-phase high-performance liquid chromatography method.

A high-performance liquid chromatographic method for the determination of 2-naphthylamine in urine using fluorescence detection was developed. The method validation analysis showed the method to be in analytical control, i.e. the distribution of the difference between the observed and true values of the method evaluation samples did not deviate significantly from the normal distribution. The recovery of the method was 85%. The entire run time of chromatography was 10 min using isocratic elution (acetonitrile-water, 35:65), and the retention time for 2-naphthylamine was 5.8 min. The relative short time of analysis in combination with the low limit of detection (0.272 nmol/l) makes the method potentially applicable for surveillance of occupational and environmental exposure to 2-nitronaphthalene. The developed method is presently used for measurement of 2-naphthylamine in urine samples from workers employed at factories, characterized by a low airborne exposure level of polycyclic aromatic hydrocarbons, i.e. in general less than 25 micrograms/m3. The urine samples of exposed workers (n = 95) showed a 2-naphthylamine range of up to 9.4 nmol/l, whereas unexposed control individuals (n = 114) showed a range of up to 0.87 nmol/l.


Trace quantitation of 4-hydroxy-2-nonenal in biological samples as its oxime-bis-tert.-butyldimethylsilyl derivative using 3-hydroxynonanal as an internal standard.

A gas chromatographic-mass spectrometric method for the determination of the lipid aldehyde 4-hydroxy-2-nonenal (4HNE) in trace quantities is described. The method utilizes the reaction of aldehydes with hydroxylamine leading to the formation of the oxime derivative. The aldehydes are recovered by octadecylsilyl solid-phase extraction and converted to the bis-tert.-butyldimethylsilyl derivatives for analysis using electron ionization. A novel 4HNE analogue, 3-hydroxynonanal, has been synthesized and is used as an internal standard. A limit of detection of approximately 1 pmol of 4 HNE in preparations of approximately 2.10(6) cells or 0.5 ml of whole blood, plasma or serum was observed. Standard addition analysis indicates that the method is accurate at these levels. Replicate analysis of the National Institutes of Standards and Technology Standard Reference Material SRM 909 indicates an average in-run precision of 8.1% and a between-run precision of 13.5% at an average concentration of 82.1 pmol/ml of reconstituted material.


Pharmacokinetic and metabolism studies on girisopam by chromatographic and spectrometric methods in humans.

Girisopam possesses selective anxiolytic action without muscle relaxant and anticonvulsive activity. After a 100-mg oral dose of 14C-labelled girisopam to seven male subjects, the mean recovery of 14C radioactivity was 51% in urine and 33% in faeces. A high-performance liquid chromatographic method has been developed for studying girisopam in single-dose pharmacokinetic studies. The serum extract was chromatographed on a normal-phase column using a mobile phase of hexane-ethanol-diethyl ether (66:9:25, v/v) and ultraviolet detection at 235 nm. The recovery was 60% and the detection limit was 3 ng/ml, using 1 ml of serum. After a 20-min delay, girisopam is rapidly absorbed. After reaching a mean serum level of 178 ng/ml at a mean time of 2.0 h, the serum concentration of girisopam decreased with a mean elimination half-time of 22.2 h. The metabolites were separated by high-performance liquid chromatography, radio thin-layer chromatography and gas chromatography. Their structures were determined by liquid chromatography-mass spectrometry, mass spectrometry and gas chromatography-mass spectrometry. Their chemical structures were confirmed by comparison with synthesized reference compounds. The major urinary metabolites were 7-demethylgirisopam (I), 4'-hydroxygirisopam (II) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in conjugated form, and 4-carboxy-4-demethylgirisopam (V), a compound with an open-chain structure (VII) and traces of 4-demethyl-4-oxogirisopam (VIII) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in non-conjugated form. The metabolic profile in the serum consisted predominantly of the glucuronides of I, II and III. The non-conjugated metabolites were the metabolite with the open-chain structure (VII), III and V. Besides the parent compound, the faeces sample contained conjugates of I and II.


In vivo sampling using loop microdialysis probes coupled to a liquid chromatograph.

Microdialysis probes with longer membranes (20-100 mm) provide increased relative recovery over traditional shorter probes (1-4 mm) developed for neuroscience applications. The characterization and optimization of "straight through" or "loop type" probes for use in subcutaneous tissue are considered. Membrane area, probe size, inlet and outlet tubing dimensions, and flow-rate are examined for their effects on relative recovery, the total collection rate, and bulk flow through the membrane wall. Polyacrylonitrile and regenerated cellulose membrane fibers with different geometries were compared. Sampling probes used fibers 3-10 cm long. Inlet and outlet tubing was varied from 25 to 110 microns I.D. with lengths of 10 to 50 cm. Probe configurations optimized for relative recovery, flow-rate, and utility for in vivo use are presented. Utilizing microdialysis probes with large membrane surface areas results in relative concentration recovery of greater than 50% at flow-rates of greater than 5 microliters/min. Therapeutic drug monitoring in subcutaneous tissue of awake animals is explored.


Determination of urinary normetanephrine, metanephrine and 3-methoxytyramine by high-performance liquid chromatography with electrochemical detection: comparison between automated column-switching and manual dual-column sample purification methods.

This report describes a high-performance liquid chromatographic method with electrochemical detection for the simultaneous quantitation of urinary metanephrine, normetanephrine and 3-methoxytyramine. This method, which involves manual dual-column purification steps for the routine determination of urinary metanephrines, is compared with the previously used spectrophotometric Pisano method and an on-line sample preparation procedure, where the automated sequential trace enrichment (ASTED) apparatus is used for the column-switching procedure. In order to automate the metanephrine assay, the enrichment technique was evaluated against the reference chromatographic method. Bio-Rad urine controls gave coefficients of variation of less than 9% at all levels for the reference method. Values of less than 19% were found in the reference range with the enrichment method, and the recovery of 3-methoxytyramine was also too poor to be measured in normal concentrations. The linearity of both methods is sufficient to determine pathological levels of these biogenic amines. Future developments should be focused on decreasing the variation of between-day assays in an on-line, automated procedure.


Application of an organic solvent extraction to the determination of catechol-O-methyltransferase activity by high-performance liquid chromatography in human mononuclear cells.

We report here a method for measuring mononuclear cell catechol-O-methyltransferase (COMT) activity which is ideally adapted to clinical studies. The method measures the O-methylation of dopamine to 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine. Whole mononuclear cell sonicate is incubated with saturating concentrations of dopamine, S-adenosyl-L-methionine and magnesium chloride in sodium-potassium phosphate buffer at pH 7.3. An organic solvent extraction using ethyl acetate is then used for product separation, followed by high-performance liquid chromatography with electrochemical detection for product separation and quantification. This method allows both O-methylated products, 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine, to be isolated and quantified separately. The apparent Michaelis constants for dopamine and S-adenosyl-L-methionine using this method are similar to values reported previously (0.51 and 14 microM, respectively). The optimal concentration of magnesium chloride is eight to ten times higher than previously reported. No endogenous inhibitors were apparent using this assay. The within-day coefficient of variation using this method is 7% when measuring 3-methoxytyramine and 5% when measuring 4-methoxy-3-hydroxyphenethylamine. The between-day coefficient of variation is 11%. Mononuclear cell COMT activity can be detected using protein concentrations as low as 0.75 mg/ml, corresponding to 2-3 ml of whole blood. The small amount of blood required per sample allows multiple sample analysis from a single patient, including infants.


Determination of 2-isopropoxyphenol in urine by capillary gas chromatography and mass-selective detection.

An analytical method for the assessment of the exposure of workers to the pesticide propoxur through biological monitoring has been developed. This study was part of a survey of occupational exposure to pesticides used in greenhouses for the growing of ornamental plants. In order to assess the actual absorbed amount of propoxur in the body, an analytical method for its metabolite 2-isopropoxyphenol in urine was required. This led to the development of a gas chromatographic-mass spectrometric assay involving hydrolysis and solvent extraction. A mass-selective detector, operated in single-ion mode, provides a selective and sensitive quantification of 2-isopropoxyphenol with a detection limit of 6 micrograms/l. The method has been validated with respect to the hydrolysis of urine samples, analytical recovery of 2-isopropoxyphenol, stability of its conjugates, limit of detection, background and precision. The analytical recovery from spiked urine was over 95%. 2-Isopropoxyphenol was excreted in urine as a conjugate and was stable for at least seven months when stored at -20 degrees C. It was not detected in urine from non-exposed persons. Between-day coefficients of variation were 20, 10, 7 and 4% for concentrations of 15, 29, 150 and 213 micrograms/l, respectively. Measured as 2-isopropoxyphenol, ca. 80% of an orally administered dose of propoxur was excreted in urine within 10 h.


Trace analysis of quinapril and its active metabolite, quinaprilat, in human plasma and urine by gas chromatography-negative-ion chemical ionization mass spectrometry.

A highly specific and sensitive method for the simultaneous determination of quinapril and its active metabolite, quinaprilat, in human plasma and urine by gas chromatography-negative-ion chemical ionization mass spectrometry is described. Quinapril and quinaprilat were extracted from human plasma and urine by using a Bond-Elut C18 cartridge. The plasma or urine extract was treated with pentafluorobenzyl bromide followed by trifluoroacetic anhydride to convert quinapril and quinaprilat into their pentafluorobenzyl-trifluoroacetyl derivatives, which were analysed by a selected-ion monitoring method using deuterium-labelled internal standards. The limits of quantitation for both quinapril and quinaprilat were 0.05 ng/ml in plasma and 0.5 ng/ml in urine. The proposed method is applicable to pharmacokinetic and clinical pharmacological studies with satisfactory reliability.


Determination of dioxopiperazine metabolites of quinapril in biological fluids by gas chromatography-mass spectrometry.

The dioxopiperazine metabolites of quinapril in plasma and urine were extracted with hexane-dichloroethane (1:1) under acidic conditions. Following derivatization with pentafluorobenzyl bromide and purification of the desired reaction products using a column packed with silica gel, the metabolites were analysed separately by capillary column gas chromatography-electron-impact mass spectrometry with selected-ion monitoring. The limits of quantitation for the metabolites were 0.2 ng/ml in plasma and 1 ng/ml in urine. The limits of detection were 0.1 ng/ml in plasma and 0.5 ng/ml in urine, at a single-to-noise ratio of greater than 3 and greater than 5, respectively. The proposed method is applicable to pharmacokinetic studies.


Measurement of azacyclonol in urine and serum of humans following terfenadine (Seldane) administration using gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method is presented for the analysis of azacyclonol (AZA), a metabolite of terfenadine in serum and urine specimens. Following an alkaline extraction, AZA and an internal standard were derivatized using heptafluorobutyric anhydride. Fourier transform infrared spectrometry suggested that two sites on the AZA molecule were derivatized. GC-MS of the extracts had a limit of quantitation (LOQ) of 1 ng/ml and linear range of 1-1000 ng/ml in urine. Four volunteers were administered a therapeutic regimen of terfenadine followed by urine and serum specimen collection(s) during the next seven days. The results indicated that following a 60-mg dose of terfenadine each 12 h for five days, (1) AZA appears in urine within 2 h, (2) urine AZA concentrations were above the LOQ 72 h following the last dose, (3) peak urine concentrations were as high as 19,000 ng/ml, and (4) mean serum concentration following the ninth dose was 59 ng/ml.


Biotransformation of diethenylbenzenes. IV. A simple high-performance liquid chromatographic method for separation of urinary metabolites of 1,3-diethenylbenzene on analytical and semi-preparative scales.

A simple ion-suppression separation on reversed-phase columns, which is applicable for both analytical and semi-preparative work, is described. Six urinary metabolites of 1,3-diethenylbenzene (I), namely 1-(3-ethenylphenyl)-1,2-dihydroxyethane beta-D-glucosiduronates (two isomers, II and III), N-acetyl-S-[1-(3-ethenylphenyl)-2-hydroxyethyl]cysteine (IV), N-acetyl-S-[2-(3-ethenylphenyl)-2-hydroxyethyl]cysteine (V), 3-ethenylphenylmandelic acid (VI) and 3-ethenylphenylglyoxylic acid (VII), were isolated (Fig. 1). Four of them, IV-VII, have been identified in our previous work; the two glucosiduronates were identified for the first time by 1H NMR spectroscopy, fast atom bombardment mass spectrometry, and enzymic hydrolysis yielding 1-(3-ethenylphenyl)-1,2-dihydroxyethane as an aglycone. The method was reproducible the concentration range 0.05-5 mg/ml, the coefficient of variation being less than 7% (n = 5). Excretion of II-VI within 24 h in the urine of rats dosed with a single intraperitoneal injection of 100, 300 and 600 mg/kg I was determined quantitatively. The utility of the method is discussed in comparison with gas chromatographic-mass spectrometric techniques used previously.


Determination of vinca alkaloids in mouse tissues by high-performance liquid chromatography.

A high-performance liquid chromatographic method is described for the determination of vinblastine in various normal mouse tissues, such as lung, heart, liver, kidney and muscles, and in implanted MO4 tumours. Vincristine was used as the internal standard. Freshly obtained mouse tissue or tumour tissue was frozen at -20 degrees C and then lyophilized. After lyophilisation, the dry tissues were pulverized and homogeneously mixed, and an aliquot was suspended in 0.1 M hydrochloric acid. The drugs of interest were then isolated from this suspension using ion-pair extraction at pH 3 with octylsulphate as counter-ion. The obtained extracts were analysed on a reversed-phase system with a cyanopropyl stationary phase. The detection limit was 1 ng/l in plasma and 10 ng/g in tissue. The extraction recoveries of vincristine and vinblastine were between 45 and 67%, and there were no interferences from blank components. Preliminary pharmacokinetic data for different mouse tissues and tumour implanted in muscle tissue are presented.


Simultaneous determination of phenolphthalein and phenolphthalein glucuronide from dog serum, urine and bile by high-performance liquid chromatography.

A procedure is described to simultaneously quantitate phenolphthalein and its glucuronide metabolite from dog serum, urine and bile using high-performance liquid chromatography. The major advantages of this over pre-existing methods include direct analysis of the parent compound and glucuronide metabolite without enzymatic hydrolysis, increased sensitivity and the potential for automation of a large number of samples. Analytes were extracted from serum and urine using a combination of liquid- and solid-phase extraction methodology. Bile samples were analyzed directly after a twenty-fold dilution with mobile phase. The components plus internal standard were separated by reversed-phase high-performance liquid chromatography using step gradient elution and quantitated by the absorbance of ultraviolet light at 230 nm. Limits of detection from 1 ml of serum, 0.1 ml of urine and 0.05 ml of bile were 0.1, 0.5 and 10 microgram/ml for phenolphthalein and 0.1, 10 and 50 microgram/ml for phenolphthalein glucuronide, respectively.


Determination of naproxen and its metabolite O-desmethylnaproxen with their acyl glucuronides in human plasma and urine by means of direct gradient high-performance liquid chromatography.

Naproxen is metabolized in humans by O-demethylation, and by acyl glucuronidation to the 1-O-glucuronide. Naproxen, its metabolite and the conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic deglucuronidation. The glucuronide conjugates were isolated by preparative chromatography from human urine samples. Mild acidic hydrolysis of one urinary conjugate resulted in naproxen. This conjugate was also formed by alkaline isomerization of isolated naproxen acyl glucuronide, indicating that the structure of this urinary conjugate must have been naproxen isoglucuronide (4-O-glucuronide). Mild acidic hydrolysis of another urinary conjugate resulted in O-desmethylnaproxen. This conjugate was also formed by alkaline isomerisation of isolated O-desmethylnaproxen acyl glucuronide, indicating that the structure of this urinary conjugate must have been O-desmethylnaproxen isoglucuronide (4-O-glucuronide). Calibriation curves were constructed by enzymic deconjugation of samples containing different concentrations of isolated naproxen acyl glucuronide, O-desmethylnaproxen acyl glucuronide, and the isoglucuronides of naproxen and O-desmethylnaproxen by mild acidic hydrolysis. The limit of quantitation of naproxen in plasma is 1.5 microgram/ml. The limits of quantitation in urine are: naproxen, O-desmethylnaproxen, naproxen acyl glucuronide and O-desmethylnaproxen acyl glucuronide, 1 microgram/ml; the isoglucuronide of naproxen and O-desmethylnaproxen, 1.5 microgram/ml. A pharmacokinetic profile of naproxen is shown, and some preliminary pharmacokinetic parameters of naproxen obtained from two human volunteers are given.


High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man.

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid-liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm x 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol-Sörensen's buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol-water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.


High-performance liquid chromatographic method for the determination of alprazolam in plasma using the column-switching technique.

A reversed-phase high-performance liquid chromatographic method is described for the quantitative determination of alprazolam in the plasma of geriatric patients in the presence of 4-hydroxyalprazolam, alpha-hydroxyalprazolam, bromazepam, oxazepam, lorazepam, clobazam, desmethylclobazam, diazepam and desmethyldiazepam. The procedure is based on the enrichment of alprazolam on a PRP-1 pre-column, followed by the transfer of the compound in a forflush mode to the analytical column. Alprazolam can be quantified reliably down to a minimum concentration of 1 ng/ml of plasma.


High-performance liquid chromatographic determination of the stereoselective biotransformation of the chiral drug praziquantel.

A selective reversed-phase high-performance liquid chromatographic method for the simultaneous quantification of praziquantel and its monohydroxylated metabolites in serum is described. The quantitative analysis was followed by determination of the enantiomeric ratio of praziquantel and trans-4-hydroxypraziquantel, the main metabolite of praziquantel in humans, on a cellulose tris-3,5-dimethyl-phenylcarbamate column (Chiralcel OD). Serum level data for five volunteers after oral administration of racemic praziquantel were compared with in vitro metabolism data for praziquantel, obtained with liver microsomes of different species.


Column switching and high-performance liquid chromatography in the analysis of amitriptyline, nortriptyline and hydroxylated metabolites in human plasma or serum.

A column-switching system for the direct injection of plasma or serum samples, followed by isocratic high-performance liquid chromatography and ultraviolet detection, is described for the simultaneous quantitation of the tricyclic antidepressant amitriptyline, its demethylated metabolite nortriptyline and the E- and Z-isomers of 10-hydroxyamitriptyline and 10-hydroxynortriptyline. The method included adsorption of amitriptyline and metabolites on a reversed-phase C8 clean-up column (10 microns; 20 mm x 4.6 mm I.D.), washing of unwanted material to waste and, after on-line column-switching, separation on a cyanopropyl analytical column (5 microns; 250 mm x 4.6 mm I.D.). The compounds of interest were separated and eluted using acetonitrile-methanol-0.01 M phosphate buffer (pH 6.8) (578:188:235, v/v) within less than 20 min. Various drugs frequently co-administered with amitriptyline or other antidepressants did not interfere with the determinations. In plasma samples spiked with 25-300 ng/ml, the recoveries were between 84 and 112% and the inter-assay coefficients of variation were 3-11%. After a minor modification, as little as 5 ng/ml could be quantitated. There were linear correlations (r greater than 0.99) between drug concentrations of 5-500 ng/ml and the detector signal. The method allows routine measurements of amitriptyline, nortriptyline and hydroxylated metabolites in blood plasma or serum of patients treated with amitriptyline or nortriptyline, and enables the results to be reported within 1 h.


Determination of N-acetylator phenotype using caffeine as a probe compound: a comparison of high-performance liquid chromatography and capillary electrophoresis methods.

A test for determining N-acetylator metabolic phenotype has been developed using caffeine as a probe drug. A spot sample of urine is taken, and the unextracted urine is then analysed by micellar electrokinetic capillary chromatography. Phenotype is determined from the peak-area ratio of urinary 5-acetylamino-6-formylamino-3-methyluracil to 1-methylxanthine. Phenotype assignments using this method were compared with those made using a standard high-performance liquid chromatography assay, with good agreement between the two methods. The advantage of the capillary electrophoresis analysis is that no sample extraction is necessary, resulting in a total analysis time of around 20 min, and removing a potential source of error.


Rapid analysis of human serum albumin by high-performance liquid chromatography.

High-performance liquid chromatographic analysis of human serum albumin, using a column containing quaternized dimethyl-aminomethylstyrene-ethylene glycol dimethacrylate, was performed by isocratic elution. This column afforded resolution of albumin components, such as human mercaptalbumin and human nonmercaptalbumin. The method is an alternative to gradient chromatography, and allows rapid determination of the albumin components.


New extraction procedure and high-performance liquid chromatographic method for analyzing polyethylene glycol-400 in urine.

We describe a new, highly efficient method for extracting polyethylene glycol-400 from urine and for its analysis by isocratic reversed-phase high-performance liquid chromatography. This method is an improvement over previously published methods in that it does not require the use of ion-exchange resins and lyophilization prior to extraction, nor does it require the separation and analysis of the individual polymers of polyethylene glycol. The procedure described in this report entails extraction with a salt-solvent combination of ammonium sulfate and dichloromethane and analysis by reversed-phase high-performance liquid chromatography. The lower limit of detection was approximately 0.25 g/l with a 2-ml urine sample. Analytical recoveries of polyethylene glycol-400 added to urine at 2.5 and 5.0 g/l averaged 97 and 96%, respectively (n = 10). Within- and between-day coefficients of variation were less than 5% at 2.5 and 5.0 g/l. Studies of various urine samples from patients receiving polyethylene glycol-400 revealed no interferences from other urine substances.


Stereoselective high-performance liquid chromatographic assay with fluorometric detection for the isomers of mivacurium in human plasma.

A high-performance liquid chromatographic assay with fluorometric detection was developed for the analysis of the stereoisomers of mivacurium, a new short-acting neuromuscular blocker, in plasma. The isomers were isolated from plasma by solid-phase extraction with C18 and anion-exchange cartridges. The extracts were chromatographed on a LiChrosphere 60 RP Select B column (125 mm x 4.6 mm I.D.) using a mobile phase of acetonitrile-water (4:6, v/v) containing 0.005 M octanesulfonic acid. The fluorescence excitation and emission wavelengths were 202 and 320 nm, respectively. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from true values and the percentage coefficient of variation, respectively, were less than or equal to 10% at all concentrations except for the percentage coefficient of variation at the lower limit of quantitation (5 ng/ml). The assay has been successfully used for the analysis of plasma samples from a pharmacokinetic study in human volunteers.


Determination of 3'-deamino-3'-[2(S)-methoxy-4-morpholinyl]doxorubicin, a new morpholinyl anthracycline, in plasma by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of 3'-deamino-3'-[2(S)-methoxy-4-morpholinyl]doxorubicin and its possible 13-dihydro metabolite in human plasma has been developed. The plasma samples were buffered and the drugs and internal standard (doxorubicin) were extracted with diethyl ether-n-butanol, back-extracted into 0.3 M phosphoric acid, then analysed by reversed-phase liquid chromatography. Quantitation was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from blank plasma sample was observed. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from female rats that had received repeated intravenous doses of the test compound.


High-performance liquid chromatographic determination of the calcium channel blocker nimodipine in monkey plasma.

A new high-performance liquid chromatographic (HPLC) assay was developed for the determination of nimodipine in monkey plasma. An ethyl acetate extraction procedure was employed with a reversed-phase HPLC separation for the analysis. Absolute recovery of nimodipine from plasma was over 95% with a lower limit of quantitation of 10 ng/ml. This method was applied to a preliminary pharmacokinetic study in which 0.25 mg/kg nimodipine was administered intravenously to three monkeys. Protein binding and stability of nimodipine in monkey plasma were also examined. The pharmacokinetic parameters of nimodipine in monkeys were similar to those obtained in humans and indicate that monkeys are an appropriate animal model for further pharmacokinetic investigations.


Determination of luxabendazole in biological fluids by high-performance liquid chromatography.

Luxabendazole, a new benzimidazole, is a highly potent broad-spectrum anthelmintic. A high-performance liquid chromatographic method has been developed for its determination in serum and urine samples. In order to optimize the clean-up of samples we compared two procedures: C18 Sep-Pak cartridges and ultrafiltration through a cellulose membrane with a 30,000 relative molecular mass cut-off. In order to obtain the most suitable mobile phase, we studied the influence of pH and acetonitrile content on the capacity factor (k'). Chromatographic separation and quantification were performed on a reversed-phase column packed with 5-microns Nucleosil C18. The mobile phase was acetonitrile-0.05 M phosphate buffer (pH 7.0), (40:60, v/v). The column effluent was monitored by ultraviolet-visible spectrophotometry at 290 nm. The method shows good recovery, precision and accuracy. The lower limit of detection for luxabendazole is 15 ng/ml in serum samples and 25 ng/ml in urine samples.


High-performance liquid chromatographic separation and detection methods for anabolic compounds.

The role of high-performance liquid chromatography (HPLC) in methods of analysis for anabolic compounds in biological samples is reviewed. Special attention is given to both the separation and detection of anabolic compounds. A distinction is made between on-line detection systems, such as ultraviolet detection and diode-array detection, and off-line detection methods with special emphasis on immunochemical detection methods using non-isotopic labels. A number of applications are given to elucidate the possibilities of HPLC in the analysis of anabolic compounds.


Drug detection in hair by chromatographic procedures.

This article reviews the analysis of 31 drugs and drug metabolites in human hair by thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gas chromatography-mass spectrometry and mass spectrometry. The most important detection method after chromatographic separation of the components is the mass spectrometry because of its sensitivity and specificity. Washing steps to exclude external contamination, extraction, derivatization, stationary phases, detection modes and detection limits of the mass spectrometric and gas chromatographic-mass spectrometric procedures are presented in five tables. Additionally, a method for a gas chromatographic-mass spectrometric screening procedure is presented.


Chromatographic methods for the determination of toxicants in faeces.

Modern chromatographic techniques and their application in the determination of toxic compounds in faeces are reviewed. Faecal analysis may be of importance in toxicokinetic studies of xenobiotics in order to determine factors such as metabolism, body burden and major routes of elimination. Compounds of interest include various food constituents, drugs and occupational or environmental factors. Further, various mutagenic or carcinogenic compounds which are excreted by faeces have been indicated to represent risk factors for colorectal cancer. In this context, the chromatographic determination of the endogenously generated fecapentaenes and bile acids, both postulated etiological factors in colorectal carcinogenesis, is reviewed. For fecapentaene determination, several high-performance liquid chromatographic (HPLC) methods are available; however, the applicability of some of these methods is limited owing to insufficient separation of various isomeric forms or discrimination between fecapentaenes and their precursors. For the determination of bile acids in faeces, many chromatographic procedures have been reported, and the characteristics of the most relevant methods are compared and discussed. It is concluded that separation by gas chromatography (GC) in combination with mass spectrometry provides the highest selectivity and sensitivity. A relatively rapid alternative analysis for the determination of total and aqueous faecal bile acids is proposed. Further, methods for the determination of polycyclic aromatic hydrocarbons (PAHs) are reviewed. Although the use of radiolabelled PAHs in animal studies has many advantages, it cannot be applied for human biological monitoring and HPLC and GC provide sensitive alternatives. An HPLC method for the determination of non-metabolized PAHs in faeces is described.


Chromatographic methods for blood alcohol determination.

This review is focused on the different chromatographic strategies for blood alcohol determination which can be adopted for clinical and/or forensic purposes. Particular attention is paid to gas chromatography and to high-performance liquid chromatography. However, other analytical techniques in common use, such as chemical and enzymic methods, are also briefly presented, together with some, at present unusual or experimental, approaches, such as enzymic reactors and catalytic electrodes, which are suitable for application in column liquid chromatography. Finally, mention is made of the methods for the determination of acetaldehyde, the major ethanol metabolite, and of some proposed markers of chronic alcohol abuse, such as acetaldehyde-protein adducts and carbohydrate-deficient transferrin. In order to give the background of knowledge for the rational choice of an analytical strategy, an updated outline of ethanol metabolism and toxicology is presented, together with basic information for the interpretation of the results. Problems concerning blood sampling and storage are also discussed.


Chromatographic separation of marine organic pollutants.

A review and discussion of the chromatographic separation of marine organic pollutants is given, including sampling and clean-up procedures, fractionation and enrichment of marine pollutants, capillary gas chromatography (cGC) and high-performance liquid chromatography applying both classical and chiral stationary phases. The potential of multi-dimensional cGC for the analysis of marine organic trace pollutants is discussed for polychlorinated biphenyls (PCBs). The chromatographic separation of coplanar PCBs and of the enantiomers of chiral pollutants provides a further insight into the toxic potential of these marine organic pollutants.


Strategies for the identification of non-polar toxicants in aqueous environmental samples using toxicity-based fractionation and gas chromatography-mass spectrometry.

Toxicity-based fractionation is a useful tool for the isolation and identification of non-polar organic compounds that are present at toxic concentrations in aqueous environmental samples. Methods for isolating such toxicants from the aqueous sample matrix and techniques for fractionating the compounds for the purpose of reducing the complexity of the sample matrix and thus facilitating identification are evaluated. Strategies for analyzing gas chromatographic-mass spectrometric data and confirming toxicant identification are presented. Studies that use toxicity-based fractionation for identifying the cause of toxicity in aqueous environmental samples such as municipal and industrial wastewater treatment plant effluents and ambient waters are discussed.


Determination of aromatic hydrocarbons and their metabolites in human blood and urine.

Methods for the biological monitoring of aromatic hydrocarbons and their metabolites in the human blood and urine are reviewed. For the determination of the unchanged aromatic hydrocarbon in blood, gas chromatographic head-space analysis is recommended. The metabolites can be monitored by photometric, thin-layer chromatographic, high-performance liquid chromatographic and gas chromatographic methods. For the assessment of health risks caused by aromatic hydrocarbons, reference values and occupational limit values, expressed as biological tolerance values and biological exposure indices, have to be considered.


Methods for the analysis of persistent chlorinated hydrocarbons in tissues.

Chlorinated hydrocarbons bioaccumulate in tissues and may have severe health consequences. These compounds occur individually, in small groups or as complex mixtures; examples of each category include aldrin, hexachlorocyclohexane and the polychlorinated biphenyls. Tissue extraction and purification schemes have been established, although new approaches such as supercritical fluid extraction are promising. Analyses often require the resolving power of capillary gas chromatography, in combination with the sensitivity and selectivity of electron-capture detection, electrolytic conductivity detection and mass spectrometry. Difficulties arise in quantitating chlorinated hydrocarbons in tissues, due to the number of components present and the fact that individual constituents may be reduced or enhanced in concentration in tissues, compared with the original formulation. Congener specific analysis and computer-assisted identification techniques have been applied to the problem.


Methods for chromatographic determination of amanitins and related toxins in biological samples.

Methods for the chromatographic determination of amanitins, toxins of Amanita phalloides (Fr.), Link mushrooms and related toxins are reviewed; particular emphasis is given to high-performance liquid chromatographic methods. The main chemical and toxicological aspects are discussed, but the focus of the present review is on the analytical problems arising in a laboratory charged with the setting up of a procedure which can direct the appropriate clinical management of an intoxicated patient or solve a forensic case.


32P-postlabelling and mass spectrometric methods for analysis of bulky, polyaromatic carcinogen-DNA adducts in humans.

There has been significant recent progress toward the development of human carcinogen-DNA adduct biomonitoring methods. 32P-Postlabelling is a technique which has found wide application in human studies. 32P-Postlabelling involves enzymatic preparation and labelling of DNA samples, followed by chromatographic separation of carcinogen-nucleotide adducts from unadducted nucleotides. Thin-layer ion-exchange and high-performance liquid chromatography (HPLC) have been utilized. This paper critically reviews 32P-postlabelling methods for analysis of bulky, polyaromatic carcinogen-DNA adducts and details a strategy to optimize this technique for monitoring human samples. Development of a human carcinogen biomonitoring method requires that the biomarker meet certain criteria: that the biomarker be responsive to exposures known to increase human cancer risk, to reductions in those exposures, and to the influence of metabolic differences. In addition, reliable samples must be available by non-invasive means. The ability of 32P-postlabelling to meet these criteria is traced in the literature and discussed. Identification of specific carcinogen-DNA adducts is a difficult task due to the low (femtomole) levels in human target tissues. Because co-chromatography in thin-layer chromatography (TLC) is generally not considered to be proof of chemical identity, both synchronous fluorescence and HPLC in conjunction with 32P-postlabelling and TLC are used to confirm the identity of specific carcinogen-DNA adducts in human samples. Mass spectrometry is a highly specific method, the sensitivity of which has been improved to the point which may allow its use to confirm the identity of carcinogen-DNA adducts isolated by 32P-postlabelling and other methods. The literature relating to the use of mass spectral techniques in carcinogen-DNA adduct analysis is reviewed.


Systematic toxicological analysis of drugs and their metabolites by gas chromatography-mass spectrometry.

Gas chromatographic-mass spectrometric (GC-MS) procedures for the systematic toxicological analysis of several categories of drugs relevant to clinical toxicology, forensic toxicology and doping control are reviewed. Papers from 1981 to 1991 are taken into consideration. They describe the detection of acute or chronic intoxication and the detection of drug abuse. Screening procedures are included for the following categories: barbiturates and other sedative-hypnotics, anticonvulsants, benzodiazepines, antidepressants, phenothiazine and butyrophenone neuroleptics, central stimulants (amphetamines, cocaine), hallucinogens (LSD, phencyclidine, tetrahydrocannabinol), opioid (narcotic) and other potent analgesics, non-opioid analgesics, antihistamines (histamine H1-receptor blockers), antiparkinsonian drugs, beta-blockers (beta-adrenoceptor blockers), antiarrhythmics (class I and IV), diuretics, laxatives and their metabolites. Methods for confirmation of results obtained by screening procedures using immunoassay or chromatographic techniques are also included. GC-MS procedures for the simultaneous detection of several categories of drugs, the so-called "general unknown analysis", are reviewed. The toxicological question to be answered and the consequence for the choice of an adequate method, the sample preparation and the chromatography itself are discussed. The basic information about the biosample assayed, work-up, GC column, mass spectral detection mode, reference data and sensitivity of each procedure are summarized in tables, arranged according to the category of drug. Examples of typical GC-MS applications are presented. Fragment ions that are suitable for mass spectral screening for particular categories of drugs and for general unknown are tabulated.


Detection of the enzymatic activity of cytochrome P-450 enzymes by high-performance liquid chromatography.

The reactions catalysed by the various cytochrome P-450 enzymes are reviewed with respect to the analysis of products by high-performance liquid chromatography (HPLC). Especially biotransformation reactions of purified cytochrome P-450 enzymes in a reconstituted system and in microsomes mainly of rat liver origin are considered. Emphasis is put on the specificity of product formation due to the individual isozymes of cytochrome P-450. It is shown that the presence of eight cytochrome P-450 isozymes can be monitored and determined by specific product formation after HPLC analysis, which is an important parameter in toxicological studies.


Gas chromatographic analysis of nicotine and cotinine in hair.

Non-invasive validation of cigarette- or cigar-smoking behaviour is necessary for large population studies. Urine or saliva samples can be used for confirmation of recent nicotine intake by analysis of cotinine, the major metabolite of nicotine. However, this test is not suitable for validation of survey data, since the quantification of cotinine in saliva only reflects nicotine exposure during the preceding week. To validate information on tobacco use, we investigated hair samples for quantifying nicotine and cotinine by gas chromatography-mass spectrometry. Hair (about 50-100 mg) was incubated in 1 M sodium hydroxide at 100 degrees C for 10 min. After cooling, samples were extracted by diethyl ether, using ketamine as an internal standard. Drugs were separated on a 12-m BP-5 capillary column, and detected using selected-ion monitoring (m/z 84, 98 and 180 for nicotine, cotinine and ketamine, respectively). Hair from non-smokers and smokers contained nicotine and cotinine. Although it is difficult to determine an absolute cut-off concentration, more than 2 ng of nicotine per milligram of hair can be used to differentiate smokers from non-smokers. Some applications of this technique are developed to determine the status of passive smokers, the gestational exposure in babies and the pattern of an individual's nicotine use by cutting strands of hair into sections of one-month intervals.


Electrophoretic analysis of snake venoms.

Electrophoretic analyses were conducted on snake venoms from 21 species representing Elapidae, Crotalidae and Viperidae. Denatured and native venoms were analyzed by polyacrylamide gel electrophoretic (PAGE) methods with sodium dodecyl sulfate (SDS) and without SDS. Both SDS-PAGE and PAGE profiles of venoms from different snake species indicate that some proteins and polypeptide components of these venoms have common electrophoretic characteristics suggesting a genetic relationship. Conversely, the electropherograms also showed the characteristic protein and polypeptide profiles that could differentiate one snake species from another. Therefore, both SDS-PAGE and PAGE profiles suggest that proteins and polypeptides with similar characteristics abound among subspecies or related species, although each venom has a unique profile that differentiates one species from the other.


Rapid assay of cocaine, opiates and metabolites by gas chromatography-mass spectrometry.

The simultaneous assay of cocaine, opiates and metabolites in small biological samples continues to be a difficult task. This report focuses upon tabulation of important techniques (extraction, derivatization, chromatographic conditions, detection mode, data acquisition) reported over the last decade that were used in the development of assays for these analytes. The most prevalent procedures for extraction of cocaine, opiates and metabolites were liquid-liquid and solid-phase extraction isolation methods. Following extraction analytes were derivatized and analyzed by gas chromatography-mass spectrometry. The technique most often used for chromatographic separation was fused-silica capillary column gas chromatography. Detection generally was performed by selected ion monitoring in the positive-ion electron-impact ionization mode, although full-scan acquisition and positive- and negative-ion chemical ionization methods have been used. It was apparent from the review that there is a continuing need for greater sensitivity and selectivity in the assay of highly potent opiates and for cocaine and metabolites.


Determination of methamphetamine enantiomer ratios in urine by gas chromatography-mass spectrometry.

Analysis of the enantiomers of methamphetamine and its metabolite amphetamine is an extremely important process for a number of scientific disciplines. From studies of biological activity and mechanisms through determination of precursor molecules in a criminal investigation are all served by this analytical procedure. Utilization of gas chromatography-mass spectrometry with chiral derivatizing reagents is the most common chiral procedure and produces excellent results. Of the chiral derivatizing reagents available, the most widely used is trifluoroacetyl-l-prolyl chloride (TPC). Utilization of other derivatives require either synthesis by the analyst or have not shown themselves to provide as good a separation as did the TPC reagent. Use of chiral stationary phases yield good results but the disadvantages of temperature limits of these columns and the narrow use to which the columns can be put has limited their utilization. A significant utility of the chiral stationary phase is the ability to determine the purity of a chiral derivatizing reagent. Even if not used for routine analysis of enantiomers, utilization of this procedure can determine the purity of a reagent such as TPC and allow for accurate calculation of actual amounts of each enantiomer. This can be estimated using chiral derivatives on an achiral column, but it is limited to the extent that it is not able to differentiate enantiomeric impurity in the reagent versus the drug itself. Description of chromatographic procedures primarily focusing on gas chromatographic-mass spectrometric techniques but also including liquid chromatographic techniques along with examples of extraction and derivatization procedures is the focus of this review.


Chromatographic and mass spectrometric methods for determination of lysergic acid diethylamide (LSD) and metabolites in body fluids.

Continued illicit use of the potent psychedelic drug lysergic acid diethylamide (LSD) has stimulated efforts to develop effective analytical methods for detection of the drug and its metabolites in body fluids from suspected LSD users. Recently reported methods based on gas and liquid chromatography, combined with single- and multiple-stage mass spectral analysis, now permit accurate detection and quantitation of LSD at sub-nanogram/milliliter concentrations.


Inclusion and fractionated release of nucleic acids using microcapsules made from plant cells.

The encapsulation and fractionated release of nucleic acids on vesicular packing (VP) materials have been investigated. The earlier described dependence of the permeation of nucleic acid molecules through the vesicle membranes on the salt concentration is a necessary precondition for both encapsulation and fractionation. Encapsulation is achieved by applying a suitable sample onto a VP column that has been equilibrated with a high-salt buffer. In that buffer the sample molecules are permeable. Immediately after sample application, elution is started with a low-salt buffer, from which the sample molecules are excluded. At the front between the two buffers the permeability changes, and some of the sample molecules distributed inside the vesicles cannot pass through the membranes. These encapsulated molecules can be released by increasing the salt concentration in the eluent. If the encapsulated nucleic acid sample is polydisperse, a stepwise or linear increase in the salt concentration leads to a fractionated release. The fractions obtained differ in their molecular size composition.


High-performance liquid chromatography and post-column derivatization with diphenyl-1-pyrenylphosphine for fluorimetric determination of triacylglycerol hydroperoxides.

Triacylglycerol monohydroperoxides (TG-mHPO) were selectively detected at the picomole levels after post-column reaction with diphenyl-1-pyrenylphosphine (DPPP). TG-mHPO were separated on two types of reserved-phase columns, an ODS column and a phenylated silica gel column, which were useful for determining TG-mHPO at their molecular species levels and their class levels, respectively. After the separation, DPPP solution was mixed with the eluent followed by reaction in a stainless-steel coil 20 m x 0.5 mm I.D. at 80 degrees C, then the fluorescence intensity of DPPP oxide was measured (lambda ex. 352 nm, lambda em. 380 nm). Using these systems, TG-mHPO were determined in the range 2-1000 pmol. The relative standard deviations were 2.3-2.8%.


Purification of serine hydroxymethyltransferase from Bacillus stearothermophilus with ion-exchange high-performance liquid chromatography.

The gene of serine hydroxymethyltransferase (SHMT) of a thermophilic bacterium Bacillus stearothermophilus was expressed in Escherichia coli, and SHMT was successfully purified from the crude extract of E. coli in two steps while maintaining the enzymatic activity. The purification steps involved ammonium sulphate precipitation followed by high-performance liquid chromatographic separation using the anion-exchange column Fractogel EMD DEAE-650(S). In addition to the DEAE column, three other types of anion- and cation-exchange columns were also studied for their ability to separate SHMT, and the performance of the four columns were compared.


Determination of tetracyclines in bovine and porcine muscle by high-performance liquid chromatography using solid-phase extraction.

A method is presented for the determination of the three tetracyclines oxytetracycline, tetracycline and chlortetracycline in muscle, spiked at 100 ng/g, using high-performance liquid chromatography (HPLC). The concentration and extraction steps are carried out using Waters Environmental Sep-Pak cartridges. The principal steps involve homogenizing the sample in EDTA-McIlvaine buffer followed by centrifugation and precipitation of the supernatant using trichloroacetic acid. After further filtration and concentration on a Sep-Pak cartridge, the sample is eluted and analysed by HPLC with UV detection and confirmation by diode-array. The column used is a Nova-Pak C18 (4 microns) cartridge (10 cm x 8 mm I.D.). A phosphate-citrate-acetonitrile buffer, utilizing ion suppression, is the mobile phase. The analytes are detectable at levels down to 10 ng/g. The analyte identity can be confirmed at 20 ng/g by the use of diode-array detection and spectral library comparison.


Determination of alendronate in pharmaceutical dosage formulations by ion chromatography with conductivity detection.

A method was developed and validated for the direct determination in pharmaceutical dosage formulations of alendronate, a non-chromophoric compound. It is based on the use of single-column ion chromatography with conductivity detection that obviates the need for the tedious chemical derivatization procedures that are required for UV and fluorescence detection. Diluted samples of 0.05 mg/ml were chromatographed directly on a Waters IC-Pak HR anion-exchange column or a Dionex OmniPac PAX-100 column with dilute nitric acid as the mobile phase followed by conductivity detection. The method was validated and shown to be precise, accurate and specific for the assay of alendronate in intravenous (i.v.) solution and tablet formulations. The ruggedness of the assay was studied by generating data from four different instruments. Also established was the equivalence between this method and a previously reported high-performance liquid chromatographic method with 9-fluorenylmethyl chloroformate derivatization and UV detection. Application of the method to the determination of alendronate in i.v. and tablet formulations is presented and the performances of the Waters IC-Pak HR and Dionex OmniPac columns are discussed.


Enzymophoresis of nucleic acids by tandem capillary enzyme reactor-capillary zone electrophoresis.

Enzymophoresis with coupled heterogeneous capillary enzyme reactor-capillary zone electrophoresis was developed and evaluated in the area of nucleic acids. Ribonuclease T1, hexokinase and adenosine deaminase were successfully immobilized on the inner walls of short fused-silica capillaries through glutaraldehyde attachment. These open-tubular capillary enzyme reactors were quite stable for a prolonged period of use under operation conditions normally used in capillary zone electrophoresis. The capillary enzyme reactors coupled in series with capillary zone electrophoresis served as peak locator on the electropherogram, improved the system selectivity, and facilitated the quantitative determination of the analytes with good accuracy. Also, they allowed the on-line digestion and mapping of minute amounts of transfer ribonucleic acids, and the simultaneous synthesis and separation of nanogram quantities of oligonucleotides.


Determination of limiting ionic mobilities and dissociation constants of some local anaesthetics.

The limiting ionic mobilities and thermodynamic acid dissociation constants were calculated from isotachophoretic experiments for the local anaesthetics procaine, tetracaine, lidocaine, trimecaine, bupivacaine, cinchocaine, diperodone, diocaine, cocaine, psicaine-neu, tropacocaine, amylocaine, beta-eucaine and leucinocaine. The pH values at which the local anaesthetics with very similar limiting ionic mobilities can be isotachophoretically separated were determined from simulated mobility curves. The measuring apparatus employed a high-frequency contactless conductivity detector.


Determination of topanol antioxidants in methacrylates using capillary gas-liquid chromatography.

This paper presents a method to identify and determine topanol A and topanol O antioxidants in methylmethacrylate and 11-bromoundecylmethacrylate. The method is both simple and rapid. Analysis is performed on a 10 m x 0.53 mm I.D. HP-1 capillary gas chromatography column with a temperature gradient and a high carrier gas flow-rate (16.5 ml/min). Quantitation is by internal standardisation. Validation of the method is described for both topanols at concentrations of approximately 50 ppm in methylmethacrylate and 250 ppm in 11-bromoundecylmethacrylate.


Support matrix effects in the reversed-phase thin-layer chromatography of some peptides.

The retentions of 28 peptides in reversed-phase thin-layer chromatography (RPTLC) were determined on cellulose and on impregnated cellulose and alumina layers with 1-propanol as the organic component of the mobile phase. Each peptide showed a support matrix effect: their RM values first decreased to a minimum, then increased with increasing 1-propanol concentration. On cellulose layers only the increasing phase was observed. The retention behaviour of peptides was adequately described with a quadratic or linear function, but the slope value of the linear function had a positive value. The results demonstrate that the support matrix effect can be observed on non-silica supports and it may occur in reversed-phase chromatography in the case of polar solutes and supports with free adsorptive centres on their surfaces. Both the intercept and slope values of the function are needed to describe the lipophilicity of peptides, but the correlation is not strong enough for the determination of the lipophilicity of peptides by RPTLC. Principal component analysis showed that the peptides form distinct clusters on the basis of their retention characteristics: peptides containing a basic amino acid, peptides with a ring structure in the amino acid side-chain and peptides containing uncharged amino acids.


Contribution of ionically immobilized bovine serum albumin to the retention of enantiomers.

The retention of the enantiomers of mandelic acid and N-benzoylalanine was studied on columns prepared by immobilizing bovine serum albumin (BSA) on an anion exchanger. The amount of BSA fixed on the column is easy to adjust and measure. The adsorption isotherms were determined. For each enantiomer, the isotherm is well accounted for by a bi-Langmuir equation. One term of the isotherm (which is the same for both enantiomers) corresponds to non-selective interactions and the other term to the chiral selective interactions. The column saturation capacity of this second term is 8% larger for the less strongly retained enantiomer. This saturation capacity corresponds approximately to one enantiomer molecule adsorbed for five BSA molecules immobilized. This result is in agreement with the assumption of the hydrophobic cavity of BSA being the chiral selective site.


Determination of erythromycin ethylsuccinate by liquid chromatography.

A method is described for the determination of erythromycin ethylsuccinate by liquid chromatography. A C18 reversed-phase column (25 cm x 4.6 mm I.D.) was used with acetonitrile-0.2 M tetrabutylammonium sulphate (pH 6.5)-0.2 M phosphate buffer (pH 6.5)-water [x:5:5:(90-x)] as mobile phase. The proportion of acetonitrile (x) has to be adapted to the type of stationary phase used. For RSil C18 LL, 42.5% was used. The column was heated at 35 degrees C, the flow-rate was 1.5 ml/min and UV detection was performed at 215 nm. The main component, erythromycin A ethylsuccinate, was separated from all other components which were present in commercial samples. The main impurities were erythromycin A and the ethylsuccinate esters of erythromycin B and C. The amide N-ethylsuccinyl-N-demethylerythromycin A was shown to be present in all the samples examined. The method was successfully applied to the analysis of specialities.


Coupled-column high-performance liquid chromatographic method for the determination of 1-hydroxypyrene in urine of subjects exposed to polycyclic aromatic hydrocarbons.

A coupled-column high-performance liquid chromatographic system for integrated, on-line sample processing and the determination of free and conjugated 1-hydroxypyrene in urine has been developed. The method is based on a "tailor-made" copper phthalocyanine-modified porous-glass precolumn packing material, which allows a direct and repeated injection of urine samples and a selective enrichment of trace amounts of particular components. The fully automated method has a low detection limit (0.01 pmol), a quantitative and matrix-independent recovery and a highly reliability, as shown by an interlaboratory comparison of methods.


Purification of DNA-derived deoxynucleotides from leukocytes involving nuclease elution of an ion-exchange column.

A method has been developed in which the DNA of leukocytes (as the buffy coat from blood) is isolated in the form of its constituent deoxynucleotides. The steps in this method are as follows: (1) lyse the leukocytes with sodium dodecyl sulfate (SDS) and enzymatically digest the proteins and RNA, (2) remove the SDS on a non-polar adsorbent (Bio-Beads SM-4) and then trap the DNA on a quaternary amine silica cartridge, (3) wash the column with 1 M NaCl-buffer, (4) digest the DNA on the column with staphylococcal nuclease and (5) elute the digested DNA with 0.5 M NaCl-buffer and digest it further with bovine spleen phosphodiesterase II to deoxynucleotide-3'-monophosphates. From a 40-microliters sample of butty coat was obtained 126 +/- 14 micrograms (two experiments, eight sample total) of deoxynucleotides. Reversed-phase high-performance liquid chromatography, which removed the added enzymes, showed only peaks for deoxynucleotides. For comparison, the amount of deoxynucleotides obtained from the leukocytes by an automated phenol extraction procedure was 101 +/- 5.4 micrograms (one experiment in triplicate).


Purification of recombinant human interferon-beta by immobilized antisense peptides.

Synthetic antisense peptides encoded in the antisense strands of DNA corresponding to the 1-14, 42-54 and 103-115 fragments of the human interferon-beta sequence were applied in the purification of recombinant human interferon-beta from a mammalian cell culture. The protein fragments were selected on the basis of their computer-predicted exposure on the surface of the protein. The antisense peptides were synthetized by the solid-phase method directly on the resin used as the stationary phase in affinity chromatography. All the tested antisense peptides showed a selective affinity for human interferon-beta, permitting a ten-fold purification of the protein.


Supercritical fluid extraction of chemical warfare agent simulants from soil.

Chemical warfare agent simulants are efficiently recovered from 2-ppm spikes in 1 g of Rocky Mountain Arsenal Standard Soil using methanol-carbon dioxide (5:95) at 300 atm for 2 min at 60 degrees C. Recoveries (n = 3) were 79 +/- 23% for dimethylmethylphosphonate, 93 +/- 14% for 2-chloroethylethyl sulfide, 92 +/- 13% for diisopropylfluorophosphate and 95 +/- 17% for diisopropylmethylphosphonate. Recoveries are higher than, but less precise than those achieved from a 5-min ultrasonic micro-scale extraction using methanol. Much less laboratory waste is generated than the current standard organic solvent extraction method (33 g of soil shaken with 100 ml of chloroform).


Capillary zone electrophoresis of linear and branched oligosaccharides.

The electrophoretic behavior of derivatized linear and branched oligosaccharides from various sources was examined in capillary zone electrophoresis with polyether-coated fused-silica capillaries. Two UV-absorbing (also fluorescent) derivatizing agents (2-aminopyridine and 6-aminoquinoline) were utilized for the electrophoresis and sensitive detection of neutral oligosaccharides, e.g., N-acetylchitooligosaccharides, high-mannose glycans and xyloglucan oligosaccharides. The oligosaccharides labelled with 6-aminoquinoline yielded eight times higher signal than those tagged with 2-aminopyridine. Plots of logarithmic electrophoretic mobilities of labelled N-acetylchitooligosaccharides with 6-aminoquinoline or 2-aminopyridine versus the number of sugar residues in the homologous series yielded straight lines in the size range studied, the slopes of which were independent of the tagging functions. The slopes of these lines are referred to as the N-acetylglucosaminyl group mobility decrement. The oligosaccharides were better resolved in the presence of tetrabutylammonium bromide in the running electrolyte. Furthermore, the electrophoretic mobilities of branched oligosaccharides were indexed with respect to linear homooligosaccharides, an approach that may prove valuable in correlating and predicting the mobilities of complex oligosaccharides.


Capillary gel electrophoresis of single-stranded DNA fragments with UV detection.

Capillary gel electrophoresis has proven to be a powerful tool in biomedical research. We report our investigation of some of the critical parameters affecting separations of single-stranded DNA fragments as monitored by ultraviolet (UV) absorbance detection. Although not as sensitive as laser-induced fluorescence (LIF), UV absorbance detection allows one to calculate quite accurately, and inexpensively, the molarity of each separated DNA fragment and, moreover, the signal "fading" effect normally observed with LIF detection can be, in many cases, substituted for fluorescence to detect the many different single-stranded DNAs, as well as for detection of sequencing reactions.


Determination of the herbicide diclofop in human urine.

A simple and sensitive method for the gas chromatographic determination of diclofop residues in human urine is described. Recoveries of diclofop, as its methyl ester, from fortified urine were greater than 85% at 100, 50, 10 and 1 micrograms kg-1, and were similar with and without the inclusion of a hydrolytic step in the analytical method. However, a hydrolytic step was necessary for analysis of 24-h urine samples collected from a male applicator following a single exposure to diclofop-methyl during application to wheat using a tractor-pulled sprayer. Diclofop residues determined with hydrolysis were approximately double those without hydrolysis, suggesting that a significant portion of diclofop was excreted in the conjugated form.


Liquid chromatography and postcolumn indirect detection of glyphosate.

Glyphosate [N-(phosphonomethyl)glycine] and its metabolite aminomethylphosphonic acid (AMPA) were separated and detected by a postcolumn indirect detection strategy. Separation can be done on a cation-exchange column, where glyphosate elutes before AMPA, or on an anion-exchange column, where the elution order is reversed. Detection was achieved by using a fluorescent Al(3+)-morin postcolumn reagent. When the postcolumn reagent combines with the column effluent in a mixing tee, the fluorescence decreases in the presence of both analytes. Variables affecting the postcolumn indirect fluorescence detection were established and optimized; the major factors were postcolumn pH and volume and temperature of the postcolumn reaction coil. Detection limits, defined as three times the background noise, for glyphosate and AMPA separated on an anion-exchange column were 14 and 40 ng, respectively.


Direct serum injection in ion chromatography on packing materials with a semi-permeable surface.

Reversed-phase packing materials with restricted access of proteins to the hydrophobic sites were tested for their applicability in the ion-interaction chromatography of anions. Especially C8-modified silica, which had been coated with a hydrophilic polymer acting as a semi-permeable barrier, could be used successfully for the separation of several anions in a proteinaceous matrix without removing the proteins prior to injection. In combination with UV or conductivity detection, this technique allows the determination of some physiologically important anions in serum samples.


Analysis of oxyhalide disinfection by-products and other anions of interest in drinking water by ion chromatography.

The US Environmental Protection Agency is developing regulations for various drinking water disinfection by-products (DBPs). This effort involves developing analytical methods for the DBPs formed as a result of different disinfection treatments and collecting occurrence data for these species. Ion chromatography is one method being used to analyze drinking water samples for the following inorganic DBPs: chlorite, chlorate and bromate. These anions, however, are difficult to separate from common interfering anions of chloride, carbonate and nitrate. A method is therefore presented by which tetraborate/boric acid is used to separate these anions. Method detection limits of the order of 10 micrograms/l, using conductivity and UV detection were obtained. Stability studies of chlorite showing the effectiveness of ethylenediamine as a preservative and summary data for an occurrence of nitrite, nitrate and the DBP precursor bromide are presented.


New approach to the analysis of low levels of anions in water.

Optimized electromigrative sample introduction improves detection limits for anions in capillary electrophoresis. Reproducible results are achieved for micromolar and nanomolar levels of concentration. The new method offers shorter runtimes, improved resolution and greater simplicity in comparison with ion chromatography. The technique was applied to water samples from the power industry. Trace levels of anions are monitored routinely in water for steam generation in conventional and nuclear power plants. Reproducible and accurate results are presented for pure water samples containing typical concentrations of anions as well as for more specific types of samples, such as water compositions in the primary and secondary circuits of a nuclear power plant.


Determination of creatinine and purine derivatives in ruminants' urine by reversed-phase high-performance liquid chromatography.

A procedure is described for the rapid and simultaneous determination of allantoin, creatinine, uric acid, hypoxanthine and xanthine in sheep urine. Separation was achieved on a Novapak C18 column under isocratic conditions. The mobile phase was potassium phosphate buffer (10 mM, pH 4.0). A flow-rate of 0.5 ml/min, detection at 218 nm and a column temperature of 25 degrees C were employed with a total analysis time of less than 15 min. Detection limits for allantoin, creatinine, uric acid, hypoxanthine and xanthine were 1.0, 0.5, 0.5, 0.5 and 0.2 micrograms/ml, respectively, at a signal-to-noise ratio of 3 in a 20-microliters injection volume of tenfold-diluted urine. This sensitivity permits the precise determination of these compounds in ruminants' urine.


Gas chromatographic retention of carbohydrate trimethylsilyl ethers. IV. Disaccharides.

Trimethylsilyl ethers of seventeen disaccharides were injected on two stationary phases and their retention indices were calculated. Multiple linear regression was used to discuss relationships between retention indices and structural features of disaccharides.


Solid-phase extraction of prostanoids using an automatic sample preparation system.

A commercial automated solid-phase extraction system for cyclooxygenase arachidonic acid metabolites in urine samples has been evaluated. Comparison of manual and automatic batch (36 samples) extraction procedures for tritium labelled prostanoids added as tracers to urine samples has shown equivalent results with recoveries greater than 90% for prostaglandins E2, F2alpha and 6-keto prostaglandin F1alpha as well as thromboxane B2. Analyte stability is not affected by the automated procedure, which uses less solvents and has a faster overall processing time than the manual method. The automated system has been applied to the extraction of prostanoids in urine samples from workers exposed to dichloroethane.


Determination of the triglyceride composition of avocado oil by high-performance liquid chromatography using a light-scattering detector.

The triglyceride composition of avocado oil was determined by high-performance liquid chromatography using a light-scattering detector. Two avocado varieties, Fuerte and Hass, were analysed, and the qualitative composition of each was found to be similar, though quantitative differences were detected. The triglyceride composition was predicted using a system of equations based on the relationship between log k' and the molecular variables equivalent carbon number, chain length and number of double bonds for each of the fatty acids in the glycerides. A total of 24 molecular species of triglycerides were identified. The chromatographic system used successfully separated the critical pairs OOO-LOS, PaPaO-LnPP and PaOO-LOP (O = olein; L = linolein; S = stearin; Pa = palmitolein; Ln = linolenin; P = palmitin). Detector response was found to have a linear relationship with the amount of sample injected over the injection range 10-70 micrograms.


Simultaneous determination of arsenic and antimony species in environmental samples using bis(trifluoroethyl)dithiocarbamate chelation and supercritical fluid chromatography.

Simultaneous separation and quantitation of arsenic(III) and antimony(III) can be achieved by extraction with lithium bis(trifluoroethyl)dithiocarbamate followed by supercritical fluid chromatographic (SFC) analysis. Arsenic(V) and antimony(V) are extracted after reduction with potassium iodide and sodium thiosulfate. Detection limits of 7 pg As and 11 pg Sb are achieved using this extraction method and SFC. Application to natural water and biological sample analysis is discussed.


Gas chromatographic determination of the pesticide dodemorph for assessment of occupational exposure.

As part of a health hazard survey of occupational exposure to pesticides in greenhouse growing of ornamentals, analytical methods are developed and validated for measurement of exposure of workers to the fungicide dodemorph. A gas chromatographic method is developed using on-column injection and nitrogen-phosphorus detection for quantification. Methods for the determination of (potential) dermal exposure by the analysis of foliar dislodgeable residues and cotton gloves are validated with respect to background, analytical recovery, stability, limit of detection, and between-day coefficients of variation. Analytical recovery from 'foliar dislodgeable residue solutions' and cotton gloves is more than 95%. Dodemorph in 'foliar dislodgeable residue solutions' and on cotton gloves is stable for at least five and six months, respectively, when stored in the refrigerator. Between-day coefficients of variation are 6% for both matrices. The limit of detection is 3 micrograms per leaf sample and 150 micrograms per pair of gloves. Institute of Occupational Medicine (IOM) samplers, designed for the collection of a defined inspirable fraction of aerosols, are tested for sampling air-borne dodemorph. IOM samplers equipped with glass-fiber or cellulose filters appear unsuitable for reliable sampling of the fungicide because of breakthrough or breakdown during sampling.


Long-term longitudinal measurements of plasma dehydroepiandrosterone sulfate in normal men.

Dehydroepiandrosterone sulfate (DS) was measured by direct tritium RIA in longitudinal plasma specimens from 97 normal healthy male participants in the Baltimore Longitudinal Study of Aging. Fasting blood was collected at regular visits (approximately 1.5 yr apart) over an average 13 yr of adulthood (cumulative age range: 32-83 yr). DS was measured in 3-4 widely spaced specimens from each subject. A decline in DS was found in 65 (67%) subjects, 13 subjects (13%) showed no change, and increases were found in the 19 remaining subjects during the study period. A plot of individual data points revealed the same pattern we had obtained previously from a cross-sectional study of a different normal male population. A plot of DS values vs. age among subjects whose DS increased during the study also revealed an age-related decline. Thus, the longitudinal decrease in circulating DS, long inferred from cross-sectional data, is confirmed for normal men in the present study. A more detailed study of every specimen collected during the study period from 12 of the Baltimore Longitudinal Study of Aging subjects (4 whose values tended to be low, 4 whose values tended to be high, and 4 whose values were near the mean) failed to reveal any patterns of variation that could be correlated with changes in life circumstances, health status, or any other discernible factors. Hence, the wide variability seen in DS among individuals within normal populations remains unexplained.


Glucocorticoid receptor abnormalities in fibroblasts from patients with idiopathic resistance to dexamethasone diagnosed when evaluated for adrenocortical disorders.

A retrospective survey was accomplished on 420 consecutive patients who had undergone dexamethasone suppression tests between 1975-1988 due to suspected adrenal disorders. We found 7 patients in whom glucocorticoid resistance was apparent. They showed 4-6 abnormalities of the 7 investigations used: insensitivity to dexamethasone inhibition (n = 7), increased urinary cortisol (n = 3), glucocorticoid receptor (GR) thermolability (n = 4), decreased number of glucocorticoid receptors (n = 4), abnormal ligand affinity of GR (n = 4), abnormal basal GR mRNA expression (n = 4), and abnormal down-regulation of basal GR mRNA levels by dexamethasone (n = 1). The four patients with GR thermolability also showed increased basal GR mRNA levels. In the other patients the number of GR per cell was decreased without an up-regulation of GR mRNA. It is concluded that the syndromes of glucocorticoid resistance vary notably, clinically as well as biochemically; in patients evaluated for adrenocortical disorders the syndrome is apparently encountered in 1-2% of the patients.


Abnormal norepinephrine and aldosterone responses to upright posture in nonmodulating hypertension.

The subgroup of patients with nonmodulating hypertension demonstrates a number of abnormalities of the renin-angiotensin-aldosterone axis. We previously identified abnormalities in plasma and urinary dopamine in nonmodulators and posited that this may be in part due to a generalized defect in sympathetic nervous system activity. In the present study we assessed the state of activation of the renin-angiotensin system and the sympathetic nervous system in normal subjects and patients with modulating, nonmodulating, and low renin essential hypertension during sodium depletion and change from supine to upright posture. Levels of plasma norepinephrine were higher in non-modulators during the posture study (P < 0.05). PRA rose with upright posture in all groups, but low renin subjects had a blunted response. Nonmodulators and low renin subjects had lower aldosterone levels both supine (P< 0.05) and upright (P< 0.01). However, the aldosterone/PRA increment ratio was increased in low renin subjects (P< 0.01), whereas it was decreased in nonmodulators. Twenty-four-hour urine collections for catecholamine determinations were obtained in a subgroup of the subjects, with nonmodulators showing higher levels of norepinephrine excretion which approached significance (P = 0.08). In vitro experiments using rat and human adrenal glomerulosa cells showed that norepinephrine does not affect aldosterone secretion per se. These observations extend the series of abnormalities observed in nonmodulating hypertension. However, it is likely that the alterations in norepinephrine levels during sodium depetion and upright posture are a secondary event and not linked to the altered aldosterone production in these patients.


Prostate visualization studies in males homozygous and heterozygous for 5 alpha-reductase deficiency.

Male pseudohermaphrodites with 5 alpha-reductase deficiency have ambiguous genitalia and nonpalpable prostates on rectal examination, suggesting the dihydrotestosterone dependency of these structures. To clearly delineate the status of the prostate, male pseudohermaphrodites with 5 alpha-reductase deficiency had transrectal sonography of the prostate performed, and the results were compared to that of age-matched male controls. In six male pseudohermaphrodites, magnetic resonance imaging studies of the prostate were also performed. Heterozygote fathers also had transrectal sonography of the prostate performed and the results compared to age-matched controls. The prostates of the male pseudohermaphrodites appeared as platelike soft tissue structures posterior to the urethra on both prostatic ultrasound and magnetic resonance imaging. Prostatic volume, as determined on prostatic ultrasound by two different methods, was significantly smaller (approximately one-tenth) than the volume of age-matched controls. Transurethral ultrasound guided biopsy of the prostate in two affected subjects revealed stromal tissue. These results correlate with undetectable prostate-specific antigen in affected subjects, suggesting atrophic epithelium or lack of epithelial differentiation. This study demonstrates the dihydrotestosterone dependence of the prostate for normal differentiation and growth. The presence of some prostatic tissue in the male pseudohermaphrodites may be due to the fact that there is a decrease and not an absence of 5 alpha-reductase activity, and/or that the increased level of testosterone in subjects with this condition partially compensates for the decreased level of dihydrotestosterone. There was no difference, however, in prostate size between heterozygous fathers and age-matched control males. The heterozygote fathers had dihydrotestosterone production sufficient for normal prostate growth and development.


Contractile effects of prostaglandins, oxytocin, and endothelin-1 in human myometrium in vitro: refractoriness of myometrial tissue of pregnant women to prostaglandins E2 and F2 alpha.

Whereas there is much evidence in support of a role for prostaglandins (PG) in the parturitional process, it has not been demonstrated unequivocally that PGs are the physiological uterotonins involved in the induction of the myometrial contractions of spontaneous labor in women. This study was conducted to evaluate the contractile responsiveness of human myometrial tissue in vitro to PGs and to compare this response with that of other uterotonins, viz. oxytocin and endothelin-1. We found that treatment of uterine smooth muscle strips obtained from nonpregnant and pregnant women with PGE2 (10(-8)-10(-6) M) caused a biphasic response characterized by an initial single contraction of increased amplitude and duration, followed by relaxation and a long period (10-15 min) of quiescence. Conversely, PGE2 acted in rat myometrium to cause a monophasic response of increased contractile frequency and force. Whereas uterine smooth muscle from nonpregnant women was responsive to PGF2 alpha, the contractile responsiveness of myometrium from pregnant women was weak. This weak response to PGF2 alpha was found in myometrium of women in labor and in myometrium of women not in labor. 15-Methyl-PGF2 alpha evoked a small response in myometrium from pregnant women. Under identical in vitro conditions, PGF2 alpha (10(-8)-10(-6) M) and 15-methyl-PGF2 alpha (10(-6) M) caused sustained contractions in human vascular smooth muscle tissues (fetal aorta and arterial smooth muscle from chorionic vessels). Similarly, oxytocin and endothelin-1 (in myometrium from pregnant women) were effective in stimulating the force and frequency of myometrial contraction in vitro. We conclude that the myometrium of pregnant women, as evaluated in vitro, is refractory to the contractile effects of PGE2 and PGF2 alpha.