Assessment of microsomal tolbutamide hydroxylation by a simple thin-layer chromatography radioactivity assay

A radio thin-layer chromatographic method is described for in vitro measurement of tolbutamide methylhydroxylation as an alternative to the commonly used HPLC assay. After the incubation experiments of [14C]tolbutamide with human liver microsomes, the supernatants were directly spotted onto standard silica gel TLC plates and developed in a horizontal chamber using a solvent system consisting of toluene-acetone-formic acid (60:39:1, v/v). Dried TLC plates were exposed to a phosphor imager plate and quantificated by use of a phosphor imager. Reaction rates were calculated from the ratio of labelled metabolite to the total radioactivity. The correlation coefficient between HPLC and the TLC method was 0.978 (n=14). The described method provides a valuable tool for the determination of tolbutamide hydroxylation activity in human liver microsomes.     

Levels of histamine and histamine-producing bacteria in smoked fish from New Zealand markets

Smoked fish has been the most commonly implicated product in presumptive cases of scombroid poisoning in New Zealand. One hundred seven samples of smoked fish were purchased from Auckland retail markets between July 1995 and March 1996, and their histamine and bacterial levels were determined. Eight samples, obtained from five of the nine retail outlets sampled, had histamine levels which exceeded 50 mg/kg, the level set by the FDA as an indicator of decomposition. Histamine levels in only 2 samples (346.4 and 681.8 mg/kg) exceeded a hazard level of 200 mg/kg. Thirty-three of the smoked fish were held at 20 degrees C for 2 days, and 8 of these developed histamine levels above 50 mg/kg with 4 exceeding 200 mg/kg (maximum 1,659.4 mg/kg). The stored samples that exceeded 200 mg/kg were all obtained from two outlets. Within or between fish species there were no consistent relationships between levels of histamine in the samples and either the total aerobic plate counts or the numbers of histamine-producing bacteria. To the contrary, there was evidence that histamine had been formed prior to smoking and that histamine-producing bacteria were eliminated during smoking.     

Interference of herbal drinks with urinalysis for drugs of abuse

Recently there have been claims among drug users that some herbal drinks interfere with urinalysis for drugs of abuse and yield false positive results. Proof of such claims has yet to be shown. Screening for drugs of abuse is usually carried out using fluorescence polarization immunoassay (FPIA) or thin-layer chromatography (TLC). Fifty herbal samples which are considered among the most purchased herbs in the consumer market were used to investigate such claims. The drug groups that were tested for included amphetamines, opiates, barbiturates, cocaine metabolite, methadone, and their analogs. The herbs were analyzed at different concentrations (0.1, 1, 3, and 5 g/100 mL of distilled water) using TLC and FPIA to determine if any interfere with urinalysis for drugs of abuse and yield false positive results. For the FPIA test, the sample infusions were analyzed directly using the automated ADX analyzer (Abbott Laboratory). For TLC, infusions of the herbs were added to a solid-phase extraction column (pH 9.25), then extracted with a methylene chloride-isopropanol solvent system. At this pH, neutral, basic, and acidic drugs of abuse are extractable. The developed chromatographic plates were sprayed sequentially with several reagents. None of the herbs in the concentration ranges screened showed any interference with TLC or FPIA, indicating the invalidity of such claims.     

Ethical implications for early pregnancy of the fetus as patient: a basic ethical concept in fetal medicine

A new and rapid method is proposed for extraction of non-polar lipids from tissues where they are present as abundant components which can interfere with the usual procedures of lipid extraction and TLC separation, and hamper, in particular, sulphatide visualization. A solvent more hydrophobic than chloroform, i.e. n-hexane, was utilized to remove the neutral lipids from samples of female rabbit parotid gland, and the n-hexane phase was used for TLC which showed considerable amounts of cholesterol esters, in addition to triglycerides, diglycerides and monoglycerides. The methanol phase, now devoid of non-polar lipids, was utilized to prepare TLC plates in order to separate and visualize the polar lipid fractions, in particular the sulphatides.     

Comparison of GC-MS and TLC techniques for asarone isomers determination

Two chromatographic methods (GC-MS and TLC) have been developed for separation and determination of alpha and beta asarone from essential oils and alcoholic extracts. The study has been performed on the Acorus calamus (I) and Asarum europaeum (II) essential oils of Romanian origin and the alcoholic extract of Acorus calcamus L (III) and it is a consequence of the International Boards exigency regarding the presence of beta asarone in food, beverages and pharmaceuticals. The isomers were determined using both internal and external standard methods. Both SIM and SCAN techniques were used and the results were compared regarding the chromatographic resolution and interference compounds. The method exhibits good repeatability and low detection limit but is expensive and time consuming. The two isomers concentrations are 5.2- 6.7 microg ml(-1) (I), 460-510 microg ml(-1) (II) and 2.7 5.7 microg ml (III) for alpha asarone and 91-98 microg ml(-1) (I), 24-29 microg ml(-1) (II) and 88 97 microg ml(-1) (III) for beta asarone. The TLC method was developed as an alternative for the GC method. The separation was performed on silica gel plates using toluene: ethyl acetate 8:2 as mobile phase. The evaluation of the chromatograms was made by densitometry using multiple wavelength. The sum of the two isomers are between 80-120 microg ml(-1) (I) and 127-145 microg ml(-1) (III) using spectrophotometric detection and between 73-93 microg ml(-1) (I) and 99-105 microg ml(-1) (III) using fluorimetric detection. The results of the two chromatographic methods were compared. Even the GC is more sensitive, mathematical computations for spots optimization and interference elimination could improves the TLC quality results.     

The effect of orally administered histamine on the weight gain and development of gizzard lesions in chicks

The results of a large scale trial confirmed preliminary findings that the feeding to chicks of a diet containing 4 mg/g of histamine can result in the production of localised lesions in the gizzard and depressed growth rate. This finding supports an earlier suggestion that when dietary fish meal is associated with gizzard erosion the condition is mediated, in part, by the histamine produced by certain types of bacterial spoilage of fish protein.     

HPTLC determination of ceftriaxone, cefixime and cefotaxime in dosage forms

The objective of this investigation was to develop a HPTLC method for the determination of ceftriaxone, cefixime and cefotaxime, cephalosporins widely used in clinical practice. High performance TLC of cephalosporins was performed on pre-coated silica gel HPTLC plates with concentrating zone (2.5 x 10 cm) by development in mobile phase ethyl acetate-acetone-methanol-water (5:2.5:2.5:1.5 v/v/v/v). A TLC scanner set at 270 nm was used for direct evaluation of the chromatograms in reflectance/absorbance mode. The calibration curves were established as dependence of peak height (linear and polynomial regression) and peak area (polynomial regression) versus ng level (125-500 ng for all cephalosporins investigated). Relative standard deviations obtained from calibration curves was compared. Precision (RSD: 1.12-2.91% (peak height versus ng) and RSD: 1.05-2.75% (peak area versus ng)), and detection limits (ng level) was validated and found to be satisfactory. The method was found to be reproducible and convenient for quantitative analysis of ceftriaxone, cefixime and cefotaxime in their raw materials and their dosage forms.     

Liquid chromatographic separation of some PTH-amino acids

Liquid chromatographic studies on the separation of ten PTH-amino acids were carried out using normal phase untreated silica gel plates, C-18 RP precoated plates and RP-HPLC. Resolution of a complex mixture of PTH-amino acids was achieved using all the three types. Certain new successful solvent systems have been worked out in each case. HPLC was carried out with Lichrosphere 100 RP-18 (5 microns) column. Acetonitrile and sodium acetate buffer of pH 4.0 was used for reversed phase chromatography while for normal phase TLC combinations of chloroform-acetonitrile and chloroform-tetrahydrofuran were applied.     

Bacillus subtilis BS 107 as an antagonist of potato blackleg and soft rot bacteria

Antimicrobial substances were produced by Bacillus subtilis BS 107 in a defined medium and isolated from culture filtrate by precipitation at pH 2.5. Active fractions were extracted in ethyl acetate, acetone, and 80% ethanol and purified by thin-layer chromatography (TLC) on silica gel plates developed with an ethanol-water mixture (2:1, v/v). In each case, a band with a Rf of 0.75 formed an inhibitory zone when the TLC plates were placed in contact with agar seeded with test cultures of the Erwinia spp. The antibiotic was released into the culture medium during early stages of growth of Bacillus subtilis BS 107 but higher amounts were released in older cultures. The antibiotic was resistant to the action of nucleases, proteases, and lipase. It was stable when autoclaved twice for 35 min at 2 atm (1 atm = 101.325 kPa) in acidic, neutral, and alkaline solutions. It remained active over the pH range of 1-14 during 1 month of observation and exhibited no loss of antimicrobial activity when stored at 4 degrees C for over 1 year. Bacillus subtilis BS 107 showed activity in vitro and in vivo against Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora, the causal agents of potato blackleg and tuber soft rot. The application of an antagonist or its antibiotic to cut potato tissues prevented or reduced symptoms of the diseases. The antibiotic was active in vitro against a broad spectrum of bacterial and fungal species.     

Genetically determined susceptibility to organophosphorus insecticides and nerve agents: developing a mouse model for the human PON1 polymorphism

Thin-layer chromatographic (TLC)-UV densitometric and gas-chromatographic-mass spectrometric detection (GC-MSD) methods were developed for simultaneous quantification of morphine and codeine in poppy capsules (Papaver somniferum). Morphine and codeine were isolated by extraction with chloroform: isopropanol (3:1, v/v) at pH = 8.5 and by solid-phase extraction on Snap-Cap cartridges at pH = 8.5. The TLC-UV densitometric quantification was performed by external standard method on silica gel plates using ethyl acetate: toluene: methanol: ammonia (68:17:10:5, v/v) as developing solvent and UV detection at 275 nm. For the GC-MSD analysis, the drugs were derivatized with acetic anhydride: pyridine (1:1, v/v) and separated on a 30 m HP5 capillary column. The quantification was performed using nalorphine as internal standard.     

Exact Buckling Solutions For Rectangular Plates Under Intermediate and End Uniaxial Loads

This paper is concerned with the elastic buckling of rectangular plates subjected to both intermediate and end uniaxial loads. The rectangular plates have two simply supported opposite edges that are perpendicular to the in-plane load direction, while the other two plate edges can have free, simply supported, or clamped edges. The solution procedure involves the use of the Levy approach, the domain decomposition technique, and the state-space concept. The method furnishes exact stability criteria; samples of which are presented in a graphical form for plates with various boundary conditions. These results will be useful to engineers who design plates (or walls) that support intermediate floors.     

Transient increase of blood histamine level induced by pentagastrin. Continuous monitoring by in vivo microdialysis

BACKGROUND: Since few studies of (penta)gastrin-induced histamine release from the gastric mucosa into blood has been performed, an effect of pentagastrin on histamine level of rat blood was examined by using the in vivo microdialysis method. METHODS: Pentagastrin was perfused through the microdialysis probe implanted into the jugular vein of urethane-anesthetized rats or in urethane-anesthetized, totally gastrectomized rats, and dialysis samples of blood were concurrently collected. Histidine decarboxylase (HDC) activities and histamine contents in the glandular stomach and gastric acid output after pentagastrin stimulation were also investigated. RESULTS: Pentagastrin induced a transient increase of blood histamine in a dose-dependent manner but failed to cause any increase of blood histamine in the totally gastrectomized rat. Pentagastrin also induced increases of the HDC activity in the glandular stomach and of the gastric acid output. The peak histamine level in blood occurred 40 min after pentagastrin perfusion, whereas the peak acid secretion occurred after 80-120 min and then leveled off. CONCLUSIONS: The transient increase of blood histamine induced by pentagastrin is attributable to the histamine released from enterochromaffin-like cells and could be monitored by using the in vivo microdialysis method.     

TLC sensitivity of six modifications of Dragendorff's reagent

The relative sensitivities of six modifications of Dragendorff's reagent were measured on TLC plates by spectrodensitometry, using compounds containing specific functional groups. A correlation between the structures of the compounds reactive to Dragendorff's reagents and the sensitivities of the reagents was made. Explanations for the variations in sensitivities between different modifications of Dragendorff's reagent are given.     

Application of a sequential analytical procedure for the detection of the beta-agonist brombuterol in bovine urine samples

The multistep analytical procedure routinely applied in our laboratory for the detection of the aryl amine beta-agonists clenbuterol, mabuterol and mapenterol in bovine matrices has been extended to the analysis in urine samples of brombuterol, a new clenbuterol-like compound. In the screening steps, the urine samples were first enzyme-linked immunosorbent assay (ELISA)-tested, then the positive samples were analyzed by thin-layer chromatography (TLC) and the beta-agonists detected with the Bratton-Marshall color reaction. The TLC spots corresponding to the suspected compounds were scraped off the plates, collected and extracted separately with methanol. The beta-agonists in the extracts were detected by HPLC-Vis (limits of detection: 0.5 ng/g). In the confirmatory step the presence and the concentration of the compounds of interest in the samples were established by GC-MS with two different ionization techniques, EI and CI (limits of detection: 1.0 ng/g). The use of this procedure has made possible the detection of brombuterol in officially sampled bovine urine.     

Diploma of continuing education from the Society of Physician of Thüringia

A rapid and sensitive high-performance thin-layer chromatographic assay has been developed for the measurement of nimesulide in human plasma. Its use for pharmacokinetic studies has been evaluated. The method includes a single-stage extraction procedure without the use of an internal standard. Analysis was performed on plasma containing known amounts of the drug, on drug-free plasma, and on plasma containing an unknown quantity of the drug. Known amounts of extract and nimesulide (100 and 200 ng, as external standard) were spotted on precoated silica-gel 60F254 plates by means of a Camag Linomat IV autosampler. Quantification was achieved using a Camag TLC scanner 3. The recovery of the method was 97.10 +/- 2.22%. The method was applied for the determination of plasma levels and pharmacokinetic parameters of nimesulide after oral administration of two formulations (100 mg) in healthy volunteers. The method is a sensitive, economical, rapid and specific assay for nimesulide in human plasma, and is suitable for pharmacokinetic studies after therapeutic doses.     

Novel role of transmembrane SCF for mast cell activation and eotaxin production in mast cell-fibroblast interactions

Mast cell activation can be induced by multiple mechanisms, including IgE-, complement-, and stem cell factor (SCF)-mediated pathways. In addition, the interaction of mast cells with particular cell populations, such as fibroblasts, have also demonstrated increased mast cell reactivity. In these studies, we have investigated the role of fibroblast-mast cell interaction for induction of histamine release and chemokine production and the specific role of SCF during this interaction. Primary pulmonary fibroblast cell lines were grown in culture and used throughout these studies. Mast cells were grown in parallel with fibroblasts by incubation of bone marrow cells with SCF and IL-3. During mast cell-fibroblast coculture, increased histamine release could be attenuated either by separation of the cell populations using a Trans-Well setup, which did not allow cellular contact, or by specific anti-SCF Ab. In addition, a significant increase in eotaxin, a potent eosinophil-specific C-C chemokine, was also observed during fibroblast-mast cell interaction. The production of eotaxin was cell contact dependent and could be inhibited using an anti-SCF Ab or specific antisense therapy. SCF was constitutively produced from fibroblasts in its transmembrane form and could be induced by TNF. SCF-coated plates induced significant mast cell-derived eotaxin production, whereas soluble SCF induced little or no eotaxin, suggesting a necessity for receptor cross-linking for activation. These studies indicate that fibroblast-mast cell contact plays a role in exacerbation of histamine release and eotaxin production.     

Histamine H1- and H2-receptor vasodilation of canine intestinal circulation

Studies were conducted in anesthetized dogs to determine whether the mesenteric vasodilator response to histamine is mediated by H1 receptors alone or whether H2 receptors are also involved in the response. Evidence favoring a role for both receptors included: 1) the vasodilator response to histamine was inhibited by either the H1-receptor antagonist, tripelennamine, or the H2-receptor antagonist, metiamide; 2) both the H1 agonist, 2-methylhistamine, and the H2 agonist, 4-methylhistamine, induced dilator responses in the mesenteric circulation; and 3) two temporal patterns of vasodilation could be distinguished, namely a transient spike and subsequent fade of blood flow (seen with either the H1 agonist or with histamine after H2-receptor blockade) and a sustained and stable increase in flow (seen with either the H2 agonist or with histamine after H1 blockade). Metiamide appeared to be a potent inhibitor of the mesenteric vasodilator response to histamine at least equal to tripelennamine.     

R-(-)-alpha-methyl-histamine has nitric oxide-mediated vasodilator activity in the mesenteric vascular bed of the cat

Responses to the histamine H3 receptor agonist R-(-)-alpha-methyl-histamine were investigated in the mesenteric vascular bed of the cat under constant-flow conditions. Injections of R-(-)-alpha-methyl-histamine and histamine caused dose-related decreases in mesenteric perfusion pressure with R-(-)-alpha-methyl-histamine being 1000-fold less potent than histamine when doses were compared on a nmol basis to take molecular weight into account. Responses to R-(-)-alpha-methyl-histamine were not altered by histamine H1 or H2 receptor antagonists at a time when responses to histamine were significantly reduced. The histamine H3 receptor antagonist thioperamide reduced responses to R-(-)-alpha-methyl-histamine but was without effect on responses to histamine [6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoro-methylphenyl)heptaneca rdoxamide dimaleate] (HTMT), or dimaprit. These data suggest the presence of histamine H1, H2 and H3 receptors mediating vasodilation in the mesenteric vascular bed. Responses to R-(-)-alpha-methyl-histamine and histamine were reduced by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) but were not altered by the cyclooxygenase inhibitor meclofenamate, the alpha-adrenoceptor blocker phentolamine, or adrenergic nerve terminal depleting agent reserpine. The present data suggest that histamine H3 receptors mediating vasodilation are present in the mesenteric vascular bed and that responses are mediated by the release of nitric oxide but not vasodilator prostaglandins or an effect on the adrenergic nervous system. These results indicate that vasodilator responses to histamine involve the activation of histamine H1 and H2 receptors and the release of nitric oxide in the mesenteric vascular bed of the cat.     

Effect of Longitudinal Stress Gradients on Elastic Buckling of Thin Plates

This paper analyzes the effect of longitudinal stress gradients on the elastic buckling of thin isotropic plates. Two types of thin plates are considered: (1) a plate simply supported on all four edges and rotationally restrained on two longitudinal edges; and (2) a plate simply supported on three edges with one longitudinal edge free and the opposite longitudinal edge rotationally restrained. These two cases illustrate the influence of longitudinal stress gradient on stiffened and unstiffened elements, respectively. A semianalytical method is derived and presented herein to calculate the elastic-buckling stress of both types of rectangular thin plates subjected to nonuniform applied longitudinal stresses. Finite-element analysis using ABAQUS is employed to validate the semianalytical model for plates with fixed and/or simple supports. Empirical formulas are produced to calculate the buckling coefficients of plates with fixed and/or simple supports under longitudinal stress gradients. The results help establish a better understanding of the effect of longitudinal stress gradients on the elastic buckling of thin plates and are intended to aid in the development of design provisions to include these effects in the strength prediction of thin-walled beams under moment gradients.