An HPLC-DAD validated method for the detection and quantification of cortisol, corticosterone and melatonin in plasma samples of two different animal species.

The monitoring of endogenous hormone plasma levels could be valuable in biomedical, veterinary and pharmaceutical research. A specific high performance liquid chromatography method with diode array detection, for the assay of cortisol, corticosterone and melatonin in animal plasma was developed and validated. The chromatographic separation was achieved on a C8 reversed phase column with a mobile phase consisting of HPLC-grade water and 35% v/v acetonitrile (pH ± 3.36). The detection was achieved through diode array detection, with two set wavelengths; 245 and 275 nm. The flow rate was at 1 ml/min and the total run time was 50 min. The method was validated according to validation guidelines (Shabir, 2006; US FDA, 2013). The method was found to be linear (R² > 0.99) over the analytical range (10 to 500 ng/ml) for all three analytes. All the other validation parameters were acceptable and within range. The method was applied to plasma samples from Sprague-Dawley rats and white rhinoceros.

Development and validation of a HPLC electrochemical detection method to measure COMT activity as a tool in drug development.

The determination of catechol-O-methyltransferase (COMT) activity is considered valuable for various pharmaceutical and biomedical research projects. A specific high performance liquid chromatography-coulometric electrochemical detection method, for the assay of COMT activity was developed by measuring the formation of normetanephrine from norepinephrine. The chromatographic separation was achieved on a C18 reversed phase column with a mobile phase consisting of 10 mM sodium dihydrogen phosphate buffer, 4 mM sodium 1-octanesulfonate, 0.17 mM ethylenediaminetetra-acetic acid disodium salt, 6 % methanol and 4 % acetonitrile (pH ± 4.0). The detection of normetanephrine was achieved through electrochemical detection, with a coulometric cell potential setting of +450 mV. The flow rate was at 1 ml/min and the total run time was 45 min. The method was validated according to validation guidelines (Shabir 2006; European Medicines Agency 2011; US FDA 2018). The method was found to be linear (R² > 0.99) over the analytical range (100 to 2500 ng/ml) for all the analytes. All the other validation parameters (sensitivity, precision, accuracy, recovery and stability) were acceptable and within range. The method was applied for the determination of COMT activity in rat liver homogenate test samples. The known selective COMT inhibitor entacapone was used as test inhibitor. The results confirmed the ability of entacapone to inhibit COMT activity by decreasing the production of all the metabolites of norepinephrine.

HPLC electrochemical detection and quantification of monoamines and their metabolites in rat brain tissue samples.

The monitoring of monoamines and their metabolites in CNS samples can be very valuable in pharmaceutical and biomedical research. A specific high performance liquid chromatography, coupled to a coulometric electrochemical detection method, for the assay of monoamines (dopamine, norepinephrine, epinephrine and serotonin) and their metabolites in rat brain tissue samples was developed. The chromatographic separation was achieved on a C8 reversed phase column with a mobile phase consisting of 0.1 M sodium formate buffer, 5 mM sodium 1-heptanesulfonate, 0.17 mM ethylenediaminetetraacetic acid disodium salt and 5% v/v acetonitrile (pH ±4.0). The detection was achieved through electrochemical detection, with a coulometric cell potential setting of +650 mV. The flow-rate was at 1 ml/min and the total run time was 50 min. The method was validated according to validation guidelines. The method was found to be linear (R2 > 0.99) over the analytical range (5 to 200 ng/ml) for all monoamines and their metabolites. All the other validation parameters were acceptable and within range. The method was applied to three rat brain areas (pre-frontal cortex, hippocampus and striatum), where the monoamines (except for epinephrine) and their metabolites were easily detected.

Crystal form conversion of nevirapine solvates subjected to elevated temperature and humidity: a qualitative study.

Some known nevirapine solvates have been reported to undergo solvent exchange in aqueous media to form a stable hemihydrate. This study aimed to determine the effects of atmospheric moisture on said nevirapine solvates and to gain insight into which factors determine the end product of transformation. Solvates were prepared by solvent recrystallisation and stored, together with the anhydrous and hemihydrate forms, in a climate chamber at 40 °C and 75% RH for a period of 28 days. Samples were analyzed using DSC, TGA, FT-IR, PXRD and Karl Fischer titration. Some solvates were observed to undergo desolvation to the anhydrous form of nevirapine (Form I), whilst others converted to the hemihydrate. It was found that water miscibility of the guest solvent determined the stable form of nevirapine, anhydrous or hemihydrate, to which each solvate eventually transformed. Transformation to the hemihydrate only occurred if the guest solvent was sufficiently water soluble to allow water molecules to enter solvent channels and displace the original guest. Solvates with hydrophobic guests desolvated to the anhydrous form. We concluded that, in the absence of a guest, solvent channels are lost during transformation to the monoclinic crystal system and space group P21/c (Form I) so that water cannot enter after desolvation.

XoxF-type methanol dehydrogenase from the anaerobic methanotroph "Candidatus Methylomirabilis oxyfera".

"Candidatus Methylomirabilis oxyfera" is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gramnegative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of "Ca. Methylomirabilis oxyfera" putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (ß) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from "Ca. Methylomirabilis oxyfera" that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of "Ca. Methylomirabilis oxyfera" cells and catalyzed methanol oxidation with appreciable specific activity and affinity (Vmax of 10 micromole min(-1) mg(-1) protein, Km of 17 microM). PQQ was present as the prosthetic group,which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from "Ca. Methylomirabilis oxyfera" is the first representative of type 1 XoxF proteins to be described.

UV spectrophotometric method for the identification and solubility determination of nevirapine.

An UV spectrophotometric method was developed for the quantitative determination of nevirapine in water, methanol and 0.1 N HCl. It was found that qualitative determination is also possible, due to a peak-shift observed with 0.1 N HCl. The solubility at 37 degrees C, for both the anhydrous and Hemihydrated forms of nevirapine, was determined in each of the three solvents.

Light and electron microscopical observations on the terminal airways and alveoli of the lung of the SA (Cape) fur seal Arctocephalus pusillus.

During activities of the Sea Fisheries Research Institute at Kleinzee, lung samples from six South African fur seals were collected. The terminal airways showed pseudostratified ciliated columnar epithelium with numerous goblet cells and occasional brush cells. Smooth muscle, cartilage and submucosal glands were also present. The epithelium changed over a short distance, in the smaller airways, through pseudostratified columnar non-ciliated to simple cuboidal epithelium with no goblet cells. The columnar non-ciliated cells contained secretory granules, which appeared to be serous. No Clara cells were found. Cartilage and muscle were present throughout, up to the origin of the alveolar ducts, but the glands disappeared together with the goblet cells. Alveoli were lined by types one and two alveolar epithelial cells, with subepithelial capillaries. They were divided by an alveolar septum with a well developed alveolar knob. This knob contained elastic fibres and fibroblasts, but not the smooth muscle cells which are present in terrestrial mammals and in Phocidae.

First report of field outbreaks of ergot-alkaloid toxicity in South Africa.

Outbreaks of clinical disease caused by the ingestion of ergotized Lolium rigidum (annual ryegrass), which resulted in a substantial loss in production, have been reported. A number of outbreaks of a hyperthermia syndrome in cattle, characterized by severe loss in milk production, loss of body mass and reduced fertility, are described. In one major outbreak in March to April 1994, a milling company reported that 2,646 dairy cows on 29 farms had developed clinical signs. In this outbreak, significant levels of ergotamine, ergosine, ergocornine and ergocryptine were found in the milled dairy rations fed to the affected cows. Barley screenings containing ergotized annual-ryegrass seed was identified as the toxic component and probable source of the ergot alkaloids in the ration. The clinical syndrome was reproduced experimentally by feeding suspected feed to a group of nine high-producing Ayrshire cows. An outbreak of gangrenous necrosis of the extremities in young cattle in the winter of 1987 was also suspected of having been caused by ergot alkaloids in grain screenings.

Death of a dog attributed to the cyanobacterial (blue-green algal) hepatotoxin nodularin in South Africa.

A bull terrier died after drinking water at the margin of Zeekoevlei near Cape Town. At the time, Zeekoevlei, a hypertrophic coastal lake, contained a bloom of the cyanobacteria Nodularia spumigena and Microcystis aeruginosa. The circumstances of the incident, clinical signs of poisoning and histopathology, which mainly revealed extensive liver damage, were consistent with cyanobacterial poisoning. The cyanobacterial bloom material contained 3.47 micrograms mg-1 dry weight of the pentapeptide hepatotoxin nodularin. It is inferred that the dog died of cyanobacterial hepatotoxicosis due to the ingestion of nodularin.

Abortion in a heifer associated with the intra-uterine transmission of Cowdria ruminantium-like organisms following heartwater vaccination.

A Nguni heifer aborted a 6-month-old foetus 35 days after intravenous inoculation with Onderstepoort heartwater vaccine. Histopathological examination of the brain revealed numerous suspected Cowdria ruminantium organisms in the endothelial cells of the blood vessels. The organisms were ultrastructurally and immunohistochemically compatible with Cowdria ruminantium, although, owing to autolysis and cross-reactions, the possibility that they represent another rickettsial or related organism could not be completely excluded. As far as we could ascertain this is the first recorded case of suspected intra-uterine transmission of Cowdria ruminantium in ruminants.

Cyanobacterial (blue-green algae) poisoning of livestock in the western Cape Province of South Africa.

Three outbreaks of cyanobacterial (blue-green algae) poisoning involving cattle and sheep are described. In 2 of these acute mortality was followed by photodermatitis in some of the surviving animals. In all 3 outbreaks the hepatotoxicity of the water collected from the dams where the animals had been drinking was confirmed following the intraperitoneal administration to mice. Nodularia spumigena was the dominant cyanobacterium in the first 2 outbreaks, and Microcystis aeruginosa in the third. The presence of the heptapeptide toxin microcystin-LR in the third outbreak was demonstrated by high pressure liquid chromatography.

New bioavailability parameters to compare the efficacy of antimicrobial drugs in dosage forms.

Pharmacokinetic parameters such as AUC, Cmax and tmax have been used to represent the rate and extent of absorption of drugs from dosage forms in comparative bioequivalence testing. None of these parameters gives a direct indication of how long the drug concentration is maintained above an acceptable level e.g. minimum inhibitory concentration (MIC) or the minimum effective concentration (MEC). This is clinically important in evaluating the onset and duration of a therapeutic effect obtained from a test dosage form in comparison to the reference dosage form. The purpose of this study is to investigate the possibility of a bioavailability parameter which relates the time that the drug concentration in the systemic circulation is maintained above a certain level (te) and the first time that the blood concentration exceeds this level (to). Two methods were used to calculate the time that drug levels are maintained above a certain minimum level. The proposed parameters proved to be valuable when the efficacy of erythromycin was used as an example. Although some problems, such as undefined MIC/MEC may arise, te and to can be used along with the conventional bioequivalence parameters to obtain a better indication of the clinical efficacy.

The application of new bioavailability parameters in the bioequivalence testing of antimicrobial agents.

Two new bioavailability parameters were recently suggested [Koeleman et al. 1991] to define (i) the time that the concentration in the blood stays above a defined minimum effective concentration, te and (ii) the onset of the effect, to. In addition to conventional bioequivalence parameters, the new bioavailabilty parameters (to and te) were calculated in this study and statistically compared for penicillin, chloroquine, oxytetracycline, amoxycillin and flucloxacillin from available bioequivalence data. For oxytetracycline, flucloxacillin and amoxycillin, the conventional bioavailability parameters indicated partial equivalence whereas using the te and to parameters, more realistic indications of the possible extent of the performance of a drug from dosage forms were obtained than with the conventional bioequivalence parameters. The new parameters gave additional information for a better evaluation of the performance of a drug from a dosage form.

The influence of different types of breakfast on the absorption of paracetamol among members of an ethnic group.

It is well known that food influences the absorption and therapeutic efficacy of many drugs. In this study, the influence of three different types of breakfast, on the absorption of paracetamol was studied in a South African ethnic population group (Tswanas). The results indicated that breakfasts with a high fat content delayed the absorption of paracetamol to the largest extent while breakfasts with a high carbohydrate content delayed the absorption of paracetamol to a lesser extent.