A novel HPLC method developed and validated for the detection and quantification of atorvastatin, fluvastatin, pitavastatin and pravastatin during transdermal delivery studies.

An HPLC method was developed and validated to quantify and identify several statins (atorvastatin, fluvastatin, pitavastatin and pravastatin) that were used during transdermal drug delivery. The method proved to be most effective with a Restek Ultra C18, 250 x 4.6 mm, 5 µm column, a flow rate of 1.0 ml/min, UV detection at 240 nm and injection volume of 10 µl. The mobile phase used was acetonitrile/Milli-Q® water with 0.1% orthophosphoric acid starting with 30% acetonitrile, which increased linearly to 70% (after 4 min) for up to 10 min and then re-equilibrated to start conditions. This HPLC method indicated linearity (correlation coefficient (R²) of 1) within the concentration range of 0.05-200.00 µg/ml and had an average recovery of 98-103%. Limit of detection (LOD) and limit of quantification (LOQ) showed that statins could still be identified at concentrations of 0.004-0.006 µg/ml with the exception of atorvastatin (quantifiable at 0.013-0.035 µg/ml). Specificity performed during method validation, confirmed that the method was suitable for accurate detection and quantification of the statins when included in the transdermal formulations with other excipients.

Determination of lovastatin, mevastatin, rosuvastatin and simvastatin with HPLC by means of gradient elution.

A novel HPLC method with UV detection was developed and validated in skin penetration (in vitro) studies to identify and quantify lovastatin, mevastatin, rosuvastatin and simvastatin. A Venusil XBP C18 (2), 150 x 4.6 mm, 5 µm column (Agela Technologies, Newark, DE) was used with gradient elution (start at 45 % acetonitrile and increase linearly to 90 % after 1 min; hold at 90 % until 6 min and then re-equilibrate at start conditions) and the mobile phase consisted of (A) Milli-Q ® water and 0.1% orthophosphoric acid, and (B) HPLC grade acetonitrile. The flow rate was set at 1 ml/min, 240 nm UV detection and an injection volume of 10 µl. Linearity was obtained over a range of 0.50-200.00 µg/ml and correlation coefficients ranging from 0.998-1.000 were obtained. Average recovery ranged from 95.9-100.6 %. The LOD and LOQ values obtained from the slope of a calibration curve and the standard deviation of the response ranged from 0.0138-0.0860 µg/ml and 0.0419-0.2615 µg/ml, respectively, where lovastatin and simvastatin could be detected at a concentration similar to the other statins, but could only be quantified at a higher concentration than the remaining statins. The specificity of the method was proved as accurate and quantification of statins was found, even within the incorporation of other compounds.

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.

Exploratory data analysis of the dependencies between skin permeability, molecular weight and log P.

Molecular weight and log P remain the most frequently used physicochemical properties in models that predict skin permeability. However, several reports over the past two decades have suggested that predictions made by these models may not be sufficiently accurate. In this study, exploratory data analysis of the probabilistic dependencies between molecular weight, log P and log Kp was performed on a dataset constructed from the combination of several popular datasets. The results suggest that, in general, molecular weight and log P are poorly correlated to log Kp. However, after employing several exploratory data analysis techniques, regions within the dataset of statistically significant dependence were identified. As an example of the applicability of the information extracted from the exploratory data analyses, a multiple linear regression model was constructed, bounded by the ranges of dependence. This model gave reasonable approximations to log Kp values obtained from skin permeability studies of selected non-steroidal ant-inflammatory drugs (NSAIDs) administered from a buffer solution and a lipid-based drug delivery system. A method of testing whether a given drug falls within the regions of statistical dependence was also presented. Knowing the ranges within which molecular weight and log P are statistically related to log Kp can supplement existing methods of screening, risk analysis or early drug development decision making to add confidence to predictions made regarding skin permeability.

Topical and transdermal delivery of L-carnitine.

BACKGROUND: The two types of skin aging (intrinsic and extrinsic) share important molecular features, while having distinct differences on the macromolecular level: both lead to increased production of reactive oxygen species, enhanced matrix metalloproteinase expression and decreased procollagen synthesis. L-Carnitine L-tartrate has been reported to have anti-aging effects. AIM AND METHODS: The delivery as well as the transport of L-carnitine to the target sites, i.e. stratum corneum and epidermis-dermis of female abdominal skin, with and without the use of Pheroid™ as delivery system, were investigated during this study by use of Franz diffusion cells and analysed by liquid chromatography/mass spectrometry. The presence of endogenous L-carnitine in human skin was also investigated. RESULTS AND CONCLUSION: The Pheroid™ delivery system enhanced the diffusion of L-carnitine through the skin, although the phosphate buffer solution (PBS) showed a higher concentration of the active agent in the skin layers. This could be because Pheroid, being more lipophilic than PBS, improved the diffusion of L-carnitine through the skin layers, consequently leading to a lesser amount of L-carnitine delivered to the target site, i.e. the epidermis-dermis.

Correlation between in vitro release from topical delivery vehicles and microbicidal activity of triclosan.

This study reports the formulation, stability, in vitro release and microbicidal activity of a cream, emulsion, foot gel, cover stick and after sun spray containing triclosan. Triclosan is a broad-spectrum antimicrobial agent with activity against a wide range of both gram-negative and gram-positive bacteria that has found increasing popular use in personal care products. These products were stable for up to 3 months when stored at 5, 25, and 40 degrees C. Antimicrobial zone inhibition tests showed that that was a liner relationship, R2 > 0.92, between the release of triclosan from these products and the size of the inhibition zones. This means the in vitro/in vivo correlation for these products was good and that release studies can be used to predict the antimicrobial activity of triclosan.

Discrimination between the functional and biochemical effects of two herbal oxytocics on the rat myometrium.

This study on the rat myometrium is the first report where the effects of herbal extracts used as oxytocics in traditional medicine have been systematically analysed in the same preparation at the level of functional (contractile) and biochemical (second messenger generation) responses. Extracts of Agapanthus africanus and Clivia miniata (used in South African traditional medicine) were compared with other uterotonic agents with regard to their ability to stimulate phosphoinositide metabolism in the rat myometrium and cause accumulation of [3H]inositol phosphates. The maximal contractile response of the isolated rat myometrium in response to stimulation by the herbal extracts and agonists was compared with the maximal contractile response to cumulative addition of acetylcholine. The rank order of intensity of stimulation of [3H]inositol phosphate generation was: oxytocin > Agapanthus > prostaglandin F2alpha(PGF2alpha) > serotonin > acetylcholine > Clivia > ergometrine. This differed from the rank order of maximum contractile response: oxytocin > acetylcholine > PGF2alpha > serotonin approximately Clivia > Agapanthus > ergometrine. Activity was also identified in chemical fractions of the plants and components common to both plants have been identified in the isolated active fractions. These results have identified that the uterotonic activity of Agapanthus is linked to increased turnover of phosphoinositides as a signal transduction mechanism, whereas this appears to play a less significant role in the uterotonic activity of Clivia. This study illustrates the benefits of using the measurement of stimulation of phosphoinositide metabolism as a bioassay in phytomedical research.

Solid-phase extraction and HPLC determination of levamisole hydrochloride in sheep plasma.

The anthelmintic, levamisole, was determined in sheep plasma by means of ion-pair solid-phase extraction (SPE) and reverse-phase liquid chromatography. The SPE columns were conditioned with 2 ml of methanol followed by 1 ml of octane sulphonic-acid buffer. After sample application, the columns were washed with 2 ml of the same buffer, followed by elution with 90/10 acetonitrile:buffer. A phenyl reverse-phase column (Spherisorb S5 Phenyl, 250 x 4.6 mm) was used with a mobile phase of acetonitrile: 0.005 M of octane sulphonic-acid sodium salt and 0.2% triethylamine in water, pH 3.5, 38/62. Extraction recoveries of 89-94% were achieved over the range from 100-3750 ng/ml. Accuracy and precision were better than 96% and 2.6%, respectively, over said range, with a limit of quantitation of 50 ng/ ml.

Fluorimetric method of analysis for D-norpseudoephedrine hydrochloride, glycine and L-glutamic acid by reversed-phase high-performance liquid chromatography.

The determination of D-norpseudoephedrine HCl, an appetite suppressant, and glycine and L-glutamic acid, both dietary supplements, in pharmaceutical formulations and dissolution media using reversed-phase high-performance liquid chromatography (HPLC) combined with fluorimetric detection is reported. A reagent solution containing omicron-phthalaldehyde and a reducing agent, mercaptoethanol, appeared to be the most favourable reagent for derivatising the three compounds. The use of this HPLC method allowed for selective and quantitatively accurate analysis and was sufficiently specific, precise and sensitive for analytical characterisation.

Paired-ion extraction and high-performance liquid chromatographic determination of diminazene in cattle plasma: a modified method.

The high-performance liquid chromatographic method published by Aliu & Odegaard (1983) was found to give poor peak separation when used to determine plasma diminazene concentrations in cattle. Before bioequivalence studies could be carried out, the method had to be modified. Solid-phase extraction with acetonitrile/0.025 M Na-octane sulphonate and 2% acetic acid as eluent, followed by sample concentration, gave recoveries of > 90% for diminazene and the internal standard. A mobile phase of acetonitrile/0,005 M Na-octane sulphonate, 0.1% triethylamine, pH 3.2 with acetic acid on a Nova Pak C18 column was used for the analysis. Wavelength switching was used to determine the internal standard (imidocarb) and diminazene at their respective wavelengths of maximum absorbance, resulting in a fivefold increase in the limit of detection for diminazene. The modified method attained a detection limit of 2 ng.m.-1 (peak 4x baseline noise), limit of quantitation of 10 ng.m.-1 (coefficient of variation < 15%) and an accuracy of > 96% over the range from 10-5000 ng.m.-1.

A bioequivalence and pharmacokinetic evaluation of two commercial diminazene aceturate formulations administered intramuscularly to cattle.

The bioequivalence of the diminazene formulation Veriben (Centaur) was determined in cattle (n = 10) by means of a single-dose, randomized cross-over experiment. The results of nine statistical procedures commonly used for bioequivalence evaluation are discussed. Veriben was found to be equivalent to Berenil (Hoechst) with respect to the area under the plasma concentration versus time curve, but not in terms of the maximum plasma drug concentration and the time to maximum plasma drug concentration. Pharmacokinetic parameters were calculated in which bioequivalence data (n = 10) together with data from an additional four cattle were used. A two-compartment model best described the pharmacokinetic behaviour of diminazene in cattle. Peak concentrations of diminazene (3.24 +/- 0.16 micrograms/ml) were reached 49.8 (+/- 7.6) min after intramuscular injection of 3.5 mg/kg drug, with absorption proceeding rapidly (t1/2 alpha = 1.93 +/- 0.95 h). Diminazene was slowly eliminated (t1/2 beta = 222 h), resulting in a mean residence time of 13.27 d. The safe interval necessary between successive treatments of diminazene or before live babesia vaccines should be administered, and a recommended pre-slaughter withdrawal period are also discussed.