Application of dermal microdialysis for the determination of bioavailability of clobetasol propionate applied to the skin of human subjects.

Dermal microdialysis was used to assess the bioavailability of a topical corticosteroid, clobetasol propionate, following application onto the skin of human subjects. The penetration of clobetasol propionate from a 4% m/v ethanolic solution applied onto 4 sites on one forearm of healthy human volunteers was studied. A lipid emulsion, Intralipid®, was used as the perfusate and linear microdialysis probes with a 2-kDa cutoff were inserted intradermally at the designated sites. The results indicated that Intralipid could be used as a suitable perfusate for in vivo microdialysis of this lipophilic drug of interest. Furthermore, the study clearly demonstrated the application of dermal microdialysis as a valuable tool to assess the bioavailability/bioequivalence of clobetasol propionate penetration into the skin following topical application.

Investigation of the antioxidant activity of African potato (Hypoxis hemerocallidea).

African potato (AP) is widely used as an immune booster for the treatment of various ailments. The norlignan glycoside hypoxoside, a major phytoconstituent of AP, its aglycon rooperol, and an aqueous preparation of lyophilized AP corms were screened for in vitro antioxidant activity using the DPPH (1,1-diphenyl-2-picryl hydrazine) and FRAP (ferric reducing ability of plasma) tests. Inhibition of quinolinic acid (QA) induced lipid peroxidation in rat liver tissue was studied in vitro using the thiobarbituric assay (TBA). Superoxide free radical scavenging activity was determined by the nitroblue tetrazolium assay. An isocratic HPLC method was developed to quantitatively determine both hypoxoside and rooperol concurrently. While rooperol and AP extracts reduced QA-induced lipid peroxidation in rat liver homogenates and significantly scavenged the superoxide anion at pharmacological doses, in comparison, hypoxoside was virtually devoid of activity. Since hypoxoside is converted to rooperol in vivo following administration of AP, the results indicate that the hypoxoside component in AP could have value as an antioxidant prodrug.

Application of reverse-flow micellar electrokinetic chromatography for the simultaneous determination of flavonols and terpene trilactones in Ginkgo biloba dosage forms.

A reverse-flow micellar electrokinetic chromatographic (RF-MEKC) method was developed for the simultaneous qualitative determination of 10 components consisting of the flavonol glycosides, rutin and quercitrin, the flavonol aglycones, isorhamnetin, kaempferol and quercetin, the terpene trilactones, ginkgolides A, B, C and J and the sesquiterpene, bilobalide. This method was used to fingerprint Ginkgo biloba solid oral dosage forms and validated for the quantitation of the marker compounds, rutin and quercetin in some commercial products. In addition to the usual variables, the influence of some essential background electrolyte (BGE) components such as sodium dodecyl sulphate (SDS) and -cyclodextrin concentrations were investigated. A polyimide fused-silica square capillary column (75 microm I.D. x 360 microm O.D.) with a total length of 60.0 cm and effective length of 45.0 cm was used for the separation. The final BGE consisted of 20 mM phosphoric acid, 40 mM SDS and 12 mM -cyclodextrin (pH 2.2) using reverse polarity with a voltage of -17.5 kV. Samples were injected electrokinetically at -5 kV for 3 s for the qualitative analysis and hydrodynamically at 20 mbar for 0.6 s for the quantitative assay. The total run time was 22 min and the limits of detection were 3.13 microg/ml and 1.88 microg/ml for rutin and quercetin, respectively. Fingerprint profiles of the solid oral dosage forms and the results of the quantitative analysis indicated that there were major discrepancies in the marker content between products and illustrates the value of this method for use as a procedure to assess product quality of commercially available Ginkgo biloba products.

The challenges of involving traditional healers in HIV/AIDS care.

In sub-Saharan Africa, traditional healers play a major role in providing for the needs of people, particularly in rural areas where western health care is unavailable. Despite a paucity of reliable figures to determine the prevalence of traditional medicine usage, it is estimated that some 70% of sub-Saharan Africans access traditional healers. There is now mounting evidence of the importance of involving traditional healers in the management of the HIV/AIDS epidemic--both for their potential benefits, although poorly researched and understood, and to reduce the impact that some traditional healing interventions may play on the spread of HIV/AIDS and unsafe treatment of infected patients. While there are few collaborative projects between traditional healers and biomedical health providers, there is an enthusiasm on the part of traditional healers to collaborate and learn from their western-trained counterparts. Collaboration is essential, given the changing epidemic of HIV and the dynamic relationship between the two health sectors.

Generic substitution: the use of medicinal products containing different salts and implications for safety and efficacy.

In their quest to gain early entry of new generic products into the market prior to patent expiration, one of the strategies pursued by generic drug product manufacturers is to incorporate different salts of an approved active pharmaceutical ingredient (API) in a brand company's marketed dosage form and subject such dosage forms to bioequivalence assessment. These initiatives present challenges to regulatory authorities where the decision to approve bioequivalent products containing such pharmaceutical alternatives must be considered in the light of safety and efficacy, and more particularly, with respect to their substitutability. This article describes the various issues and contentions associated with the concept of pharmaceutical alternatives, specifically with respect to the uses of different salts and the implications for safety, efficacy and generic substitution.

High-performance liquid chromatographic method for the quantitative determination of Hypoxoside in African potato (Hypoxis hemerocallidea) and in commercial products containing the plant material and/or its extracts.

Hypoxoside is a norlignan diglucoside present in the corms of African potato (Hypoxis hemerocallidea). The latter is used as a popular African traditional medicine for it's nutritional and immune-boosting properties. A reverse phase high-performance liquid chromatography method was developed and validated for the determination of hypoxoside using a mobile phase consisting of acetonitrile:water (20:80, v/v). The method was linear throughout the range of 10-100 microg/mL and provided a high degree of accuracy (100 +/- 4%). The recovery of the method was found to be 100 +/- 5%, and the precision of the study, % relative standard deviation intraday and interday (over three separate days), was better than 6.15 and 5.64%, respectively. The limits of detection and quantification were calculated to be 0.75 and 3.5 microg/mL, respectively. This method was applied to the analysis and quality control of African potato corms as well as 12 commercially available products. The daily intake of hypoxoside through traditionally prepared African potato decoction was also evaluated.

Determination of terpene trilactones in Ginkgo biloba solid oral dosage forms using HPLC with evaporative light scattering detection.

A reversed phase high performance liquid chromatographic method with evaporative light scattering detection (RP-HPLC-ELSD) was developed for the quantitative determination of the terpene trilactones, ginkgolide A, B, C and J and the sesquiterpene, bilobalide in Ginkgo biloba solid oral dosage forms. Separation was achieved using a minibore Phenomenex Luna (5 microm) C18 column with dimensions 250 mm x 2.00 mm maintained at a temperature of 45 degrees C. A simple gradient method using a mobile phase of methanol:water and a flow rate of 350 microl/min facilitated baseline separation of the selected marker compounds within 14 min. The ELSD parameters affecting the detector response were optimized prior to the validation. The limits of detection and quantification were 31.25 and 62.50 ng, respectively. The percentage relative errors of the recovery ranged between -3.16 and +1.88 and both intra-day and inter-day percentage standard deviations were all better than 6%. This method was used to assay commercially available Ginkgo biloba products and proved to be suitable for the routine analysis of such products for quality control purposes.

Determination of stigmasterol, beta-sitosterol and stigmastanol in oral dosage forms using high performance liquid chromatography with evaporative light scattering detection.

A validated and repeatable high performance liquid chromatography (HPLC) method with online evaporative light scattering (ELSD) was developed for the analysis of two sterols, stigmasterol, beta-sitosterol and a stanol, stigmastanol, found to be common in many herbal formulations and health care supplements. The method is based on the separation of the three marker compounds on a C8 column (Phenomenex Luna, 5 microm, 150 mmx4.6 mm i.d.) using methanol:water (95:5 v/v) as the mobile phase, and a flow rate of 1 ml/min to separate all the marker compounds within 12 min. Cholesterol (50 microg/ml) was used as internal standard and methanol as the extraction solvent. The ELSD response parameters were optimised and the limits of detection (LOD) and quantification (LOQ) were calculated to be 2 and 5 microg/ml, respectively, which is more sensitive than obtained by photo diode array detection (5 and 7 microg/ml). Using ELSD, the percentage relative standard deviation (%R.S.D.) of intra-day and inter-day (3 days) precision for each marker was better than 3%, the accuracy data were within 97-103% and the recovery data were found to be within 95-107% for the five commercially available products examined. This method was used to assay commercially available products formulated as oral dosage forms purported to contain African Potato and associated sterols and stanol and proved to be suitable for the routine analysis and quality control of such products.

An LC-MS-MS method for the determination of cyclizine in human serum.

Cyclizine is a piperazine derivative with anti-emetic activity that is useful in the prevention and treatment of nausea and vomiting associated with motion sickness. A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method is presented for the quantitation of cyclizine in serum. Sample pretreatment involved liquid-liquid extraction of 200 microl of serum with dichloromethane after the addition of 100 microl each of ammonium hydroxide and internal standard solutions. The extracts were analyzed by HPLC on a Luna C18 reversed-phase column and an ion-trap mass spectrometer with an electrospray interface. A limit of detection of 1 ng/ml was determined which allowed for the reliable measurement of cyclizine in the serum of human subjects. The method was found to be linear over the calibration range of 2.5-100 ng/ml. The applicability of this method was demonstrated by the analysis of serum obtained from a human volunteer following administration of a single 50 mg cyclizine hydrochloride tablet. The reported method was observed to have the necessary sensitivity, selectivity, precision and accuracy for monitoring cyclizine concentrations in human subjects following oral administration.

The simultaneous determination of selected flavonol glycosides and aglycones in Ginkgo biloba oral dosage forms by high-performance liquid chromatography-electrospray ionisation-mass spectrometry.

An accurate, precise and sensitive liquid chromatography-tandem mass spectrometric (LC-MS-MS) method was developed for the determination of two flavonol glycosides, rutin and quercitrin, together with the algycone markers, quercetin, kaempferol and isorhamnetin in several Ginkgo biloba solid oral dosage forms. In addition, a novel quercetin glycoside, not yet reported in Ginkgo extracts, was identified. Liquid chromatography was performed using a minibore high-performance liquid chromatography (HPLC) column (150 mm x 2.0mm i.d.) and a one step gradient of acetonitrile-formic acid (0.3%) at a flow rate of 0.5 ml/min. Baseline separation of the five selected flavonol marker compounds was achieved within 20 min at 45 degrees C. Tandem mass spectrometry was performed using electrospray ionisation (ESI) in the negative ion mode. The marker compounds exhibited linearity over the range of 3-26 microg/ml and intra- and inter-day standard deviations were better than 7% and 16%, respectively. All Ginkgo products investigated were found to contain varying amounts of target analytes.

Steady state pharmacokinetics and dose-proportionality of phenylpropanolamine in healthy subjects.

The pharmacokinetics of phenylpropanolamine (PPA) were studied in five healthy male volunteers after single oral doses of 25, 50 and 100 mg of the drug as well as at steady state after seven, 4-hourly doses of PPA. The peak serum concentrations and AUC infinity values increased linearly with an increase in dose, whereas the time to reach peak serum concentrations did not vary significantly between doses. The half-life remained relatively constant with an increase in dose (t1/2 = 3.8 to 4.3 hours), as did renal clearance (ClR = 0.41 to 0.44 l/kg/h). The percentage of unchanged PPA excreted in the urine over a 14 hour period was 64%, 63% and 73% for the 25, 50 and 100 mg doses, respectively. The pharmacokinetics of PPA were found to be linear in the dosage range 25 to 100 mg. Steady state serum concentrations were significantly higher than single dose concentrations, with the mean peak serum concentration increasing from 113 ng/ml after a single dose to 183 ng/ml at steady state. The time at which these were attained decreased from 1.47 hours after a single dose to 0.73 hours at steady state. Both clearance and volume of distribution were significantly different after a single dose compared to steady state (P less than 0.05), whereas no significant differences were found between the other parameters.

Pharmacokinetics of oral decongestants.

Only three drugs are commonly used as oral decongestants--phenylpropanolamine (PPA), pseudoephedrine (PDE), and phenylephrine (PE). They are all chiral drugs that exist as stereoisomers. It is possible that each enantiomer can reflect significant enantioselective differences with regard to both pharmacokinetic and pharmacodynamic effects. Both PPA and PDE are readily and completely absorbed, whereas PE, with a bioavailability of only approximately 38%, is subject to gut wall metabolism and is thought to be absorbed erratically. Peak concentrations are reached between 0.5 and 2 hours after administration. All three drugs are extensively distributed into extravascular sites (apparent volume of distribution between 2.6 and 5.0 L/kg). No protein-binding data in humans are available. Whereas PPA and PDE are not substantially metabolized, PE undergoes extensive biotransformation in the gut wall and the liver. Elimination of PPA and PDE is predominantly renal, with urinary excretion being pH dependent. Half-lives are relatively short, approximately 2.5 hours for PE, 4 hours for PPA, and 6 hours for PDE. Elimination of PPA and PDE may be rapid in children, and the agents should be used with caution in patients with renal impairment. In addition, PPA increases caffeine plasma levels and decreases theophylline clearance. Reduced metabolism of PE occurs with concurrent administration of monoamine oxidase inhibitors. No direct relationship between nasal decongestant effect and plasma concentration has been established.

Analysis of macrolide antibiotics.

The following macrolide antibiotics have been covered in this review: erythromycin and its related substances, azithromycin, clarithromycin, dirithromycin, roxithromycin, flurithromycin, josamycin, rokitamycin, kitasamycin, mycinamycin, mirosamycin, oleandomycin, rosaramicin, spiramycin and tylosin. The application of various thin-layer chromatography, paper chromatography, gas chromatography, high-performance liquid chromatography and capillary zone electrophoresis procedures for their analysis are described. These techniques have been applied to the separation and quantitative analysis of the macrolides in fermentation media, purity assessment of raw materials, assay of pharmaceutical dosage forms and the measurement of clinically useful macrolide antibiotics in biological samples such as blood, plasma, serum, urine and tissues. Data relating to the chromatographic behaviour of some macrolide antibiotics as well as the various detection methods used, such as bioautography, UV spectrophotometry, fluorometry, electrochemical detection, chemiluminescence and mass spectrometry techniques are also included.

Variability in the absorption and disposition of erythromycin estolate in humans.

The inter- and intrapersonal variability in the absorption and disposition of erythromycin estolate in humans was assessed by comparing total erythromycin serum concentrations in five subjects who each received a single erythromycin estolate tablet on three separate occasions under identical experimental conditions. Coefficients of variation for the pharmacokinetic parameters Cmax, Tmax, AUC0-12, MRT, and t1/2 were of a similar magnitude when calculated between subjects in any one administration phase or between phases. In addition, serum concentrations of both erythromycin base (active component) and erythromycin propionate were selectively determined in the same subjects only during the final two phases of the study. Comparison of AUC0-12 values showed that the percentage of the total area (base and ester components) present as erythromycin base remained relatively constant between administrations (mean values of 14.5 and 11.2%), with low coefficients of variation between subjects (10.8 and 11.6%). Discrepancy value calculations revealed that the variability in serum concentration-time profiles between the three phases of the study fell into the "small"-to-"moderate" classification. From these data it is apparent that intrapersonal variations in the absorption and disposition of erythromycin estolate are of a similar magnitude to interpersonal variations and should be borne in mind in the design and interpretation of comparative bioavailability studies involving similar formulations of this compound. The relative proportions of erythromycin base and propionate did, however, remain fairly constant both between treatments and between subjects.

Determination of erythromycin in serum and urine by high-performance liquid chromatography with ultraviolet detection.

A high-performance liquid chromatographic analysis of erythromycin in human serum and urine with UV detection at 200 nm is presented. The method involves a solid-phase extraction procedure followed by a simple phase separation step and chromatography on a reversed-phase column. The method has sensitivity limits of 0.25 and 1.0 micrograms/mL in serum and urine, respectively, and is sufficiently sensitive to monitor concentrations of erythromycin in human serum and urine after the administration of a single 500-mg erythromycin stearate tablet.

Determination of phenylpropanolamine in serum and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic analysis of phenylpropanolamine in human serum and urine without prior derivatization is presented. Using direct UV detection the method is sufficiently sensitive to detect 25 ng of drug/ml of serum or urine; the coefficients of variation at 25 ng/ml and 500 ng/ml were 5. 16 and 2.12, respectively, in serum. The method involves serum and urine extraction at a basic pH with chloroform, a single back-extraction, and chromatography on a reverse-phrase column. Serum and urine data following administration of a single 150-mg sustained-release tablet of phenylpropanolamine hydrochloride in six healthy volunteers demonstrates the suitability of the analytical method.

Determination of picogram nitroglycerin plasma concentrations using capillary gas chromatography with on-column injection.

A specific, sensitive, and precise capillary gas chromatographic (GC) assay capable of analyzing picogram concentrations of nitroglycerin in human plasma was developed. The analytical procedure involves a double extraction of 1 mL of plasma with pentane, after the addition of internal standard (1 ng of 2,6-dinitrotoluene), followed by evaporation and reconstitution in 50 microL of heptane. The extract (1 microL) was injected onto a capillary column using the on-column injection technique. The GC oven temperature was programmed from 120 degrees C to 180 degrees C at a rate of 5 degrees C/min. The oven temperature was then programmed to 250 degrees C and was maintained for 10 min. The nitroglycerin and internal standard retention times were 8.6 and 11.4 min, respectively. The position of the end of the capillary column inside the detector is a critical determinant of sensitivity: the column exit must be positioned such that nitroglycerin adsorption to the detector is minimized (i.e., sensitivity maximized). The assay limit of quantitation was 25 pg/mL (CV = 7.6%) using 1 mL of plasma. This GC assay, specific for nitroglycerin in the presence of its metabolites, isosorbide dinitrate, and several other drugs, may be used to quantitate plasma levels obtained after therapeutic nitroglycerin doses.

A capillary zone electrophoresis (CZE) method for the determination of cyclizine hydrochloride in tablets and suppositories.

Current compendial methods of assay for the analysis of cyclizine tablets involve the use of UV spectrophotometry. Since this is a non-selective technique its application to more complex dosage forms, such as suppositories, is unlikely to be appropriate. There is therefore a need for the development of a highly specific quantitative analytical method, such as high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). The latter technique was chosen in view of some specific advantages over HPLC, such as the use of relatively non-toxic aqueous buffers, as opposed to organic solvents, which obviates the use of expensive HPLC grade solvents making CE more cost effective. Cyclizine was analyzed in 50mM phosphate buffer (pH 2.3) and run at an applied voltage 25 kV. Detection sensitivity was enhanced by using a wavelength of 200 nm and samples were loaded hydrodynamically onto an uncoated fused-silica capillary (60 cm x 50 mm i.d.). Chlorcyclizine was used as the internal standard and resolution of both compounds was achieved in less than 7 min. Stress testing was undertaken in order to investigate the appearance of breakdown products. The method has the requisite accuracy, selectivity, sensitivity and precision to assay cyclizine in tablets and suppositories. Degradation products resulting from the stress studies did not interfere with the detection of cyclizine and the assay is thus stability-indicating.

Multidrug assay method for antimalarials.

A general separation strategy, involving solid-phase extraction followed by reversed-phase ion-pairing HPLC with an organic counter ion for a set of 11 widely used antimalarial drugs and metabolites has been developed. The basis underlying the separation has been explored and work, including quantitative data, has been carried out on illustrative separations which form the basis of novel quantitative assays of groups of antimalarials which are relevant to current prophylaxis and treatment of malaria.