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.

A capillary zone electrophoresis method for the quantitative determination of hypoxoside in commercial formulations of African potato (Hypoxis hemerocallidea).

Hypoxoside is a norlignan diglucoside present in the corms of African potato, Hypoxis hemerocallidea, used as a popular African traditional medicine for its nutritional and immune boosting properties. A specific analytical method employing capillary zone electrophoresis has been developed and validated for the quantitative determination of this analyte. Sulfafurazole was used as internal standard, and electrophoretic separation of both analytes could be achieved within 12 min. Linearity of the method was established within the range 5-60 microg/mL and provided a high degree of accuracy (100 +/- 3%). The recovery of the method was found to be 100 +/- 5% and the RSDs of the intra- and inter-day precision were better than 5.19 and 2.52%, respectively. The limits of detection and quantification were calculated to be 0.5 and 2 microg/mL, respectively. The described method was used for the analysis and quality control of two commercially available products containing African potato. The method can also be used to determine product stability since it could separate the hypoxoside peak from its degraded products obtained from degradation studies.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

Comparative bioavailability of josamycin, a macrolide antibiotic, from a tablet and solution and the influence of dissolution on in vivo release.

The bioavailability of josamycin from a tablet formulation (2 x Josacine 500 mg tablets) was investigated and compared with the bioavailability of a solution (containing 1 g drug and buffered at pH 4.0) following administration to six healthy human volunteers. Bioavailability profiles for the solution indicated that the drug was inherently rapidly absorbed with a mean Cmax of 1.64 +/- 0.67 mg L-1 attained at a mean tmax of 0.39 +/- 0.08 h. The AUC0-last was 1.510 +/- 0.687 mg h L-1. Bioavailability was significantly lower from the tablets than from the solution. Highly variable serum concentration-time profiles were obtained from the tablets and Cmax values ranged from 0.05 to 0.71 mg L-1 with a tmax range of 0.33-2.0 h. AUC0-last values ranged from 0.03 to 0.95 mg h L-1. Dissolution of josamycin from the tablets was generally unaffected at low pH (pH 1.2-5.0), but, rather, limited predominantly by tablet disintegration. However, dissolution was increasingly limited as the pH increased from 5.0 to 9.0. Besides poor disintegration, the particularly low intrinsic dissolution rate and solubility of josamycin at these pH values is likely to further reduce the dissolution rate. Comparison of the solution and tablet serum concentration-time profiles suggests that the absorption of josamycin from the tablets was dissolution rate limited. This is supported by the in vitro dissolution-pH topogram, which suggests that dissolution will be particularly rate limiting if dissolution of whole or parts of tablets occurs in gastro-intestinal fluid above pH 5.0.

Evaluation of the proposed FDA pilot dose-response methodology for topical corticosteroid bioequivalence testing.

PURPOSE: The American FDA has recently released a Guidance document for topical corticosteroid bioequivalence testing. The purpose of this study was to evaluate the recommendations of this document for appropriateness. The new specifications require a dose-vasoconstriction response estimation by the use of a Minolta chromameter in a preliminary pilot study to determine the parameters for use in a pivotal bioequivalence study. METHODS: The visually-assessed human skin balancing assay methodology routinely practiced in our laboratories was modified to comply with the requirements of the pilot study so that visual and chromameter data could be compared. Two different cream formulations, each containing 0.12% betamethasone 17-valerate, were used for this comparison. RESULTS: Visual data showed the expected rank order of AUC values for most dose durations whereas the chromameter data did not show similar results. The expected rank order of AUC values for both chromameter and visual data was not observed at very short dose durations. In fitting the data to pharmacodynamic models, equivalent goodness of fit criteria were obtained when several different parameter estimates were used in the model definition, however the visual data were best described by the sigmoid Emax model while the chromameter data were best described by the simple Emax model. CONCLUSIONS: The Emax values predicted by the models were close to the observed values for both data sets and in addition, excellent correlation between the AUC values and the maximum blanching response (Rmax) (r > 0.95) was noted for both methods of assessment. The chromameter ED50 values determined in this study were approximately 2 hours for both preparations. At this dose duration the instrument would not be sensitive enough to distinguish between weak blanching responses and normal skin for bioequivalence assessment purposes.

Development of a capillary electrophoretic method for the separation of the macrolide antibiotics, erythromycin, josamycin and oleandomycin.

Capillary electrophoresis (CE) provides high separation efficiency and thus is suitable for the analysis of complex mixtures of structurally similar compounds. The versatile nature of CE can be realised by controlling the chemistry of the inner capillary wall, by modifying the electrolyte composition and by altering the physicochemical properties of the analyte. A CE method has been developed for the separation of three macrolide antibiotics, erythromycin, oleandomycin and josamycin. A systematic approach was used to maximise analyte differential electrophoretic mobility by manipulating electrolyte pH, molarity and composition. In addition, some instrumental parameters such as capillary length and diameter and applied voltage were varied. The effect of the sample solvent and on-capillary concentrating techniques such as field amplified sample injection were investigated. Also, the influence of the injection of a water plug on the quantity of sample injected was demonstrated. The macrolides were completely resolved in less than 30 min in a 100 cm x 75 microm I.D. fused-silica uncoated capillary with a Z-shaped flow cell of path-length 3 mm. The analysis was performed in a 75 mM phosphate buffer (pH 7.5) with 50% (v/v) methanol and an applied voltage of 25 kV was selected to effect the separation.

Determination of erythromycin and related substances by capillary electrophoresis.

Current compendial methods of assay for the analysis of erythromycin and its related substances involve the use of microbiological techniques. These techniques are non-selective and tedious, thus there is a need for the development of highly specific, quantitative analytical methods. Erythromycin was analysed in a 50 mM phosphate buffer (pH 7.5) and run at an applied voltage of 20 kV. Detection sensitivity was enhanced by using a wavelength of 200 nm and selecting an injection solvent of lower conductivity than the electrolyte: acetonitrile-water (20:80, v/v). In order to facilitate the separation of erythromycin and its related substances, the organic solvent ethanol (35%, v/v) was incorporated into a modified 150 mM phosphate buffer (pH 7.5) and run at an applied voltage of 30 kV. Resolution of all the compounds was achieved in approximately 45 min. The methods described are accurate and precise and thus suitable for the quantitative determination of erythromycin and the related substances, erythromycin C, anhydroerythromycin and N-demethylerythromycin A.