UV spectrophotometric method for simultaneous estimation of betamethasone valerate and tazarotene with absorption factor method: Application for in-vitro and ex-vivo characterization of lipidic nanocarriers for topical delivery.

The present study elucidates the development of an accurate, precise and simple simultaneous estimation method for the routine analysis of Betamethasone Valerate (BV) and Tazarotene (TZ). This combination is widely used in the treatment of psoriasis. No method has been reported so far for the simultaneous estimation of BV and TZ in topical dosage forms. The method proposed by this study for the quantification of BV and TZ is the Absorption factor method. The developed method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline. The validated method was found to be linear in a concentration range of 10-38 µg/mL and 4-14 µg/mL for BV and TZ respectively with a regression coefficient >0.990. The method was validated for accuracy and precision which revealed the recovery of >99.80% with RSD <2.0. The method was found to be precise with RSD <2% for inter and intraday. The developed method was employed for quantification of BV and TZ in lipid based nanocarriers formulation and their in-vitro drug release samples. Further, the developed method was successfully applied for the estimation of BV and TZ in the ex-vivo skin matrix. This showed that the method can sensitively determine the drugs in aqueous and biological samples.

Stability indicating liquid chromatographic method for simultaneous quantification of betamethasone valerate and tazarotene in in vitro and ex vivo studies of complex nanoformulation.

Low-potency corticosteroid betamethasone valerate and vitamin-A tazarotene are used in combination for effective treatment of psoriasis. There is no robust high-performance liquid chromatography analytical technique available for simultaneous estimation of betamethasone valerate and tazarotene in conventional and nanocarriers based formulations. A simple, accurate, robust isocratic high-performance liquid chromatography method was developed for simultaneous estimation of betamethasone valerate and tazarotene in topical pharmaceutical formulations. The developed method was validated as per the regulatory guidelines. The validated method was linear over the concentration range of 150-6000 ng/mL (r2  > 0.999) at 239 nm wavelength. Limits of detection and quantification of two analytes were 50 and 150 ng/mL, respectively. The %relative standard deviation for intraday and interday precision was less than 2%. The method was also evaluated in the presence of forced degradation conditions. The developed method was successfully applied for in vitro and ex vivo drug release studies of in-house designed nanoformulations.

Simultaneous HPLC Determination of Betamethasone Esters-Containing Mixtures: Analysis of Their Topical Preparations.

Topical pharmaceutical preparations containing betamethasone esters are widely prescribed for treatment of severe inflammatory skin conditions. Some betamethasone esters-containing preparations are formulated with either an antibacterial or an antifungal agent or a vitamin D3 derivative. A fast reversed-phase high-performance liquid chromatography method has been developed for the simultaneous determination of three betamethasone esters-containing binary mixtures along with the excipients of their dosage forms using clobetasone butyrate as internal standard. The first mixture was betamethasone valerate and fusidic acid (Mixture I) with chlorocresol as preservative. The second mixture was betamethasone dipropionate (BTD) and clotrimazole (Mixture II) with benzyl alcohol as preservative. The third mixture was BTD and calcipotriol monohydrate (Mixture III). Optimized chromatographic separation was achieved on a Discovery® C18 (4.6 × 250 mm, 5 µm) column, using water: acetonitrile (35:65, v/v) as mobile phase at flow rate of 1 mL/min with UV detection at 230 nm. The method was validated according to ICH guidelines. The regression coefficients were > 0.999 for all drugs. The method was successfully applied for the determination of the studied drugs in bulk, synthetic mixtures and dosage forms. The developed method is accurate, sensitive, selective and precise and can be used for routine analysis in quality control laboratories.

Simultaneous Determination of Cinchocaine Hydrochloride and Betamethasone Valerate in Presence of Their Degradation Products.

Cinchocaine hydrochloride (CIN) and betamethasone valerate (BMV) are co-formulated in pharmaceutical formulations that could be used for local treatment of hemorrhoids. Both drugs are susceptible to hydrolytic degradation. Two sensitive and precise stability-indicating chromatographic methods were developed for the simultaneous determination of both active pharmaceutical ingredients. The developed methods were applied for quantitation of CIN and BMV in their pure forms, in presence of their corresponding degradation products and in their pharmaceutical formulation. The first method was a high performance liquid chromatographic (HPLC) one, separation and quantitation was achieved using a Waters Spheriosorb® 5 µm ODS2 C18 analytical column and an isocratic mobile phase formed of acetonitrile-acetate buffer (pH 6.5 ± 0.1) in a ratio of (55:45, v/v). The mobile phase was pumped at a flow rate of 1.2 mL/min. UV-detection was done at 240 nm using photodiode array detector. The second method was based on thin layer chromatography (TLC) fractionation coupled with densitometric determination. Separation was done on high performance thin layer chromatography (HPTLC) silica gel 60F254 plates using a developing system formed of chloroform-toluene-ethanol-acetic acid in a ratio of (4.5:4.5:1:1, by volume). The separated bands were scanned densitometrically at 240 nm. For the HPLC method, linearity was confirmed over concentration ranges of 4-300 and 4-350 µg/mL for CIN and BMV, respectively. For the HPTLC-densitometric method, the obtained ranges were 0.5-12 and 0.5-10 µg/band for CIN and BMV, respectively. The developed methods were optimized and validated according to the ICH guidelines. CIN acid degradation products were separated and identified by mass spectroscopy. The developed HPLC method was used to study the kinetics of acid and alkali degradation of the both drugs. The results obtained were statistically analyzed and compared with those obtained by applying the official methods for both drugs.

Development and validation of an UHPLC method for the determination of betamethasone valerate in cream, gel, ointment and lotion.

An ultra high performance liquid chromatographic method has been developed and validated for the determination of betamethasone valerate (BMV) in topical dermatologic formulations. For the development of the method, response surface methodology based on a three-level full factorial design was used. The eluent composition, the column dimension and the flow rate were chosen as relevant experimental parameters to investigate. The response surface plots revealed an optimum separation by using a RP column (30 mm × 2 mm i.d., 2.2 µm particle size), at 30 °C; isocratic mobile phase consisting of acetonitrile:water (60:40) at a flow rate of 0.2 mL min(-1) and a wavelength set at 254 nm. The proposed method was validated for four types of matrices according to ICH guidelines requirements. Dexamethasone acetate (DMA) was used as internal standard. Linearity was studied in the range of 5-200 µg mL(-1) for BMV in spiked matrix samples. Recoveries were in the range of 95-105% and precision was better than 5% for both analytes, either in cream, gel, ointment, or lotion formulations, when using simple sample preparation. Retention times were 0.95 min for DMA and 1.40 min for BMV, demonstrating a short method run time. The method was successfully applied for routine analysis of dermatological formulations containing betamethasone valerate.

An RP-HPLC Method for the Stability-Indicating Analysis of Impurities of Both Fusidic Acid and Betamethasone-17-Valerate in a Semi-Solid Pharmaceutical Dosage Form.

A topical pharmaceutical cream containing the active pharmaceutical ingredients (APIs) betamethasone-17-valerate and fusidic acid has been developed for the treatment of inflammatory skin conditions and associated secondary infections. In this work, a novel stability-indicating RP-HPLC method has been developed for the simultaneous quantitation of impurities of both APIs present in this cream. The HPLC column was a 150 mm × 4.6 mm I.D. YMC-Pack Pro C18 column with 3 µm particles. The column-oven temperature was maintained at 40°C and UV detection at 235 nm was used. A gradient programme was employed at a flow rate of 0.7 mL/min. Mobile phase A comprised of a 16:21:21:42 (v/v/v/v) mixture of methanol, 10 g/L phosphoric acid, HPLC grade water and acetonitrile. Mobile phase B comprised of a 24:5:5:66 (v/v/v/v) mixture of methanol, 10 g/L phosphoric acid, HPLC grade water and acetonitrile. The method has been validated according to current International Conference on Harmonisation (ICH) guidelines and applied during formulation development and stability studies. The procedure has been shown to be stability-indicating for the topical cream.

Formulation considerations in the design of topical, polymeric film-forming systems for sustained drug delivery to the skin.

Polymeric film-forming systems (FFSs) are potential drug delivery systems for topical application to the skin. The FFSs form thin and transparent polymeric films in situ upon solvent evaporation. Their application convenience and cosmetic attributes, superior to conventional semi-solids, may offer improved patient compliance. This study represents the first phase of an investigation into the use of FFSs for prolonged dermal drug delivery. FFS formulations were distinguished based on their ability to sustain the release of betamethasone 17-valerate (BMV) in vitro over 72 h. The effect of film-forming polymer (hydrophilic: hydroxypropyl cellulose (Klucel™ LF); hydrophobic: polymethacrylate copolymers (Eudragit® NE and Eudragit® RS), and polyacrylate copolymer (Dermacryl® 79) was first determined, and then the impact of incorporation of plasticisers (triethyl citrate, tributyl citrate, and dibutyl sebacate) was examined. The Klucel film released a significantly higher amount of BMV than the hydrophobic FFS, 42 versus 4 µg/cm(2), respectively. The release was increased when a plasticiser was incorporated, and with higher enhancement ratios achieved with the more lipophilic plasticisers. In conclusion, the results show that FFSs can sustain drug release (hence representing useful systems for prolonged dermal therapy) and emphasise the importance of the formulation on drug delivery, with the type of polymer being of greatest significance.

Spectrophotometric methods for simultaneous determination of betamethasone valerate and fusidic acid in their binary mixture.

Five spectrophotometric methods were successfully developed and validated for the determination of betamethasone valerate and fusidic acid in their binary mixture. Those methods are isoabsorptive point method combined with the first derivative (ISO Point--D1) and the recently developed and well established methods namely ratio difference (RD) and constant center coupled with spectrum subtraction (CC) methods, in addition to derivative ratio (1DD) and mean centering of ratio spectra (MCR). New enrichment technique called spectrum addition technique was used instead of traditional spiking technique. The proposed spectrophotometric procedures do not require any separation steps. Accuracy, precision and linearity ranges of the proposed methods were determined and the specificity was assessed by analyzing synthetic mixtures of both drugs. They were applied to their pharmaceutical formulation and the results obtained were statistically compared to that of official methods. The statistical comparison showed that there is no significant difference between the proposed methods and the official ones regarding both accuracy and precision.

Development and validation of a novel stability-indicating HPLC method for the simultaneous assay of betamethasone-17-valerate, fusidic acid, potassium sorbate, methylparaben and propylparaben in a topical cream preparation.

A novel stability-indicating reversed phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous assay of betamethasone-17-valerate, fusidic acid and potassium sorbate as well as methyl- and propylparaben in a topical cream preparation has been developed. A 100mm×3.0mm ID. Ascentis Express C18 column maintained at 30°C and UV detection at 240nm were used. A gradient programme was employed at a flow-rate of 0.75ml/min. Mobile phase A comprised of an 83:17 (v/v) mixture of acetonitrile and methanol and mobile phase B of a 10g/l solution of 85% phosphoric acid in purified water. The method has been validated according to current International Conference on Harmonisation (ICH) guidelines and applied during formulation development and stability studies. The procedure has been shown to be stability-indicating for the topical cream.

In vitro penetration properties of solid lipid nanoparticles in intact and barrier-impaired skin.

Treatment of skin diseases implies application of a drug to skin with an impaired epidermal barrier, which is likely to affect the penetration profile of the drug substance as well as the carrier into the skin. To elucidate this, the effect of skin barrier damage on the penetration profile of a corticosteroid applied in solid lipid nanoparticles (SLN) composed of different lipids, varying in polarity, was studied. The studies were carried out in vitro using impaired and intact porcine ear skin, and the SLN were compared with a conventional ointment. It was shown that a significantly higher amount of corticosteroid remained in the skin, intact as well as barrier impaired, when SLN was used as a vehicle. In general, the penetration profile of the drug substance into the skin was affected by the type of lipid used in the formulation and related to lipid polarity and drug substance solubility. When formulated in SLN and applied to intact skin, the permeation of the drug substance across the skin was significantly reduced, as compared to the ointment. Altogether, in both barrier-impaired and intact skin, a higher amount of drug substance remained in the skin during application of SLN for 6, 16, and 24h, as compared to the ointment. These results emphasize the applicability of SLN to create a drug reservoir in skin, with the drug localized distinctively in the stratum corneum.

Static headspace gas chromatographic method for the determination of low and high boiling residual solvents in Betamethasone valerate.

Currently, there are no analytical methods available in the literature that can simultaneously separate and quantitate residual levels of acetone, methylene chloride, n-butyl ether and dimethylsulfoxide in Betamethasone valerate active pharmaceutical ingredient (API). This paper describes the development and validation of a simple, efficient, accurate and robust static headspace gas chromatography method for the determination of high and low boiling residual solvents, namely acetone, methylene chloride, n-butyl ether and dimethylsulfoxide, in Betamethasone valerate API. This method has been demonstrated to be accurate, linear, precise, reproducible, specific and robust for its intended purpose. Quantitation limits (QL) for acetone, methylene chloride and n-butyl ether are 20 ppm (20 µg/g of API) and 50 ppm (50 µg/g of API) for dimethylsulfoxide. Several other APIs (Loratadine and a few other corticosteroid compounds) were analyzed using the conditions of this method to evaluate and assess the versatility of this method for the purpose of residual solvents analysis for a wide range of APIs. The results of this evaluation strongly indicates that this method can be readily used (as-is or with minor modifications) to determine both low and high boiling residual solvents present in a wide range of APIs.

Application of LC-MS(n) in conjunction with mechanism-based stress studies in the elucidation of drug impurity structure: rapid identification of a process impurity in betamethasone 17-valerate drug substance.

Through a case study, the use of LC-MS(n) technique in conjunction with a mechanism-based stress study is shown to be a very effective way in the rapid elucidation of unknown drug impurities. In this case, the drug substance sample was first analyzed using LC-MS(n) through which the unknown species was found to be a valeryl-containing, isomeric impurity of the active pharmaceutical ingredient (API), betamethasone 17-valerate, based on its molecular ion and major fragments. Since a substantial knowledge regarding a large number of isomeric impurities of betamethasone has been accumulated in the literature as well as in our laboratory, a hydrolytic stress study (forced degradation) of the isolated unknown species was then designed and carried out accordingly in order to remove the valeryl group from the unknown species. During the stress study, a betamethasone isomer was generated as expected. However, a new unknown species isomeric to betamethasone 17-valerate was also formed unexpectedly. By comparing the UV spectra and more importantly MS(n) fragmentation patterns of the two newly formed species with those of betamethasone, dexamethasone, betamethasone 17-valerate, and betamethasone 21-valerate, these two unknown species generated in the stress study were identified as dexamethasone and dexamethasone 21-valerate, respectively. Based on the plausible reaction mechanism of the forced degradation, the original impurity present in betamethasone 17-valerate drug substance was then identified as dexamethasone 17-valerate; the structure assignment was later confirmed by various 1D and 2D NMR experiments. The efficient conversion from dexamethasone 17-valerate to dexamethasone 21-valerate was also observed during a 2D NMR acquisition of the isolated dexamethasone 17-valerate sample.

Differential-pulse polarography (DPP) determination of betamethasone valerate in dosage form.

The electrochemical reduction of betamethasone valerate (BV) in a pharmaceutical formulation containing neomycin has been carried out in Britton-Robinson buffer (BRB) (0.04 mol L(-1)) by differential-pulse polarography (DPP). BV exhibits a well-defined irreversible reduction peak at -1.03 V/ref. The influence of pH on the reduction of BV was studied in Britton-Robinson buffer (pH range 1.7-10). A method for the analysis of BV in BRB (0.04 mol L(-1)), which allows quantification over the range 3.9x10(-6)-1.1x10(-4) mol L(-1), was proposed and successfully applied to the determination of BV in tablets with mean recovery and relative standard deviation of 100.81% and 0.45%, respectively.

A case of contact dermatitis due to impurities of cetyl alcohol.

A 29-year-old man being treated for itchy lesions on the amputation stump of the thigh became allergic to betamethasone valerate and gentamicin sulfate cream (Rinderon VG). Closed patch tests with all the ingredients of the cream revealed positive reactions to cetyl alcohol 30% to 5% pet. Gas chromatographic analysis of the cetyl alcohol in the cream base detected stearyl alcohol (C18), myristyl alcohol (C14) and lauryl alcohol (C12) in addition to the main component of cetyl alcohol (C16). Patch testing with 99% pure analytical reagent grade saturated alcohols (C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20) showed negative reactions. Thus, it is concluded that some minor impurities in cetyl alcohol not detected by gas chromatography might be the cause of this dermatitis.

On-line post-column photochemical derivatization in liquid chromatographic--diode-array detection analysis of binary drug mixtures.

HPLC methods were developed for the analysis of pharmaceutical creams containing binary drug mixtures (betamethasone valerate-chlorocresol; hydrocortisone-miconazole nitrate; desonide pivalate-chlorhexidine; dexamethasone-clotrimazole; triamcinolone acetonide-econazole nitrate). The chromatographic separations were performed on C-18 and cyano columns under reversed-phase conditions. A post-column on-line photochemical reactor (irradiation at 254 nm) was arranged between the analytical column and the diode-array detector to enhance the performance of the method. Two UV spectra (photoreactor on and off) were obtained for each analyte and these additional sources of information proved to be useful for the unambiguous identification of the various analytes. The method was applied to the quality control of commercial creams using a solid-phase extraction procedure for the sample clean-up.

[Betamethasone valerate reference standard (Control 941) of the National Institute of Health Sciences].

The raw material for betamethasone valerate was tested for preparation of the "Betamethasone Valerate Reference Standard (Control 941)". Analytical data obtained were as follows: melting point, 194.3 degrees C (decomposition); UV and infrared spectra, the same as those for JP Betamethasone Valerate Reference Standard (Control 844); optical rotation, [alpha]20D = +79.4 degrees; thin-layer chromatography and high-performance liquid chromatography (HPLC), no impurities were detected; assay, 100.0% by HPLC. Based on the above results, the candidate material was authorized as the JP Betamethasone Valerate Reference Standard (Control 941).

Are generic formulations equivalent to trade name topical glucocorticoids?

Trade name glucocorticoid formulations triamcinolone acetonide, fluocinolone acetonide, and betamethasone valerate were compared with their generic equivalents because of increasing substitution of generic formulations for trade name formulations. The vasoconstrictor assay was the method used for these comparisons. Large differences were found between generic and trade name formulations containing the same steroid in the same concentration in both cream and ointment vehicles. If generic substitutions are to be used for trade name formulations, the physician must be aware that significant differences in therapeutic effectiveness may be expected.

The stability and blanching efficiency of some Betnelan-V cream dilutions.

In this study Betnelan-V cream was diluted with Beeler's basis and Cold cream. Cold Cream reduced the skin-blanching activity of the original cream more than Beeler's base. The stability of betamethasone-17-valerate was better in a Beeler's basis dilution than in a Cold cream dilution.