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

Simultaneous HPLC Determination of Betamethasone Esters-Containing Mixtures: Analysis of Their... Abstract 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. Introduction Topical preparations containing a combination of different drug classes, having different mechanisms of action, acquires increasing interest for achieving treatment goal. Betamethasone is a corticosteroid with mainly glucocorticoid activity used either in the free alcohol form or in one of the esterified forms. Betamethasone valerate (BTV, Figure 1A) and betamethasone dipropionate (BTD, Figure 1B) are extensively used for topical application in the treatment of various skin disorders. Fusidic acid, (FSA, Figure 1C), is a steroidal antibacterial with a bacteriostatic or bactericidal activity mainly against Gram-positive bacteria. Clotrimazole, (CLO, Figure 1D), is an imidazole antifungal drug with antimicrobial activity, used in superficial candidiasis and in other skin infections as dermatophytosis. Calcipotriol monohydrate, (CAL, Figure 1E), is a vitamin D3 derivative that induces differentiation and suppresses proliferation of keratinocytes in the management of psoriasis (1). Figure 1. View largeDownload slide Chemical structures and chemical names of betamethasone valerate (A), betamethasone dipropionate (B), fusidic acid (C), clotrimazole (D), calcipotriol monohydrate (E), chlorocresol (F), benzyl alcohol (G) and clobetasone butyrate (IS) (H). Figure 1. View largeDownload slide Chemical structures and chemical names of betamethasone valerate (A), betamethasone dipropionate (B), fusidic acid (C), clotrimazole (D), calcipotriol monohydrate (E), chlorocresol (F), benzyl alcohol (G) and clobetasone butyrate (IS) (H). Betamethasone esters are widely used for their anti-inflammatory (glucocorticoid) and immunosuppressant properties in various skin conditions. They are used either alone or in combination with FSA in order to provide effective anti-inflammatory and antibacterial activities (2). Such combination is formulated with chlorocresol, (CHC, Figure 1F), a potent disinfectant and antiseptic agent which has bactericidal activity against both Gram-positive and Gram-negative bacteria. It is also used as a preservative in creams and in cosmetics (1). In addition, combination of BTD with CLO is widely used for treatment of superficial mycoses by acting as anti-inflammatory, antibacterial and fungistatic (3). It contains also benzyl alcohol, (BA, Figure 1G), used as a solubilizer and preservative. It has a bacteriostatic activity mainly against Gram-positive bacteria (1). Also, a combination of BTD with CAL showed higher efficacy in the management of mild to moderate psoriasis than each of them alone (4). Literature survey revealed that the simultaneous determination of BTV and FSA mixture has been carried out by spectrophotometric method (5), high-performance liquid chromatography (HPLC) method for drug determinations in pharmaceutical dosage form (6, 7) and in plasma (8). Spectrophotometric (9) and HPLC methods (10, 11) have been also described for analysis of BTD and CLO in binary mixture. Ternary mixture containing BTD, CLO and BZA was quantified by three spectrophotometric methods (12–14), HPLC method (15) and micellar electrokinetic chromatography method (15). Besides, BTD and CAL mixture has been determined by several HPLC methods(16–21). CHC and BTD has been determined by HPLC method (22). Many of the reported spectrophotometric methods lack sensitivity (5, 12), while the reported HPLC methods suffers from either long retention time and hence long analysis time (6, 7, 20, 21) or the use of complex gradient mobile phases, thus consuming time and cost (16, 20, 21) or lack sensitivity (19). Based on literature survey of the aforementioned reported methods, there was a need to develop and validate a reliable HPLC method for the simultaneous separation and quantification of three of the most commonly prescribed topical preparations containing betamethasone either as valerate or dipropionate ester in combination with FSA, CLO and CAL when one of the components is present in low concentration relative to the other which encounters an analytical problem in their determination. In addition, our objective was to achieve the analysis of the cited drugs in a short run time with high sensitivity and selectivity using a simple mobile phase in an isocratic elution mode and by utilizing the most commonly used reversed-phase column. The developed method succeeded in separating five drugs representing three binary mixtures in addition to two preservatives present in their dosage forms and the internal standard (IS) with good resolution factor in relatively short run time. The method also proved to be sensitive, simple, rapid and selective so it can be used for routine analysis of the cited mixtures in quality control laboratories either in bulk or in pharmaceutical preparations. Experimental Instrumentation The HPLC (Young Lin instrument Acme 9000, Korea) system was equipped with a vacuum degasser, a mixer, a gradient pump and a UV/Vis detector. Young Lin Autochrome-3000 program was used for the instrument control, data acquisition and analysis. Separation and quantitation were carried out on Discovery® C18 (4.6 × 250 mm, 5 μm) column. The mobile phase was filtered through 0.45 μm Chromtech® nylon membrane filter (UK) and degassed using Powersonic 405 (Korea). Materials and reagents BTV was kindly supplied by Amriya Pharmaceutical Ind. (its purity was certified as 99.80%). BTD and CAL were kindly supplied by Sabaa International Company (their purities were certified as 99.83% and 99.99%, respectively). Clobetasone butyrate, used as IS (Figure 1H), was kindly supplied by GlaxoSmithKline (its purity was certified as 99.75%). CLO was kindly supplied by Borg Pharmaceutical Ind. (its purity was certified as 99.73%). FSA was kindly supplied by Julphar Pharmaceutical Ind. (its purity was certified as 99.77%). CHC and BZA were kindly supplied by the National Organization of Drug Control and Research (NODCAR), Egypt (their purities were certified as 100.52% and 99.75%, respectively). Topical preparations were purchased from local pharmacy in Egypt as follows: Fucicort® cream (Batch No. GGE2353), each gram nominally contains BTV (1 mg) and FSA (20 mg); Clotrisone® cream (Batch No. 018074), each gram nominally contains BTD (0.64 mg) and CLO (10 mg) and Calcipoheal-Cort® ointment (Batch No. 50388), each gram nominally contains BTD (0.5 mg) and Cal (50 μg). HPLC grade methanol and acetonitrile were used (Sigma-Aldrich Co., France). Bi-distilled water was produced in-house (Aquatron Water Still, A4000D, UK). Chromatographic conditions Chromatographic separation was achieved using a Discovery® C18 (4.6 × 250 mm, 5 μm) column and a mobile phase consisting of water: acetonitrile (35:65, v/v). The mobile phase was filtered through 0.45 μm membrane filter and degassed for 30 min in an ultrasonic bath prior to its use. The flow rate was maintained at 1 mL/min. Analyses were performed at ambient temperature and the UV detection was carried out at 230 nm. The injection volume was 20 μL. Standard stock and working solutions Standard stock solutions (1 mg/mL) of BTV, CAL BTD, FSA and CLO were prepared separately by dissolving 100 mg of each drug in 100 mL methanol. Standard working solutions (50 μg/mL) of BTD, BTV and CAL and (40 μg/mL) of BTD, FSA and CLO were prepared by further dilution of the corresponding stock solutions with the mobile phase. Clobetasone butyrate (IS) stock solution (0.5 mg/mL) was prepared by dissolving 125 mg of clobetasone butyrate in 250 mL methanol. Samples preparation Accurate weights of fucicort® cream (1.2 g), clotrisone® cream (2 g) and calcipoheal-cort ointment (5 g) were dissolved separately and transferred by 50 mL hot n-hexane in separating funnels, mixed for 2 min then cooled. Twenty milliliters of methanol were added with shaking for 10 min. The extraction process was repeated for two more times using 10 mL methanol. At the end of the extraction process, the methanolic layer was collected in 50-mL volumetric flask and the three sample solutions were cooled at 8°C for 30 min to get rid of any traces of the hexane layer. The extraction method was guided by the British Pharmacopoeia (2016). The solutions were completed to volume with methanol to obtain a concentration equivalent to 24 μg/mL of BTV and 480 μg/mL of FSA in fucicort® sample solution, 25.6 μg/mL of BTD and 400 μg/mL of CLO in clotrisone® sample solution, and 50 μg/mL of BTD and 5 μg/mL of CAL in calcipoheal-cort® sample solution. The three sample solutions were then filtered using cotton previously washed with methanol and the first 10 mL of the filtrate were discarded. Procedure Linearity Accurately measured aliquots of standard solutions equivalent to 5–200 μg of BTV and 0.02–4 mg of FSA for (Mixture I), 5–200 μg of BTD and 0.02–3 mg of CLO for (Mixture II) and 0.005–2 mg of BTD and 10–200 μg of CAL for (Mixture III) were separately transferred into three series of 10-mL volumetric flasks. A constant aliquot (1 mL) of IS stock solution was added and solutions were completed to volume with the mobile phase. A volume of 20 μL of each solution was injected into the chromatograph in triplicates. The previously mentioned chromatographic conditions were adjusted with a run time for 12 min. A calibration curve for each drug was obtained by plotting peak area ratio (peak area of the studied drug/peak area of IS) (PAR) against concentration (C) in μg/mL and the corresponding regression equations were obtained. Assay of laboratory prepared mixtures The procedure mentioned in linearity section was repeated for the analysis of laboratory prepared mixtures containing BTV and FSA equivalent to 10–130 μg and 0.2–2.6 mg, respectively (Mixture I) and BTD and CLO equivalent to 16–176 μg and 0.25–2.7 mg, respectively (Mixture II) and BTD and CAL equivalent to 0.2–1.9 mg and 20–190 μg, respectively (Mixture III). Assay of pharmaceutical preparations For the determination of the examined drugs in Fucicort® cream, Clotrisone® cream and Calcipoheal-Cort® ointment, the sample solutions, prepared in Samples preparation section, were further diluted with the mobile phase to prepare sample working solutions containing (24–84 μg BTV and 0.48–1.68 mg FSA), (25.6–89.6 μg BTD and 0.4–1.4 mg CLO) and (0.125–0.25 mg BTD and 12.5–25 μg CAL), respectively. Then, 20 μL of each sample working solution were injected into the chromatograph, in triplicates. Results Method development Both Discovery® C18 (4.6 × 250 mm, 5 μm) and Discovery® phenyl (4.6 × 250 mm, 5 μm) columns showed good separation of all mixture components. Discovery® C18 column was the most suitable one as it achieved the intended aim showing better peaks shape with good symmetry giving reasonable retention times for the mixture components when operating under optimum conditions. Various mobile phase compositions containing different ratios of organic and aqueous phases were attempted in an isocratic mode. Both acetonitrile and methanol in different ratios were tried as organic modifier. Acetonitrile was found optimum for the elution as it showed good separation, better peaks shape and it shortened the analysis time. Phosphate buffer (25 mM KH2PO4) with different pH (3, 4.5 and 7) were tried but no more privilege was obtained in comparison with the use of water. Based on these investigations, an isocratic elution using a mobile phase composed of water: acetonitrile (35:65, v/v) was used. Good separation of the seven components in the mixture along with clobetasone butyrate (the chosen IS) was obtained at a flow rate of 1 mL/min. UV detection was carried out at 230 nm. The retention times were found to be 2.80, 3.30, 4.55, 6.05, 7.01, 8, 9.28 and 10.20 min for FSA, BZA, CHC, BTV, CAL, BTD, CLO and IS, respectively; as presented in Figure 2. Figure 2. View largeDownload slide A typical LC chromatogram of synthetic mixture of fusidic acid (FSA), benzyl alcohol (BZA), chlorocresol (CHC), betamethasone valerate (BTV), calcipotriol monohydrate (CAL), betamethasone dipropionate (BTD), clotrimazole (CLO) and clobetasone butyrate (IS). Figure 2. View largeDownload slide A typical LC chromatogram of synthetic mixture of fusidic acid (FSA), benzyl alcohol (BZA), chlorocresol (CHC), betamethasone valerate (BTV), calcipotriol monohydrate (CAL), betamethasone dipropionate (BTD), clotrimazole (CLO) and clobetasone butyrate (IS). System suitability The parameters of these tests are column efficiency represented as number of theoretical plates, peak resolution factor, tailing of chromatographic peak, selectivity factor, capacity factor, repeatability as %RSD of peak area for six injections and reproducibility of retention as %RSD of retention time. The results of these tests for the developed method are listed in Table I. Table I. System Suitability Tests for the Proposed HPLC Method Item FSA BZA CHC BTV CAL BTD CLO IS N 2570 3400 8434 9020 9828 10424 11947 12249 R     2.25     4.88     7.38     3.60     3.14      4.10      2.54 T 1.05 1.10 1.10 1.00 1.00 0.98 1.20 0.98 α     1.28     1.54     1.42     1.19     1.16      1.18      1.11 K′ 1.80 2.30 3.55 5.05 6.01 7.00 8.28 9.20 %RSD of peak area for six injections 1.03 0.84 0.95 0.87 0.65 1.01 %RSD of retention time 1.01 1.09 0.71 0.70 0.72 0.84 0.86 1.00 Item FSA BZA CHC BTV CAL BTD CLO IS N 2570 3400 8434 9020 9828 10424 11947 12249 R     2.25     4.88     7.38     3.60     3.14      4.10      2.54 T 1.05 1.10 1.10 1.00 1.00 0.98 1.20 0.98 α     1.28     1.54     1.42     1.19     1.16      1.18      1.11 K′ 1.80 2.30 3.55 5.05 6.01 7.00 8.28 9.20 %RSD of peak area for six injections 1.03 0.84 0.95 0.87 0.65 1.01 %RSD of retention time 1.01 1.09 0.71 0.70 0.72 0.84 0.86 1.00 N, number of theoretical plates; R, resolution factor; T, tailing factor; α, selectivity factor; K′, capacity factor. Table I. System Suitability Tests for the Proposed HPLC Method Item FSA BZA CHC BTV CAL BTD CLO IS N 2570 3400 8434 9020 9828 10424 11947 12249 R     2.25     4.88     7.38     3.60     3.14      4.10      2.54 T 1.05 1.10 1.10 1.00 1.00 0.98 1.20 0.98 α     1.28     1.54     1.42     1.19     1.16      1.18      1.11 K′ 1.80 2.30 3.55 5.05 6.01 7.00 8.28 9.20 %RSD of peak area for six injections 1.03 0.84 0.95 0.87 0.65 1.01 %RSD of retention time 1.01 1.09 0.71 0.70 0.72 0.84 0.86 1.00 Item FSA BZA CHC BTV CAL BTD CLO IS N 2570 3400 8434 9020 9828 10424 11947 12249 R     2.25     4.88     7.38     3.60     3.14      4.10      2.54 T 1.05 1.10 1.10 1.00 1.00 0.98 1.20 0.98 α     1.28     1.54     1.42     1.19     1.16      1.18      1.11 K′ 1.80 2.30 3.55 5.05 6.01 7.00 8.28 9.20 %RSD of peak area for six injections 1.03 0.84 0.95 0.87 0.65 1.01 %RSD of retention time 1.01 1.09 0.71 0.70 0.72 0.84 0.86 1.00 N, number of theoretical plates; R, resolution factor; T, tailing factor; α, selectivity factor; K′, capacity factor. Method validation Validation was assessed according to ICH guidelines (23). Linearity A linear relationship between peak area ratio (PAR) and component concentration (C) was obtained for each drug. The regression equation for each mixture component was also calculated. In this work, six concentrations were chosen for BTV, CAL and BTD. Seven concentrations of both FSA and CLO were used. Each concentration was repeated three times. The linearity of the calibration curves was validated by the high value of regression coefficients (>0.999). The analytical data of the calibration curve of each drug including standard deviations for the slope and intercept (Sb, Sa) are presented in Tables II–IV. Table II. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Fusidic Acid and Betamethasone Valerate (Mixture I) Item FSA BTV Retention time (min) 2.80 6.05 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 2–400 0.5–20 Regression equation PAR230nm = 0.0065 CFSA + 0.0038 PAR230nm = 0.0174 CBTV + 0.0072 Regression coefficient (r2) 0.9998 0.9992 LOD (μg/mL) 0.6364 0.1449 LOQ (μg/mL) 1.9285 0.4389 Sb 4.17 × 10−5 2.42 × 10−4 Sa 0.0090 0.0029 Confidence limit of the slope 0.0065 ± 0.0001 0.0174 ± 0.0007 Confidence limit of the intercept 0.0038 ± 0.0231 0.0072 ± 0.0081 Standard error of estimation 0.0144 0.0040 aIntra-day %RSD 0.04–0.12 0.38–0.48 bInter-day %RSD 0.06–0.13 0.21–0.62 Drug in laboratory prepared mixture 100.29 ± 0.70 100.03 ± 0.37 Drug added to dosage form 99.88 ± 0.60 100.15 ± 0.38 Drug in dosage form 100.46 ± 0.53 99.99 ± 0.70 Item FSA BTV Retention time (min) 2.80 6.05 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 2–400 0.5–20 Regression equation PAR230nm = 0.0065 CFSA + 0.0038 PAR230nm = 0.0174 CBTV + 0.0072 Regression coefficient (r2) 0.9998 0.9992 LOD (μg/mL) 0.6364 0.1449 LOQ (μg/mL) 1.9285 0.4389 Sb 4.17 × 10−5 2.42 × 10−4 Sa 0.0090 0.0029 Confidence limit of the slope 0.0065 ± 0.0001 0.0174 ± 0.0007 Confidence limit of the intercept 0.0038 ± 0.0231 0.0072 ± 0.0081 Standard error of estimation 0.0144 0.0040 aIntra-day %RSD 0.04–0.12 0.38–0.48 bInter-day %RSD 0.06–0.13 0.21–0.62 Drug in laboratory prepared mixture 100.29 ± 0.70 100.03 ± 0.37 Drug added to dosage form 99.88 ± 0.60 100.15 ± 0.38 Drug in dosage form 100.46 ± 0.53 99.99 ± 0.70 aThe intra-day (n = 3), average of three concentrations of FSA (160, 200 and 240 μg/mL) and BTV (8, 10 and 12 μg/mL) repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of FSA (160, 200 and 240 μg/mL) and BTV (8, 10 and 12 μg/mL) repeated three times in three successive days. Table II. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Fusidic Acid and Betamethasone Valerate (Mixture I) Item FSA BTV Retention time (min) 2.80 6.05 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 2–400 0.5–20 Regression equation PAR230nm = 0.0065 CFSA + 0.0038 PAR230nm = 0.0174 CBTV + 0.0072 Regression coefficient (r2) 0.9998 0.9992 LOD (μg/mL) 0.6364 0.1449 LOQ (μg/mL) 1.9285 0.4389 Sb 4.17 × 10−5 2.42 × 10−4 Sa 0.0090 0.0029 Confidence limit of the slope 0.0065 ± 0.0001 0.0174 ± 0.0007 Confidence limit of the intercept 0.0038 ± 0.0231 0.0072 ± 0.0081 Standard error of estimation 0.0144 0.0040 aIntra-day %RSD 0.04–0.12 0.38–0.48 bInter-day %RSD 0.06–0.13 0.21–0.62 Drug in laboratory prepared mixture 100.29 ± 0.70 100.03 ± 0.37 Drug added to dosage form 99.88 ± 0.60 100.15 ± 0.38 Drug in dosage form 100.46 ± 0.53 99.99 ± 0.70 Item FSA BTV Retention time (min) 2.80 6.05 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 2–400 0.5–20 Regression equation PAR230nm = 0.0065 CFSA + 0.0038 PAR230nm = 0.0174 CBTV + 0.0072 Regression coefficient (r2) 0.9998 0.9992 LOD (μg/mL) 0.6364 0.1449 LOQ (μg/mL) 1.9285 0.4389 Sb 4.17 × 10−5 2.42 × 10−4 Sa 0.0090 0.0029 Confidence limit of the slope 0.0065 ± 0.0001 0.0174 ± 0.0007 Confidence limit of the intercept 0.0038 ± 0.0231 0.0072 ± 0.0081 Standard error of estimation 0.0144 0.0040 aIntra-day %RSD 0.04–0.12 0.38–0.48 bInter-day %RSD 0.06–0.13 0.21–0.62 Drug in laboratory prepared mixture 100.29 ± 0.70 100.03 ± 0.37 Drug added to dosage form 99.88 ± 0.60 100.15 ± 0.38 Drug in dosage form 100.46 ± 0.53 99.99 ± 0.70 aThe intra-day (n = 3), average of three concentrations of FSA (160, 200 and 240 μg/mL) and BTV (8, 10 and 12 μg/mL) repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of FSA (160, 200 and 240 μg/mL) and BTV (8, 10 and 12 μg/mL) repeated three times in three successive days. Table III. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Betamethasone Dipropionate and Clotrimazole (Mixture II) Item BTD CLO Retention time (min) 8.00 9.28 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 0.5–20 2–300 Regression equation PAR230nm = 0.0147 CBTD + 0.0016 PAR230nm = 0.0155 CCLO + 0.0163 Regression coefficient (r2) 0.9996 0.9997 LOD (μg/mL) 0.0648 0.5031 LOQ (μg/mL) 0.1964 1.5245 Sb 0.0002 0.0001 Sa 0.0018 0.0198 Confidence limit of the slope 0.0147 ± 0.0004 0.0155 ± 0.0003 Confidence limit of the intercept 0.0016 ± 0.0050 0.0163 ± 0.0509 Standard error of estimation 0.0025 0.0289 aIntra-day %RSD 0.16–0.35 0.04–0.09 bInter-day %RSD 0.24–0.66 0.15–0.17 Drug in laboratory prepared mixture 100.08 ± 0.47 100.27 ± 0.78 Drug added to dosage form 100.25 ± 0.52 100.16 ± 0.34 Drug in dosage form 100.81 ± 0.70 100.41 ± 0.80 Item BTD CLO Retention time (min) 8.00 9.28 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 0.5–20 2–300 Regression equation PAR230nm = 0.0147 CBTD + 0.0016 PAR230nm = 0.0155 CCLO + 0.0163 Regression coefficient (r2) 0.9996 0.9997 LOD (μg/mL) 0.0648 0.5031 LOQ (μg/mL) 0.1964 1.5245 Sb 0.0002 0.0001 Sa 0.0018 0.0198 Confidence limit of the slope 0.0147 ± 0.0004 0.0155 ± 0.0003 Confidence limit of the intercept 0.0016 ± 0.0050 0.0163 ± 0.0509 Standard error of estimation 0.0025 0.0289 aIntra-day %RSD 0.16–0.35 0.04–0.09 bInter-day %RSD 0.24–0.66 0.15–0.17 Drug in laboratory prepared mixture 100.08 ± 0.47 100.27 ± 0.78 Drug added to dosage form 100.25 ± 0.52 100.16 ± 0.34 Drug in dosage form 100.81 ± 0.70 100.41 ± 0.80 aThe intra-day (n = 3), average of three concentrations of BTD (8, 10 and 12 μg/mL) and CLO (125, 156.25 and 187.5 μg/mL) and repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of BTD (8, 10 and 12 μg/mL) and CLO (125, 156.25 and 187.5 μg/mL) repeated three times in three successive days. Table III. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Betamethasone Dipropionate and Clotrimazole (Mixture II) Item BTD CLO Retention time (min) 8.00 9.28 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 0.5–20 2–300 Regression equation PAR230nm = 0.0147 CBTD + 0.0016 PAR230nm = 0.0155 CCLO + 0.0163 Regression coefficient (r2) 0.9996 0.9997 LOD (μg/mL) 0.0648 0.5031 LOQ (μg/mL) 0.1964 1.5245 Sb 0.0002 0.0001 Sa 0.0018 0.0198 Confidence limit of the slope 0.0147 ± 0.0004 0.0155 ± 0.0003 Confidence limit of the intercept 0.0016 ± 0.0050 0.0163 ± 0.0509 Standard error of estimation 0.0025 0.0289 aIntra-day %RSD 0.16–0.35 0.04–0.09 bInter-day %RSD 0.24–0.66 0.15–0.17 Drug in laboratory prepared mixture 100.08 ± 0.47 100.27 ± 0.78 Drug added to dosage form 100.25 ± 0.52 100.16 ± 0.34 Drug in dosage form 100.81 ± 0.70 100.41 ± 0.80 Item BTD CLO Retention time (min) 8.00 9.28 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 0.5–20 2–300 Regression equation PAR230nm = 0.0147 CBTD + 0.0016 PAR230nm = 0.0155 CCLO + 0.0163 Regression coefficient (r2) 0.9996 0.9997 LOD (μg/mL) 0.0648 0.5031 LOQ (μg/mL) 0.1964 1.5245 Sb 0.0002 0.0001 Sa 0.0018 0.0198 Confidence limit of the slope 0.0147 ± 0.0004 0.0155 ± 0.0003 Confidence limit of the intercept 0.0016 ± 0.0050 0.0163 ± 0.0509 Standard error of estimation 0.0025 0.0289 aIntra-day %RSD 0.16–0.35 0.04–0.09 bInter-day %RSD 0.24–0.66 0.15–0.17 Drug in laboratory prepared mixture 100.08 ± 0.47 100.27 ± 0.78 Drug added to dosage form 100.25 ± 0.52 100.16 ± 0.34 Drug in dosage form 100.81 ± 0.70 100.41 ± 0.80 aThe intra-day (n = 3), average of three concentrations of BTD (8, 10 and 12 μg/mL) and CLO (125, 156.25 and 187.5 μg/mL) and repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of BTD (8, 10 and 12 μg/mL) and CLO (125, 156.25 and 187.5 μg/mL) repeated three times in three successive days. Table IV. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Calcipotriol and Betamethasone Dipropionate (Mixture III) Item CAL BTD Retention time (min) 7.01 8.00 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 1–20 0.5–200 Regression equation PAR230nm = 0.0145 CCAL + 0.0050 PAR230nm = 0.0160 CBTD + 0.0071 Regression coefficient (r2) 0.9997 0.9996 LOD (μg/mL) 0.0394 0.1326 LOQ (μg/mL) 0.1195 0.4018 Sb 0.0001 0.0002 Sa 0.0014 0.0169 Confidence limit of the slope 0.0145 ± 0.0003 0.0160 ± 0.0004 Confidence limit of the intercept 0.0050 ± 0.0039 0.0071 ± 0.0470 Standard error of estimation 0.0019 0.0259 aIntra-day %RSD 0.15–0.36 0.03–0.08 bInter-day %RSD 0.51–0.62 0.06–0.22 Drug in laboratory prepared mixture 100.24 ± 0.61 100.04 ± 0.45 Drug added to dosage form 100.09 ± 0.53 100.27 ± 0.74 Drug in dosage form 103.08 ± 0.61 100.96 ± 0.57 Item CAL BTD Retention time (min) 7.01 8.00 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 1–20 0.5–200 Regression equation PAR230nm = 0.0145 CCAL + 0.0050 PAR230nm = 0.0160 CBTD + 0.0071 Regression coefficient (r2) 0.9997 0.9996 LOD (μg/mL) 0.0394 0.1326 LOQ (μg/mL) 0.1195 0.4018 Sb 0.0001 0.0002 Sa 0.0014 0.0169 Confidence limit of the slope 0.0145 ± 0.0003 0.0160 ± 0.0004 Confidence limit of the intercept 0.0050 ± 0.0039 0.0071 ± 0.0470 Standard error of estimation 0.0019 0.0259 aIntra-day %RSD 0.15–0.36 0.03–0.08 bInter-day %RSD 0.51–0.62 0.06–0.22 Drug in laboratory prepared mixture 100.24 ± 0.61 100.04 ± 0.45 Drug added to dosage form 100.09 ± 0.53 100.27 ± 0.74 Drug in dosage form 103.08 ± 0.61 100.96 ± 0.57 aThe intra-day (n = 3), average of three concentrations of CAL (8, 10 and 12 μg/mL) and BTD (80, 100 and 120 μg/mL) repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of CAL (8, 10 and 12 μg/mL) and BTD (80, 100 and 120 μg/mL) repeated three times in three successive days. Table IV. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Calcipotriol and Betamethasone Dipropionate (Mixture III) Item CAL BTD Retention time (min) 7.01 8.00 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 1–20 0.5–200 Regression equation PAR230nm = 0.0145 CCAL + 0.0050 PAR230nm = 0.0160 CBTD + 0.0071 Regression coefficient (r2) 0.9997 0.9996 LOD (μg/mL) 0.0394 0.1326 LOQ (μg/mL) 0.1195 0.4018 Sb 0.0001 0.0002 Sa 0.0014 0.0169 Confidence limit of the slope 0.0145 ± 0.0003 0.0160 ± 0.0004 Confidence limit of the intercept 0.0050 ± 0.0039 0.0071 ± 0.0470 Standard error of estimation 0.0019 0.0259 aIntra-day %RSD 0.15–0.36 0.03–0.08 bInter-day %RSD 0.51–0.62 0.06–0.22 Drug in laboratory prepared mixture 100.24 ± 0.61 100.04 ± 0.45 Drug added to dosage form 100.09 ± 0.53 100.27 ± 0.74 Drug in dosage form 103.08 ± 0.61 100.96 ± 0.57 Item CAL BTD Retention time (min) 7.01 8.00 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 1–20 0.5–200 Regression equation PAR230nm = 0.0145 CCAL + 0.0050 PAR230nm = 0.0160 CBTD + 0.0071 Regression coefficient (r2) 0.9997 0.9996 LOD (μg/mL) 0.0394 0.1326 LOQ (μg/mL) 0.1195 0.4018 Sb 0.0001 0.0002 Sa 0.0014 0.0169 Confidence limit of the slope 0.0145 ± 0.0003 0.0160 ± 0.0004 Confidence limit of the intercept 0.0050 ± 0.0039 0.0071 ± 0.0470 Standard error of estimation 0.0019 0.0259 aIntra-day %RSD 0.15–0.36 0.03–0.08 bInter-day %RSD 0.51–0.62 0.06–0.22 Drug in laboratory prepared mixture 100.24 ± 0.61 100.04 ± 0.45 Drug added to dosage form 100.09 ± 0.53 100.27 ± 0.74 Drug in dosage form 103.08 ± 0.61 100.96 ± 0.57 aThe intra-day (n = 3), average of three concentrations of CAL (8, 10 and 12 μg/mL) and BTD (80, 100 and 120 μg/mL) repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of CAL (8, 10 and 12 μg/mL) and BTD (80, 100 and 120 μg/mL) repeated three times in three successive days. Accuracy Satisfactory results of the analysis of laboratory prepared mixtures of the drugs, including the mean of the recovery and standard deviation, are displayed in Tables II–IV. By applying standard addition technique, good results obtained for the added drugs proved that there was no interference from the co-formulated drug or the frequently encountered excipients (Tables II–IV). Precision The repeatability of the method was validated by analyzing three separate mixtures containing (10 and 200 μg/mL of BTV and FSA, respectively), (100 and 10 μg/mL of BTD and CAL, respectively) and (10 and 156.25 μg/mL of BTD and CLO, respectively), (n = 6). The values of the intra-day and inter-day precision (%RSD), (using three different concentrations in triplicates for three consecutive days), for the analytes are presented in Tables II–IV. Selectivity The selectivity of a method is checked by its ability to determine the analyte(s) of interest in their laboratory prepared mixtures without any interference. Selectivity was also checked by assessing whether any interference arisen from common excipients and it was verified that the signals measured was attributed only to the analytes. The three analyzed dosage forms also contain preservatives (BZA, CHC) and inactive ingredients such as: white soft paraffin, liquid paraffin, tocopherol acetate and ethoxy castor oil. It was found that the excipients did not interfere with the obtained results. As shown in (Figures 3–5) the chromatograms of the pharmaceutical formulations did not show any interference with the separated mixtures components, thus confirming the selectivity of the developed method. Figure 3. View largeDownload slide A typical LC chromatogram of Fucicort® cream (BTV (4.80 μg/mL) and FSA (96 μg/mL)). Figure 3. View largeDownload slide A typical LC chromatogram of Fucicort® cream (BTV (4.80 μg/mL) and FSA (96 μg/mL)). Figure 4. View largeDownload slide A typical LC chromatogram of Clotrisone® cream (BTD (6.40 μg/mL) and CLO (100 μg/mL)). Figure 4. View largeDownload slide A typical LC chromatogram of Clotrisone® cream (BTD (6.40 μg/mL) and CLO (100 μg/mL)). Figure 5. View largeDownload slide A typical LC chromatogram of Calcipoheal-cort® ointment (BTD (20 μg/mL) and CAL (2 μg/mL)). Figure 5. View largeDownload slide A typical LC chromatogram of Calcipoheal-cort® ointment (BTD (20 μg/mL) and CAL (2 μg/mL)). Robustness Robustness of the present analytical procedure was examined by variation of some chromatographic conditions such as the flow rate of the mobile phase by ±0.1 mL/min and organic strength of the mobile phase by ±2% were evaluated. The most important parameter to be studied was the resolution factor between the eight peaks. As can be revealed in Table V, good values of the resolution factor were obtained for all these variations, indicating good robustness of the proposed HPLC method. Table V. Robustness Study of the Proposed RP-HPLC Method Parameter Drug Standard Flow rate (mL/min) Mobile phase composition 0.9 1.1 33:67 37:63 Resolution FSA/BZA 2.25 2.37 2.12 2.20 2.28 BZA/CHC 4.88 5.00 4.63 4.80 4.95 CHC/BTV 7.38 7.60 7.20 7.31 7.43 BTV/CAL 3.60 3.76 3.44 3.56 3.69 CAL/BTD 3.14 3.35 3.00 3.04 3.20 BTD/CLO 4.10 4.33 3.88 4.01 4.19 CLO/IS 2.54 2.63 2.41 2.48 2.61 Parameter Drug Standard Flow rate (mL/min) Mobile phase composition 0.9 1.1 33:67 37:63 Resolution FSA/BZA 2.25 2.37 2.12 2.20 2.28 BZA/CHC 4.88 5.00 4.63 4.80 4.95 CHC/BTV 7.38 7.60 7.20 7.31 7.43 BTV/CAL 3.60 3.76 3.44 3.56 3.69 CAL/BTD 3.14 3.35 3.00 3.04 3.20 BTD/CLO 4.10 4.33 3.88 4.01 4.19 CLO/IS 2.54 2.63 2.41 2.48 2.61 Table V. Robustness Study of the Proposed RP-HPLC Method Parameter Drug Standard Flow rate (mL/min) Mobile phase composition 0.9 1.1 33:67 37:63 Resolution FSA/BZA 2.25 2.37 2.12 2.20 2.28 BZA/CHC 4.88 5.00 4.63 4.80 4.95 CHC/BTV 7.38 7.60 7.20 7.31 7.43 BTV/CAL 3.60 3.76 3.44 3.56 3.69 CAL/BTD 3.14 3.35 3.00 3.04 3.20 BTD/CLO 4.10 4.33 3.88 4.01 4.19 CLO/IS 2.54 2.63 2.41 2.48 2.61 Parameter Drug Standard Flow rate (mL/min) Mobile phase composition 0.9 1.1 33:67 37:63 Resolution FSA/BZA 2.25 2.37 2.12 2.20 2.28 BZA/CHC 4.88 5.00 4.63 4.80 4.95 CHC/BTV 7.38 7.60 7.20 7.31 7.43 BTV/CAL 3.60 3.76 3.44 3.56 3.69 CAL/BTD 3.14 3.35 3.00 3.04 3.20 BTD/CLO 4.10 4.33 3.88 4.01 4.19 CLO/IS 2.54 2.63 2.41 2.48 2.61 Limit of detection and limit of quantitation Limit of detection (LOD) and limit of quantitation (LOQ) were calculated according to ICH recommendations (23). Both LOD and LOQ were calculated based on standard deviation of the intercept and the slope of the regression equation, Tables II–IV. Statistical analysis A statistical analysis of the results obtained by the proposed method was carried out. The results for the analysis of the studied drugs in their synthetic mixtures are in good agreement with those obtained using the reference method for each drug.The significant difference between groups were tested using Student’s t-test and variance ratio F-test at p = 0.05 (23) as shown in Table VI. The tests revealed that there was no significant difference among the methods regarding the accuracy and precision. Table VI. Statistical Comparison Between the Results of the Proposed Method and the Reference Methods for the Determination of Fusidic Acid, Betamethasone Valerate, Betamethasone Dipropionate, Clotrimazole and Calcipotriol Statistical term Reference method for FSAa FSA by the proposed method Reference method for BTVb BTV by the proposed method Reference method for BTDc BTD by the proposed method in Mixture II BTD by the proposed method in Mixture III Reference method for CLOd CLO by the proposed method Reference method for CALe CAL by the proposed method Mean 99.77 100.29 99.80 100.03 99.83 100.08 100.04 99.73 100.27 99.99 100.24 ± SD 0.87 0.70 0.54 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 ± SE 0.36 0.29 0.22 0.15 0.12 0.19 0.18 0.18 0.32 0.19 0.25 %RSD 0.87 0.70 0.55 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 n 6 6 6 6 6 6 6 6 6 6 6 V 0.76 0.49 0.30 0.14 0.09 0.22 0.20 0.20 0.61 0.22 0.37 t (2.23)* 1.12 0.86 1.11 0.97 1.47 0.80 F (5.05)* 1.55 2.14 2.44 2.22 3.05 1.68 Statistical term Reference method for FSAa FSA by the proposed method Reference method for BTVb BTV by the proposed method Reference method for BTDc BTD by the proposed method in Mixture II BTD by the proposed method in Mixture III Reference method for CLOd CLO by the proposed method Reference method for CALe CAL by the proposed method Mean 99.77 100.29 99.80 100.03 99.83 100.08 100.04 99.73 100.27 99.99 100.24 ± SD 0.87 0.70 0.54 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 ± SE 0.36 0.29 0.22 0.15 0.12 0.19 0.18 0.18 0.32 0.19 0.25 %RSD 0.87 0.70 0.55 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 n 6 6 6 6 6 6 6 6 6 6 6 V 0.76 0.49 0.30 0.14 0.09 0.22 0.20 0.20 0.61 0.22 0.37 t (2.23)* 1.12 0.86 1.11 0.97 1.47 0.80 F (5.05)* 1.55 2.14 2.44 2.22 3.05 1.68 *Figures in parentheses are the theoretical t and Fvalues at (p = 0.05). aBritish Pharmacopoeia 2016: Titration. bBritish Pharmacopoeia 2016: Spectrophotometric method. cBritish Pharmacopoeia 2016: HPLC method. dBritish Pharmacopoeia 2016: Non-aqueous titration. eBritish Pharmacopoeia 2016: HPLC method. Table VI. Statistical Comparison Between the Results of the Proposed Method and the Reference Methods for the Determination of Fusidic Acid, Betamethasone Valerate, Betamethasone Dipropionate, Clotrimazole and Calcipotriol Statistical term Reference method for FSAa FSA by the proposed method Reference method for BTVb BTV by the proposed method Reference method for BTDc BTD by the proposed method in Mixture II BTD by the proposed method in Mixture III Reference method for CLOd CLO by the proposed method Reference method for CALe CAL by the proposed method Mean 99.77 100.29 99.80 100.03 99.83 100.08 100.04 99.73 100.27 99.99 100.24 ± SD 0.87 0.70 0.54 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 ± SE 0.36 0.29 0.22 0.15 0.12 0.19 0.18 0.18 0.32 0.19 0.25 %RSD 0.87 0.70 0.55 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 n 6 6 6 6 6 6 6 6 6 6 6 V 0.76 0.49 0.30 0.14 0.09 0.22 0.20 0.20 0.61 0.22 0.37 t (2.23)* 1.12 0.86 1.11 0.97 1.47 0.80 F (5.05)* 1.55 2.14 2.44 2.22 3.05 1.68 Statistical term Reference method for FSAa FSA by the proposed method Reference method for BTVb BTV by the proposed method Reference method for BTDc BTD by the proposed method in Mixture II BTD by the proposed method in Mixture III Reference method for CLOd CLO by the proposed method Reference method for CALe CAL by the proposed method Mean 99.77 100.29 99.80 100.03 99.83 100.08 100.04 99.73 100.27 99.99 100.24 ± SD 0.87 0.70 0.54 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 ± SE 0.36 0.29 0.22 0.15 0.12 0.19 0.18 0.18 0.32 0.19 0.25 %RSD 0.87 0.70 0.55 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 n 6 6 6 6 6 6 6 6 6 6 6 V 0.76 0.49 0.30 0.14 0.09 0.22 0.20 0.20 0.61 0.22 0.37 t (2.23)* 1.12 0.86 1.11 0.97 1.47 0.80 F (5.05)* 1.55 2.14 2.44 2.22 3.05 1.68 *Figures in parentheses are the theoretical t and Fvalues at (p = 0.05). aBritish Pharmacopoeia 2016: Titration. bBritish Pharmacopoeia 2016: Spectrophotometric method. cBritish Pharmacopoeia 2016: HPLC method. dBritish Pharmacopoeia 2016: Non-aqueous titration. eBritish Pharmacopoeia 2016: HPLC method. Discussion Two challenges were encountered in this study. First, the investigated mixtures contain betamethasone either as valerate or dipropionate ester in combination with FSA, CLO and CAL when one of the components is present in low concentration relative to the other which represented an analytical problem in their determination. Second, the separation of betamethasone esters (BTV, BTD) and FSA which are closely related in structures and could not be easily separated simultaneously in a single HPLC run. Therefore, the aim of this study was to successfully overcome the two encountered challenges and to develop a simple and sensitive method for the simultaneous separation and quantification of three of the most commonly prescribed topical preparations containing betamethasone in a short time to be suitable for routine high capacity work in quality control laboratories either in bulk or in pharmaceutical dosage form. So, to achieve this aim, different chromatographic conditions including different reversed-phase columns and different mobile phases were attempted. Various mobile phase compositions containing different ratios of organic and aqueous phases were tried in an isocratic mode. Good separation and best peak shape were obtained upon using Discovery® C18 column and an isocratic mobile phase composed of water: acetonitrile (35:65, v/v), Figure 2. According to USP 2015 (11), system suitability tests are a fundamental part of liquid chromatographic methods in the optimization of the conditions of the proposed method. They are used to verify that the resolution and reproducibility were adequate for the performed analysis. The developed HPLC system was found to be suitable for the determination of all components, Table I. Method validation was carried out in accordance with ICH guidelines (23). The linearity of the proposed HPLC method was assessed. Linear relationships between PAR and component concentration (C) and high regression coefficients were obtained for each drug, Tables II–IV. Accuracy of the proposed method was assessed by calculating % recovery of six different concentrations of laboratory prepared mixtures of the drugs analyzed by the proposed method. The accuracy was also confirmed by recovery studies from sample solution at different levels of standard additions. Intra-day and inter-day precision were also evaluated. The developed method was found to be accurate and precise, Tables II–IV. In addition, good results in terms of LOD, LOQ, robustness and selectivity were obtained, Tables II–V. Statistical analysis of the results revealed no significance difference between the results of the developed method and that of the reported ones, Table VI. As a conclusion, the developed method succeeded in separating five drugs in addition to two preservatives and the IS, simultaneously, with good resolution factor in relatively short run time. The method also proved to be sensitive, simple, rapid and selective so it can be used for routine analysis of the cited mixtures. Conclusion In this study, a simple, sensitive, highly selective and reliable HPLC method has been developed for the simultaneous determination of three binary mixtures containing betamethasone esters in combination with FSA or CAL or CLO in laboratory prepared mixtures and pharmaceutical preparations, without any interference from the excipients. The analytes were successfully resolved and quantified in relatively short run time using isocratic mode of elution and simple mobile phase consisting of water and acetonitrile. Consequently, the developed method can be considered time- and cost-effective beside the other advantages including accuracy and sensitivity. The method was validated showing satisfactory results for all the method validation parameters tested. It can be conveniently used in quality control laboratories and routine analysis of the cited drugs. References 1 Sweetman , S.C. ; Martindale: The Complete Drug Reference . Pharmaceutical Press , London, ( 2011 ). 2 Girolomoni , G. , Mattina , R. , Manfredini , S. , Vertuani , S. , Fabrizi , G. ; Fusidic acid betamethasone lipid cream ; International Journal of Clinical Practice , ( 2016 ); 70 : 4 – 13 . Google Scholar CrossRef Search ADS PubMed 3 Schaller , M. , Friedrich , M. , Papini , M. , Pujol , R.M. , Veraldi , S. ; Topical antifungal‐corticosteroid combination therapy for the treatment of superficial mycoses: conclusions of an expert panel meeting ; Mycoses , ( 2016 ); 59 : 365 – 373 . Google Scholar CrossRef Search ADS PubMed 4 Murphy , G. , Reich , K. ; In touch with psoriasis: topical treatments and current guidelines ; Journal of the European Academy of Dermatology and Venereology , ( 2011 ); 25 : 3 – 8 . Google Scholar CrossRef Search ADS PubMed 5 Lotfy , H.M. , Salem , H. , Abdelkawy , M. , Samir , A. ; Spectrophotometric methods for simultaneous determination of betamethasone valerate and fusidic acid in their binary mixture ; Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy , ( 2015 ); 140 : 294 – 304 . Google Scholar CrossRef Search ADS 6 Byrne , J. , Velasco-Torrijos , T. , Reinhardt , R. ; 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 ; Journal of Chromatographic Science , ( 2015 ); 53 ( 9 ): 1498 – 1503 . 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Chem., Trabzon, Turkey, ( 2013 ). 15 Lin , M. , Wu , N. ; Comparison between micellar electrokinetic chromatography and HPLC for the determination of Betamethasone Dipropionate, Clotrimazole and their related substances ; Journal of Pharmaceutical and Biomedical Analysis , ( 1999 ); 19 ( 6 ): 945 – 954 . Google Scholar CrossRef Search ADS PubMed 16 Simonsen , L. , Høy , G. , Didriksen , E. , Persson , J. , Melchior , N. , Hansen , J. ; Development of a new formulation combining calcipotriol and betamethasone dipropionate in an ointment vehicle ; Drug Development and Industrial Pharmacy , ( 2004 ); 30 ( 10 ): 1095 – 1102 . 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Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

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

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Abstract

Abstract 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. Introduction Topical preparations containing a combination of different drug classes, having different mechanisms of action, acquires increasing interest for achieving treatment goal. Betamethasone is a corticosteroid with mainly glucocorticoid activity used either in the free alcohol form or in one of the esterified forms. Betamethasone valerate (BTV, Figure 1A) and betamethasone dipropionate (BTD, Figure 1B) are extensively used for topical application in the treatment of various skin disorders. Fusidic acid, (FSA, Figure 1C), is a steroidal antibacterial with a bacteriostatic or bactericidal activity mainly against Gram-positive bacteria. Clotrimazole, (CLO, Figure 1D), is an imidazole antifungal drug with antimicrobial activity, used in superficial candidiasis and in other skin infections as dermatophytosis. Calcipotriol monohydrate, (CAL, Figure 1E), is a vitamin D3 derivative that induces differentiation and suppresses proliferation of keratinocytes in the management of psoriasis (1). Figure 1. View largeDownload slide Chemical structures and chemical names of betamethasone valerate (A), betamethasone dipropionate (B), fusidic acid (C), clotrimazole (D), calcipotriol monohydrate (E), chlorocresol (F), benzyl alcohol (G) and clobetasone butyrate (IS) (H). Figure 1. View largeDownload slide Chemical structures and chemical names of betamethasone valerate (A), betamethasone dipropionate (B), fusidic acid (C), clotrimazole (D), calcipotriol monohydrate (E), chlorocresol (F), benzyl alcohol (G) and clobetasone butyrate (IS) (H). Betamethasone esters are widely used for their anti-inflammatory (glucocorticoid) and immunosuppressant properties in various skin conditions. They are used either alone or in combination with FSA in order to provide effective anti-inflammatory and antibacterial activities (2). Such combination is formulated with chlorocresol, (CHC, Figure 1F), a potent disinfectant and antiseptic agent which has bactericidal activity against both Gram-positive and Gram-negative bacteria. It is also used as a preservative in creams and in cosmetics (1). In addition, combination of BTD with CLO is widely used for treatment of superficial mycoses by acting as anti-inflammatory, antibacterial and fungistatic (3). It contains also benzyl alcohol, (BA, Figure 1G), used as a solubilizer and preservative. It has a bacteriostatic activity mainly against Gram-positive bacteria (1). Also, a combination of BTD with CAL showed higher efficacy in the management of mild to moderate psoriasis than each of them alone (4). Literature survey revealed that the simultaneous determination of BTV and FSA mixture has been carried out by spectrophotometric method (5), high-performance liquid chromatography (HPLC) method for drug determinations in pharmaceutical dosage form (6, 7) and in plasma (8). Spectrophotometric (9) and HPLC methods (10, 11) have been also described for analysis of BTD and CLO in binary mixture. Ternary mixture containing BTD, CLO and BZA was quantified by three spectrophotometric methods (12–14), HPLC method (15) and micellar electrokinetic chromatography method (15). Besides, BTD and CAL mixture has been determined by several HPLC methods(16–21). CHC and BTD has been determined by HPLC method (22). Many of the reported spectrophotometric methods lack sensitivity (5, 12), while the reported HPLC methods suffers from either long retention time and hence long analysis time (6, 7, 20, 21) or the use of complex gradient mobile phases, thus consuming time and cost (16, 20, 21) or lack sensitivity (19). Based on literature survey of the aforementioned reported methods, there was a need to develop and validate a reliable HPLC method for the simultaneous separation and quantification of three of the most commonly prescribed topical preparations containing betamethasone either as valerate or dipropionate ester in combination with FSA, CLO and CAL when one of the components is present in low concentration relative to the other which encounters an analytical problem in their determination. In addition, our objective was to achieve the analysis of the cited drugs in a short run time with high sensitivity and selectivity using a simple mobile phase in an isocratic elution mode and by utilizing the most commonly used reversed-phase column. The developed method succeeded in separating five drugs representing three binary mixtures in addition to two preservatives present in their dosage forms and the internal standard (IS) with good resolution factor in relatively short run time. The method also proved to be sensitive, simple, rapid and selective so it can be used for routine analysis of the cited mixtures in quality control laboratories either in bulk or in pharmaceutical preparations. Experimental Instrumentation The HPLC (Young Lin instrument Acme 9000, Korea) system was equipped with a vacuum degasser, a mixer, a gradient pump and a UV/Vis detector. Young Lin Autochrome-3000 program was used for the instrument control, data acquisition and analysis. Separation and quantitation were carried out on Discovery® C18 (4.6 × 250 mm, 5 μm) column. The mobile phase was filtered through 0.45 μm Chromtech® nylon membrane filter (UK) and degassed using Powersonic 405 (Korea). Materials and reagents BTV was kindly supplied by Amriya Pharmaceutical Ind. (its purity was certified as 99.80%). BTD and CAL were kindly supplied by Sabaa International Company (their purities were certified as 99.83% and 99.99%, respectively). Clobetasone butyrate, used as IS (Figure 1H), was kindly supplied by GlaxoSmithKline (its purity was certified as 99.75%). CLO was kindly supplied by Borg Pharmaceutical Ind. (its purity was certified as 99.73%). FSA was kindly supplied by Julphar Pharmaceutical Ind. (its purity was certified as 99.77%). CHC and BZA were kindly supplied by the National Organization of Drug Control and Research (NODCAR), Egypt (their purities were certified as 100.52% and 99.75%, respectively). Topical preparations were purchased from local pharmacy in Egypt as follows: Fucicort® cream (Batch No. GGE2353), each gram nominally contains BTV (1 mg) and FSA (20 mg); Clotrisone® cream (Batch No. 018074), each gram nominally contains BTD (0.64 mg) and CLO (10 mg) and Calcipoheal-Cort® ointment (Batch No. 50388), each gram nominally contains BTD (0.5 mg) and Cal (50 μg). HPLC grade methanol and acetonitrile were used (Sigma-Aldrich Co., France). Bi-distilled water was produced in-house (Aquatron Water Still, A4000D, UK). Chromatographic conditions Chromatographic separation was achieved using a Discovery® C18 (4.6 × 250 mm, 5 μm) column and a mobile phase consisting of water: acetonitrile (35:65, v/v). The mobile phase was filtered through 0.45 μm membrane filter and degassed for 30 min in an ultrasonic bath prior to its use. The flow rate was maintained at 1 mL/min. Analyses were performed at ambient temperature and the UV detection was carried out at 230 nm. The injection volume was 20 μL. Standard stock and working solutions Standard stock solutions (1 mg/mL) of BTV, CAL BTD, FSA and CLO were prepared separately by dissolving 100 mg of each drug in 100 mL methanol. Standard working solutions (50 μg/mL) of BTD, BTV and CAL and (40 μg/mL) of BTD, FSA and CLO were prepared by further dilution of the corresponding stock solutions with the mobile phase. Clobetasone butyrate (IS) stock solution (0.5 mg/mL) was prepared by dissolving 125 mg of clobetasone butyrate in 250 mL methanol. Samples preparation Accurate weights of fucicort® cream (1.2 g), clotrisone® cream (2 g) and calcipoheal-cort ointment (5 g) were dissolved separately and transferred by 50 mL hot n-hexane in separating funnels, mixed for 2 min then cooled. Twenty milliliters of methanol were added with shaking for 10 min. The extraction process was repeated for two more times using 10 mL methanol. At the end of the extraction process, the methanolic layer was collected in 50-mL volumetric flask and the three sample solutions were cooled at 8°C for 30 min to get rid of any traces of the hexane layer. The extraction method was guided by the British Pharmacopoeia (2016). The solutions were completed to volume with methanol to obtain a concentration equivalent to 24 μg/mL of BTV and 480 μg/mL of FSA in fucicort® sample solution, 25.6 μg/mL of BTD and 400 μg/mL of CLO in clotrisone® sample solution, and 50 μg/mL of BTD and 5 μg/mL of CAL in calcipoheal-cort® sample solution. The three sample solutions were then filtered using cotton previously washed with methanol and the first 10 mL of the filtrate were discarded. Procedure Linearity Accurately measured aliquots of standard solutions equivalent to 5–200 μg of BTV and 0.02–4 mg of FSA for (Mixture I), 5–200 μg of BTD and 0.02–3 mg of CLO for (Mixture II) and 0.005–2 mg of BTD and 10–200 μg of CAL for (Mixture III) were separately transferred into three series of 10-mL volumetric flasks. A constant aliquot (1 mL) of IS stock solution was added and solutions were completed to volume with the mobile phase. A volume of 20 μL of each solution was injected into the chromatograph in triplicates. The previously mentioned chromatographic conditions were adjusted with a run time for 12 min. A calibration curve for each drug was obtained by plotting peak area ratio (peak area of the studied drug/peak area of IS) (PAR) against concentration (C) in μg/mL and the corresponding regression equations were obtained. Assay of laboratory prepared mixtures The procedure mentioned in linearity section was repeated for the analysis of laboratory prepared mixtures containing BTV and FSA equivalent to 10–130 μg and 0.2–2.6 mg, respectively (Mixture I) and BTD and CLO equivalent to 16–176 μg and 0.25–2.7 mg, respectively (Mixture II) and BTD and CAL equivalent to 0.2–1.9 mg and 20–190 μg, respectively (Mixture III). Assay of pharmaceutical preparations For the determination of the examined drugs in Fucicort® cream, Clotrisone® cream and Calcipoheal-Cort® ointment, the sample solutions, prepared in Samples preparation section, were further diluted with the mobile phase to prepare sample working solutions containing (24–84 μg BTV and 0.48–1.68 mg FSA), (25.6–89.6 μg BTD and 0.4–1.4 mg CLO) and (0.125–0.25 mg BTD and 12.5–25 μg CAL), respectively. Then, 20 μL of each sample working solution were injected into the chromatograph, in triplicates. Results Method development Both Discovery® C18 (4.6 × 250 mm, 5 μm) and Discovery® phenyl (4.6 × 250 mm, 5 μm) columns showed good separation of all mixture components. Discovery® C18 column was the most suitable one as it achieved the intended aim showing better peaks shape with good symmetry giving reasonable retention times for the mixture components when operating under optimum conditions. Various mobile phase compositions containing different ratios of organic and aqueous phases were attempted in an isocratic mode. Both acetonitrile and methanol in different ratios were tried as organic modifier. Acetonitrile was found optimum for the elution as it showed good separation, better peaks shape and it shortened the analysis time. Phosphate buffer (25 mM KH2PO4) with different pH (3, 4.5 and 7) were tried but no more privilege was obtained in comparison with the use of water. Based on these investigations, an isocratic elution using a mobile phase composed of water: acetonitrile (35:65, v/v) was used. Good separation of the seven components in the mixture along with clobetasone butyrate (the chosen IS) was obtained at a flow rate of 1 mL/min. UV detection was carried out at 230 nm. The retention times were found to be 2.80, 3.30, 4.55, 6.05, 7.01, 8, 9.28 and 10.20 min for FSA, BZA, CHC, BTV, CAL, BTD, CLO and IS, respectively; as presented in Figure 2. Figure 2. View largeDownload slide A typical LC chromatogram of synthetic mixture of fusidic acid (FSA), benzyl alcohol (BZA), chlorocresol (CHC), betamethasone valerate (BTV), calcipotriol monohydrate (CAL), betamethasone dipropionate (BTD), clotrimazole (CLO) and clobetasone butyrate (IS). Figure 2. View largeDownload slide A typical LC chromatogram of synthetic mixture of fusidic acid (FSA), benzyl alcohol (BZA), chlorocresol (CHC), betamethasone valerate (BTV), calcipotriol monohydrate (CAL), betamethasone dipropionate (BTD), clotrimazole (CLO) and clobetasone butyrate (IS). System suitability The parameters of these tests are column efficiency represented as number of theoretical plates, peak resolution factor, tailing of chromatographic peak, selectivity factor, capacity factor, repeatability as %RSD of peak area for six injections and reproducibility of retention as %RSD of retention time. The results of these tests for the developed method are listed in Table I. Table I. System Suitability Tests for the Proposed HPLC Method Item FSA BZA CHC BTV CAL BTD CLO IS N 2570 3400 8434 9020 9828 10424 11947 12249 R     2.25     4.88     7.38     3.60     3.14      4.10      2.54 T 1.05 1.10 1.10 1.00 1.00 0.98 1.20 0.98 α     1.28     1.54     1.42     1.19     1.16      1.18      1.11 K′ 1.80 2.30 3.55 5.05 6.01 7.00 8.28 9.20 %RSD of peak area for six injections 1.03 0.84 0.95 0.87 0.65 1.01 %RSD of retention time 1.01 1.09 0.71 0.70 0.72 0.84 0.86 1.00 Item FSA BZA CHC BTV CAL BTD CLO IS N 2570 3400 8434 9020 9828 10424 11947 12249 R     2.25     4.88     7.38     3.60     3.14      4.10      2.54 T 1.05 1.10 1.10 1.00 1.00 0.98 1.20 0.98 α     1.28     1.54     1.42     1.19     1.16      1.18      1.11 K′ 1.80 2.30 3.55 5.05 6.01 7.00 8.28 9.20 %RSD of peak area for six injections 1.03 0.84 0.95 0.87 0.65 1.01 %RSD of retention time 1.01 1.09 0.71 0.70 0.72 0.84 0.86 1.00 N, number of theoretical plates; R, resolution factor; T, tailing factor; α, selectivity factor; K′, capacity factor. Table I. System Suitability Tests for the Proposed HPLC Method Item FSA BZA CHC BTV CAL BTD CLO IS N 2570 3400 8434 9020 9828 10424 11947 12249 R     2.25     4.88     7.38     3.60     3.14      4.10      2.54 T 1.05 1.10 1.10 1.00 1.00 0.98 1.20 0.98 α     1.28     1.54     1.42     1.19     1.16      1.18      1.11 K′ 1.80 2.30 3.55 5.05 6.01 7.00 8.28 9.20 %RSD of peak area for six injections 1.03 0.84 0.95 0.87 0.65 1.01 %RSD of retention time 1.01 1.09 0.71 0.70 0.72 0.84 0.86 1.00 Item FSA BZA CHC BTV CAL BTD CLO IS N 2570 3400 8434 9020 9828 10424 11947 12249 R     2.25     4.88     7.38     3.60     3.14      4.10      2.54 T 1.05 1.10 1.10 1.00 1.00 0.98 1.20 0.98 α     1.28     1.54     1.42     1.19     1.16      1.18      1.11 K′ 1.80 2.30 3.55 5.05 6.01 7.00 8.28 9.20 %RSD of peak area for six injections 1.03 0.84 0.95 0.87 0.65 1.01 %RSD of retention time 1.01 1.09 0.71 0.70 0.72 0.84 0.86 1.00 N, number of theoretical plates; R, resolution factor; T, tailing factor; α, selectivity factor; K′, capacity factor. Method validation Validation was assessed according to ICH guidelines (23). Linearity A linear relationship between peak area ratio (PAR) and component concentration (C) was obtained for each drug. The regression equation for each mixture component was also calculated. In this work, six concentrations were chosen for BTV, CAL and BTD. Seven concentrations of both FSA and CLO were used. Each concentration was repeated three times. The linearity of the calibration curves was validated by the high value of regression coefficients (>0.999). The analytical data of the calibration curve of each drug including standard deviations for the slope and intercept (Sb, Sa) are presented in Tables II–IV. Table II. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Fusidic Acid and Betamethasone Valerate (Mixture I) Item FSA BTV Retention time (min) 2.80 6.05 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 2–400 0.5–20 Regression equation PAR230nm = 0.0065 CFSA + 0.0038 PAR230nm = 0.0174 CBTV + 0.0072 Regression coefficient (r2) 0.9998 0.9992 LOD (μg/mL) 0.6364 0.1449 LOQ (μg/mL) 1.9285 0.4389 Sb 4.17 × 10−5 2.42 × 10−4 Sa 0.0090 0.0029 Confidence limit of the slope 0.0065 ± 0.0001 0.0174 ± 0.0007 Confidence limit of the intercept 0.0038 ± 0.0231 0.0072 ± 0.0081 Standard error of estimation 0.0144 0.0040 aIntra-day %RSD 0.04–0.12 0.38–0.48 bInter-day %RSD 0.06–0.13 0.21–0.62 Drug in laboratory prepared mixture 100.29 ± 0.70 100.03 ± 0.37 Drug added to dosage form 99.88 ± 0.60 100.15 ± 0.38 Drug in dosage form 100.46 ± 0.53 99.99 ± 0.70 Item FSA BTV Retention time (min) 2.80 6.05 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 2–400 0.5–20 Regression equation PAR230nm = 0.0065 CFSA + 0.0038 PAR230nm = 0.0174 CBTV + 0.0072 Regression coefficient (r2) 0.9998 0.9992 LOD (μg/mL) 0.6364 0.1449 LOQ (μg/mL) 1.9285 0.4389 Sb 4.17 × 10−5 2.42 × 10−4 Sa 0.0090 0.0029 Confidence limit of the slope 0.0065 ± 0.0001 0.0174 ± 0.0007 Confidence limit of the intercept 0.0038 ± 0.0231 0.0072 ± 0.0081 Standard error of estimation 0.0144 0.0040 aIntra-day %RSD 0.04–0.12 0.38–0.48 bInter-day %RSD 0.06–0.13 0.21–0.62 Drug in laboratory prepared mixture 100.29 ± 0.70 100.03 ± 0.37 Drug added to dosage form 99.88 ± 0.60 100.15 ± 0.38 Drug in dosage form 100.46 ± 0.53 99.99 ± 0.70 aThe intra-day (n = 3), average of three concentrations of FSA (160, 200 and 240 μg/mL) and BTV (8, 10 and 12 μg/mL) repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of FSA (160, 200 and 240 μg/mL) and BTV (8, 10 and 12 μg/mL) repeated three times in three successive days. Table II. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Fusidic Acid and Betamethasone Valerate (Mixture I) Item FSA BTV Retention time (min) 2.80 6.05 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 2–400 0.5–20 Regression equation PAR230nm = 0.0065 CFSA + 0.0038 PAR230nm = 0.0174 CBTV + 0.0072 Regression coefficient (r2) 0.9998 0.9992 LOD (μg/mL) 0.6364 0.1449 LOQ (μg/mL) 1.9285 0.4389 Sb 4.17 × 10−5 2.42 × 10−4 Sa 0.0090 0.0029 Confidence limit of the slope 0.0065 ± 0.0001 0.0174 ± 0.0007 Confidence limit of the intercept 0.0038 ± 0.0231 0.0072 ± 0.0081 Standard error of estimation 0.0144 0.0040 aIntra-day %RSD 0.04–0.12 0.38–0.48 bInter-day %RSD 0.06–0.13 0.21–0.62 Drug in laboratory prepared mixture 100.29 ± 0.70 100.03 ± 0.37 Drug added to dosage form 99.88 ± 0.60 100.15 ± 0.38 Drug in dosage form 100.46 ± 0.53 99.99 ± 0.70 Item FSA BTV Retention time (min) 2.80 6.05 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 2–400 0.5–20 Regression equation PAR230nm = 0.0065 CFSA + 0.0038 PAR230nm = 0.0174 CBTV + 0.0072 Regression coefficient (r2) 0.9998 0.9992 LOD (μg/mL) 0.6364 0.1449 LOQ (μg/mL) 1.9285 0.4389 Sb 4.17 × 10−5 2.42 × 10−4 Sa 0.0090 0.0029 Confidence limit of the slope 0.0065 ± 0.0001 0.0174 ± 0.0007 Confidence limit of the intercept 0.0038 ± 0.0231 0.0072 ± 0.0081 Standard error of estimation 0.0144 0.0040 aIntra-day %RSD 0.04–0.12 0.38–0.48 bInter-day %RSD 0.06–0.13 0.21–0.62 Drug in laboratory prepared mixture 100.29 ± 0.70 100.03 ± 0.37 Drug added to dosage form 99.88 ± 0.60 100.15 ± 0.38 Drug in dosage form 100.46 ± 0.53 99.99 ± 0.70 aThe intra-day (n = 3), average of three concentrations of FSA (160, 200 and 240 μg/mL) and BTV (8, 10 and 12 μg/mL) repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of FSA (160, 200 and 240 μg/mL) and BTV (8, 10 and 12 μg/mL) repeated three times in three successive days. Table III. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Betamethasone Dipropionate and Clotrimazole (Mixture II) Item BTD CLO Retention time (min) 8.00 9.28 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 0.5–20 2–300 Regression equation PAR230nm = 0.0147 CBTD + 0.0016 PAR230nm = 0.0155 CCLO + 0.0163 Regression coefficient (r2) 0.9996 0.9997 LOD (μg/mL) 0.0648 0.5031 LOQ (μg/mL) 0.1964 1.5245 Sb 0.0002 0.0001 Sa 0.0018 0.0198 Confidence limit of the slope 0.0147 ± 0.0004 0.0155 ± 0.0003 Confidence limit of the intercept 0.0016 ± 0.0050 0.0163 ± 0.0509 Standard error of estimation 0.0025 0.0289 aIntra-day %RSD 0.16–0.35 0.04–0.09 bInter-day %RSD 0.24–0.66 0.15–0.17 Drug in laboratory prepared mixture 100.08 ± 0.47 100.27 ± 0.78 Drug added to dosage form 100.25 ± 0.52 100.16 ± 0.34 Drug in dosage form 100.81 ± 0.70 100.41 ± 0.80 Item BTD CLO Retention time (min) 8.00 9.28 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 0.5–20 2–300 Regression equation PAR230nm = 0.0147 CBTD + 0.0016 PAR230nm = 0.0155 CCLO + 0.0163 Regression coefficient (r2) 0.9996 0.9997 LOD (μg/mL) 0.0648 0.5031 LOQ (μg/mL) 0.1964 1.5245 Sb 0.0002 0.0001 Sa 0.0018 0.0198 Confidence limit of the slope 0.0147 ± 0.0004 0.0155 ± 0.0003 Confidence limit of the intercept 0.0016 ± 0.0050 0.0163 ± 0.0509 Standard error of estimation 0.0025 0.0289 aIntra-day %RSD 0.16–0.35 0.04–0.09 bInter-day %RSD 0.24–0.66 0.15–0.17 Drug in laboratory prepared mixture 100.08 ± 0.47 100.27 ± 0.78 Drug added to dosage form 100.25 ± 0.52 100.16 ± 0.34 Drug in dosage form 100.81 ± 0.70 100.41 ± 0.80 aThe intra-day (n = 3), average of three concentrations of BTD (8, 10 and 12 μg/mL) and CLO (125, 156.25 and 187.5 μg/mL) and repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of BTD (8, 10 and 12 μg/mL) and CLO (125, 156.25 and 187.5 μg/mL) repeated three times in three successive days. Table III. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Betamethasone Dipropionate and Clotrimazole (Mixture II) Item BTD CLO Retention time (min) 8.00 9.28 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 0.5–20 2–300 Regression equation PAR230nm = 0.0147 CBTD + 0.0016 PAR230nm = 0.0155 CCLO + 0.0163 Regression coefficient (r2) 0.9996 0.9997 LOD (μg/mL) 0.0648 0.5031 LOQ (μg/mL) 0.1964 1.5245 Sb 0.0002 0.0001 Sa 0.0018 0.0198 Confidence limit of the slope 0.0147 ± 0.0004 0.0155 ± 0.0003 Confidence limit of the intercept 0.0016 ± 0.0050 0.0163 ± 0.0509 Standard error of estimation 0.0025 0.0289 aIntra-day %RSD 0.16–0.35 0.04–0.09 bInter-day %RSD 0.24–0.66 0.15–0.17 Drug in laboratory prepared mixture 100.08 ± 0.47 100.27 ± 0.78 Drug added to dosage form 100.25 ± 0.52 100.16 ± 0.34 Drug in dosage form 100.81 ± 0.70 100.41 ± 0.80 Item BTD CLO Retention time (min) 8.00 9.28 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 0.5–20 2–300 Regression equation PAR230nm = 0.0147 CBTD + 0.0016 PAR230nm = 0.0155 CCLO + 0.0163 Regression coefficient (r2) 0.9996 0.9997 LOD (μg/mL) 0.0648 0.5031 LOQ (μg/mL) 0.1964 1.5245 Sb 0.0002 0.0001 Sa 0.0018 0.0198 Confidence limit of the slope 0.0147 ± 0.0004 0.0155 ± 0.0003 Confidence limit of the intercept 0.0016 ± 0.0050 0.0163 ± 0.0509 Standard error of estimation 0.0025 0.0289 aIntra-day %RSD 0.16–0.35 0.04–0.09 bInter-day %RSD 0.24–0.66 0.15–0.17 Drug in laboratory prepared mixture 100.08 ± 0.47 100.27 ± 0.78 Drug added to dosage form 100.25 ± 0.52 100.16 ± 0.34 Drug in dosage form 100.81 ± 0.70 100.41 ± 0.80 aThe intra-day (n = 3), average of three concentrations of BTD (8, 10 and 12 μg/mL) and CLO (125, 156.25 and 187.5 μg/mL) and repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of BTD (8, 10 and 12 μg/mL) and CLO (125, 156.25 and 187.5 μg/mL) repeated three times in three successive days. Table IV. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Calcipotriol and Betamethasone Dipropionate (Mixture III) Item CAL BTD Retention time (min) 7.01 8.00 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 1–20 0.5–200 Regression equation PAR230nm = 0.0145 CCAL + 0.0050 PAR230nm = 0.0160 CBTD + 0.0071 Regression coefficient (r2) 0.9997 0.9996 LOD (μg/mL) 0.0394 0.1326 LOQ (μg/mL) 0.1195 0.4018 Sb 0.0001 0.0002 Sa 0.0014 0.0169 Confidence limit of the slope 0.0145 ± 0.0003 0.0160 ± 0.0004 Confidence limit of the intercept 0.0050 ± 0.0039 0.0071 ± 0.0470 Standard error of estimation 0.0019 0.0259 aIntra-day %RSD 0.15–0.36 0.03–0.08 bInter-day %RSD 0.51–0.62 0.06–0.22 Drug in laboratory prepared mixture 100.24 ± 0.61 100.04 ± 0.45 Drug added to dosage form 100.09 ± 0.53 100.27 ± 0.74 Drug in dosage form 103.08 ± 0.61 100.96 ± 0.57 Item CAL BTD Retention time (min) 7.01 8.00 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 1–20 0.5–200 Regression equation PAR230nm = 0.0145 CCAL + 0.0050 PAR230nm = 0.0160 CBTD + 0.0071 Regression coefficient (r2) 0.9997 0.9996 LOD (μg/mL) 0.0394 0.1326 LOQ (μg/mL) 0.1195 0.4018 Sb 0.0001 0.0002 Sa 0.0014 0.0169 Confidence limit of the slope 0.0145 ± 0.0003 0.0160 ± 0.0004 Confidence limit of the intercept 0.0050 ± 0.0039 0.0071 ± 0.0470 Standard error of estimation 0.0019 0.0259 aIntra-day %RSD 0.15–0.36 0.03–0.08 bInter-day %RSD 0.51–0.62 0.06–0.22 Drug in laboratory prepared mixture 100.24 ± 0.61 100.04 ± 0.45 Drug added to dosage form 100.09 ± 0.53 100.27 ± 0.74 Drug in dosage form 103.08 ± 0.61 100.96 ± 0.57 aThe intra-day (n = 3), average of three concentrations of CAL (8, 10 and 12 μg/mL) and BTD (80, 100 and 120 μg/mL) repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of CAL (8, 10 and 12 μg/mL) and BTD (80, 100 and 120 μg/mL) repeated three times in three successive days. Table IV. Validation Parameters and Results Obtained by the Proposed Method for Simultaneous Determination of Calcipotriol and Betamethasone Dipropionate (Mixture III) Item CAL BTD Retention time (min) 7.01 8.00 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 1–20 0.5–200 Regression equation PAR230nm = 0.0145 CCAL + 0.0050 PAR230nm = 0.0160 CBTD + 0.0071 Regression coefficient (r2) 0.9997 0.9996 LOD (μg/mL) 0.0394 0.1326 LOQ (μg/mL) 0.1195 0.4018 Sb 0.0001 0.0002 Sa 0.0014 0.0169 Confidence limit of the slope 0.0145 ± 0.0003 0.0160 ± 0.0004 Confidence limit of the intercept 0.0050 ± 0.0039 0.0071 ± 0.0470 Standard error of estimation 0.0019 0.0259 aIntra-day %RSD 0.15–0.36 0.03–0.08 bInter-day %RSD 0.51–0.62 0.06–0.22 Drug in laboratory prepared mixture 100.24 ± 0.61 100.04 ± 0.45 Drug added to dosage form 100.09 ± 0.53 100.27 ± 0.74 Drug in dosage form 103.08 ± 0.61 100.96 ± 0.57 Item CAL BTD Retention time (min) 7.01 8.00 Wavelength of detection (nm) 230 230 Range of linearity (μg/mL) 1–20 0.5–200 Regression equation PAR230nm = 0.0145 CCAL + 0.0050 PAR230nm = 0.0160 CBTD + 0.0071 Regression coefficient (r2) 0.9997 0.9996 LOD (μg/mL) 0.0394 0.1326 LOQ (μg/mL) 0.1195 0.4018 Sb 0.0001 0.0002 Sa 0.0014 0.0169 Confidence limit of the slope 0.0145 ± 0.0003 0.0160 ± 0.0004 Confidence limit of the intercept 0.0050 ± 0.0039 0.0071 ± 0.0470 Standard error of estimation 0.0019 0.0259 aIntra-day %RSD 0.15–0.36 0.03–0.08 bInter-day %RSD 0.51–0.62 0.06–0.22 Drug in laboratory prepared mixture 100.24 ± 0.61 100.04 ± 0.45 Drug added to dosage form 100.09 ± 0.53 100.27 ± 0.74 Drug in dosage form 103.08 ± 0.61 100.96 ± 0.57 aThe intra-day (n = 3), average of three concentrations of CAL (8, 10 and 12 μg/mL) and BTD (80, 100 and 120 μg/mL) repeated three times within the day. bThe inter-day (n = 3), average of three concentrations of CAL (8, 10 and 12 μg/mL) and BTD (80, 100 and 120 μg/mL) repeated three times in three successive days. Accuracy Satisfactory results of the analysis of laboratory prepared mixtures of the drugs, including the mean of the recovery and standard deviation, are displayed in Tables II–IV. By applying standard addition technique, good results obtained for the added drugs proved that there was no interference from the co-formulated drug or the frequently encountered excipients (Tables II–IV). Precision The repeatability of the method was validated by analyzing three separate mixtures containing (10 and 200 μg/mL of BTV and FSA, respectively), (100 and 10 μg/mL of BTD and CAL, respectively) and (10 and 156.25 μg/mL of BTD and CLO, respectively), (n = 6). The values of the intra-day and inter-day precision (%RSD), (using three different concentrations in triplicates for three consecutive days), for the analytes are presented in Tables II–IV. Selectivity The selectivity of a method is checked by its ability to determine the analyte(s) of interest in their laboratory prepared mixtures without any interference. Selectivity was also checked by assessing whether any interference arisen from common excipients and it was verified that the signals measured was attributed only to the analytes. The three analyzed dosage forms also contain preservatives (BZA, CHC) and inactive ingredients such as: white soft paraffin, liquid paraffin, tocopherol acetate and ethoxy castor oil. It was found that the excipients did not interfere with the obtained results. As shown in (Figures 3–5) the chromatograms of the pharmaceutical formulations did not show any interference with the separated mixtures components, thus confirming the selectivity of the developed method. Figure 3. View largeDownload slide A typical LC chromatogram of Fucicort® cream (BTV (4.80 μg/mL) and FSA (96 μg/mL)). Figure 3. View largeDownload slide A typical LC chromatogram of Fucicort® cream (BTV (4.80 μg/mL) and FSA (96 μg/mL)). Figure 4. View largeDownload slide A typical LC chromatogram of Clotrisone® cream (BTD (6.40 μg/mL) and CLO (100 μg/mL)). Figure 4. View largeDownload slide A typical LC chromatogram of Clotrisone® cream (BTD (6.40 μg/mL) and CLO (100 μg/mL)). Figure 5. View largeDownload slide A typical LC chromatogram of Calcipoheal-cort® ointment (BTD (20 μg/mL) and CAL (2 μg/mL)). Figure 5. View largeDownload slide A typical LC chromatogram of Calcipoheal-cort® ointment (BTD (20 μg/mL) and CAL (2 μg/mL)). Robustness Robustness of the present analytical procedure was examined by variation of some chromatographic conditions such as the flow rate of the mobile phase by ±0.1 mL/min and organic strength of the mobile phase by ±2% were evaluated. The most important parameter to be studied was the resolution factor between the eight peaks. As can be revealed in Table V, good values of the resolution factor were obtained for all these variations, indicating good robustness of the proposed HPLC method. Table V. Robustness Study of the Proposed RP-HPLC Method Parameter Drug Standard Flow rate (mL/min) Mobile phase composition 0.9 1.1 33:67 37:63 Resolution FSA/BZA 2.25 2.37 2.12 2.20 2.28 BZA/CHC 4.88 5.00 4.63 4.80 4.95 CHC/BTV 7.38 7.60 7.20 7.31 7.43 BTV/CAL 3.60 3.76 3.44 3.56 3.69 CAL/BTD 3.14 3.35 3.00 3.04 3.20 BTD/CLO 4.10 4.33 3.88 4.01 4.19 CLO/IS 2.54 2.63 2.41 2.48 2.61 Parameter Drug Standard Flow rate (mL/min) Mobile phase composition 0.9 1.1 33:67 37:63 Resolution FSA/BZA 2.25 2.37 2.12 2.20 2.28 BZA/CHC 4.88 5.00 4.63 4.80 4.95 CHC/BTV 7.38 7.60 7.20 7.31 7.43 BTV/CAL 3.60 3.76 3.44 3.56 3.69 CAL/BTD 3.14 3.35 3.00 3.04 3.20 BTD/CLO 4.10 4.33 3.88 4.01 4.19 CLO/IS 2.54 2.63 2.41 2.48 2.61 Table V. Robustness Study of the Proposed RP-HPLC Method Parameter Drug Standard Flow rate (mL/min) Mobile phase composition 0.9 1.1 33:67 37:63 Resolution FSA/BZA 2.25 2.37 2.12 2.20 2.28 BZA/CHC 4.88 5.00 4.63 4.80 4.95 CHC/BTV 7.38 7.60 7.20 7.31 7.43 BTV/CAL 3.60 3.76 3.44 3.56 3.69 CAL/BTD 3.14 3.35 3.00 3.04 3.20 BTD/CLO 4.10 4.33 3.88 4.01 4.19 CLO/IS 2.54 2.63 2.41 2.48 2.61 Parameter Drug Standard Flow rate (mL/min) Mobile phase composition 0.9 1.1 33:67 37:63 Resolution FSA/BZA 2.25 2.37 2.12 2.20 2.28 BZA/CHC 4.88 5.00 4.63 4.80 4.95 CHC/BTV 7.38 7.60 7.20 7.31 7.43 BTV/CAL 3.60 3.76 3.44 3.56 3.69 CAL/BTD 3.14 3.35 3.00 3.04 3.20 BTD/CLO 4.10 4.33 3.88 4.01 4.19 CLO/IS 2.54 2.63 2.41 2.48 2.61 Limit of detection and limit of quantitation Limit of detection (LOD) and limit of quantitation (LOQ) were calculated according to ICH recommendations (23). Both LOD and LOQ were calculated based on standard deviation of the intercept and the slope of the regression equation, Tables II–IV. Statistical analysis A statistical analysis of the results obtained by the proposed method was carried out. The results for the analysis of the studied drugs in their synthetic mixtures are in good agreement with those obtained using the reference method for each drug.The significant difference between groups were tested using Student’s t-test and variance ratio F-test at p = 0.05 (23) as shown in Table VI. The tests revealed that there was no significant difference among the methods regarding the accuracy and precision. Table VI. Statistical Comparison Between the Results of the Proposed Method and the Reference Methods for the Determination of Fusidic Acid, Betamethasone Valerate, Betamethasone Dipropionate, Clotrimazole and Calcipotriol Statistical term Reference method for FSAa FSA by the proposed method Reference method for BTVb BTV by the proposed method Reference method for BTDc BTD by the proposed method in Mixture II BTD by the proposed method in Mixture III Reference method for CLOd CLO by the proposed method Reference method for CALe CAL by the proposed method Mean 99.77 100.29 99.80 100.03 99.83 100.08 100.04 99.73 100.27 99.99 100.24 ± SD 0.87 0.70 0.54 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 ± SE 0.36 0.29 0.22 0.15 0.12 0.19 0.18 0.18 0.32 0.19 0.25 %RSD 0.87 0.70 0.55 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 n 6 6 6 6 6 6 6 6 6 6 6 V 0.76 0.49 0.30 0.14 0.09 0.22 0.20 0.20 0.61 0.22 0.37 t (2.23)* 1.12 0.86 1.11 0.97 1.47 0.80 F (5.05)* 1.55 2.14 2.44 2.22 3.05 1.68 Statistical term Reference method for FSAa FSA by the proposed method Reference method for BTVb BTV by the proposed method Reference method for BTDc BTD by the proposed method in Mixture II BTD by the proposed method in Mixture III Reference method for CLOd CLO by the proposed method Reference method for CALe CAL by the proposed method Mean 99.77 100.29 99.80 100.03 99.83 100.08 100.04 99.73 100.27 99.99 100.24 ± SD 0.87 0.70 0.54 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 ± SE 0.36 0.29 0.22 0.15 0.12 0.19 0.18 0.18 0.32 0.19 0.25 %RSD 0.87 0.70 0.55 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 n 6 6 6 6 6 6 6 6 6 6 6 V 0.76 0.49 0.30 0.14 0.09 0.22 0.20 0.20 0.61 0.22 0.37 t (2.23)* 1.12 0.86 1.11 0.97 1.47 0.80 F (5.05)* 1.55 2.14 2.44 2.22 3.05 1.68 *Figures in parentheses are the theoretical t and Fvalues at (p = 0.05). aBritish Pharmacopoeia 2016: Titration. bBritish Pharmacopoeia 2016: Spectrophotometric method. cBritish Pharmacopoeia 2016: HPLC method. dBritish Pharmacopoeia 2016: Non-aqueous titration. eBritish Pharmacopoeia 2016: HPLC method. Table VI. Statistical Comparison Between the Results of the Proposed Method and the Reference Methods for the Determination of Fusidic Acid, Betamethasone Valerate, Betamethasone Dipropionate, Clotrimazole and Calcipotriol Statistical term Reference method for FSAa FSA by the proposed method Reference method for BTVb BTV by the proposed method Reference method for BTDc BTD by the proposed method in Mixture II BTD by the proposed method in Mixture III Reference method for CLOd CLO by the proposed method Reference method for CALe CAL by the proposed method Mean 99.77 100.29 99.80 100.03 99.83 100.08 100.04 99.73 100.27 99.99 100.24 ± SD 0.87 0.70 0.54 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 ± SE 0.36 0.29 0.22 0.15 0.12 0.19 0.18 0.18 0.32 0.19 0.25 %RSD 0.87 0.70 0.55 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 n 6 6 6 6 6 6 6 6 6 6 6 V 0.76 0.49 0.30 0.14 0.09 0.22 0.20 0.20 0.61 0.22 0.37 t (2.23)* 1.12 0.86 1.11 0.97 1.47 0.80 F (5.05)* 1.55 2.14 2.44 2.22 3.05 1.68 Statistical term Reference method for FSAa FSA by the proposed method Reference method for BTVb BTV by the proposed method Reference method for BTDc BTD by the proposed method in Mixture II BTD by the proposed method in Mixture III Reference method for CLOd CLO by the proposed method Reference method for CALe CAL by the proposed method Mean 99.77 100.29 99.80 100.03 99.83 100.08 100.04 99.73 100.27 99.99 100.24 ± SD 0.87 0.70 0.54 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 ± SE 0.36 0.29 0.22 0.15 0.12 0.19 0.18 0.18 0.32 0.19 0.25 %RSD 0.87 0.70 0.55 0.37 0.30 0.47 0.45 0.45 0.78 0.47 0.61 n 6 6 6 6 6 6 6 6 6 6 6 V 0.76 0.49 0.30 0.14 0.09 0.22 0.20 0.20 0.61 0.22 0.37 t (2.23)* 1.12 0.86 1.11 0.97 1.47 0.80 F (5.05)* 1.55 2.14 2.44 2.22 3.05 1.68 *Figures in parentheses are the theoretical t and Fvalues at (p = 0.05). aBritish Pharmacopoeia 2016: Titration. bBritish Pharmacopoeia 2016: Spectrophotometric method. cBritish Pharmacopoeia 2016: HPLC method. dBritish Pharmacopoeia 2016: Non-aqueous titration. eBritish Pharmacopoeia 2016: HPLC method. Discussion Two challenges were encountered in this study. First, the investigated mixtures contain betamethasone either as valerate or dipropionate ester in combination with FSA, CLO and CAL when one of the components is present in low concentration relative to the other which represented an analytical problem in their determination. Second, the separation of betamethasone esters (BTV, BTD) and FSA which are closely related in structures and could not be easily separated simultaneously in a single HPLC run. Therefore, the aim of this study was to successfully overcome the two encountered challenges and to develop a simple and sensitive method for the simultaneous separation and quantification of three of the most commonly prescribed topical preparations containing betamethasone in a short time to be suitable for routine high capacity work in quality control laboratories either in bulk or in pharmaceutical dosage form. So, to achieve this aim, different chromatographic conditions including different reversed-phase columns and different mobile phases were attempted. Various mobile phase compositions containing different ratios of organic and aqueous phases were tried in an isocratic mode. Good separation and best peak shape were obtained upon using Discovery® C18 column and an isocratic mobile phase composed of water: acetonitrile (35:65, v/v), Figure 2. According to USP 2015 (11), system suitability tests are a fundamental part of liquid chromatographic methods in the optimization of the conditions of the proposed method. They are used to verify that the resolution and reproducibility were adequate for the performed analysis. The developed HPLC system was found to be suitable for the determination of all components, Table I. Method validation was carried out in accordance with ICH guidelines (23). The linearity of the proposed HPLC method was assessed. Linear relationships between PAR and component concentration (C) and high regression coefficients were obtained for each drug, Tables II–IV. Accuracy of the proposed method was assessed by calculating % recovery of six different concentrations of laboratory prepared mixtures of the drugs analyzed by the proposed method. The accuracy was also confirmed by recovery studies from sample solution at different levels of standard additions. Intra-day and inter-day precision were also evaluated. The developed method was found to be accurate and precise, Tables II–IV. In addition, good results in terms of LOD, LOQ, robustness and selectivity were obtained, Tables II–V. Statistical analysis of the results revealed no significance difference between the results of the developed method and that of the reported ones, Table VI. As a conclusion, the developed method succeeded in separating five drugs in addition to two preservatives and the IS, simultaneously, with good resolution factor in relatively short run time. The method also proved to be sensitive, simple, rapid and selective so it can be used for routine analysis of the cited mixtures. Conclusion In this study, a simple, sensitive, highly selective and reliable HPLC method has been developed for the simultaneous determination of three binary mixtures containing betamethasone esters in combination with FSA or CAL or CLO in laboratory prepared mixtures and pharmaceutical preparations, without any interference from the excipients. The analytes were successfully resolved and quantified in relatively short run time using isocratic mode of elution and simple mobile phase consisting of water and acetonitrile. Consequently, the developed method can be considered time- and cost-effective beside the other advantages including accuracy and sensitivity. The method was validated showing satisfactory results for all the method validation parameters tested. It can be conveniently used in quality control laboratories and routine analysis of the cited drugs. References 1 Sweetman , S.C. ; Martindale: The Complete Drug Reference . Pharmaceutical Press , London, ( 2011 ). 2 Girolomoni , G. , Mattina , R. , Manfredini , S. , Vertuani , S. , Fabrizi , G. ; Fusidic acid betamethasone lipid cream ; International Journal of Clinical Practice , ( 2016 ); 70 : 4 – 13 . Google Scholar CrossRef Search ADS PubMed 3 Schaller , M. , Friedrich , M. , Papini , M. , Pujol , R.M. , Veraldi , S. ; Topical antifungal‐corticosteroid combination therapy for the treatment of superficial mycoses: conclusions of an expert panel meeting ; Mycoses , ( 2016 ); 59 : 365 – 373 . 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Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Published: Sep 1, 2018

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