Stability Indicating RP- HPLC Method for Simultaneous Estimation of Salicylic Acid and Ketoconazole in Shampoo Formulation

70 Available online at www.derpharmachemica.com ISSN 0975-413X CODEN (USA): PCHHAX Der Pharma Chemica, 2023, 15(3): 70-79 (http://www.derpharmachemica.com/archive.html) Stability Indicating RPHPLC Method for Simultaneous Estimation of Salicylic Acid and Ketoconazole in Shampoo Formulation Dilip Kowndinya B1*, Basava Rao VV1 and Sailu CH1 1Department of Pharmacy, University College of Technology, Osmania, Hyderabad-500007, Telangana, India *Corresponding author: Dilip Kowndinya B, Department of Pharmacy, University College of Technology, Osmania, Hyderabad-500007, Telangana, India, E-mail: [email protected] Received: 01-May-2023, Manuscript no: dpc-23-101157, Editor assigned: 05-May-2023, PreQC No: dpc-23-101157, Reviewed: 19-May-2023, QC No: dpc-23-101157, Revised: 26-May-2023, Manuscript No: dpc-23-101157, Published: 31-May-2023, DOI: 10.4172/0975-413X.15.3.70-79 ABSTRACT A reverse phase HPLC method was developed and validated for the simultaneous estimation of Salicylic acid and Ketoconazole in shampoo formulation. Separation of the drugs was done using Kromasil C18 (250×4.6mm, 5µ) column at 322 nm using a mobile phase comprising of Acetonitrile: Buffer (0.01 N Potassium dihydrogen orthophosphate adjusted to a PH of 5.4 using dilute Orthophosphoric acid) in the ratio of 50:50 at a flow rate of 1.0 mL/min. Retention times of Salicylic acid and Ketoconazole were found to be 2.307 min, 3.342 min respectively. Method was found to be linear in the range of 5-30 µg/ml. Developed method was validated and stability studies were performed according to ICH guidelines and applied for the assay of the drugs in the shampoo formulation. Keywords: Salicylic acid; Ketoconazole; Validation; HPLC; Shampoo INTRODUCTION Ketoconazole1-[4-(4-{[(2R, 4S)-2-(2, 4-Dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1, 3-dioxolan-4-yl] methoxy} phenyl) piperazin-1-yl] ethan1-one is antifungal drug used to prevent and treat fungal skin infections (Figure1). Ketoconazole is commercially available in the form of antidandruff shampoo, topical creams and lotions. Salicylic acid is a (2-hydroxyl benzoic acid), and naturally occurring in the bark of willow tree (Salix Alba) (Figure 2) The salts and esters of salicylic acid are known as salicylates that are widely used as rubefacient and analgesic in several topical formulations. Salicylic acid alleviates peeling of intercellular cement and binds with scales in the stratum corneum, thereby loosening the Dilip Kowndinya B, et al 71 Der Pharma Chemica, 2023, 15(3): 70-79 keratin. This keratolytic effect also renders an antifungal effect as removal of the stratum corneum suppresses the fungal growth. It is widely used for the treatment of several skin diseases like acne, psoriasis, seborrhoeic dermatitis, calluses, keratosis pilaris, and warts due to its keratolytic, fungicidal, bacteriostatic, and photo protective properties. HPLC methods for estimation of Salicylic acid and Ketoconazole were published individually and in combination with other drugs like pythione. Literature review [1-3] revealed that there are no HPLC methods published till date for the simultaneous estimation Salicylic Acid and Ketoconazole; hence there is a scope for development of a new method for the simultaneous estimation of both the drugs in combined dosage forms. The main objective of present study is to develop a simple, accurate, precise, sensitive, selective, reproducible and rapid analytical technique for simultaneous estimation of Salicylic Acid and Ketoconazole, in bulk and shampoo [4, 5]. Figure 1: Structure of Ketoconazole. Figure 2: Structure of Salicylic acid. EXPERIMENTAL Materials Required (2) Salicylic Acid and Ketoconazole pure drugs (API) received from MSN Pharma Ltd. Combined form of Salicylic Acid and Ketoconazole shampoo purchased from local market (Table 12) Table 1: Equipment’s and Apparatus. S. No Instruments Make and model 1 HPLC Waters HPLC system outfitted with 515 dual head reciprocating pump and a 2489 UV Visible detector. The software used was Empower-2. Enable C-18 column (5µm, 250mm×4.6mm i.d) 2 UV-VIS Spectrophotometer Model-2201, SYSTRONICS. 3 Digital pH meter Model-MKVI, SYSTRONICS. 4 Electronic balance Model-AY 220, SHIMADZU. 5 Ultrasonicator BIOTECHNICS, India. 6 Vacuum filtration apparatus Borosil 5340 Vacuum pressure pump TID-75-S. Dilip Kowndinya B, et al 72 Der Pharma Chemica, 2023, 15(3): 70-79 Table 2: Chemicals and Reagents. S. No Chemical/Reagent Grade Make 1 Methanol HPLC Merck 2 Acetonitrile HPLC Merck 3 Orthophosphoric acid AR Merck 4 Potassium dihydrogen phosphate AR Merck 5 Water HPLC In house 6 Sodium hydroxide AR SD fine Chemicals 7 Hydrochloric acid AR SD fine Chemicals Preparation of Solutions (3) Preparation of Stock solutions (3.1) Accurately weighed 100 mg of Ketoconazole and 100 mg of Salicylic Acid and transferred to 100 ml volumetric flask. And 3/4th of diluent was added to these flask and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution. (1000 µg/ml of Ketoconazole and 1000µg/ml of Salicylic Acid) Preparation of Cc standards (3.2) Calibration curve standards were prepared by pipetting suitable aliquots of secondary stock solution containing 200µg/ml of Ketoconazole and 200µg/ml of Salicylic Acid into separate 10 ml volumetric flasks and the volume was made up to the mark with diluent to obtain the CC standards in the range of 5 to 30 µg/ ml concentrations for both drugs (Table 3) Table 3: Preparation of standard Solutions for linearity. S.No Pipetted from stock (mL) Volume of flask (mL) Concentration in ppm (Sali) Concentration in ppm (Keto) %Linearity Level 1 0.25 10 5 5 25 2 0.5 10 10 10 50 3 0.75 10 15 15 75 4 1 10 20 20 100 5 1.25 10 25 25 120 6 1.5 10 30 30 150 Preparation of diluent (3.2.1) Acetonitrile and Water taken in the ratio of 50:50 %v/v. It was ultra sonicated for 5min. Preparation of Cc standards (3.2.2) Shampoo equivalent to 20mg of Salicylic Acid & 20mg of Ketoconazole was transferred into a 100 ml of volumetric flask, 50ml of diluents was added and sonicated for 25min, and further the volume was made up with diluent and filtered. Resulting solution contain 200µg/ml of Salicylic Acid & 200µg/ml Ketoconazole. 1ml of filtered sample stock solution was transferred in to a 10ml volumetric flask and made up the volume with diluent, resulting solution contain 20µg/ml of Salicylic Acid & 20µg/ml Ketoconazole. Dilip Kowndinya B, et al 73 Der Pharma Chemica, 2023, 15(3): 70-79 METHODOLOGY Chromatographic conditions (4) The method was developed and validated using Kromosil C-18, (250 x 4.5 mm, 5.) column. Mobile phase is prepared by mixing HPLC grade Acetonitrile and 0.01N Potassium phosphate buffer (adjusted to pH 5.4) in the ratio of 50:50 % v/v as mobile phase. Separation was done in isocratic mode at 1.0 mL/min flow rate. Detection wavelength was selected at 322 nm System suitability (4.1) The system suitability parameters were determined by using standard solutions of concentration 20 ppm for both Ketoconazole and Salicylic Acid. Six replicate injections of the standards were injected and the parameters like USP peak tailing, resolution and USP plate count were determined. Method validation (4.2) The method validation was performed in accordance with ICH guidelines Linearity (4.2.1) Prepared standard dilutions of Ketoconazole and Salicylic Acid (5-30µg/ml) were injected and chromatograms were recorded. A calibration curve was constructed by taking concentration on Xaxis and peak area on Yaxis. Accuracy (4.2.2) Accuracy was determined by the recovery studies of the analyte. It is determined by standard addition method, where the test solution of known quantity is spiked with standard solutions at three levels i.e., 50%, 100% & 150% in triplicate. Percentage recoveries at all the levels were calculated. Precision (4.2.3) Precision of the method was established at two levels i.e., system precision and method precision. System precision was performed by injecting the working standard solution. Six injections were given from the same standard solution and the peak areas were obtained. Average area, standard deviation and % RSD were calculated for two drugs. Method precision was established by taking multiple sampling from a sample stock solution. Six working sample solutions of same concentrations were prepared, single injection from each working sample solution was given. Average peak area, standard deviation and % RSD were calculated for two drugs. Robustness (4.2.4) Small deliberate changes in method like flow rate, mobile phase ratio, and temperature are made. Robustness conditions like Flow minus (0.9ml/min), Flow plus (1.1ml/min), mobile phase minus, mobile phase plus, temperature minus (25°C) and temperature plus (35°C) was maintained and samples were injected in sextet. System suitability parameters are evaluated by making the deliberate changes. Specificity (4.2.5) The specificity was established by performing stress degradation studies for the analyte under various conditions like acid degradation, alkali degradation, oxidative degradation, thermal degradation and photo degradation. Acid Degradation Studies (4.2.5.1) To 1 ml of stock solution Salicylic Acid & Ketoconazole 1ml of 2N Hydrochloric acid was added and refluxed for 30mins.The resultant solution was diluted to obtain (20ppm & 20ppm) solution and 10 µl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample Alkali Degradation Studies (4.2.5.2) To 1 ml of stock solution Salicylic Acid & Ketoconazole 1 ml of 2N sodium hydroxide was added and refluxed for 30mins. The resultant solution was diluted to obtain (20ppm& 20ppm) solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample. Thermal Degradation Studies (4.2.5.3) The standard drug solution was placedinovenat1050C for 6h to study dry heat degradation. For HPLC study, the resultant solution was diluted to (20ppm& 20ppm) solutionand10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample. Photo Stability studies (4.2.5.4) The photochemical stability of the drug was also studied by exposing the (200ppm & 200ppm) solution to UV Light by keeping the beaker in UV Chamber for 7days or 200 Watt hours/m2 in photo stability chamber. For HPLC study, the resultant solution was diluted to obtain (20ppm & 20ppm) solutions and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample. Oxidative degradation (4.2.5.5) To 1 ml of stock solution of Ketoconazole and Salicylic Acid, 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were Dilip Kowndinya B, et al 74 Der Pharma Chemica, 2023, 15(3): 70-79 kept for 30 min at 60 0C. For HPLC study, the resultant solution was diluted to obtain 60 µg/ ml & 40 µg/ ml solution.10 µl was injected into the system and the chromatograms were recorded to assess the stability of sample. RESULTS AND DISCUSSION Assay of formulation (5.1) Assay of the formulation is performed in triplicate. The amount of drug present in the formulation was calculated. The % assay of Salicylic Acid and Ketoconazole obtained was 99.02 and 99.51 % respectively. Representative chromatograms for standard and test were given in (Figure 34). Results were given in the below (Table 4) Figure 3: Representative Chromatogram of working standard solution. Figure 4: Representative Chromatogram of working sample solution. Table 4: Assay of Salicylic Acid and Ketoconazole. S.NO Peak area of Salicylic Acid Peak area of Ketoconazole 1 1205637 1248455 2 1201125 1245480 3 1211505 1242130 4 1206432 1245404 5 1214450 1249068 6 1213795 1242086 Dilip Kowndinya B, et al 75 Der Pharma Chemica, 2023, 15(3): 70-79 Avg 1208824 1245437 Regression equation y = 60256x + 11786 y = 6134.x + 432.1 % Assay 99.02% 99.51% System suitability (5.2) System suitability parameters were determined according to ICH guidelines. Plate count should be more than 2000, tailing factor should be less than 2 and resolution must be more than 2. All the system suitable parameters were passed and were within the limits. The results showing system suitability parameters were given in below (Table 5) Table 5: System suitability parameters for Salicylic Acid and Ketoconazole. Salicylic Acid Ketoconazole Inj RT(min) USP Plate Count Tailing RT(min) USP Plate Count Tailing Resolution 1 2.305 5069 1.43 3.339 6721 1.43 6.8 2 2.307 5115 1.48 3.341 6759 1.43 6.9 3 2.307 5169 1.47 3.341 6777 1.43 6.9 4 2.308 5101 1.51 3.342 6631 1.42 6.9 5 2.309 5197 1.5 3.346 6650 1.42 6.8 6 2.3 5235 1.49 3.346 6560 1.44 6.8 Validation (5.3) Linearity (5.3.1) The linearity was determined at concentration range of 5-30µg/ml for Ketoconazole and Salicylic Acid. Graph was plotted using Peak areas against concentration. The calibration curve was shown in (Figure 5-7). The Correlation coefficient (r2) was greater than 0.99 within the concentration range for both the drugs. The results for linearity were given in the below (Table 6) Table 6: Linearity data of Salicylic Acid and Ketoconazole. Salicylic Acid Ketoconazole Conc (μg/mL) Peak area Conc (μg/mL) Peak area 5 320230 5 325672 10 623245 10 635101 15 919360 15 998492 20 1217201 20 1259266 25 1505773 25 1606603 30 1823620 30 1885676 Dilip Kowndinya B, et al 76 Der Pharma Chemica, 2023, 15(3): 70-79 Figure 5: Calibration curve of Salicylic Acid. Figure 6: Calibration curve of Ketoconazole. Figure 7: Overlay chromatogram of linearity. y = 60256x + 11786 R² = 0.9998 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 2000000 0 5 10 15 20 25 30 35 Peak Area Concentration Linearity of Salicylic acid y = 63165x + 11217 R² = 0.999 0 500000 1000000 1500000 2000000 2500000 0 5 10 15 20 25 30 35 Peak Area Concentration Linearity of Ketoconazole Dilip Kowndinya B, et al 77 Der Pharma Chemica, 2023, 15(3): 70-79 Accuracy (5.3.2) Accuracy of the method was established at three levels of concentrations by standard addition method. Triplicate injections were given at each level of accuracy and percentage recoveries were calculated. The mean % Recovery was obtained was 99.09 to 99.46% for Salicylic Acid and 99.1499.46 % for Ketoconazole. The results for accuracy was given in the below (Table 7-8) Table 7: Accuracy results of Salicylic Acid. %Level(N=3) Spiked Concentration(μg/mL) Standard concentration (μg/mL) Amount recovered(μg/mL) % Recovery Mean % Recovery 50% 10 20 9.960559 99.61 99.48. 10 20 10.02872 100.29 10 20 9.85448 98.54 100% 20 20 19.62445 98.12 99.09 20 20 19.87763 99.39 20 20 19.95562 99.78 150% 30 20 30.14734 100.49 99.96 30 20 29.91877 99.73 30 20 29.8999 99.67 Table 8: Accuracy results of Ketoconazole. % Level (N=3) Spiked Concentration (μg/mL) Standard concentration (μg/mL) Amount recovered(μg/mL) % Recovery Mean % Recovery 50% 10 20 9.939957 99.4 99.44 10 20 9.842787 98.43 10 20 9.920437 99.2 100% 20 20 19.8967 99.48 99.44 20 20 19.85804 99.29 20 20 19.91413 99.57 150% 30 20 29.75129 99.17 99.16 30 20 29.68003 98.93 30 20 29.81963 99.4 Precision (5.3.3) The precision of the method was studied by considering system precision and method precision. System precision was studied by taking six replicate injections from same homogenous standard solution and peak areas were determined. Average area, standard deviation and % RSD were calculated for two drugs. % RSD obtained as 0.9% and 1.0 % respectively for Ketoconazole and Salicylic Acid. The results for system precision were given in (Table 6). Method precision was studied by preparing six test solutions of same concentration and injected once from each solution and peak areas were determined. Average area, standard deviation and % RSD were calculated for two drugs and obtained as 0.4 % and 0.2 % respectively for Ketoconazole and Salicylic Acid. The results for method precision were given in the (Table 9-10) Dilip Kowndinya B, et al 78 Der Pharma Chemica, 2023, 15(3): 70-79 Table 9: System Precision data of Salicylic Acid and Ketoconazole. System precision S. No Area of Area of Salicylic Acid Ketoconazole 1 1065046 1223116 2 1076051 1220824 3 1084787 1218110 4 1078299 1227569 5 1058304 1247270 6 1074445 1213756 Mean 1072822 1225108 S.D 9565.1 11809.9 %RSD 0.9 1 Table 10: Method Precision results of Salicylic Acid and Ketoconazole. Salicylic Acid Ketoconazole S. No Area of Standard solution Area of test solution % Assay Area of Standard solution Area of test solution % Assay 1 1193010 1205637 98.75 1252212 1248455 99.39 2 1225777 1201125 98.39 1242571 1245480 99.16 3 1215780 1211505 99.24 1258468 1242130 98.89 4 1206484 1206432 98.82 1250160 1245404 99.15 5 1225638 1214450 99.48 1256925 1249068 99.44 6 1212195 1213795 99.42 1253390 1242086 98.89 Mean 1217175 1208824 99.02 1252288 1245437 99.15 S.D 12436 5266.9 0.431 5654.4 2982 0.24 %RSD 1 0.4 0.4 0.5 0.2 0.2 Robustness (5.3.4) Robustness of the method was studied by making deliberate changes in flow rate, mobile phase ratio and column oven temperature. After making each change in the conditions, chromatograms were recorded by injecting the standard solutions in six replicates. System suitability parameters were checked at each level. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit. Results were given in the below (Table 11). Table 11: Robustness data for Ketoconazole and Salicylic Acid. S. No Robustness Condition Salicylic Acid %RSD (n= 6) Ketoconazole %RSD (n= 6) 1 Flow rate (-) 0.9ml/min 1 0.7 2 Flow rate (+) 1.1ml/min 0.9 1 3 Mobile phase (-) 60B:40A 0.9 0.8