A simple stability indicating high performance thin layer chromatography (HPTLC) method was developed and

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ISSN: 0975766X CODEN: IJPTFI Available Online through Research Article www.ijptonline.

1 ISSN: X CODEN: IJPTFI Available Online through Research Article DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF IFENESIN AND BUTAMOL SULPHATE Dipali D. Chavan, Mrinalini C. Damle *, Purushotam Sinha Department of Quality Assurance, AISSMS College of Pharmacy, Kennedy Road, Near R.T.O., Pune411001, Maharashtra, India. Received on Accepted on Abstract A simple stability indicating high performance thin layer chromatography (HPTLC) method was developed and validated for determination of Guaifenesin and Salbutamol. Study was performed on precoated silica gel HPTLC plates using Toluene: Methanol: Formic acid (6.5:3:0.5 v/v/v) as the mobile phase. Detection wavelength chosen was 240 nm. Retention factor (Rf) of Guaifenesin and salbutamol sulphate were 0.60 ± 0.02 and 0.29 ± 0.03 respectively. Guaifenesin and salbutamol were subjected to acid, base, neutral, oxidation, heat and photo degradation studies. The degradation products obtained were well resolved from the pure drugs. Method was validated according to ICH Q2R1 guidelines. The correlation coefficients of calibration curves were found to be for Guaifenesin and for Salbutamol in the concentration range of ng/band for both the drugs. The method had an accuracy in the range of % for Guaifenesin and %for Salbutamol sulphate. As the method could effectively separate the drugs from its degradation products, it can be used for stabilityindicating analysis. Keywords: Guaifenesin, High performance thin layer chromatography, Salbutamol sulphate, Stability indicating method. Introduction: Guaifenesin () is chemically 3(2methoxyphenoxy)propane1,2diol. It is a white crystalline powder with molecular weight of and is sparingly soluble in water and soluble in alcohol. It acts as an expectorant in the symptomatic management of coughs associated with the common cold, bronchitis, influenza, pharyngitis, measles IJPT July2015 Vol. 7 Issue No Page 8183

2 etc.salbutamol sulphate () is a shortacting β 2 adrenergic receptor agonist. It is used for the relief of bronchospasm in conditions such as asthma, chronic obstructive pulmonary disease (COPD) and in prevention of exercise induced bronchospasm [13]. is chemically (RS)1(4hydroxy3hydroxymethylphenyl) 2 (tertbutylamino)ethanol sulphate. It is a white crystalline powder with a molecular weight of The combinations of and are available in the market and used to relieve respiratory obstruction and improve pulmonary ventilation [4, 5]. Chemical structure of and are shown in Figure 1. Fig 1: Chemical structures of (a) and (b) Several methods have been reported in literature for individual estimation of the drugs but very few methods have been reported for simultaneous estimation of and in combined dosage forms, which includes, HPLC [6,7], stability indicating RPHPLC [8], HPTLC [9], UV [10]. But there was no report of stability indicating HPTLC method. Therefore the aim of this study was to develop and validate simple and specific stability indicating HPTLC method for simultaneous estimation of and in pure forms and their combined dosage forms. The proposed method was successfully applied to marketed Ventorlin syrup and the contents were determined without any interference of excipients. Experimental Instrumentation and chromatographic conditions The samples were spotted in the form of bands of 4 mm with a Camag 100 µl sample syringeusing a Linomat 5 applicator, aluminumplates precoated with silica gel 60 F 254 (10 cm x 10 cm, 250 µm layer thickness) were used. Development was performed in a Camag twin trough glass chamber (10 cm x 10cm) saturated with the mobile phase. The mobile phase was composed of Toluene:Mathanol:Formic acid (6.5:3:0.5 v/v/v) with saturation time of 15 min and10 ml of mobile phase was used per run. Densitometric scanning was performed on Camag TLC scanner III in the IJPT July2015 Vol. 7 Issue No Page 8184

3 range nm, operated by WinCATS software, slit dimensions were 3.00 x 0.45 mm and Duterium lamp was used as a radiation source. Materials Guaifenesin and Salbutamol sulphate were kindly supplied as gift sample by Wockhardt pharmaceuticals Aurangabad and Cipla pharmaceuticals Ltd. Mumbai respectively. Toluene, methanol and formic acid of AR grade purchased from Merck Pvt. Ltd. Mumbai. Hydrochloric acid, hydrogen peroxide and sodium hydroxide were purchased from LOBA CHEMIE PVT. LTD. Mumbai.Ventorlin syrup (GSK Pharmaceutical Ltd, India) was purchased from local market. Preparation of standard solutions Standard stock solution of Guaifenesin was prepared separately by dissolving 10 mg of drug in 10 ml of methanol to get concentration of 1000 µg/ml. And solution of Salbutamol sulphate was similarly prepared using water. From the respective standard stock solution, working standard solution was prepared containing 100 µg/ml of Guaifenesin and Salbutamol sulphate separately in methanol. Preparation of sample solution (syrup formulation analysis) Extraction of from syrup: To 5 ml of syrup (equivalent to 1 mg of ) 5 ml of NaOH (5% w/v) was added. The solution was mixed and extracted with 2.5 ml of chloroform twice. Aqueous layer was discarded by filtration through cotton plug. The organic layer was evaporated and residue was dissolved in 10 ml methanol to get conc. of 100 µg/ml. Extraction of Guaifenesin from syrup: 5 ml of syrup (equivalent to 50 mg of ) was extracted using 2.5 ml chloroform twice. The organic layer was evaporated and remaining residue was diluted suitably with methanol to get final conc. (100 µg/ml). Selection of analytical wavelength: 10 µg/ml of standard solutions of and were scanned over the range of nm. The spectra were obtained. It was observed that max of was 226 nm and max of was 224 nm. As both the drugs have nearly same max, 224 nm was selected for simultaneous scanning of and. Optimization of HPTLC densitometric method Chromatographic separation studies were carried out on the working standard solution of (100 µg/ml) and (100 µg/ml). Initially, trials were carried out using various solvents in various proportions on normal TLC plates, to IJPT July2015 Vol. 7 Issue No Page 8185

4 obtain the desired system suitability parameters.the optimized mobile phase comprising of toluene: methanol: formic acid (6.5:3:0.5 v/v/v) showed as acceptable resolution and gave compact spots together with sharp and well defined symmetrical peaks for both drugs.other chromatographic conditions like chamber saturation time and sample application volume were optimized. Stress degradation studies of bulk drug Stress degradation studies were carried out as per ICH Q1A (R2) under condition of acid/ base/ neutral hydrolysis, oxidation, dry heat and photolysis. For each study, three samples were prepared: stressed sample, stressed sample and the blank subjected to stress in the same manner as the drug solution. Dry heat and photolytic degradation were carried out in solid state. Acidic degradation: Acid induced degradation was performed by adding 2.5 ml of 0.01 N and 0.1 N HCL (methanolic) separately to each volumetric flask containing 2.5 ml of 1000 µg/mlof and respectively. The volume was made upto 25 ml with methanol and refluxed for 30 min at 50 0 C. 15 µl of the resultant solution was then applied on TLC plate and densitogram was developed. Neutral degradation: 2.5 ml standard solution of (1000 µg/ml) was mixed with 2.5 ml water and volume was made upto 25 ml with methanol. The solution was refluxed for 1 hr at 60 0 C. was treated in similar manner as. Alkaline degradation: Base induced degradation was performed by adding 2.5 ml of 0.1 N NaOH to each containing 2.5 ml of 1000 µg/mlof and. The volume was made upto 25 ml with methanol and refluxed for 2 hr at 50 0 C. Oxidative degradation: Hydrogen peroxide (10 %) was prepared and 2.5 ml was added each containing2.5 mlof 1000 µg/ml of and. The volume was made upto 25 ml with methanol and refluxed at 60 0 C for 30 min. Degradation under dry heat: For dry heat degradation, the standard drugs were ( for 6 hr and for 24 hr) placed in an oven at 80 0 C. Samples were withdrawn, dissolved in methanol separately and diluted to get 100 g/ml. IJPT July2015 Vol. 7 Issue No Page 8186

5 Photodegradation studies: Photolytic studies were carried out by exposure of and to UV light up to 200 watt hours/square meter and subsequently to cool fluorescent light to achieve an illumination of 1.2 million Lux hours. After exposuresamples were weighed, dissolved and diluted to get 100 µg/ml. Validation of the proposed method The proposed method was validated according to ICH Q2A(R1). The following parameters were used for validation of the developed method. Specificity: The specificity of the method was ascertained by peak purity profiling studies. The peak purity values were found to be more than , indicating the noninterference of any other peak of degradation product or impurity. Linearity and range Calibration curves were plotted over the concentration range of ng/band for both and. From 100 µg/ml standard solutions of and (5, 10, 15, 20, 25, and 30 µl) were overspottedon TLC plate. The calibration curves were prepared by plotting peak area versus concentration (ng/band). Each reading was an average of five determinations. Precision The precision of the method was demonstrated by intraday and interday studies. In the intraday precision six independent sample preparations of (100 µg/ml) and (100 µg/ml) were prepared,15 µl of these (1500 ng/band) were over spotted. Interday precision of the system was evaluated by analysing 3 independent standard preparations of (100 µg/ml) and (100 µg/ml) on three consecutive days and 10, 15, 20 µl of solutions were over spotted. Accuracy Accuracy of the method was determined by standard addition method and calculating the recoveries of and. Standard addition done at three different levels 80, 100 and 120%. Hence 800, 1000 and 1200 ng/band of and were spiked to the sample solution containing 1000 ng/band of and after sample dilution. The recovery of and were calculated by using linearity equations of and respectively. IJPT July2015 Vol. 7 Issue No Page 8187

6 Limit of detection (LOD)and limit of quantification(loq) The limit of detection and quantification of the developed method were calculated from the standard deviation of the response and slope of calibration curves of and using the formulae as given below. LOD = 3.3 x σ / S LOQ = 10 x σ / S Where,σ = standard deviation of the response S = slope of calibration curve Robustness The effect of deliberate variations in method parameters like the chamber saturation time, composition of the mobile phase, time from spotting to development and time from development to scanning were evaluated in this study. The effect of these changes on peak areas was evaluatedby calculating the relative standard deviations (RSD) for each parameter. Results and Discussion Forced degradation studies: Results of forced degradation studies are given in Table 1. According to the forced degradation studies carried out for and, it was found that, although % recovery was reduced for under all stress conditions but no peak of degradation product was observed in chromatograph. when exposed to 0.01 N HCL, 23.75% degradation was observed with degradation product at Rf of 0.45 while was found to be stable. When subjected to higher stress condition with 0.1 N HCL, 9.8% degradation was recorded. Table 1: Results of forced degradation studies. Drug Stress Degradation Condition % Recovery Acid (HCl) 0.1 N, reflux at 50 0 C for 30 min 0.01 N, reflux at 50 0 C for 30 min Neutral Reflux at 60 0 C for 1 hr Reflux at 60 0 C for 1 hr R f of Degradant 0.45 Peak purity r(s,m) r(m,e) Base (NaOH) 0.1 N, reflux at 50 0 C for 2 hrs IJPT July2015 Vol. 7 Issue No Page 8188

7 0.1 N, reflux at 50 0 C for 2 hrs Oxidative (H 2 O 2 ) 10% v/v, reflux at 60 0 C for 30min 10% v/v, reflux at 60 0 C for 30min Heat 80 0 C for 24 hrs 80 0 C for 6 hrs Florescence (UV, 200 watt hrs/square meter, Florescence, 1.2million Lux.Hrs) Under alkaline conditions showed 14.93% degradation while showed 21.09% degradation and separate degradation peak was obtained for at Rf of The only representative chromatograms of drugs showing degradation and overlaid UV spectra of respective degradation products are presented in Figure 3. showed 7.7%, 15.84%, 10.59%, and 5.62% degradation under neutral, oxidative, thermal and photo degradation conditions respectively. showed %, 9.48 %, % and1.02 % degradation under neutral, oxidative, thermal and photo degradation conditions respectively. The degradation peaks were well resolved form the main peak. Figure 3: I) Chromatogram of Acid treated, II) overlaid spectra of std., acid treated and its degradant, III) chromatogram of NaOH treated, IV) overlaid UV spectra of Std., NaOH treated and it s degradant. IJPT July2015 Vol. 7 Issue No Page 8189

8 Results of stress degradation are comparable to the one reported by Walode et.al. And the developed HPTLC method has advantage of high throughput over reported stability indicating HPLC method. Method validation Linearity Peak areas were found to have better linear relationship with the concentration, linearity 3D Densitogram of and shown in figure 4. For the r 2 was found to be , and for the r 2 was Calibration curves (Figure 5) were constructed in concentration range of ng/band for and both. Figure 2: Overlay UV Spectrum of (10 µg/ml) and (10 µg/ml) Figure 4: 3D Chromatogram of linearity of and ( ng/band) at 224 nm y = x R² = Conc ng/band Area y = x Area R² = Conc. ng/band Figure 5: Calibration curves of and IJPT July2015 Vol. 7 Issue No Page 8190

9 Accuracy: Mrinalini C. Damle* et al. International Journal Of Pharmacy & Technology The recovery studies were carried out at 80%, 100% and 120% of the test concentration. The % recovery of and at all the three levels was found to be satisfactory (Table 2). For, the % recovery was found between % and % and for between % and % respectively. Table2: Recovery studies of and. Level (%) Therapeutic Conc. (ng/band) Area Recovered Conc. (ng/band) % Recovery Precision The intra and interday precision of the developed method were expressed in terms of relative standard deviation (% RSD) of the peak area. The values of RSD for intraday and interday variations1.80 % and 1.35 % for and 0.92 % and 1.39 % for were within the acceptable range (Table 3 and 4). Table3: Intraday precision of and. Replicate 1500 ng/band 1500 ng/band Mean Area Std. Dev % RSD IJPT July2015 Vol. 7 Issue No Page 8191

10 Table4: Interday precision of and. Mrinalini C. Damle* et al. International Journal Of Pharmacy & Technology Concentration (ng/band) Area Mean Area SD % RSD LOD and LOQ The LOD and LOQ were found to be and ng/band for and and ng/band for indicating sensitivity of the developed method. Robustness The robustness of the method evaluated by assessing the effect of variations in method parameters on peak areas showed low RSD values (less than 2%) indicating robustness of the method (Table 5). Table5: Robustness Study. Sr. No. Parameters Robust condition % RSD 1. Chamber saturation time(15min) ± 2 min. 13min min IJPT July2015 Vol. 7 Issue No Page 8192

11 2. Mobile phase Toluene: MeOH: Formic acid composition (85%) (6.3:3.2:0.5 v/v/v) Toluene: MeOH: Formic acid (85%) (6.5:3:0.5 v/v/v) Toluene: MeOH: Formic acid (85%) (6.7:2.9:0.5 v/v/v) Time from spotting to development (immediate) 4 Time from development to scanning (immediate) After 30min After 1hr After 30min After 1hr Solution Stability Freshly prepared solutions of both the drugs were kept in a refrigerator (cool condition) after use. UV absorbance of refrigerated solutions were compared with absorbance of fresh solutions daily. It was observed that solution is stable for 4 days whereas is stable for 5 days. Table6: Summary of validation parameters. Sr. No. Validation parameters 1. Linearity Equation (r2) Range 2. Precision (% RSD) Interday Intraday 3. Accuracy y=2.6775x R2= ng/band y=1.8017x R2= ng/band Limit of Detection Limit of Quantitation Specificity Specific Specific 7. Robustness Robust Robust IJPT July2015 Vol. 7 Issue No Page 8193

12 Conclusion: The developed HPTLC method is found to be simple, sensitive, precise, specific and robust.the stability indicating properties established as per the recommendations of ICH guidelines also indicated that the drugs could be evaluated in presence of their degradation products and thereby can be employed forroutine quality control as well as assessing the stability of and in bulk drug and pharmaceutical dosage form. Acknowledgement: The authors would like to thank Wockhardt Pharmaceuticals Ltd. (Aurangabad, India) and Cipla Pharmaceuticals Ltd. (Mumbai, India) for providing gift samples of drugs. Authors are also thankful to the Principal and Management, AISSMS College of Pharmacy, Pune for providing required facilities for research work. References 1. Indian pharmacopoeia, The Controller of Publication, New Delhi, 2007, Vol II, British pharmacopoeia, London, United Kingdom: Stationary office on behalf of Medicine and health care products regulatory agency, 2005, Vol II, United State Pharmacopoeia (USP30/NF25), 2007, Indian Pharmacopoeia,The Controller Publication, Govt of India. New Delhi, 2007, Vol. III, United States Pharmacopoeia (USP30/NF25),2007, 1310, N. Dubey, S. Sahu, G.N. Singh, Development of HPLC method for simultaneous estimation of ambroxol, Guaifenesin and salbutamol in single dosage from, Indian Journal of Chemistry, Nov 2012, 51B, S. S. Kamatham, S. Kolli, D. D. Joga, K. R. Vasa, B. D. Yanamadala, Simultaneous Estimation Of Salbutamol, Ambroxol And Guaifenesin In Tablet Dosage Forms By Using RPHPLC, International Journal Of Pharmaceutical Research And Biomedical Analysis, Sept 2013, 2(3), S. G. Walode, S. D. Deshpande, A. V. Deshpande, Stability indicating RPHPLC method for simultaneous estimation of salbutamol sulphate and Guaifenesin, Pelagia Research Library, 2013, 4(2), S. B. Ezhava, K. D. Bhalara, I. S. Rathod, D. D Bhalara, Simultaneous Estimation of Salbutamol Sulphate and guaiphenesin in their Combined Liquid Dosage Form by HPTLC Method, International Journal of Pharma Research and Health Sciences, Apr 2014, 2(2), A. A. Bankar, S. R. Lokhande, R. L. Sawant, A. R. Bhagat, Spectrophotometric estimation of guaifenesin and salbutamol in pure and tablet dosage form by using different methods, Der Pharma Chemica, 2013, 5(3), IJPT July2015 Vol. 7 Issue No Page 8194

13 11. ICH Guidelines, Q1A (R2) Stability Testing of New Drug Substances and Products, Geneva, Switzerland ICH Guidelines, Q1B Stability Testing: Photo stability Testing of New Drug Substances and Products, Geneva, Switzerland ICH Guidelines, Q2 (R1) Validation of Analytical Procedures: Text and Methodology, Geneva, Switzerland Corresponding Author: Mrinalini C. Damle *, IJPT July2015 Vol. 7 Issue No Page 8195

Development and Validation of Stability Indicating HPTLC Method for Estimation of Seratrodast

ARC Journal of Pharmaceutical Sciences (AJPS) Volume 2, Issue 3, 2016, PP 15-20 ISSN 2455-1538 DOI: http://dx.doi.org/10.20431/2455-1538.0203004 www.arcjournals.org Development and Validation of Stability