Professional Documents
Culture Documents
Stability indicating RP-HPLC method for the determination of Terbutaline sulphate, Guaifenesin, Ambroxol hydrochloride and preservatives content in liquid formulations
Hanimi Reddy Bapatu 1*, Maram Ravi Kumar2, Useni Reddy Mallu 3, Hari kishan Reddy Ganthi 3, Chandra Mohan Rao Kota 4 and Viswanath Reddy Pyreddy 3 1 Department of Chemistry, JNT University, Kukatpally, Hyderabad, AP, India-500072. 2 AR&D, Custom Pharmaceutical Services, Dr. Reddys Laboratories Ltd, Bachupally, Hyd-72, India. 3 Department of Chemistry, Sri Krishnadevaraya University, Anantapur, AP, India-515003. 4 Ideal College of Arts and Sciences, Kakinada, East Godhavari, AP, India-533464.
Key words:Terbutaline sulphate, Guaifenesin, ambroxol, Methyl paraben, Propyl paraben, Liquid formulations and RP-HPLC method.
INTRODUCTION Each 5 mL syrup contains Ambroxol hydrochloride IP Terbutaline sulphate IP Guaiphenesin IP
: 20 mg : 1.25 mg : 50 mg
Terbutaline (1-6) is a 2-receptor agoinst. It helps the relaxation of the smooth muscle found principally in bronchial, vascular and uterine tissue; wheezing and shortness of breathe troubled breathing caused by asthma, chronic bronchitis, emphysema and other lung diseases. Terbutaline has little effect on 1 receptors; thus direct cardiovascular stimulation occurs. However, terbutaline should be used carefully in cats with pre-existing cardiac disease, such as cardiomyopathy. Terbutaline use during pregnancy is associated with development of autism in humans. Terbutaline side effects are drowsiness and headaches, increased heart rate, diabetes, anxiety and worsening breathing problems. Guaiphenesin is also called as guaifenesin (7-11). It is used to reduce chest congestion caused by the common cold, infections, or allergies, including medical, veterinary, and personal, women to facilitiate conception by thinning and increasing the amount of cervical mucus, treatment of primary dysmenorrhea . Guaifenesin may cause side effects, headache, nausea and vomiting. Ambroxol is an active mucolytic agent. Ambroxol is used in the treatment of respiratory diseases, pain relief in acute sore throat (12-15). Ambroxol is a very potent inhibitor of the neuronal Na+ channels. Chemical structures of all ingredients were represented in figure-1.All three ingredients are available in liquid pharmaceutical dosage forms.
Ambroxol hydrochloride
Terbutaline sulphate
Propyl paraben
Figure-1: Chemical structure of all ingredients All ingredients have reported methods for individual and other combination products (16-18) and there is no method reported for the simultaneous estimation of Terbutaline Sulphate, Guaifenesin, Ambroxol hydrochloride, methyl paraben and propyl paraben in combined liquid dosage forms. The objective of the present study is to develop a single RP-HPLC method for the estimation of Terbutaline Sulphate, Ambroxol hydrochloride, Guaifenesin and preservatives in liquid formulation. MATERIALS AND METHODS
*Corresponding author.
Hanimi Reddy Bapatu H. No: 4-22-51/25, Koritepadu, Guntur, Pin No: 522 007, Andhra Pradesh, India. Tel.: + 91-9490310239 E-mail:hanimi.b@gmail.com
Selection of mobile phase: Various buffer salts, pH values were tried with different organic solvents (acetonitrile or methanol) for the optimization of mobile phase. Finally well shaped and high resolution was achieved with pH 6.8 phosphate buffer and acetonitrile with gradient program. Chemicals and reagents: Potasssium di hydrogen orthophosphate, triethyl amine (AR Grade) were pro-
4117-4122
The %RSD of the area of analyte peaks in standard chromatograms should be NMT 2.0 %; Theoretical plates of analyte peaks in Standard chromatograms should be NLT 2000; Tailing Factor of analyte peaks in Standard Chromatograms should be NMT 2.0. Calculation: mg/5 ml = Test solution area x Std. Dilution factor x Std. Potency Standard solution area x Test dilution factor % of active = mg/5 ml x 100_______ Labeled amount in mg / 5 ml
RESULTS AND DISCUSSION METHOD DEVELOPMENT All active ingredients have UV activity. All pure standard solutions were prepared with diluent and scanned the UV absorbance from 200nm to 400nm. UV spectrums of all ingredients (Source: HPLC/PDA analysis) were represented in figure-2. Based on the UV spectrum of all actives, samples absorbance was measured at 214nm. Initial steps in method development trials were performed with water and acetonitrile, C8 250mm column but the elution of active peaks and separation was poor further trials were done with ammonium acetate and perchlorate buffers but the elution of ambroxol and propyl paraben was poor and baseline also not acceptable. Finally, optimized the chromatographic conditions with phosphate buffer, acetonitrile, C18, 250x4.6mm, 5 column and absorbance was measured at 214nm. System suitability: The retention times of Terbutaline Sulphate, Guaifenesin, Methyl Paraben, Ambroxol and Propyl Paraben were found at ~ 3.6, 15.1, 18.7, 23.2and 24.3min, respectively. These retention times did not vary to any considerable changes during the analysis. Percent (%) RSD of five replicate injections of standard retention time and area were found to be within the limit (%RSD of RT is not more than 1.0% and area is not more than 2.0%). Resolution between all active peaks is more than 3.0 (limit is note less than 2.0). Diluent and placebo have no interference with all active peaks. Diluent, standard solution and test solution chromatograms were represented in figure-3 to 6. Table-1 represents the system suitability parameters.
AU
276.8
0.00 16.433 Ambroxol
223.5
AU 1.00 0.00 20.057 Methyl Paraben
273.2
255.4
AU 0.50
0.00 24.481 Guaiphenesin 1.00 AU 0.50 0.00 0.06 AU 0.04 0.02 0.00 220.00 240.00 260.00 280.00 300.00 nm 320.00 340.00 360.00 380.00 400.00 26.035 Propyl Paraben
247.1 309.0
255.4
Figure-2: UV spectrum of all ingredinets minutes & make up the volume with diluent. Filter the solution through Whatman 0.45 membrane filter paper rejecting first few ml of filtrate. (Concentration of Terbutaline in solution is 12.5ppm, Ambroxol is 200ppm, Guaifenesin is 500ppm, methyl paraben 100ppm and propyl paraben 10ppm). System Suitability: System suitability parameters were finalyzed as per ICH and FDA guidelines. Figure-4: Placebo chromatogram
4117-4122
Guaifenesin Methyl paraben 7699035 7665054 7680969 7718547 7925415 7737804 3108443 3093421 3082145 3091245 3096314 3094314
METHOD VALIDATION Validation is a process of establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics. The method was validated with Linearity, Accuracy, Precision, Specificity and Robustness as per ICH and FDA guidelines (19-23). Table-2: Method Precision Studies
Sample % Assay of Label content (SET- I: Precision; SET-II: Intermediate precision) Ambroxol HCl Terbutaline sulphate Guaifenesin Methyl Paraben Propyl paraben Number SET- I SET- II SET- I SET- II SET-I SET-II SET-I SET-II SET-I SET-II 1 2 3 4 5 6 Average Average % RSD 99.8 99.9 99.9 99.9 100.0 99.8 99.9 100.1 0.2 100.2 100.3 100.0 100.6 100.1 100.2 100.2 98.6 101.3 99.8 99.7 100.5 99.3 99.9 99.4 1.0 97.6 99.0 98.5 99.9 99.4 99.4 99.0 99.5 99.9 99.9 99.5 100.4 99.9 99.9 99.6 0.4 99.8 99.7 99.4 99.6 99.1 98.9 99.4 99.0 99.1 98.9 99.0 99.5 100.2 99.3 100.0 0.8 100.7 100.7 100.2 100.8 100.8 100.8 100.7 101.0 97.7 98.9 98.4 98.5 97.9 98.7 99.0 0.9 98.8 98.6 99.7 99.5 99.4 99.0 99.2
Precision Method precision was evaluated by six separately prepared samples of the same batch of syrup and injected. Results were found to be satisfactory. Precision (setI) and intermediate precision (set-II) results were tabulated in table-2.
4117-4122
Peroxide (3%H2O2, Water (water 15min @ 60 C) 15 min @ 60C) 0.157 0.299 Pass 0.302 0.333 Pass
Peroxide (3%H2O2, Water (water 15min @ 15 min @ 60 C) 60C) 0.200 0.234 Pass Peroxide (3%H2O2, 15min @ 60 C) 0.047 0.224 Pass 0.196 0.237 Pass Water 15 min @ 60C) 0.059 0.228 Pass
Sun-Light UV-Light (1.2 million Lux (200 watt hours) hr/m 2 ) 0.163 0.259 Pass Sun-Light (1.2 million Lux hours) 0.055 0.250 Pass Sun-Light (1.2 million Lux hours) 0.124 0.215 Pass 0.261 0.269 Pass UV-Light (200 watt hr/m 2 ) 0.059 0.251 Pass UV-Light (200 watt hr/m 2 ) 0.178 0.214 Pass
Peroxide (3%H2O2, Water (water 15min @ 60 C) 15 min @ 60C) 0.239 0.248 Pass 0.199 0.266 Pass
4117-4122
Linearity: Linearity solutions were prepared from stock solution at six concentration levels from 50 to 150% of analyte concentrations (98.7 to 296.1 ppm for Ambroxol hydrochloride, 6.2 to 18.6 ppm for Terbutaline Sulphate, 246.4 to 747.3 ppm for Guaifenesin, 44.0 to 136.1 ppm for Methyl paraben and 5.5 to 14.6 ppm for Propyl paraben). The linear regression analysis of Ambroxol hydrochloride, Terbutaline Sulphate, Guaifenesin, Methyl paraben and Propyl paraben were constructed by plotting the peak area of the analytes (y) versus analytes concentration in (x) axis. The calibration curves were linear in the range of 98.7 to 296.1 ppm for Ambroxol hydrochloride, 6.3 to 18.6 ppm for Terbutaline Sulphate, 246.4 to 747.3 ppm for Guaifenesin, 44.0 to 136.1 ppm for Methyl paraben and 5.5 to 14.6 ppm for Propyl paraben a correlation coefficient of more than 0.999 for all the three drugs. The slope, Y-intercept and correlation coefficient were calculated and summarized in Table-5. Overlaid chromatograms of the all linearity solutions were represented in figure-8. Table-5: Linearity Results
Linearity Results Ambroxol HCl Level Conc Area (ppm) 1 (50%) 98.7 2 (80%) 157.9 3 (100%) 197.4 4 (110%) 217.1 5 (120%) 236.9 6 (150%) 296.1 Correlation Coefficient: 0.9999 4516957 7163944 8976039 9855655 10776103 13312612 Terbutaline sulphate Guaifenesin Conc Area Conc Area (ppm) (ppm) 6.2 9.9 12.4 13.6 14.9 18.6 0.9991 109932 170137 215433 239655 264855 321265 246.4 397.5 496.9 548.5 596.2 747.3 0.9999 3986794 6209405 7744331 8531503 9277393 11571142 Methyl Paraben Conc Area (ppm) 44.0 80.1 100.1 110.1 120.1 136.1 0.9995 1556601 2574380 3088601 3447198 3652614 4115342
Propyl paraben Conc Area (ppm) 5.5 7.0 9.6 10.6 11.6 14.6 0.9994 201017 253005 328887 366570 402909 490836
50%
98.7506 98.7506 98.7506 100% 197.5012 197.5012 197.5012 150% 294.2768 294.2768 294.2768 Terbutaline sulphate 50% 6.5203 6.5203 6.5203 100% 13.0406 13.0406 13.0406 150% 19.2504 19.2504 19.2504 Guaifenesin 50% 249.8590 249.8590 257.7910 100% 495.7520 495.7520 495.7520 150% 745.6110 745.6110 745.6110 Methyl Paraben 50% 50.1046 50.1046 50.1046 100% 100.2092 100.2092 100.2092 150% 150.3139 150.3139 150.3139 Propyl Paraben 50% 5.0533 5.0533 5.0533 100% 10.1065 10.1065 10.1065 150% 14.6545 14.6545 14.6545
0.2 0.1
2 0.0
Ambroxol HCl 0.2-0.5 0.1-0.6 1.1-1.4 Terbutaline Sulphate 0.8-1.8 0.4-0.8 0.7-1.5 Guaifenesin 0.6-1.4 0.5-1.6 0.9-1.9 Methyl paraben 0.3-0.8 0.1-0.0 1.3-1.2 Propyl paraben 0.7-1.5 0.4-1.5 0.8-1.0 Tailing factor Ambroxol HCl 1.0-1.7 1.0-1.4 1.1-1.1 Terbutaline Sulphate 1.1-1.5 1.1-1.4 1.2-1.1 Guaifenesin 1.0-1.5 1.0-1.2 0.9-0.9 Methyl paraben 1.7-1.2 1.4-1.4 1.1-1.1 Propyl paraben 1.7-1.1 1.3-0.8 1.0-1.0 Theoretical plates Ambroxol HCl 72324-182062 90269-182062 184299-181576 Terbutaline Sulphate 8673-17324 7682-17324 16446-16995 Guaifenesin 96478-253651 142821-253651 140082-144252 Methyl paraben 94086-156986 139632-130733 205329-19896 Propyl paraben 102019-211064 180800-30407 155298-145938
NMT 2.0
NMT 2.0
NLT 2000
REFERENCES
1. 2. 3. R Ellul-Micallef, Effect of terbutaline sulphate in chronic allergic cough, British Medical Journal, 1983, 287 (6397), 940-943. Rau HL, Aroor AR and PG Rao, Fluorimetric estimation of terbutaline sulphate in dosage forms, Inidan Journal of Pharmaceutical Sciences, 1990, 52 (5), 255-256. Hultquist C, Wollmer P, Eklundh G, Jonson B, Effect of inhaled terbutaline sulphate in relation to its deposition in the lungs, Pulmonary Pharmacology, 1992, 5 (2), 127132. Newman SP, Morn F, Trofast E, Talaee N and SW Clarke , Deposition and clinical efficacy of terbutaline sulphate from Turbuhaler, a new multi-dose powder inhaler, The European Respiratory Journal, 1989, 2 (3), 247-252. Pramod K and HG Shivakumar, Novel core in cup buccoadhesive system and films of Terbutaline Sulphatedevelopment and in vitro evaluation, Asian Journal of pharmaceutical Science, 2006, 175-187. Lotufo JP, Ejzenberg B, Vieira S, Mukai L, Macedo H, Yamashita C, Ventura G, Baldacci ER, Okay Y, Continuous nebulization with terbutaline sulfate under tent inhalation, evaluation of the efficacy in children 2 to 5 years of age in asthmatic crises, Revue De Maladies Respiratories, 1998, 15 (3), 255-261. Guaifenesin, Drugs.com. http://www.drugs.com/ppa/Guaifenesin-glycerylguaiacolate.html. Retrieved 2008-10-29 john. Gutierrez K, Pharmacotherapeutics: Clinical Reasoning in Primary Care. W.B. Saunders Co, 2007. Goldstein, Jacob, FDA Bumps Phlegm-Fighters From Market, The Wall Street Journal, 2007. Marsden JS, Strickland CD, Clements TL, Guaifenesin as a treatment for primary dysmenorrhea, J Am Board Fam Pract, 2004, 17 (4), 240246. Smith SM, Schroeder K, Fahey T, Over-the-counter medications for acute cough in children and adults in ambulatory settings, Cochrane Database Syst Rev, 2008. Kido H et al. Secretory leukoprotease inhibitor and pulmonary surfactant serve as principal defenses against influenza A virus infection in the airway and chemical
4.
5.
6.
98.1 99.2 99.1 1.7 1.7 0.6
Accuracy Accuracy of the method was studied by recovery evaluation. Known concentration of each active ingredient and preservatives was spiked in to separate 50 ml aliquots of placebo solution to give mixture solutions of approximately 50, 100 and 150 % of stated standard concentration. These samples were analyzed according to procedure and percentage recoveries calculated. For all analytes at the different concentration levels (50, 100 and 150 %), the recovery values
4117-4122
13.
19. 20.
14. 15.
21.
16.
22. 23.
17.
4117-4122