Hanimi Reddy Bapatu et al.

/ Journal of Pharmacy Research 2011,4(11),4117-4122

Research Article ISSN: 0974-6943

Available online through www.jpronline.info

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.

Received on: 19-05-2011; Revised on: 08-06-2011; Accepted on:01-07-2011 ABSTRACT

Objective: To develop a single RP-HPLC method for determination of Terbutaline sulphate, Guaifenesin, Ambroxol hydrochloride and preservatives (methyl paraben and propyl paraben) contents in liquid formulation. Method: Chromatographic separation was achieved on a Sunfire C18, 250 x4.6mm, 5 column. Mobile phase composed of Sol-A: phosphate buffer (0.01M Potassium dihydrogen orthophosphate buffer pH 6.0 0.1) and Sol-B: Acetonitrile with a simple gradient program (0-5min, sol-A:87-87; 5-10min- sol-A:87-80; 10-20min- sol-A:80-50; 20-25min- sol-A:50-50, 25-30min- sol-A:50-87and 30-35min- solA:87-87). 1.0ml per min flow rate and detection was at 214 nm. Results: High resolution was achieved with the simple gradient program and retention time of terbutaline sulphate, Guaifenesin, methyl paraben, ambroxol hydrochloride and propyl paraben are about 3.68min, 15.17min, 18.71min, 23.27min and 24.38min, respectively. The area of all ingredient peaks were a linear function of concentration in the range 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 and the corelation co-efficient value of all active ingredients within the limit (0.999). Conclusion: Proposed gradient HPLC method was validated with specificity, linearity, accuracy, reproducibility ruggedness and it is applicable for regular analysis.

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

Methyl paraben Guaifenesin

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-

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cured from S.D fine chemicals. High pure (NLT 98.5%) standards (Terbutaline Sulphate, Guaifenesin, ambroxol hydrochloride, methyl paraben and propyl paraben) were used for this study. HPLC grade Methanol and acetonitrile were procured from Spectrochem Pvt. Ltd. Water is prepared by mili Q system (Milli-pore). Buffer preparation (Mobile Phase-A): 0.01M Potassium dihydrogen orthophosphate buffer adjusted pH 6.0 with Triethylamine. Filtered through 0.45 membrane filter and degassed. Mobile phase B: Filtered and degassed Acetonitrile as mobile phase-B. Diluent: water and Methanol in the ratio of 50:50 v/v. HPLC conditions: Column: Sunfire C18 (250X4.6mm, 5m) Wavelength: 214nm Injection volume: 10 L Flow rate: 1.0 mL/min Temperature: 40C Run time: 35min Mobile phase: Sol-A: Buffer and Sol-B: Acetonitrile Gredient program: (0-5min, sol-A:87-87; 5-10min- sol-A:87-80; 10-20minsol-A:80-50; 20-25min- sol-A:50-50, 25-30min- sol-A:50-87and 30-35minsol-A:87-87) Standard Preparation: Prepared the standard solution with high pure standards consist of ambroxol 200ppm, Terbutaline sulphate 12.5ppm, Guaifenesin 500ppm, methyl paraben 100ppm and propyl paraben 10ppm with diluent. Sample preparation: Weigh accurately sample quantity equivalent to 5 ml of syrup sample and transfer into a 100 ml volumetric flask. Add 50 ml diluent, sonicate for about 5
4.283Terbutaline 0.05

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

Figure-3: Diluent chromatogram

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

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Specificity The specificity of the method has been evaluated with respect to diluent, placebo and degradation impurities. Degration studies were performed with acid, base, peroxide, water, thermal, sunlight and UV light, results were satisfactory and tabulated in table-3. Peak purity also checked for all peaks and found to be satisfactory. Peak purity chromatograms were represented in figure-7 and results were represented in table-4. Table-3: Forced degradation study
Stress Condition Ambroxol HCl Acid (0.1N HCl,15 min @ 60C) Alkali (0.1N NaOH,15 min @ 60C) Peroxide (3%H2O2,15min @ 60 C) Water (water 15 min @ 60C) Thermal (15 min @ 60C) Sun-Light (1.2 million Lux hours) UV-Light (200 watt hours / m 2 ) 6.74 7.95 7.95 8.17 9.15 6.54 5.40 % Degradation Terbutaline Guaifenesin Methyl Propyl Sulfate paraben paraben 3.77 0.94 0.94 7.55 1.89 7.55 6.60 7.50 10.39 11.18 11.89 12.07 9.34 8.10 0.2 3.97 6.31 7.22 6.31 4.29 4.68 8.67 6.3 6.9 8.67 9.85 5.33 6.67

Figure-5: Standard chromatogram.

Figure-6: Test solution chromatogram Table 1: System suitability

System Suitability parameter (five replicate injections) % RSD Theoretical plates(avg) Tailing factor (avg) USP Resolution Peak area response Std. Inj. 1 2 3 4 5 Average Observations Terbutaline Sulphate 0.8 17324 1.1 NA Ambroxol HCl 8993170 8957507 8978468 8951595 8988266 8973801 Guaifenesin 1.4 253651 1.0 89.5 Terbutaline sulphate 208995 206121 208777 205639 209297 207766 Methyl paraben 0.31 222081 1.0 24.4 Ambroxol hydrochloride 0.2 182062 1.0 27.1 Propyl paraben 0.30 283356 1.1 6.1 Propyl paraben 309812 308452 309541 307562 308214 308716

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.

Figure-7: Peak purity graphs of all active peaks

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Table-4: Peak purity results of all active peaks
Ambroxol hydrochloride Purity-1 Angle Purity-1 Threshold Peak purity Terbutaline sulphate Acid (0.1N HCl, 15 min @ 60C) 0.08 0.260 Pass Acid (0.1N HCl,15 min @ 60C) 0.162 0.295 Pass Acid (0.1N HCl,1 5 min @ 60C) 0.184 0.234 Pass Acid (0.1N HCl, 15 min @ 60C) Purity-1 Angle Purity-1 Threshold Peak purity Propyl Paraben 0.047 0.221 Pass Acid (0.1N HCl, 15 min @ 60C) 0.125 0.242 Pass Alkali (0.1N NaOH, 15 min @ 60C) 0.095 0.263 Pass Alkali (0.1N NaOH, 15 min @ 60C) 0.186 0.286 Pass Alkali (0.1N NaOH, 15 min @ 60C) 0.138 0.233 Pass Alkali (0.1N NaOH, 15 min @ 60C) 0.048 0.223 Pass Alkali (0.1N NaOH, 15 min @ 60C) 0.183 0.238 Pass Peroxide (3%H2O2,15min @ 60 C) 0.096 0.265 Pass Water 15 min @ 60C) 0.091 0.266 Pass Thermal (15 min @60C) 0.094 0.267 Pass Thermal (15 min @ 60C) 0.154 0.303 Pass Thermal (15 min @ 60C) 0.166 0.235 Pass Thermal (15 min @ 60C) 0.067 0.227 Pass Thermal (15 min @ 60C) 0.192 0.259 Pass Sun-Light (1.2 million Lux hours) 0.330 0.943 Pass Sun-Light (1.2 million Lux hours) 0.169 0.242 Pass UV-Light (200 watt hr/m 2 ) 0.355 0.990 Pass UV-Light (200 watt hr/m 2 ) 0.175 0.240 Pass

Peroxide (3%H2O2, Water (water 15min @ 60 C) 15 min @ 60C) 0.157 0.299 Pass 0.302 0.333 Pass

Purity-1 Angle Purity-1 Threshold Peak purity Guaifenesin

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

Purity-1 Angle Purity-1 Threshold Peak purity Methyl Paraben

Peroxide (3%H2O2, Water (water 15min @ 60 C) 15 min @ 60C) 0.239 0.248 Pass 0.199 0.266 Pass

Purity-1 Angle Purity-1Threshold Peak purity

Figure-8: Overlaid chromatograms of linearity solutions

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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

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were within the limit 100 2.5%. These recovery results reveals that developed RP-HPLC method can determine Terbutaline Sulphate, Guaifenesin, Ambroxol HCl, methyl paraben and propyl paraben. Recovery results were tabulated in table-6. Ruggedness and Robustness: Ruggedness of the method was validated by using different analysts, instruments and columns with same experimental conditions these results were tabulated in table-2. Robustness of the method was studied by changes in experimental conditions like pH, flow rate, temperature. The most important aspect of robustness is to allow for expected variations in method parameters. According to ICH guidelines, robustness should be considered early in the development stage of a method. The typical variations studied under this parameter are Flow rate, Wavelength, Mobile phase composition, Temperature, pH of the mobile phase. Table-7 represents the robustness results. CONCLUSION The complete study results reveals that the developed and validated RP-HPLC method is accurate, precise, robust and stability indicating. This method has wide applicability for preservatives such as methyl paraben and propyl paraben and useful for regular quality control analysis of formulation samples.

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

Table-6: Accuracy results

Level Ambroxol hydrochloride Qty Added Qty Recovered % (ppm) (ppm) Recovery 99.6746 99.9734 99.9449 197.1341 197.3208 197.3213 289.1785 289.1706 289.1754 6.4654 6.6149 6.6089 12.8607 13.2047 13.0171 19.0878 19.2766 19.2108 251.2659 254.2009 262.1058 493.4650 495.1577 495.4416 751.8942 760.7437 760.8692 49.9319 49.8573 49.7651 99.2224 99.2641 99.0638 150.9569 151.8228 152.5114 5.0531 4.9097 4.9038 10.2098 9.8697 9.9946 14.5565 14.5888 14.4369 100.9 101.2 101.2 99.8 99.9 99.9 98.3 98.3 98.3 99.2 101.5 101.4 98.6 101.3 99.8 99.2 100.1 99.8 100.6 101.7 101.7 99.5 99.9 99.9 100.8 102.0 102.0 99.7 99.5 99.3 99.0 99.1 98.9 100.4 101.0 101.5 100.0 97.2 97.0 101.0 97.7 98.9 99.3 99.6 98.5 Mean Recovery (%) 101.1 99.9 98.3 % RSD Sr. No. System Suitability parameter The % RSD

Table-7: Robustness results

Active names Variations system suitability results Flow Rate Temp Buffer Concn (0.9 to1.1 (35 to45C) (5%) mL/min) Limits

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

100.7 99.9 99.7

1.3 1.4 0.5

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

101.3 99.8 101.6

0.6 0.2 0.7

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99.5 99.0 101.0

0.2 0.1 0.5

4.

5.

6.
98.1 99.2 99.1 1.7 1.7 0.6

7. 8. 9. 10. 11. 12.

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

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13.

19. 20.

14. 15.

21.

16.

22. 23.

17.

Source of support: Nil, Conflict of interest: None Declared

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