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Stability indicating RP-HPLC method for the determination of Terbutaline sulphate,Guaifenesin, Ambroxol hydrochloride and preservatives content in liquid formulations

Stability indicating RP-HPLC method for the determination of Terbutaline sulphate,Guaifenesin, Ambroxol hydrochloride and preservatives content in liquid formulations

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Published by Hanimi Reddy
Stability indicating RP-HPLC method for the determination of Terbutaline sulphate,
Guaifenesin, Ambroxol hydrochloride and preservatives content in liquid formulations
Stability indicating RP-HPLC method for the determination of Terbutaline sulphate,
Guaifenesin, Ambroxol hydrochloride and preservatives content in liquid formulations

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Published by: Hanimi Reddy on Jan 23, 2012
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 Journal of Pharmacy Research Vol.4.Issue 11.November 2011 Hanimi Reddy Bapatu et al. / Journal of Pharmacy Research 2011,4(11),4117-4122 4117-4122
Research ArticleISSN: 0974-6943
Available online throughwww.jpronline.info
*Corresponding author.
 Hanimi Reddy Bapatu H. No: 4-22-51/25, Koritepadu, Guntur, Pin No: 522 007, Andhra Pradesh, India.
Tel.: + 91-
Each 5 mL syrup containsAmbroxol hydrochloride IP: 20 mgTerbutaline sulphate IP: 1.25 mgGuaiphenesin IP: 50 mg
is aß
-receptor agoinst.It helps the relaxation of the smoothmuscle found principally in bronchial, vascular and uterine tissue; wheezing andshortness of breathe troubled breathing caused by asthma, chronic bronchitis,emphysema and other lung diseases. Terbutaline has little effect on ß
receptors;thus direct cardiovascular stimulation occurs. However, terbutaline should beused carefully in cats with pre-existing cardiac disease, such ascardiomyopathy.Terbutaline use during pregnancy is associated with development of autisminhumans. Terbutaline side effects are drowsiness and headaches, increasedheart rate, diabetes, anxiety and worsening breathing problems.
is also called as guaifenesin
. It is used to reduce chest con-gestion caused by the common cold, infections, or allergies, including medical,veterinary, and personal, women to facilitiate conception by thinning and in-creasing the amount of cervical mucus, treatment of primary dysmenorrhea
Guaifenesin may cause side effects, headache, nausea and vomiting.
is an active mucolytic agent. Ambroxol is used in the treatment of respiratory diseases, pain relief in acute sore throat
. Ambroxol is a verypotent inhibitor of the neuronal Na+ channels.Chemical structures of all ingredients were represented in figure-1.All threeingredients are available in liquid pharmaceutical dosage forms.
Stability indicating RP-HPLC method for the determination of Terbutaline sulphate,Guaifenesin, Ambroxol hydrochloride and preservatives content in liquid formulations
Hanimi Reddy Bapatu
, Maram Ravi Kumar
, Useni Reddy Mallu
, Hari kishan Reddy Ganthi
,Chandra Mohan Rao Kota
and Viswanath Reddy Pyreddy
 Department of Chemistry, JNT University, Kukatpally, Hyderabad, AP, India-500072.
AR&D, Custom Pharmaceutical Services, Dr. Reddys Laboratories Ltd, Bachupally, Hyd-72, India.
 Department of Chemistry, Sri Krishnadevaraya University, Anantapur, AP, India-515003.
 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
To develop a single RP-HPLC method for determination of Terbutaline sulphate, Guaifenesin, Ambroxol hydrochloride and preservatives (methylparaben and propyl paraben) contents in liquid formulation.
Chromatographic separation was achieved on a Sunfire C18, 250 x4.6mm, 5µ 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 simplegradient 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- sol-A:87-87). 1.0ml per min flow rate and detection was at 214 nm.
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 18.6ppm 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 thecorelation co-efficient value of all active ingredients within the limit (0.999).
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.
Ambroxol hydrochlorideMethyl parabenTerbutaline sulphateGuaifenesin Propyl parabenFigure-1: Chemical structure of all ingredients
All ingredients have reported methods for individual and other combinationproducts
and there is no method reported for the simultaneous estimation of Terbutaline Sulphate, Guaifenesin, Ambroxol hydrochloride, methyl parabenand propyl paraben in combined liquid dosage forms. The objective of thepresent study is to develop a single RP-HPLC method for the estimation of Terbutaline Sulphate, Ambroxol hydrochloride, Guaifenesin and preservatives inliquid formulation.
MATERIALS AND METHODSSelection of mobile phase:
Various buffer salts, pH values were tried with different organic solvents (aceto-nitrile or methanol) for the optimization of mobile phase. Finally well shapedand high resolution was achieved with pH 6.8 phosphate buffer and acetonitrilewith gradient program.
Chemicals and reagents:
Potasssium di hydrogen orthophosphate, triethyl amine (AR Grade) were pro-
 Journal of Pharmacy Research Vol.4.Issue 11.November 2011 Hanimi Reddy Bapatu et al. / Journal of Pharmacy Research 2011,4(11),4117-4122 4117-4122
cured from S.D fine chemicals. High pure (NLT 98.5%) standards (TerbutalineSulphate, Guaifenesin, ambroxol hydrochloride, methyl paraben and propylparaben) were used for this study. HPLC grade Methanol and acetonitrile wereprocured 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 Tri-ethylamine. Filtered through 0.45µ membrane filter and degassed.
Mobile phase B:
Filtered and degassed Acetonitrile as mobile phase-B.
water and Methanol in the ratio of 50:50 v/v.
HPLC conditions:
Column:Sunfire C18 (250X4.6mm, 5µm)Wavelength:214nmInjection volume:10 µLFlow rate:1.0 mL/minTemperature:40°CRun time:35minMobile phase:Sol-A: Buffer and Sol-B: AcetonitrileGredient 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-sol-A:87-87)
Standard Preparation:
Prepared the standard solution with high pure standards consist of ambroxol200ppm, Terbutaline sulphate 12.5ppm, Guaifenesin 500ppm, methyl paraben100ppm and propyl paraben 10ppm with diluent.
Sample preparation:
Weigh accurately sample quantity equivalent to
5 ml of syrup sample andtransfer into a 100 ml volumetric flask. Add 50 ml diluent, sonicate for about 5
4.283 Terbutaline
      A      U
0.000.0516.433 Ambroxol
      A      U
0.001.0020.057 Methyl Paraben
      A      U
0.000.5024.481 Guaiphenesin
      A      U
0.000.501.0026.035 Propyl Paraben
      A      U 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00
The %RSD of the area of analyte peaks in standard chromatograms should beNMT 2.0 %; Theoretical plates of analyte peaks in Standard chromatogramsshould be NLT 2000; Tailing Factor of analyte peaks in Standard Chromato-grams should be NMT 2.0.
mg/5 ml = Test solution area x Std. Dilution factor x Std. PotencyStandard solution area x Test dilution factor% of active = mg/5 ml x 100_______Labeled amount in mg / 5 ml
All active ingredients have UV activity. All pure standard solutions were pre-pared with diluent and scanned the UV absorbance from 200nm to 400nm. UVspectrums of all ingredients (Source: HPLC/PDA analysis) were represented infigure-2. Based on the UV spectrum of all actives, samples absorbance wasmeasured at 214nm. Initial steps in method development trials were performedwith water and acetonitrile, C8 250mm column but the elution of active peaksand separation was poor further trials were done with ammonium acetate andperchlorate buffers but the elution of ambroxol and propyl paraben was poorand baseline also not acceptable. Finally, optimized the chromatographic condi-tions with phosphate buffer, acetonitrile, C18, 250x4.6mm, 5µ column andabsorbance was measured at 214nm.
Figure-2: UV spectrum of all ingredinets
minutes & make up the volume with diluent. Filter the solution through Whatman0.45µ 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.
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 changesduring the analysis. Percent (%) RSD of five replicate injections of standardretention time and area were found to be within the limit (%RSD of RT is notmore than 1.0% and area is not more than 2.0%). Resolution between all activepeaks is more than 3.0 (limit is note less than 2.0). Diluent and placebo have nointerference with all active peaks. Diluent, standard solution and test solutionchromatograms were represented in figure-3 to 6. Table-1 represents the systemsuitability parameters.
Figure-3: Diluent chromatogram
Figure-4: Placebo chromatogram
 Journal of Pharmacy Research Vol.4.Issue 11.November 2011 Hanimi Reddy Bapatu et al. / Journal of Pharmacy Research 2011,4(11),4117-4122 4117-4122
Figure-5: Standard chromatogram.Figure-6: Test solution chromatogram
System Suitability parameterObservations(five replicate injections)TerbutalineGuaifenesinMethylAmbroxolPropylSulphateparabenhydrochlorideparaben
% RSD0.81.40.310.20.30Theoretical plates(avg)17324253651222081182062283356Tailing factor (avg) ResolutionNA89.524.427.16.1
Peak area responseStd. Inj.AmbroxolTerbutalineGuaifenesinMethylPropylHClsulphateparabenparaben
Table 1: System suitabilityMETHOD VALIDATION
Validation is a process of establishing documented evidence, which provides ahigh degree of assurance that a specific activity will consistently produce adesired result or product meeting its predetermined specifications and qualitycharacteristics. The method was validated with Linearity, Accuracy, Precision,Specificity and Robustness as per ICH and FDA guidelines
Table-2: Method Precision Studies
Sample% Assay of Label content (
SET- I: Precision; SET-II: Intermediate precision
)Ambroxol HClTerbutaline sulphateGuaifenesinMethyl Paraben Propyl parabenNumberSET- ISET- IISET- ISET- IISET-ISET-IISET-ISET-II SET-I SET-II1
99.8100.298.697.699.599.899.0100.7101.0 98.8
99.9100.3101.399.099.999.799.1100.797.7 98.6
99.9100.099.898.599.999.498.9100.298.9 99.7
99.9100.699.799.999.599.699.0100.898.4 99.5
100.0100.1100.599.4100.499.199.5100.898.5 99.4
99.8100.299.399.499.998.9100.2100.897.9 99.0
Average99.9100.299.999.099.999.499.3100.798.7 99.2Average100.199.499.6100.099.0% RSD0.
Method precision was evaluated by six separately prepared samples of the samebatch of syrup and injected. Results were found to be satisfactory. Precision (set-I) and intermediate precision (set-II) results were tabulated in table-2.
The specificity of the method has been evaluated with respect to diluent, pla-cebo and degradation impurities. Degration studies were performed with acid,base, peroxide, water, thermal, sunlight and UV light, results were satisfactoryand tabulated in table-3. Peak purity also checked for all peaks and found to besatisfactory. Peak purity chromatograms were represented in figure-7 and re-sults were represented in table-4.
Table-3: Forced degradation study
Figure-7: Peak purity graphs of all active peaks
Stress Condition % DegradationAmbroxolTerbutalineGuaifenesinMethylPropylHClSulfateparabenparaben
Acid (0.1N HCl,15 min @ 60°C) 6.743.777.500.28.67Alkali (0.1N NaOH,15 min @ 60°C) 7.950.9410.393.976.3Peroxide (3%H2O2,15min @ 60 °C) 7.950.9411.186.316.9Water (water 15 min @ 60°C) 8.177.5511.897.228.67Thermal (15 min @ 60°C) 9.151.8912.076.319.85Sun-Light (1.2 million Lux hours) 6.547.559.344.295.33UV-Light (200 watt hours / m
) 5.406.608.104.686.67

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