International Journal of Pharma Sciences Vol. 5, No. 2 (2015): 991-1001 Research Article Open Access

ISSN: 2320-6810

A Validated Stability Indicating RP-HPLC Method for Estimation of Ambroxol, Salbutamol and Theophylline in Bulk and Pharmaceutical Dosage Form Sreedhar Lade1* and Y. Rajendra Prasad2 1 2

University College of Pharmaceutical Sciences, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur-522510 (A.P) India. University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam-530003 (A.P) India.

* Corresponding author: Sreedhar Lade; email: [email protected]

Received: 22 January 2015

Accepted: 11 March 2015

Online: 16 March 2015

ABSTRACT

A new simple, precise, selective, accurate and rapid reverse phase high performance liquid chromatographic stability indicating method had been developed and validated for simultaneous quantitative determination of Ambroxol, Salbutamol and Theophylline in bulk and pharmaceutical dosage form. The chromatographic separation was achieved with Inertsil ODS C-18, (250×4.6 mm) and 5µm particle size column. The optimized mobile phase consisting of phosphate buffer: Acetonitrile (55:45%v/v) and adjusted the mobile phase to pH 3.0 with o-phosphoric acid. The flow rate was 1.0 mL/min and eluents were detected at 225 nm using PDA detector. The retention time of Salbutamol, Theophylline and Ambroxol were found to be 2.371, 3.808 and 5.863 respectively. The percentage recoveries for three molecules were found to be in the range of 98-102%. The calibration curve was constructed between peak area vs. concentration and demonstrated good linear in the range of 7.5-45 μg/ml for Ambroxol, 0.5-3 μg/ml for Salbutamol and 25-150 μg/ml for Theophylline. Degradation studies were carried for Ambroxol, Salbutamol and Theophylline under various stress conditions such as acid hydrolysis, base hydrolysis, oxidation, thermal, photochemical and UV. All the degradation peaks were resolved effectively from Ambroxol, Salbutamol and Theophylline peaks. The developed method was validated according to ICH guidelines. As the method could effectively separates the degradation products from active ingredient, it can be used for routine analysis of drug both in bulk and pharmaceutical dosage form.

Keywords: Ambroxol, Salbutamol and Theophylline, Acetonitrile, Buffer, RP-HPLC. 1. INTRODUCTION

Salbutamol sulphate 2 mg, Theophylline 100 mg and Ambroxol HCl 30 mg tablets are available with trade name of AMBROLITE-ST tab from “Nicholas Piramal”. Ambroxol hydrochloride is chemically, 1 ({[2 –Amino – 3, 5 dibromo phenyl] methyl} amino) cyclohexanol monohydrochloride. This is semi synthetic derivative of vasicine from the Indian shrub “Adhatodavasica”. It is an expectoration improver and a mucolytic agent used in the treatment of bronchial asthma and chronic bronchitis. Ambroxol hydrochloride can be used for cough suppressing effect and anti-inflammatory action. Recently the inhibition of nitric oxide dependent activation of soluble guanylate cyclase was suggested one of the molecular mechanism of the therapeutic

http://ijps.aizeonpublishers.net/content/2015/2/ijps991-1001.pdf

action of Ambroxol hydrochloride, also used in pulmonary alveolar proteinosis in pulmonary distress and infant respiratory distress [1]. Its molecular formula is C13H18Br2N2O; Molecular weight is 378.10

Figure 1: Ambroxol

Salbutamol is chemically 1-(4-hydroxy-3-hydroxymethylphenyl)-2-(tert-butyl amino) ethanol (C13H21

991

Sreedhar Lade et al. / Int J Pharma Sci. 2015, 5(2): 991-1001

NO3), which is a short acting β2-adrenergic receptor agonist and contains a phenyl ethanolamine nucleus having a β-hydroxylamine group on the side chain, as shown in Figure-2. It is used for the relief of bronchospasm in conditions such as asthma and chronic obstructive pulmonary diseases. Salbutamol sulfate is usually administered by the inhaled route for direct effect on bronchial smooth muscle. It can also be administered orally or intravenously. Although Salbutamol sulfate is well absorbed, its systemic bioavailability is only 50 % due to extensive presystemic metabolism in the gastrointestinal tract and liver [2-4]. Its molecular formula is C13H21NO3; Molecular weight is 239.31

Figure 2: Salbutamol

Theophylline is used to prevent and treat wheezing, shortness of breath, and difficulty breathing caused by asthma, chronic bronchitis, emphysema, and other lung diseases. It relaxes and opens air passages in the lungs, making it easier to breathe5. Theophylline is well absorbed throughout the gastrointestinal tract, half-life in adults varies considerably. In healthy adults, it ranges from 3 to 12 h. In children, the half-life is usually significantly shorter than in adults, averaging about 3.5 h. Thus, the drug finds wide application in pediatrics in immediate release as well as sustained release dosage forms [6-10]. For pediatric application the dosage form should be palatable. Hence, microspheres were formulated by solvent evaporation method for masking the bitter taste of the drug. The chemical name of Theophylline is 1, 3-dimethyl-2, 3, 6, 7-tetrahydro-1Hpurine-2, 6-Dione. Its molecular formula is C7H8N4O2; Molecular weight is 180.16.

Figure 3: Theophylline

A stability indicating Reversed phase high performance liquid chromatographic method for simultaneous determination of Ambroxol, Salbutamol and Theophylline in bulk drugs and Pharmaceutical Dosage form.

2. MATERIALS AND METHODS

2.1 Chemicals and solvents Ambroxol, Salbutamol and Theophylline were obtained as gift samples from spectrum research labs, Hyderabad, India. The commercial Pharmaceutical Preparation AMBROLITE-ST tab containing 30 mg, 2 mg and 100 mg of Ambroxol, Salbutamol and

http://ijps.aizeonpublishers.net/content/2015/2/ijps991-1001.pdf

Theophylline respectively (Marketed by NICHOLAS PIRAMAL) were procured from local pharmacy. Acetonitrile, and water used are of HPLC grade.

2.2 Instrumentation The chromatographic separations were performed using HPLC-Waters alliance (Model-2695) consisting of an in-built auto sampler, a column oven and 2996 PDA detector. The data was acquired through Empower-2software. The column used was Inertsil ODS (250mm x 4.6 mm), 5µ. Meltronics sonicator was used for enhancing dissolution of the compounds. Elico pH meter was used for adjusting the pH of buffer solution. All weighing’s were done on Sartorius balance (Model AE-160).

2.3 Chromatographic conditions The mobile phase consists of Buffer and Acetonitrile in the ratio of 55:45(v/v). The mobile phase was pumped from solvent reservoir in the ratio of 55:45 %v/v to the column at the flow rate of 1.0 ml/min whereas run time set was 11 min. The separation was performed on Inertsil ODS (250mm x 4.6 mm), 5µ. column and the column was maintained the temperature of 30°C and the volume of each injection was 10 µL. Prior to injection, the column was equilibrated for at least 30 min with mobile phase flowing through the system. The eluents were monitored at 225 nm. 2.4 Preparation of buffer solution (pH 3.0) Accurately weighed 1.36gm of Potassium dihydrogen orthophosphate in a 1000ml volumetric flask and added about 900ml of Milli-Q water and degassed by sonication and finally made up the volume with water and adjusted pH to 3 with diluted orthophosphoric acid.

2.5 Preparation of mobile phase Buffer and Acetonitrile were mixed in the ratio 55:45(v/v) and filtered through 0.45µ filter under vacuum.

2.6 Preparation of standard solution (30µg/ml Ambroxol, 2µg/ml Salbutamol and 100µg/ml of Theophylline) Accurately weighed and transferred 3mg of Ambroxol into 10ml clean dry volumetric flask, 2mg of Salbutamol into 100ml clean dry volumetric flask and 10mg of Theophylline working Standard into 10ml clean dry volumetric flasks. Added 3/4th volume of diluent, sonicated for 30 minutes and make up to the final volume with diluent. From the above stock solutions, 1 ml of each solution was pipetted in to a 10ml volumetric flask and then made up to the final volume with diluent.

2.7 Preparation of sample solution 5 tablets were weighed and the average weight of each tablet was calculated; then the weight equivalent to 5 tablets was transferred into a 100ml volumetric flask, 20ml of diluent added and sonicated for 30 min, further the volume made up with diluent and filtered. From the 992

Sreedhar Lade et al. / Int J Pharma Sci. 2015, 5(2): 991-1001

filtered solution 0.2ml was pipetted out into a 10 ml volumetric flask and made up to 10ml with diluent.

Label Claim: 30mg of Ambroxol + 2mg of Salbutamol+ 100mg of Theophylline 2.8 Validation of Proposed method The developed method was validated as per the ICH (International Conference on Harmonization) guidelines with respect to System suitability, Precision, Specificity, Forced degradation studies, Linearity, Accuracy, Limit of detection and Limit of quantification. 2.9 Linearity

Aliquots of 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 ml were taken from stock solution of concentration 300µg/ml Ambroxol, 20µg/ml Salbutamol and 1000µg/ml of Theophylline and then diluted up to mark with diluent. Such that the final concentrations were in the range of 7.5 ppm-45 ppm for Ambroxol, 0.5 ppm-3 ppm for Salbutamol and 25 ppm-150 ppm for Theophylline. Volume of 10µl of each sample was injected in five times for each concentration level and calibration curve was constructed by plotting the peak area versus drug concentration. A linear relationship between peak areas vs. concentration was observed in the range of study. The observations and calibration curve were shown in Table-1 and Fig. 4, 5.

Table 1: Optimized Chromatographic conditions and system suitability parameters for proposed HPLC method for Ambroxol, Salbutamol and Theophylline

Parameter Instrument Flow rate Column Detector wave length Column temperature Injection volume Run time Diluent Mode of separation

: : : : : : : : :

Chromatographic conditions Waters 2695, High performance Liquid chromatography 1 ml/min Inertsil ODS 250 x 4.6 mm, 5µ. 225nm 30°C 10µL 11 min At first dissolved in methanol than make up with buffer Isocratic mode

S. No

Conc. of Ambroxol in ppm

Ambroxol Area

1 2 3 4 5 6

7.5 15 22.5 30 37.5 45

155685 315833 484073 630290 779708 937413

Table 2: Linearity

Conc. of Salbutamol in ppm 0.5 1 1.5 2 2.5 3

2.10 System precision Precision is the measure of closeness of the data values to each other for a number of measurements under the same analytical conditions. Standard solution of 30µg/ml Ambroxol, 2µg/ml Salbutamol and 100µg/ml of Theophylline were prepared as per test method and injected for 6 times. Results are shown in Table-3.

2.11 PRECISION Intraday and interday precision study of Ambroxol, Salbutamol and Theophylline were carried out by estimating corresponding responses for 6 times on the same day and on consecutive days for the concentration of 30µg/ml Ambroxol, 2µg/ml Salbutamol and 100µg/ml of Theophylline. The percent relative standard deviation (%RSD) was calculated which was within the acceptable criteria of not more than 2. The results were shown in table no.3 and 4.

http://ijps.aizeonpublishers.net/content/2015/2/ijps991-1001.pdf

Salbutamol area 33661 72611 108892 141279 173083 210977

Conc. of Theophylline in ppm 25 50 75 100 125 150

Theophylline area 723116 1356285 2103704 2700836 3478029 4146869

2.12 Accuracy (Recovery studies) To determine the accuracy in sample preparation method of standard additions was made for measuring the recovery of the drugs. A fixed amount of sample was taken and standard drug was added at 50%, 100% and 150% levels. The results were analyzed and the results were found to be within the limits. The accuracy was expressed as the percentage of the analytes recovery. The results were shown in table no.5.

2.13 Specificity The specificity of the method was performed by injecting blank solution( without any sample) and then a drug solution of 10µl injected into the column, under Optimized chromatographic conditions, to demonstrate the separation of three molecules Ambroxol, Salbutamol and Theophylline from any of the impurities, if present. As there was no interference of impurities and also no considerable change in the retention time, the method was found to be specific.

993

Sreedhar Lade et al. / Int J Pharma Sci. 2015, 5(2): 991-1001

% Conc 50 50 50 100 100 100 150 150 150

Amount Added (µg/ml) 15 15 15 30 30 30 45 45 45 Average SD RSD

Ambroxol Amount Found (µg/ml) 15.1285 14.8791 15.1298 30.2532 29.8506 29.7813 45.2561 45.4045 44.4286

% Recovery 100.86 99.19 100.87 100.84 99.50 99.27 100.57 100.90 98.73 100.08 0.890 0.889

INJECTIONS 1 2 3 4 5 6 AVG S.D %RSD

Sample Preparations Sample-1 Sample-2 Sample-3 Sample-4 Sample-5 Sample-6 AVG S.D %RSD

Sample Preparations Sample-1 Sample-2 Sample-3 Sample-4 Sample-5 Sample-6 AVG S.D %RSD

Table 3: Accuracy

Salbutamol Amount Amount Added Found (µg/ml) (µg/ml) 1 0.9877 1 0.9929 1 1.0085 2 1.9853 2 2.0153 2 2.0056 3 2.9823 3 2.9607 3 3.0343 Average SD RSD

% Recovery 98.77 99.29 100.85 99.26 100.77 100.28 99.41 98.69 101.14 99.83 0.941 0.939

Table 4: System precision

Ambroxol 619879 619944 621487 620062 620555 621361 620548 719.9 0.1

Areas Salbutamol 138161 139493 138409 139096 138636 138245 138673 523.0 0.38

Table 5: Precision Table 5A: Intra-day precision Ambroxol 98.42 98.78 100.23 100.72 100.09 99.80 99.67 0.89 0.89

%Assay Salbutamol 100.48 99.03 98.42 100.63 99.59 99.49 99.61 0.845 0.85

Table 5B: Inter-day precision Ambroxol 99.09 99.27 99.21 99.04 98.76 99.14 99.09 0.179 0.181

2.14 Limit of Detection and Limit of Quantification LOD and LOQ were calculated using the following formula LOD = 3.3(SD)/S and LOQ= 10 (SD)/S, where SD = standard deviation of response (peak area) and S= slope of the calibration curve. Limit of Detection and Limit of Quantification were found to be 0.027μg/ml, 0.08μg/ml respectively for Amboxol, 0.0004 μg/ml and 0.005 μg/ml respectively for Salbutamol and 0.09μg/ml

http://ijps.aizeonpublishers.net/content/2015/2/ijps991-1001.pdf

%Assay Salbutamol 98.29 99.73 99.41 99.38 99.09 98.43 99.06 0.577 0.582

Theophylline Amount Amount Added Found (µg/ml) (µg/ml) 50 49.4792 50 50.5652 50 49.8096 100 99.0089 100 100.5313 100 101.5594 150 149.4921 150 150.8383 150 151.4702 Average SD RSD

% Recovery 98.96 101.13 99.62 99.01 100.53 101.56 99.66 100.56 100.98 100.22 0.945 0.942

Theophylline 2654116 2659465 2664685 2662711 2674263 2657815 2662176 6985.8 0.3

Theophylline 99.99 101.23 100.75 99.33 100.14 99.67 100.19 0.70 0.70

Theophylline 99.17 99.42 99.52 99.62 99.98 99.34 99.51 0.278 0.279

and 0.28μg/ml respectively for Theophylline as per ICH guidelines. The results were shown in table no.6.

2.15 Robustness Robustness was carried by varying three parameters from the optimized chromatographic conditions such as making small changes in flow rate (±0.1ml/min), mobile phase composition (±5%) and column 994

Sreedhar Lade et al. / Int J Pharma Sci. 2015, 5(2): 991-1001

temperature (±5ºC). It was observed that the small changes in these operational parameters did not lead to considerable changes of retention time of the peak of interest and the %RSD was not more than 2. The degree of reproducibility of the results proven that the method is robust. The results were shown in table no.7. 2.16 System suitability & System precision The system suitability was determined by making six replicate injections from freshly prepared standard solutions. The observed RSD values were well within usually accepted limits (≤2%). Theoretical plates, tailing factor of Ambroxol, Salbutamol and Theophylline were determined. The results are all within acceptable limits summarized in Table-4,5,6.

2.17 FORCED DEGRADATION STUDUES Forced degradation studies were performed to demonstrate the optimized method is stability indicating. To prove the method which can be able to measure accurately active pharmaceutical ingredient in presence of degradants which are expected to be formed during different types of degradations applied to the drug sample. For forced degradation analysis, aliquots of stock (300µg/ml Ambroxol, 20µg/ml Salbutamol and 1000µg/ml of Theophylline) were separately treated with 1ml of 2N HCl (Acid stability), 1ml of 2N NaOH (Alkaline stability), 1ml of 20% H2O2 (Oxidative degradation) at 60°C for 30 minutes, exposure of standard drug solution at 105ºC for 6 hrs (dry heat degradation), photo stability degradation (exposure of drug at 200 watt/m2) and neutral degradation (refluxing with water at 60ºC for 6 hours. Stability of these samples was compared with fresh sample on the day of analysis. The HPLC chromatograms of degraded products show no interference at the analyte peaks, hence the method was specific and stability indicating. The chromatograms were shown in figures 5 to 8 and the results were shown in table no.8. The detailed degradation for each condition is as follows: 2.17.1 Oxidation: To 1 ml of stock solution of Ambroxol, Salbutamol and Theophylline 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 min at 600c. For HPLC study, the resultant solution was diluted to obtain 30µg/ml, 2µg/ml and 100µg/ml of all Sr.No

1 2 3

Parameter

Flow Rate (±0.1 ml/min) Column Temperature (±5ºC) Mobile phase composition

Optimized

1.0 ml/min 30ºC

Buffer:ACN:: 45:55v/v

components and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

2.17.2 Acid Degradation Studies: To 1 ml of stock s solution Ambroxol, Salbutamol and Theophylline, 1ml of 2N Hydrochloric acid was added and refluxed for 30mins at 600c .The resultant solution was diluted to obtain 30µg/ml, 2µg/ml and 100µg/ml of all components and 10 µl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.

2.17.3 Alkali Degradation Studies: To 1 ml of stock solution Ambroxol, Salbutamol and Theophylline, 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 600c. The resultant solution was diluted to obtain 30µg/ml, 2µg/ml and 100µg/ml of all components and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample. 2.17.4 Dry Heat Degradation Studies: The standard drug solution was placed in oven at 1050c for 6 h to study dry heat degradation. For HPLC study, the resultant solution was diluted obtain 30µg/ml, 2µg/ml and 100µg/ml of all components and10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

2.17.5 Photo Stability studies: The photochemical stability of the drug was also studied by exposing the 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 30µg/ml, 2µg/ml and 100µg/ml of all components and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

2.17.6 Neutral Degradation studies: Stress testing under neutral conditions was studied by refluxing the drug in water for 6hrs at a temperature of 60º. For HPLC study, the resultant solution was diluted to obtain 30µg/ml, 2µg/ml and 100µg/ml of all components and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Table 6A: Robustness_Paracetamol Used

Peak Area

0.9 ml/min 1.0 ml/min 25ºC 30ºC 35 ºC 40:60 45:55 50:50

1.1 ml/min

http://ijps.aizeonpublishers.net/content/2015/2/ijps991-1001.pdf

Plate count

846445 620548

Retention Time 8.295 5.825

8519 7704

Tailing Factor 1.16 1.16

684563 620548 670507 769260 620548 693555

6.566 5.825 6.313 6.480 5.825 7.242

7969 7704 8284 7382 7704 7576

1.15 1.16 1.15 1.15 1.16 1.15

660354

6.238

6612

1.17

995

Sreedhar Lade et al. / Int J Pharma Sci. 2015, 5(2): 991-1001

Sr.No

1 2 3 Sr.No

1 2 3

Parameter

Flow Rate (±0.1 ml/min) Column Temperature (±5ºC)

Optimized

Table 6B: Robustness_ Salbutamol

1.0 ml/min 30ºC

Buffer:ACN::45:5 5v/v

Parameter

Optimized

Mobile phase composition

Plate count

189291 138673

Retention Time 3.216 2.362

3525 4172

Tailing Factor 1.34 1.28

0.9 ml/min 1.0 ml/min 25ºC 30ºC 35 ºC 40:60 45:55 50:50

153483 138673 149635 172489 138673 157556

2.438 2.362 2.569 2.584 2.362 2.573

3149 4172 4386 4410 4172 2599

1.31 1.28 1.29 1.29 1.28 1.27

148406

Table 6C: Robustness_ Theophylline

1.0 ml/min 30ºC

Peak Area

1.1 ml/min

Mobile phase composition

Flow Rate (±0.1 ml/min) Column Temperature (±5ºC)

Used

Buffer:ACN::45:5 5v/v

Used

Peak Area

0.9 ml/min 1.0 ml/min 25ºC 30ºC 35 ºC 40:60 45:55 50:50

1.1 ml/min

2.368

3785

1.31

Plate count

3556245 2662176

Retention Time 6.344 3.797

9571 6569

Tailing Factor 1.18 1.16

2952204 2662176 2889170 3416914 2662176 3076379

4.092 3.797 4.416 4.228 3.797 4.282

7850 6569 9720 8429 6569 8647

1.17 1.16 1.15 1.17 1.16 1.16

2826334

3.878

8204

1.16

Figure 4: HPLC Chromatogram of Standard

Figure 5: HPLC Chromatogram of Placebo http://ijps.aizeonpublishers.net/content/2015/2/ijps991-1001.pdf

996

Sreedhar Lade et al. / Int J Pharma Sci. 2015, 5(2): 991-1001

Figure 6: HPLC Chromatogram of Sample

Table 7: Forced Degradation Studies Table 7A: Forced Degradation studies_ Ambroxol

Sr.No

Injection

1 2 3 4 5 6 7

Controlled sample Acid Degradation Base Degradation Peroxide Degradation Thermal Degradation UV Degradation Water Degradation

Sr.No

Injection

1 2 3 4 5 6 7

%Assay

%Degradation

Purity Angle

99.67 93.41 94.25 92.34 96.78 98.77 99.35

-6.26 5.42 7.33 2.89 0.9 0.32

0.763 0.189 0.181 0.684 0.203 0.194 0.197

Table 7B: Forced Degradation studies_ Salbutamol %Assay

%Degradation

Purity Angle

Controlled sample Acid Degradation Base Degradation Peroxide Degradation Thermal Degradation UV Degradation Water Degradation

99.61 94.11 93.58 92.55 97.57 98.39 99.29

--5.50 6.03 7.06 2.04 1.22 0.32

0.561 1.378 0.301 0.711 1.458 0.815 0.967

Sr.No

Injection

%Assay

%Degradation

Purity Angle

1 2 3 4 5 6 7

Controlled Sample Acid Degradation Base Degradation Peroxide Degradation Thermal Degradation UV Degradation Water Degradation

100.19 93.77 94.39 93.01 97.48 98.90 99.33

--6.42 5.80 7.18 2.71 1.29 0.86

0.322 0..301 0.372 0.280 0.198 0.236 0.236

Table 7C: Forced Degradation studies_ Theophylline

Figure 7A: Acid Degradation

http://ijps.aizeonpublishers.net/content/2015/2/ijps991-1001.pdf

Purity Threshold 1.342 0.399 0.393 0.896 0.405 0.402 0.405

Purity Flag No No No No No No No

Purity Threshold 1.447 2.332 0.438 0.884 1.582 1.635 1.749

Purity Flag

Purity Threshold 1.847 0.510 0.664 0.468 0.381 0.414 0.415

Purity Flag

No No No No No No No

No No No No No No No

997

Sreedhar Lade et al. / Int J Pharma Sci. 2015, 5(2): 991-1001

Figure 7B: Base Degradation

Figure 7C: Peroxide Degradation

Figure 7D: Thermal Degradation

Figure 7E: UV Degradation

http://ijps.aizeonpublishers.net/content/2015/2/ijps991-1001.pdf

998

Sreedhar Lade et al. / Int J Pharma Sci. 2015, 5(2): 991-1001

Figure 7F: Water Degradation

Figure 8A: HPLC Chromatogram of LOD

Figure 8B: HPLC Chromatogram of LOQ Drug Salbutamol

Table 8: Characteristics of HPLC method Parameters defined Linearity range (ppm) Regression coefficient(r2) Intercept Slope LOD (ppm) LOQ (ppm) Tailing factor Plate count

http://ijps.aizeonpublishers.net/content/2015/2/ijps991-1001.pdf

Obtained value 0.5 – 3.0 ppm 0.9993 738.74 70031 0.0176 0.0532 1.28 4172

999

Sreedhar Lade et al. / Int J Pharma Sci. 2015, 5(2): 991-1001

Theophylline

Ambroxol

Linearity range (ppm) Regression coefficient(r2) Intercept Slope LOD (ppm) LOQ (ppm) Tailing factor Plate count Linearity range (ppm) Regression coefficient(r2) Intercept Slope LOD (ppm) LOQ (ppm) Tailing factor Plate count

25-150 ppm 0.9995 5371.7 27564 0.0911 1.2762 1.16 8635 7.5-45 ppm 0.9997 3135.2 20832 0.0898 0.2721 1.16 7704

Figure 9A: Linearity of Salbutamol

Figure 9B: Linearity of Theophylline

Figure 9C: Linearity of Ambroxol

http://ijps.aizeonpublishers.net/content/2015/2/ijps991-1001.pdf

1000

Sreedhar Lade et al. / Int J Pharma Sci. 2015, 5(2): 991-1001

3. RESULTS AND DISCUSSION

The main aim for development of chromatographic method was to get reliable method for quantification of Ambroxol, Salbutamol and Theophylline from bulk and pharmaceutical dosage form and which will be applicable for the degradation products also. Different chromatographic conditions were employed for the analysis of the Ambroxol, Salbutamol and Theophylline in both bulk and pharmaceutical dosage form. Finally the analysis was performed by using Phosphate Buffer: Acetonitrile in the ration of 55:45 (v/v) at a flow rate 1.0 ml/min. Samples were analyzed at 225 nm at an injection volume of 10 μL and separation was carried by using Inertisil ODS, (250 x 4.6 mm), 5µ column. The retention time and tailing factor were calculated. The retention time of Ambroxol, Salbutamol and Theophylline were found to be 5.863, 3.808 & 2.371 min respectively. The proposed column was selected which gave a sharp and symmetrical peak with 1.16 tailing factor and theoretical plates of 7704 for Ambroxol, and, 1.28 tailing factor and theoretical plates of 4172 for Salbutamol and 1.17 tailing factor and theoretical plates of 8635 for Theophylline. The calibration curve was linear over the concentration range of 7.5ppm-45ppm (Ambroxol), 0.5ppm-3ppm (Salbutamol) and 25ppm-150ppm (Theophylline). Six different concentrations of Ambroxol, Salbutamol and Theophylline in the given range were prepared and injected into HPLC. The linearity of the method was statistically confirmed. RSD values for accuracy and precision studies obtained were less than 2% which revealed that developed method was accurate and precise. The system suitability parameters were given in table-5.

Forced degradation studies of different conditions shows that all the degradants were well resolved from these main drug peaks and able to quantify the Ambroxol, Salbutamol and Theophylline in presence of degradants & excipients which proved that the method is found to be stability indicating. Hence the proposed method can be conveniently adopted for the routine quality control analysis in the bulk and combined formulations.

5. Acknowledgements

The authors gratefully acknowledge Spectrum Pharma Research Solutions, Hyderabad, India for providing necessary facilities to carry out this research work.

6. REFERENCES

Forced degradation studies concluded that the all the degradant peaks obtained during degradation were well resolved from the main drugs i.e. Ambroxol, Salbutamol and Theophylline. And the peak purity was passed i.e. purity angle was less than purity threshold as per Empower-2 software. Hence the method is found to be stability indicating. The slope (m) and intercept(c) obtained were shown in the table-5. Therefore proposed validated stability indicating method was successfully applied to determine Ambroxol, Salbutamol and Theophylline in Bulk and Pharmaceutical dosage form.

4. CONCLUSION

The developed method is accurate, simple, rapid and selective & proved to be stability indicating for the simultaneous estimation of Ambroxol, Salbutamol and Theophylline in Bulk and pharmaceutical dosage form. The sample preparation is simple, the analysis time is short and the elution is by isocratic method. The retention time of Ambroxol, Salbutamol and Theophylline were found to be 5.863, 3.808 & 2.371 min respectively. The excipients of the commercial sample analyzed did not interfere in the analysis, which proved the specificity of the method for these drugs. http://ijps.aizeonpublishers.net/content/2015/2/ijps991-1001.pdf

*****

1.

2. 3. 4. 5.

6. 7.

8.

9. 10. 11. 12. 13.

14.

Ilangovan Ponnilavarasan, C. Sunil narendra kumar and P.Asha. (2011) Simultaneous estimation of Ambroxol Hydrochloride and Loratadine in tablet dosage form by using UV Spectrophotometric method, International Journal of Pharma and Bio Sciences 2(2) 338-344. Selvadurai Muralidharan, and Jayaraj Kumar (2012) High Performance Liquid Chromatographic Method Development and Its Validation for Salbutamol. British Journal of Pharmaceutical Research 2(4): 228-237 Sanjay G. Walode, Shruti D. Deshpande and Avinash V. Deshpande (2013) Stability indicating RP-HPLC method for simultaneous estimation of salbutamol sulphate and guaifenesin, Der Pharmacia Sinica 4(2): 61-67 Ghulam Murtaza, Mahmood Ahmad, Muhammad Asadullah Madni and Muhammad Waheed Asghar (2009) A new reverse phase hplc method with fluorescent detection for the determination of salbutamol sulfate in human plasma, Bull. Chem. Soc. Ethiop. 23(1), 1-8 http://www.nlm.nih.gov/medlineplus/druginfo/meds/a681 006.html Mutasem G (2000) Comparative bioavailability of two test brands of Theophylline - Tablets and a reference, Quibron® under fasting and Limited food conditions. Med J Isl Acad Sci 13:109-114. Sanghavi NM, Kamath PR, Amin DS (1990) Sustained Release Tablets of Theophylline. Drug Dev Ind Pharm 16:1843-8. Evelyn O, Edna M, Tais C, Vladi O (2005) Formulation and in vitro evaluation of Theophylline-Eudragit® sustainedrelease tablets. Braz J Pharm Sci. 41:377-84. Meshali MM (1996) Preparation and evaluation of Theophylline sustained-release tablets. Drug Dev Ind Pharm 22:373-376. Ravi Gowda, Padmakar A. Sathe and Sudesh Bhure (2013) Simultaneous Quantitative Determination of Terbutaline and Theophyline from Drug Product by RP-HPLC Method. Biological Forum – An International Journal 5(1): 3-41 Dave Hiral N, Mashru Rajeshree C, Patel Alpesh K (2010) Thin Layer Chromatographic Method For the determination of ternary mixture containing salbutamol sulphate, ambroxol hydrochloride and Theophylline. International Journal of Pharmaceutical Sciences 2(1): 390-394 Quantitative analysis of drugs in pharmaceutical formulation. 3rd Edition. CBS Publisher and Distributors. New Delhi. India. Pages: 51 – 64. 15. B.K.Sharma, Instrumental method of chemical Analysis. Willard, H.H., Merit Instrumental method of analysis., 6th edition. L.Loyd R. Synder Practical HPLC method development I edition.

© 2015; AIZEON Publishers; All Rights Reserved

This is an Open Access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1001