Simultaneous Determination of Amoxicillin Trihydrate and Ambroxol Hydrochloride in Solid Dosage Form By Spectrophotometric and Stability Indicating RP-HPLC Method

 

Sohan S. Chitlange*, Sneha R. Tawargeri and Kaushalendra K. Chaturvedi

Padm. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research , Pimpri, Pune-411018, M.H., India.

*Corresponding Author E-mail: sohanchitlange@rediffmail.com

 

ABSTRACT:

Simple, accurate, and reproducible UV spectrophotometric and a stability indicating RP-HPLC method for simultaneous estimation of Amoxicillin Trihydrate (AMOX) and Ambroxol Hydrochloride (AMBRO) have been developed in the present work. The first developed method is Simultaneous equation method, wavelength selected are 228nm for amoxicillin and 245nm for ambroxol respectively. Linearity was observed in concentration range of 2-12µg/ml for ambroxol and 20-120 µg/ml for amoxicillin. Second developed  method  is RP-HPLC method using Thermo C18 column (4.6 mm i.d × 250 mm) and acetonitrile: 0.025M potassium dihydrogen orthophosphate buffer (pH adjusted to 3  with orthophosphoric acid) in the ratio of 28:72% v/v  as a mobile phase at a flow rate of 0.8 ml/min in the detection wavelength of 237 nm. For HPLC method, linearity was observed in the concentration range of 4-24µg/ml for ambroxol and 25-200 μg/ml  for amoxicillin. The correlation coefficients for both the  methods obtained were near to 1. The accuracy of these methods were evaluated by recovery studies and good recovery results were obtained from 98 % to 102% for both the methods and the relative standard deviation of below 2% were achieved. The drugs were subjected to oxidation, hydrolysis, and heat to apply stress condition for degradation studies.

 

KEYWORDS: Amoxicillin Trihydrate, Ambroxol hydrochloride, Simultaneous equation method, RP-HPLCmethod, Degradation studies .

 


 

1. INTRODUCTION:

Amoxicillin trihydrate (AMOX) is chemically a (2S, 5R,6R) [[( 2R )- 2 - amino-2(4- hydoxyphenyl ) acetyl ]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]heptane-2-carboxyic acid, and it belongs to class of broad spectrum antibiotics. AMOX  is official in IP, BP, Eur Pharmacopoeia and USP 1-4 .Literature survey reveals that  AMOX has been determined by spectrophotometry 5-7, HPLC8-11 , HPLC with fluorimetric detection12 , HPLC with photo diode array detection13, voltametry14.

 

Ambroxol hydrochloride (AMBRO) [trans-4-(2-amino-3,5-dibromobenzylamino) cyclohexanol Hydrochloride]15  is semi-synthetic derivative of vasicine obtained from Indian shrub Adhatoda vasica. It is a metabolic product of bromhexine. It is used as broncho secretolytic and expectorant drug.

 

It stimulates the transportation of the viscous secretions in the respiratory organs and reduces the stand stillness of the secretions16 .It is official in IP1. Few methods have been reported in the literature for the determination of AMBRO individually or in combination with other drugs17-19

 

Combination of AMOX and AMBRO  is used as Respiratory system and anti-allergics. The chemical structures of AMOX and AMBRO are shown in Fig.1

 

Amoxicillin

 

Ambroxol

Fig.1: Chemical structures of Amoxicillin and   Ambroxol

 

Study has been reported on simultaneous determination of AMOX and AMBRO in human plasma by LC-MS/MS 20  and HPLC method21. In the present work a successful attempt has been made to estimate both these drugs simultaneously by UV spectrophotometric method  and  HPLC method. The HPLC method was extended to analyze the drugs after forced degradation under various stress conditions(thermal, oxidation, acid and base hydrolysis)22,23 The proposed methods were optimized and validated as per ICH guidelines 24.

 

2. MATERIALS AND METHODS:

2.1. Materials:

Gift samples of AMOX  and AMBRO were provided by  Elder Pharmaceuticals  Pvt.Ltd.,Mumbai. Acetonitrile and Methanol (both HPLC Grade) was obtained from Qualigens laboratory, Mumbai. Doubled distilled water was used to prepare phosphate buffer solutions for HPLC method. All solutions were prepared daily.

 

2.2. UV-Spectrophotometry:

2.2.1. Instrument: The UV method was performed on a Double-beam Shimadzu UV- Visible spectrophotometer, 1700, with spectral bandwidth of 2 nm, wavelength accuracy ± 0.5 nm and a pair of 1-cm matched quartz cells was used to measure absorbance of solution.

 

2.2.2 Solvent used: Methanol-AR grade, distilled water

2.2.3 Preparation of stock solution:

Accurately weighed quantity of AMOX (25 mg) and AMBRO (25 mg)   were transferred to two separate 50.0 ml volumetric flask. They were dissolved by adding 10 ml methanol and then both the drug solutions were diluted up to the mark with methanol (Stock solution 500 μg/ml) .Further dilutions were carried out in distilled water.

 

2.2.4 Simultaneous equation method:

From the overlain spectra of both the drugs, the wavelength was fixed for spectrophotometric measurement of AMOX  at 228 nm and AMBRO at 245 nm for the simultaneous equation as shown in (Fig. 2). The calibration curves for AMOX and AMBRO were plotted in the concentration range of 20-120 μg/ml  and 2-12 μg/ml respectively. The method employs solving of simultaneous equations using Cramer’s rule and matrices.

 

 

The simultaneous equations were;

A1 = Є1amox × C1 + Є1amb × C2…………(1)

A2 = Є2amox× C1 + Є2amb × C2………….(2)

 

Є1amb and Є2amb  ,absorptivity values of AMBRO at 245 nm wavelength,

Є1amox and Є2amox , absorptivity values of AMOX at 228 nm wavelength,

C1 and C2 concentrations of AMOX and AMBRO respectively in sample solution

 

Fig. 2: UV-overlain spectra of AMOX and AMBRO

 

2.2.5 Assay of tablet formulation by simultaneous equation method

Twenty tablets were weighed and finely powdered; an accurately weighed powder equivalent to one tablet containing 3 mg of AMBRO and 25 mg of AMOX was transferred to 25.0 ml volumetric flask , added 15 ml methanol, ultrasonicated for 20 min., volume was made up to the mark with methanol . Diluted solution was filtered through Whatman filter paper No. 42. The sample filtrate was further diluted with a final concentration of AMBRO 5 µg/ml and AMOX 40 µg/ml which was used for analysis. Absorbance of these solutions was measured at 228 nm and 245 nm of both wavelengths as A1 and A2 respectively and concentrations of these two drugs in each sample were calculated using simultaneous equation. Results of tablet analysis shown in table no.1

 

Table No. 1: Result Of Marketed Formulation Analysis

Method

Content

Label claim

(mg/tab)

Amount found*

RSD*

(In mg)

(In %)

UV-method

AMOX

25.0

24.8

99.2

0.6694

AMBRO

3.0

3.04

101.3

0.5527

HPLC-method

AMOX

25.0

24.7

98.8

0.1474

AMBRO

3.0

3.01

100.3

0.0689

* Mean of six estimations

 

2.3 HPLC method:

2.3.1 Instrumentation and Chromatographic conditions:

HPLC method was performed on HPLC system (Merck Hitachi) consisting of quaternary gradient pump, column oven, and UV detector (L-7400) was employed for analysis. Chromatographic data was acquired using Winchrome software. Thermo C18 column (4.6 mm i.d × 250 mm) was used as stationary phase. AMOX and AMBRO were eluted isocratically with a flow rate 0.8 ml/min using a mobile phase consisting of 0.025M phosphate buffer (pH was adjusted to 3 using orthophosphoric acid) and acetonitrile in a proportion of 72:28 v/v respectively. The wavelength of UV detector was set to 237nm. The mobile phase was prepared daily, filtered through 0.45µm  membrane filter (Milipore) and sonicated before use. The HPLC system was operated at 25± 1şC.

 

2.3.2 Preparation of solutions:

Standard sock solution: For measurement by HPLC, standard stock solutions of 400 μg/ml of AMOX and AMBRO were prepared separately using the mobile phase. Different concentrations of standard solutions were prepared from 4 to 24 μg/ml of AMBRO and from 25 to 200 μg/ml of AMBRO and calibration curves were plotted.

 

Sample solution:

Twenty tablets were weighed and average weight was calculated. The tablets were crushed to obtain fine powder. Tablet powder equivalent to 3 mg AMBRO and 25 mg of AMOX was taken and dissolved in mobile phase and sonicated for 20min. and  then volume was made up to the mark with mobile phase. The resulting solution was mixed and filtered through Whatmann filter paper No.42 and filtrate was appropriately diluted to get approximate concentration of 9µg/ml of AMBRO and 75 µg/ml of AMOX. The diluted solutions were filtered through 0.20µm  membrane filter to get clear solutions. A steady baseline was recorded; the typical chromatogram was recorded for sample as shown in Fig. 3.The results of tablet analysis are shown in table no.1.The retention times of sample AMOX and AMBRO were found to be 3.45 min and 6.79 min respectively.

 

Fig. 3 Chromatogram of AMOX (RT=3.45 min)  and AMBRO  ( RT=6.79 min)  extracted from tablet by HPLC

2.4 Procedure for forced degradation study

Decomposition studies were performed in solutions containing drug at a concentration of  9µg/ml for AMOX and 75µg/ml for AMBRO. Samples were withdrawn and subjected to HPLC analysis, after suitable dilution. The stress conditions were as follows:

 

2.4.1 Stress degradation by hydrolysis under acidic conditions

For acid degradation study 1ml of 0.5M HCl  was added to final drug solution and it was refluxed for 2hr at 800 C. After 2hr this solution was injected in stabilized chromatographic condition. The chromatogram   recorded is shown in Fig.4

 

Fig.4 Chromatogram of AMOX and AMBRO  in 0.5M HCl

 

2.4.2 Stress degradation by hydrolysis under alkaline conditions

For alkali degradation study 1ml of 0.5M NaOH was added to final drug solution and it was refluxed for 2hr at 800 C. After 2hr this solution was injected in stabilized chromatographic condition. The chromatogram recorded is shown in Fig.5

 

Fig.5 Chromatogram of AMOX and AMBRO  in 0.5M NaOH

 

2.4.3 Oxidative degradation

For oxidation study 1ml of  5% v/v H2O2 was added to final drug solution and it was refluxed for 2hr at 800 C. After 2hr this solution was injected in stabilized chromatographic condition. The chromatogram recorded is shown in Fig.6

 

Fig.6 Chromatogram of AMOX and AMBRO in 5% v/v H2O2

 

2.4.4 Temperature stress studies:

For temperature stress studies final drug solution was refluxed for 2hr at 800C and then after 2hr this solution was injected in stabilized chromatographic condition. The chromatogram recorded is shown in Fig.7

 

Fig.7. Chromatogram of AMOX and AMBRO in temperature stress condition

 

2.5 Method Validation:

The accuracy studies of UV (Simultaneous equation) and HPLC method were carried out by adding known amount of each drug corresponding to three concentration levels 80% , 100%  and 120% of the label claim along with the excipients in triplicate.

 

Precision of these methods was checked by analyzing the samples at three different time intervals of the same day (intraday precision) as well as on different days (interday precision).Robustness for HPLC method was performed by deliberately changing the chromatographic conditions. The flow rate of the mobile phase was changed from 0.8 ml/min to 0.7 ml/min and 0.9 ml/min while the ratio of the mobile phase was changed by ± 1%.

LOD and LOQ are calculated by using the values of slopes and intercepts of the calibration curves for both the drugs.

 

3.RESULTS AND DISCUSSIONS:

Both, UV spectrophotometric and HPLC methods were found to be simple, accurate and rapid for routine simultaneous estimation of amoxicillin and ambroxol, in tablet dosage forms.

 

3.1 UV spectrophotometry (Simultaneous equation method):

For simultaneous equation method the linearity was observed in the concentration range of  20-120 µg/ml for AMOX and 2-12µg/ml for AMBRO. Commercial formulations containing AMOX and AMBRO were analyzed by the proposed method. Six replicate analysis of the formulation was carried out and the mean assay values were found close to 100%. The proposed method was validated as per ICH guidelines. The accuracy of the proposed method was determined by recovery studies. It was confirmed from results that the method is highly accurate(Table no. 2). Precision was calculated as intraday and interday variations for both the drugs. Percent relative standard deviation are well within the acceptable limit of 2%(Table no. 3).

 

3.2 HPLC method:

For HPLC method the linearity was observed in the concentration range of 4-24µg/ml for ambroxol and 25-200 μg/ml  for amoxicillin. Commercial formulations containing AMOX and AMBRO were analyzed by the proposed method. A typical chromatogram of the marketed formulation is shown in fig.3. Six replicate analysis of the formulation was carried out and the mean assay values were found close to 100%. The tailing factor  were less than 2% for both the peaks. The elution order was AMOX (RT=3.45 min)  and  AMBRO( RT=6.79 min) , at a flow rate of 0.8 ml/min. The chromatogram was recorded at 237nm as the overlain UV spectrum of  AMOX and AMBRO in the mobile phase showed maximum response at this wavelength.

 


Table 2: Results of recovery studies

Content

UV-Method

RP-HPLC method

Level of Recovery(% Recovery ,RSD*)

80

100

120

80

100

120

AMOX

98.55,

0.6405

101.02,

1.2740

100.33,

0.7404

99.18,

0.3567

98.80,

0.133

98.66,

0.1154

AMBRO

98.12,

0.4611

98.99,

0.995

98.28,

0.6341

100.74,

0.5389

101.22,

0.0996

100.56,

0.1312

RSD=Relative standard deviation, * Mean of six estimation

Table 3: Validation parameters for determination of AMOX and AMBRO by proposed methods

VALIDATION

PARAMETERS

UV-Method

RP-HPLC

AMBRO

AMOX

AMBRO

AMOX

Linearity and range ( µg/ml)

2-12

20-120

4-24

25-200

Correlation coefficient

0.998

0.999

0.998

0.998

Standard Deviation

0.033

0.071

0.971

1.125

LOD (µg/ml)

0.187

0.247

0.024

0.064

LOQ (µg/ml)

0.56

0.75

0.074

0.190

Accuracy(%)

98.12

101.33

98.66

100.81

Inter-day Precision RSD(%)

0.923

0.815

1.021

1.033

Intra-day Precision RSD(%)

1.212

0.966

1.334

1.468

LOD-Limit of detection , LOQ- Limit of Quantitation , RSD-Relative standard deviation

 

 

 


The accuracy of the proposed method was determined by recovery studies. It was confirmed from results that the  method is highly accurate(Table no. 2). Precision was calculated as intraday and interday variations for both  the drugs. Percent relative standard deviation are well within the acceptable limit of 2%(Table no. 3). For  robustness studies in all deliberately varied conditions, the RSD of the contents of AMOX and AMBRO was found to be well within the acceptable limit of 2%. System suitability was established by injecting standard solution. The resolution obtained was 3.095 and the tailing factors were 1.16 and 1.125 for AMOX and AMBRO respectively. No.of theoretical plates were more than 2000.

 

AMOX  and  AMBRO  underwent acid hydrolysis. It is depicted in Fig.4. It also underwent  degradation  in alkaline condition. And decrease in peak area of both the drug was observed and it is shown in Fig.5. The drug degraded in the presence of  hydrogen peroxide as  shown in Fig.6. Upon refluxing the drug for 2hr at 80şC, fall in the original peak area of both  the drugs and additional peaks was observed and it is shown in Fig.7.Initially milder conditions were used for degradation studies but no significant degradation was observed. Then  stepwise increase in the degradation conditions were carried out till sufficient degradation was obtained which was between 10-20% degradation. The percent amount of drug degraded after degradation studies are given in Table No.4

 

Table 4: Result Of Stress Degradation Studies Of AMOX And AMBRO By RP-HPLC Method

Type of degradation

AMOX

(% degradation)

AMBRO

 (% degradation)

Acid degradation (0.5N HCl)

(2 hrs of reflux)

 

15.88

 

13.67

Alkali degradation

(0.5M NaOH)

(2 hrs of reflux)

 

12.76

 

14.86

Oxidation (5% H2O2)

(2 hrs of reflux)

 

8.67

 

16.78

Thermal (80 0C)

(2 hrs of reflux)

 

9.34

 

11.59

 

4. CONCLUSION:

A novel stability indicating RP-HPLC method and a spectrophotometric method  were developed and validated for the simultaneous determination of Amoxicillin trihydrate and Ambroxol HCl in solid dosage form. These methods assured the  satisfactory precision and accuracy and have high analytical potential. These methods were found to be simple, accurate, and reproducible and can be applied for routine analysis in laboratories. The stability indicating RP-HPLC method is suitable for the quality control of the raw materials, formulations, dissolution studies and can be employed for bioequivalence studies for the same formulation.

 

5. ACKNOWLEDGEMENT:

The authors gratefully acknowledge Dr. P. D. Patil, Chairman, Dr. D. Y. Patil Vidya Pratishthan Society and Dr. A. D. Deshpande, Director of Pharmacy for providing excellent infrastructure facility to carry out this research work. Thanks also go to Elder Pharmaceutical Pvt. Ltd. Mumbai for providing pure drug samples.

 

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Received on 17.02.2011        Modified on 24.03.2011

Accepted on 05.05.2011        © AJRC All right reserved

Asian J. Research Chem. 4(6): June, 2011; Page 1025-1030