In Vitro New Dissolution Method for the Evaluation of Roxithromycin using pH 6.0 Phosphate Buffer and Determination of its Content by Validated UV Spectrophotometric Method

 

Shilpa Jagtap1, Smrutidevi Sonavane1, Shrikrishna Baokar1*, Vinod Pawar2, Patil R. N.1

1Department of Pharmaceutical Chemistry, Shivnagar Vidya Prasarak Mandal’s College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist -Pune, Maharashtra, India – 413115.

2Department of Pharmacology, Shivnagar Vidya Prasarak Mandal’s College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist -Pune, Maharashtra, India – 413115.

 

ABSTRACT:

A rapid dissolution method was developed for the evaluation of Roxithromycin tablets and the determination of Roxithromycin content was done by UV-spectrophotometer. The proposed method comprises the measurement absorbance of standard concentrations of Roxithromycin in the pH 6.0 phosphate buffer solution against wavelength maximum [λmax] of 205 nm. Beer's Law limits were obeyed in the concentration range of 1µg/ml – 10µg/ml. The linear regression coefficient was estimated to be 0.999 and the linear regression equation obtained was y=0.0954x-0.0014. The solutions were stable for more than 12 hours. The method has been extended for the determination of Roxithromycin content in tablets after the dissolution for 60 minutes at 50 rpm using USP Apparatus II [Paddle apparatus] under experimental conditions. The unknown concentration of Roxithromycin in the solution was estimated from the standard curve and it was found to be within the range of the labeled claim. The relative standard deviation of six replicate solutions was determined to be 0.55. No interference of excipient and coating material was found in this method. This method is economical, precise and accurate. Hence, this method can be employed for the routine evaluation of Roxithromycin tablets by dissolution and dissolution profile by using pH 6.0 phosphate buffer as a medium.

 

KEYWORDS: Roxithromycin, Spectrophotometric, Validation, Dissolution.

 

INTRODUCTION:

Roxithromycin (ROX) is a semi-synthetic macrolide antibiotic.[1] Chemically is (3R,4S,5S,6R,7R,9R,11S,12R,13S,14R)-6-[(2S,3R,4S,6R)-4-d-3-hydroxy-6methyloxan-2-yl]oxy-14-ethyl-7,12,13-trihydroxy-4-[(2R,4R, 5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyloxan-2yl]oxy-10-(2-methoxy ethoxy methoxy imino)-3,5,7,9,11,13-hexamethyl-1-oxacyclo tetradecan-2-one. [1] It acts on gram-positive and gram-negative bacteria [2,3]. It is used to treat respiratory tract, urinary and soft tissue infections. ROX is derived from erythromycin, containing the same 14-membered lactone ring.


However, an N-oxime side chain is attached to the lactone ring. It is also currently undergoing clinical trials for the treatment of male-pattern hair loss [4]. ROX is official in British Pharmacopoeia [5] European Pharmacopoeia [6] and it is assayed by HPLC method. Literature survey reveals that ROX is estimated in pharmaceutical and biological fluids by Near IR reflectance spectroscopy [7] HPLC with fluorescence and UV detection [8] RP HPLC [9,10] HPLC/MS [11] LC/MS [12] Capillary electrophoresis [13,14] LCESI- MS [15] analysis were reported for the estimation of ROX.

 

Reviewing the literature revealed that nothing has been published concerning the proposed dissolution method using pH 6.0 phosphate buffer as medium and determination of its content by UV spectrophotometer. The purpose of applying UV Spectrophotometric to the assay of an analyte is to facilitate faster analysis of the analyte at different time intervals in the dissolution method at the analytical wavelength in the suitable buffering medium. The UV absorption spectra of many substances containing ionisable functional groups are dependent on the state of ionization of that particular functional group(s) and on the pH of the solution. At a particular pH, an ionisable substance has a characteristic absorption spectrum. Active ingredients do not show a significant absorption because of their poor solubility and lesser ionizing capacity. The present method describes the development of a new dissolution method using pH 6.0 buffer as medium followed by determination of ROX content in tablets by UV Spectrophotometer. This method was based on the UV-spectrum of ROX in pH 6.0 phosphate buffer at its absorption maximum of 251 nm. The experimental conditions like rpm, time intervals, buffering medium, buffer volume and dissolution apparatus were optimized. The best suitable final procedure was optimized to determine the ROX content in tablets using pH 6.0 as buffering medium.

 

MATERIAL AND METHODS:

Apparatus:

Schimadzu1700 Double beam UV Spectrophotometer with 1 cm quartz cell, Electrolab Dissolution testing apparatus (8 Vessels), pH Meter and calibrated volumetric Borosilicate glassware’s were used for experiment.

 

Chemicals and Reagent:

ROX was gifted by Naproid and other reagents like Hydrochloric acid, various phosphate buffers were prepared from AR grade materials. Water was obtained from Milli ORQ water purification system.

 

Selection of Wavelength:

The wavelength for the analysis of ROX (3 ppm) was selected from the UV spectrum and maximum wavelength found at 205 nm.

 

Preparation of a Series of Phosphate Buffers:

Reagents like 0.1N Potassium Dihydrogen Phosphate, 0.2M Sodium Hydroxide are mixed in different proportions as per USP to get required pH of the buffer. Each buffer was tested for its accurate pH with a calibrated pH meter and their actual pH values were noted down.    

 

Solubility Characterization:

The pH solubility profile of ROX at room temperature was determined. Excess raw material was suspended in 10 ml series of buffer adjusted with different pH and the suspension was equilibrated by shaking in water bath for 12 hr at designated temperature. Aliquots were withdrawn and filtered through 0.45 µm hydrophilic filters. The solubility of ROX in presence of excipient was also studied in various buffers using above procedure for their quantification. It was revealed that solubility of ROX were high in a pH 6.0 phosphate buffer.

 

General Procedure:

Aliquots of 250 mg of ROX in different buffers were prepared. The absorption spectra were studied and different absorbance values were noted against wavelength range of 205 nm. The observation of various spectra in different buffers and solubility studies indicates a high solubility of ROX in pH 6.0 phosphate buffer. Hence pH 6.0 phosphate buffer was chosen as a medium.

 

Study of Dissolution Parameters:

Dissolution parameter such as medium volume rpm time interval and USP apparatus, have been evaluated. Four different method conditions were adapted to conduct single and multiple vessel dissolution apparatus which are mentioned below.

Method - 1 Volume = 900 ml, 50 rpm, Time = 5, 10, 30, 45 min, USP Apparatus II

Method - 2 Volume = 900 ml, 100 rpm, Time = 5, 10, 30, 45 min, USP Apparatus II

Method - 3 Volume = 900 ml, 50 rpm, Time = 5, 10, 30, 45 min, USP Apparatus I

Method - 4 Volume = 900 ml, 100 rpm Time = 5, 10, 30, 45 min, USP Apparatus I

 

Final Method:

The dissolution samples were analyzed by UV spectrophotometer and the most suitable dissolution method condition were optimized and shown in   Table No. 1

 

Table No. 1 Dissolution Parameters

Medium

pH 6.0 phosphate buffer

Volume

900 ml

Temperature

37o+0.5oC

Rpm

50

Apparatus

USP Apparatus II[paddle]

Time Intervals

5, 10,15, 20, 30, 45,60 min.

Q value

60 minutes

 

Quantitative Determination of the Drugs Using the Developed Method

Label claim -        Each film coated tablet contains ROX equivalent to ROX 200 mg.

Brand Name - Roxibest 150, Roxid 150.

 

Procedure for Dissolution of ROX Raw Material and Tablets:

Electrolab dissolution test apparatus was set for equilibrium after adjusting the dissolution parameter. A series of 10mg of pure ROX raw material were accurately weighed and placed in 8 vessels separately. Dissolution testing was run continuously for 60 min with intermittent samples withdrawn for successive time interval of 5, 10, 15, 20, 25, 30, 45, 60 min.

 

RESULTS AND DISCUSSIONS:

The spectrophotometric determination of ROX was studied using UV spectrophotometer. This method was optimized by measuring absorbance of ROX in pH 6.0 phosphate buffer at wavelength of 205 nm. The parameter were optimized and fixed by evaluating the effect of excipient and coating material. Interference studies were also conducted in pH 6.0 phosphate buffer. The proposed method has been further validated for its linearity, precision and accuracy. Different aliquots of standard solution between 1-10 µg/ml were prepared. Absorbance values were noted with solution of ROX in pH 6.0 phosphate buffer. A linear plot was established with concentration of ROX on X axis and absorbance (A) on Y axis. The Beer’s law limits were found to be 1-10 ppm.

 

Fig. No. 1 Dissolution Profile of Commercially Available ROX Tablets with Two Different Label Claim

 

Evaluation of Dissolution and Dissolution Profile of ROX Tablet:

Under fixed dissolution parameters, a series of ROX with different strength were evaluated for the determination of Q value and dissolution profile at different time intervals using ph 6.0 phosphate buffer.

 

 

Table No. 2 Drug Release of ROX Tablet by Dissolution

Time (Min)

Abs

Conc. in 1ml (mcg)

Conc. in 5ml (mcg)

Conc. in 900ml (mcg)

Drug Release

% Released

5

0.216

1.372

6.861

1235.069

1235.07

12.350

10

0.536

3.405

17.026

3047.776

3040.915

30.409

15

0.879

5.584

27.922

4998.125

4981.125

49.810

30

1.195

7.592

37.960

6794.949

6767.027

67.670

45

1.304

8.284

41.423

7414.739

7376.779

73.767

60

1.561

9.917

49.587

8876.080

8834.657

88.346

 

Table No. 3 Drug Release of ROX Bulk Drug by Dissolution

Time (Min)

Abs

Conc. in 1ml (mcg)

Conc. in 5ml (mcg)

Conc. in 900ml (mcg)

Drug Released

% Released

5

0.241

1.53

7.65

1378.01

1378.01

13.78

10

0.564

2.27

17.91

3206.98

3206.88

32.06

15

0.816

5.18

25.92

4639.89

4639.88

46.39

30

1.007

6.39

31.98

5725.95

5725.95

57.25

45

1.194

7.58

37.92

6789.26

6789.26

67.89

60

1.531

9.72

48.63

8705.49

8705.49

87.05

 

Table No. 4 Dissolution of commercial ROX by proposed methods

Product Label Claim

% Found

( ±%RSD)

Average %RSD

Roxid

150 mg

87.90±0.21

89.06

89.72±0.40

89.57±0.25

 

Roxibest 150 mg

90.40±0.19

88.54

87.10±0.14

88.14±0.16

 

VALIDATION:

Validation of an analytical method is a process to establish that the performance characteristics of the developed method meet the requirement of the intended analytical application [16].

 

Linearity [17, 18]:

Various aliquots were prepared from secondary stock solution ranging from 1-10 ppm. The samples were scanned in UV VIS spectrophotometer against 6.0 phosphate buffer as blank. It was found that selected drug shows linearity between the ranges of 1-10 ppm. Correlation Coefficient and Standard Deviations were found as 0.9970 and 0.481 respectively.

 

Precision:

The precision of an analytical procedure expresses the closeness of agreement [19] (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision of the method was demonstrated by intraday and interday variation studies.

 

Intraday Precision:

In intraday variation six different solution of same conc. 8 ppm were analyzed three times in a day i. e. from morning, afternoon and evening. The %RSD was found as 0.4980

 

Inter day Precision:

In the inter day variation study, solution of same conc. 10 ppm were analyze three times for the consecutive days. The absorbance result mean, S.D. and % RSD was calculated.

 

Robustness:

The Robustness for variations in wavelength of detection (+2 nm and -2 nm) was measured. The % RSD 0.42 and 0.21 was calculated respectively.

 

Ruggedness:

Ruggedness of the method was determined by carrying out the analysis by different analyst in different laboratories using two different UV spectrophotometers [20] and the respective absorbance of 20µg/ml was noted. The result was indicated as % RSD.

 

CONCLUSION:

The proposed dissolution method is more precise, accurate and rapid than most of the reported methods and characterized by instrumental simplicity, cost effective in the use of reagents and time. No interference of excipient and coating material was found in this method. All aliquot solutions prepared were stable for 12 hrs. Hence this method can be employed for routine evaluation and validation of ROX tablet during the small scale, pilot scale and large production scale batches.

 

ACKNOWLEDGMENT:

We would like to thanks Principal and Management of Shivnagar Vidya Prasarak Mandal’s College of Pharmacy for providing all the facilities to complete this work successfully.

 

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Received on 03.03.2013

Modified on 02.08.2013

Accepted on 10.08.2013     

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(5): September-October, 2013, 277-281