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Current Pharmaceutical Analysis, 2013, 9, 310-317
Development and Validation of a Simple and Sensitive Spectrometric Method for Estimation of Azithromycin Dihydrate in Tablet Dosage Forms: Application to Dissolution Studies Vinay Kumar, Sachin Kumar Singh*, Monica Gulati, Renuka, Ranjith Anishetty, Tamilvanan Shunmugaperumal School of Pharmaceutical Sciences, Lovely Professional University, Phagwara- 144411, Punjab, India Abstract: Azithromycin dihydrate is a semi-synthetic macrolide antibiotic drug, effective against a wide variety of bacteria. It is primarily used to treat the bacterial infections associated with weaker immune system. A simple, accurate, rapid and cost effective spectrometric method for the estimation of azithromycin has been developed and validated by the acidic hydrolysis of the drug with sulphuric acid and monitoring the absorbance at 482nm. The validated method was successfully applied for dissolution studies of azithromycin dihydrate tablets. The developed method was validated as per ICH Q2 (R1) guidelines, which include linearity, precision, accuracy, specificity, robustness, detection and quantitation limits. The method has shown good linearity over the range of 10.0 to 50.0 g/mL with a correlation coefficient of 0.9995. The percentage recovery of 99.98 % showed that the method was highly accurate. The precision demonstrated relative standard deviation of less than 2.5 %. The specificity of the method was proven as the excipients present in the tablets did not interfere with the assay. The developed and validated method was successfully applied for dissolution studies with a cumulative release of 99.3 % in 45 minutes. The proposed method might be applied in routine quality control in the pharmaceutical industries as it has shown very good precision, accuracy and simplicity.
Keywords: Azithromycin dihydrate, acid hydrolysis, validation, ICH, UV-Vis spectrometry, quality control 1. INTRODUCTION Azithromycin [1] is chemically (2R, 3S, 4R, 5R, 8R, 10R,11R,12S,13S,14R)-13-[(2,6-dideoxy -3 - C - methyl - 3 O - methyl - - L - ribo - hexopyranosyl) oxy]-2-ethyl-3, 4, 10-trihydroxy-3, 5, 6, 8, 10, 12, 14- heptamethyl - 11 - [[3, 4, 6-trideoxy-3-(dimethylamino)--D-xylo- exopyranosyl]oxy]1-oxa- 6-azacyclopentadecan-15-one. It is derived from erythromycin; however, it differs chemically from erythromycin in that methyl-substituted nitrogen atom is incorporated into the lactone ring. It is widely marketed in the form of azithromycin dihydrate. Azithromycin, like all macrolide antibiotics, prevent bacteria from growing by interfering with their ability to make protein. Due to the differences in the way proteins are made in bacteria and humans, the macrolide antibiotics do not interfere with human’s ability to make proteins. It binds to the 50s rRNA subunit of the 70s bacterial ribosome’s, therefore inhibits RNA-dependent protein synthesis. It inhibits the translation of mRNA in bacterial cells at the chain elongation step; result in the blockage of transpeptidation. It is used in respiratory tract infections [2] like pharyngitis, pneumonia, chronic bronchitis, and bronchopneumonia. The recommended dosage for azithromycin is 100-500 mg per day. Azithromycin [2] is official in the United States Pharmacopoeia, and it is assayed by the high performance liquid chromatographic method. The structure of azithromycin dihydrate is shown in Fig. (1). *Address correspondence to this author at the School of Pharmaceutical Sciences, Lovely Professional University, Phagwara- 144411, Punjab, India; Tel: +919888720835; Fax: +91 1824501900; E-mail:
[email protected] 1875-676X/13 $58.00+.00
Fig. (1). Chemical structure of azithromycin dihydrate (CAS 117772-70-0).
Azithromycin has been analyzed in biological samples by microbiological method [3] and high performance liquid chromatography using electrochemical detector [4-7] and fluorescence [8]. Till date, there is noticeable shortage of methods described in the literature for its determination in pharmaceutical dosage forms. Chromatographic methods developed for azithromycin quantitation [9-11] demand expensive equipment and could not be available in many laboratories. An alternative electrochemical method has been described in literature [12], but it is based on reduction of azithromycin in strongly basic media at mercury electrode. Suhagia (2006) developed a colorimetric method for the estimation of azithromycin in pharmaceutical dosage forms. In the proposed method, azithromycin was oxidized with potassium permanganate (excess potassium permanganate is decolourized with ox© 2013 Bentham Science Publishers
Colorimetric Estimation of Azithromycin
alic acid) to liberate formaldehyde. The liberated Formaldehyde was determined in situ, using acetyl acetone, in the presence of ammonium acetate, which gives a yellow coloured chromogen with absorption maxima 412 nm. The procedure used was time consuming and use of residual solvents like acetyl acetone limits the use of this method. However, in this study potassium permanganate was used as an oxidizing agent which is susceptible to photolytic degradation [2]. Sultana et al. (2006) reported the degradative behavior and determination of azithromycin by spectrophotometry. The following research work was based on the observation that macrolide antibiotics like erythromycin undergo hydrolytic cleavage at glycosidic linkage when reacted with 13.5 mol/L sulphuric acid and produce aglycone moiety that is erythronolide that exhibits strong absorption in visible region at 482 nm [13]. The same reaction formed the basis of the present study. The method was validated as per ICH Q2 (R1) guidelines [14] and applied successfully for the assay and dissolution studies of azithromycin tablets. 2. EXPERIMENTAL 2.1. Materials
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cells at a fast scan speed by using mixture of 0.01M phosphate buffer pH 7.5 and 13.5 mol/L sulphuric acid (1:9) as a blank solution as shown in Fig. (2). 2.2.2. Analytical method Development 2.2.2.1. Development of Colour and Preparation of Calibration Curve One hundred milligrams of azithromycin dihydrate working standard was weighed accurately and transferred to 100 mL standard volumetric flask and the volume was adjusted to 100 mL using 0.01M phosphate buffer pH 7.5 in order to get a concentration of 1 mg/mL. 10 mL of sample aliquot was withdrawn from azithromycin dihydrate standard stock solution was diluted to obtain the known standard concentrations of 100 g/mL. From this, 1, 2, 3, 4 and 5 mL of aliquots were withdrawn in 10 mL standard volumetric flask and the volume was adjusted to 10 mL using 13.5 mol/L sulphuric acid in order to get concentrations of 10, 20, 30, 40 and 50 μg/mL, respectively. The prepared dilutions were heated on a water bath at 50°C for 30 minutes in order to get yellow colour solution and finally measured at 482 nm using UV-Visible spectrophotometer. 2.2.2.2. Sample Solutions
Azithromycin dihydrate was a gift sample from Ranbaxy Laboratories, Gurgaon, India. Potassium dihydrogen orthophosphate was purchased from CDH Laboratories, Mumbai, India. Sulphuric Acid was purchased from Molychem, Mumbai, India. Sodium hydroxide flakes were purchased from Loba Chemie, Mumbai, India. All other chemicals and reagents used were of analytical grade. Triple distilled water was used throughout the study. Analysis was done using double beam UV – Visible spectrophotometer (Shimadzu 1800, Japan).
Twenty tablets of (Azipro® 500 mg) were used. The tablets were weighed, triturated and the contents were thoroughly mixed. The mass equivalent to 40 mg of azithromycin dihydrate was weighed into a 200 mL volumetric flask, and phosphate buffer pH 7.4 was added to fill to volume. Appropriate dilutions were made using 13.5 mol/L sulphuric acid. Solutions were heated at 50°C for 30 minutes in order to get yellow colour solution.
2.2. Methods
The stability of azithromycin dihydrate standard stock solution in a mixture of 0.01 M phosphate buffer pH 7.5 and 13.5 mol/L sulphuric acid (1:9) at a concentration of 30 g/mL was investigated at different time intervals using the experimental conditions.
2.2.1. Spectrophotometric Measurements The spectra of different concentrations of reference solutions of azithromycin dihydrate were recorded in 1 cm glass
2.2.2.3. Stability of Azithromycin in Solution
Fig. (2). Spectrum of different concentrations of azithromycin dihydrate in a mixture of 0.01 M phosphate buffer pH 7.5 and 13.5 mol/L sulphuric acid.
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2.2.3. Validation of the Developed Method
2.2.3.4. Precision
2.2.3.1. Linearity and Range
Precision was evaluated with respect to both repeatability and intermediate precision. Repeatability was tested by seven determinations at three levels, 50 %, 100 % and 150 % of the method concentration (30 g/mL) of the same day and under the same experimental conditions. Intermediate precision was evaluated by performing the analysis on three different days (interday) on the same levels as mentioned above and by three analysts performing the analysis in the same laboratory and under the same experimental conditions (between analysts). Seven replicates at a concentration of 15, 30 and 45 g/mL were prepared and assayed at 482 nm. The percentage of relative standard deviation (R.S.D.) of the analytical responses was calculated.
Linearity had been accessed by calibration curve in which solutions of 10, 20, 30, 40 and 50 g/mL respectively Measurements were taken on samples prepared on 3 consecutive days (n = 3). The values are reported as the mean ± confidence interval of the calibration curves. The data was analyzed at a wavelength of 482 nm. Evaluation parameters such as the correlation coefficient were calculated and are presented. 2.2.3.2. Specificity Studies Specificity was evaluated by an analysis of the visible spectrum of placebo solutions, azithromycin dihydrate working standard solution and azithromycin sample solution at concentration of 30 g/mL. The placebo solution was prepared with the same composition as the pharmaceutical formulation without the addition of azithromycin dihydrate and treated in the same manner as the commercial sample solution and the reference solution Fig. (3). 2.2.3.3. Accuracy The method of accuracy was determined by measuring the reference standard recovery in triplicate at three levels, 50 %, 100 % and 150 % of the method concentration (30 g/mL). A standard stock solution 1 mg/mL of azithromycin dihydrate was prepared as described in section 2.2.2.1. In volumetric flasks of 100 mL, aliquots of 1.5, 3.0 and 4.5 mL of this solution (which would yield concentrations of 15, 30 and 45 g/mL respectively) were combined with 15 mL of the 200 g/mL sample solution from section 2.2.2.2. (this would yield a concentration of 30 g/mL). The volume was made upto 100mL using 13.5 mol/L sulphuric acid solution. Thus the final concentrations were 45.0, 60.0 and 75 g/mL, which correspond to 50, 100 and 150 % of the target concentration, respectively. The mean recoveries of Azipro® 500 mg, expressed in terms of the percentage recovery and relative standard deviation (% RSD), were determined.
2.2.3.5. Robustness The robustness of analytical method is measure of its ability to remain unaffected by small changes. In the present study robustness has been calculated by varying the pH and taking the absorbance at pH 7.2, pH 7.4 and pH 7.6 of the sample solution (Azipro® 500 mg). Moreover, the robustness of the method was determined by analyzing a change of 2nm in the wavelength of analysis. Seven replicates of the working standard solution and sample solution were prepared at the same concentration (30 g/mL), and the assays were carried out at 480, 482 and 484 nm. The percentage relative standard deviation (R.S.D.) of the quantitation of azithromycin dihydrate in the tablets was calculated. 2.2.3.6. Limit of Detection (LOD) and Limit of Quantification (LOQ) Estimation of LOD and LOQ were determined by standard deviation of response () and slope of calibration curve (S). Standard deviation of Y intercepts of regression line was used as standard deviation. Equations 1 and 2 for LOD and LOQ respectively are as follow. LOD= 3.3/S
(1)
LOQ=10/S
(2)
Fig. (3). Absorption spectra of azithromycin dihydrate working standard solution, sample solution and placebo solution.
Colorimetric Estimation of Azithromycin
Current Pharmaceutical Analysis, 2013, Vol. 9, No. 3
3.3. Stability of Azithromycin in Solution
2.2.4. Application of Validated Method to Assay and Dissolution Studies of Azithromycin Dihydrate Tablet
The results from the stability study indicated that the azithromycin dihydrate stock standard solution was stable at room temperature for at least one week (Table 1).
2.2.4.1. Assay of Azithromycin Dihydrate Tablets The validated UV- spectrometric method was applied to azithromycin dihydrate quantitation of the (Azipro® tablets 500 mg). The results were obtained by comparing the sample spectrophotometric measurements (n = 7) with those obtained from the azithromycin dihydrate standard solutions (n = 7) at the same concentration levels.
3.4. Method Validation After the method development, the analytical method was validated according to ICH recommendations [14, 15]. 3.4.1. Linearity and Range
2.2.4.2. Dissolution Studies
The analytical curves, obtained on three consecutive days (n = 3) by plotting the mean of absorbance at 482 nm against the concentration, were found to be linear in the 10 to 50 g/mL range and yielded a correlation coefficient (r) of 0.9995 (Table 2) and Fig. (4).
The dissolution study of azithromycin dihydrate tablet (Azipro® tablets 500 mg) was carried out in 900 mL of 0.1 M phosphate buffer pH 6.0 at 75 rpm. From the dissolution medium 5 mL samples were withdrawn at 10, 20, 30 and 45 min respectively. From this 1 mL of solution was pipetted out and diluted to 10 mL using 13.5 mol/L sulphuric acid. After the dilution all the dissolution samples were heated at 50°C for 30 min and then absorbance were taken at 482 nm. The study was conducted for 6 tablets and the mean % cumulative drug release was recorded (n = 6).
3.4.2. Selectivity The spectra analysis show that the formulation excipient of the pharmaceutical tablet product Azipro® tablets 500 mg did not interfere with the developed colorimetric method Fig. (3). The spectrum showed that the placebo did not have absorbance in the wavelength used in this method.
3. RESULTS AND DISCUSSIONS
3.4.3. Accuracy
3.2. Method Development
The accuracy of the proposed method was assessed by determining the average recoveries of samples using the standard addition method. As shown in Table 3, the mean percentage recovery of Azipro® 500 mg was 99.98 % and the % relative standard deviation was 0.73 %. The results were in accordance with fixed limits of 98.0 % to 102.0 %, indicating the suitability of the developed method in quantifying the concentration of azithromycin dihydrate in tablets. The accuracy value of the current method was found excellent.
The reported methods for the determination of azithromycin dihydrate are complex, time consuming or require large amount of organic solvents, some methods developed on sophisticated instruments like HPLC which have higher costs. Precolumn derivatization of azithromycin dihydrate (as it lacks chromophore) followed by HPLC analysis makes the process tedious and expensive. In this manuscript, phosphate buffer pH 7.4 and sulphuric acid were chosen to obtain an inexpensive, simple and environment friendly colorimetric method for the quantification of azithromycin dihydrate in tablets. Also this method uses simple instruments.
Table 1.
3.4.4. Precision The precision, evaluated as the repeatability of the analytical method, was studied by calculating the % relative standard deviation for the seven determination of the 15, 30,
Stability of the azithromycin dihydrate stock standard solution at a concentration of 30 g/mL in mixture of 0.01 M phosphate buffer pH 7.5 and 13.5 mol/L sulphuric acid (1:9)
Responses at 482 nm
Table 2.
313
0h
1h
5h
6h
24 h
1 week
Mean
R.S.D. (%)
0.442
0.453
0.462
0.434
0.444
0.419
0.442
0.81
0.444
0.435
0.442
0.451
0.447
0.440
0.443
1.25
0.438
0.448
0.445
0.434
0.446
0.443
0.442
1.20
Linearity Parameters for the Determination of Azithromycin Dihydrate Parameter
Result
Linearity range (g/mL)
10 to 50
Slope
0.027 ± 0.004
Intercept
0.012 ± 0.002
Correlation coefficient (r)
0.9995
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Fig. (4). Calibration curve of azithromycin dihydrate in a mixture of 0.01 M phosphate buffer and 13.5 mol/L sulphuric acid (1:9). Table 3.
Method Accuracy Results for Azithromycin Dihydrate Tablets
Sample (g/mL) (Azipro®)
30
Reference Standard Concentration added (g/mL)
Total Concentration of solution (g/mL)
Concentration of drug found (g/mL)
Recovery (%)
R.S.D. (%) (n= 3)
15
45
44.94
99.86
0.17
30
60
60.12
100.20
1.30
99.89
0.71
45
75
45 g/mL working standard solution performed on the same day and under the same experimental conditions. The obtained % R.S.D. value was less than 2.5 %. The intermediate precision was assessed by analyzing a sample of the pharmaceutical formulation on 3 different days (interday precision) and by analyzing the samples with three analysts (between – analysts precision). The % R.S.D. values were less than 2.5 % confirming that the method is sufficiently precise (Table 4). 3.4.5. Robustness The robustness was found reliable, as determined by % R.S.D. (< 2%). It was observed that the constancy of the absorbance with deliberative changes in the experimental parameter of wavelength resulted in a % R.S.D. of 0.77 and change in pH resulted in a % R.S.D. of 0.68 (Table 5). The minor changes that occurred during the analysis did not affect the absorbance intensity of the samples. 3.4.6. Limits of Detection and Quantification LOD and LOQ values were found to be 0.66 ± 0.065 and 2.02 ± 0.061 g/mL, respectively, for Azipro® tablets (500 mg). These results demonstrate that the analyses were being performed in a region above the quantitation limit value. 3.5.1. Assay of Azithromycin Dihydrate Tablets The validated method was applied to the determination of azithromycin dihydrate in tablets. Three samples of Azipro® tablets (500 mg) were analyzed. The results, expressed as the percentage drug as related to the content label claim, are shown in Table 6. The results showed that the percentage drug
74.92
Mean Recovery (%)
99.98
is in accordance with the Indian, British and United States Pharmacopoeias, which permit azithromycin dihydrate in tablets to be in a range of 92.5 to 110.0 % of the content label claim. 3.5.2. Dissolution Studies An analytical method should be developed in such a way that it should be able to evaluate all the parameters quantitatively during the early product development stage till the final product release. Dissolution studies play a major role in order to provide drug release testing during early preformulation studies as well as in vitro – in vivo correlation of the compound during the bioavailability studies and clinical trials. Therefore dissolution studies were conducted in order to check the feasibility of this developed and validated method for quantification of dissolution samples. The mean % cumulative drug release was found to be 99.3 % after 45 min. The dissolution profile of Azipro® tablets (500 mg) is shown in Fig. (5). This shows that the developed method can be successfully applied to dissolution studies. UV, UV – VIS and derivative spectrophotometry are broadly used techniques to quantify antibiotics and other drugs [16-24] because they are cost effective, simple and do not require time consuming sample preparation compared to other techniques [25]. Moreover, UV- Visible spectrophotometry produces very low amount of residues and solvents, which is an important ecological aspect as per regulatory point of view and currently discussed in routine laboratory analysis. Because of these reasons and the careful validation of this method, this technique can be recommended for the routine laboratory analysis of the antibiotic azithromycin dihydrate in tablets.
Colorimetric Estimation of Azithromycin
Table 4.
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315
Method Precision Results for Azithromycin Dihydrate Tablets Analytical Responses Concentration (g/mL)
1
2
3
4
5
6
7
R.S.D. (%)
15
0.243
0.234
0.238
0.247
0.238
0.240
0.245
1.88
30
0.438
0.440
0.435
0.434
0.446
0.433
0.443
1.11
45
0.523
0.537
0.532
0.545
0.537
0.525
0.527
1.48
15
0.248
0.249
0.236
0.241
0.246
0.237
0.239
2.2
30
0.438
0.442
0.445
0.435
0.449
0.452
0.447
1.36
45
0.537
0.532
0.540
0.546
0.540
0.538
0.541
0.79
15
0.244
0.241
0.251
0.246
0.255
0.248
0.250
1.88
30
0.435
0.440
0.448
0.432
0.441
0.443
0.437
1.21
45
0.543
0.541
0.555
0.547
0.549
0.551
0.547
0.86
15
0.242
0.251
0.238
0.246
0.241
0.251
0.249
2.11
30
0.442
0.446
0.438
0.440
0.445
0.436
0.447
0.95
45
0.545
0.541
0.538
0.544
0.545
0.546
0.540
0.56
15
0.246
0.239
0.241
0.241
0.243
0.252
0.248
1.89
30
0.445
0.450
0.438
0.437
0.446
0.448
0.442
1.11
45
0.540
0.545
0.539
0.549
0.551
0.541
0.546
0.85
15
0.243
0.249
0.241
0.235
0.238
0.246
0.241
1.94
30
0.438
0.442
0.447
0.449
0.440
0.451
0.444
1.07
45
0.547
0.542
0.548
0.541
0.538
0.549
0.545
0.75
15
0.238
0.253
0.246
0.248
0.241
0.242
0.249
2.12
30
0.446
0.439
0.439
0.441
0.439
0.448
0.451
1.12
45
0.547
0.543
0.549
0.540
0.541
0.547
0.540
0.69
Repeatability
Day 1
Day 2 Intermediate Precision Day 3
Analyst A
Analyst B
Analyst C
Table 5.
Robustness Test Results Sample
pH
Content (mg)a
Content (%)a
506.51
101.30
498.65
99.73
503.46
100.69
480
505.51
101.10
482
501.12
100.22
484
497.89
99.57
Wavelength (nm)
7.2 7.4 Azipro 500 mg tablet
7.6 *NA
a
NA
mean of seven replicates ; *NA = Not Applicable
R.S.D. (%)
0.68
0.77
316 Current Pharmaceutical Analysis, 2013, Vol. 9, No. 3
Table 6.
Kumar et al.
Assay of Azithromycin Tablet (Azipro® 500mg Tablets) Samples A, B and C Sample
Content (mg)a
Content (%)a
1
523.16
104.63
2
518.12
103.62
3
534.36
106.87
R.S.D. (%)
1.58
Fig. (5). Mean % cumulative drug release profile of Azipro 500 mg Tablets (n = 6).
4. CONCLUSION The validated analytical method for quantitative determination of azithromycin dihydrate in tablets has the advantage of simplicity, speed, low cost conditions and a lack of polluting reagents. All validation parameters were found to be highly satisfactory, including linearity, accuracy, precision, selectivity, robustness and adequate detection and quantitation limits. The validated method could be a good alternative for routine quality control of azithromycin dihydrate by the pharmaceutical industries and quality control laboratories. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest.
[4] [5]
[6] [7]
[8]
ACKNOWLEDGEMENTS
[9]
The authors are grateful to shree Ashok Mittal, Honourable Chancellor, Lovely Professional University, Punjab, India for providing facilities to carry out the aforementioned research. We also owe our thanks to Ranbaxy Laboratories, Gurgaon, India, for providing the gift samples of reference standard of azithromycin dihydrate.
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Revised: January 31, 2013
Accepted: February 06, 2013
