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310 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 bacte-
ria. 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 success-
fully 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 per-
centage 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 inter-
fere with the assay. The developed and validated method was successfully applied for dissolution studies with a cumula-
tive release of 99.3 % in 45 minutes. The proposed method might be applied in routine quality control in the pharmaceuti-
cal 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 erythro-
mycin in that methyl-substituted nitrogen atom is incorpo-
rated 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 Fig. (1). Chemical structure of azithromycin dihydrate (CAS
the way proteins are made in bacteria and humans, the mac- 117772-70-0).
rolide antibiotics do not interfere with human’s ability to
make proteins. It binds to the 50s rRNA subunit of the 70s Azithromycin has been analyzed in biological samples
bacterial ribosome’s, therefore inhibits RNA-dependent pro- by microbiological method [3] and high performance liq-
tein synthesis. It inhibits the translation of mRNA in bacte- uid chromatography using electrochemical detector [4-7]
rial cells at the chain elongation step; result in the blockage and fluorescence [8]. Till date, there is noticeable short-
of transpeptidation. It is used in respiratory tract infections age of methods described in the literature for its determi-
[2] like pharyngitis, pneumonia, chronic bronchitis, and nation in pharmaceutical dosage forms. Chromatographic
bronchopneumonia. The recommended dosage for azithro- methods developed for azithromycin quantitation [9-11]
mycin is 100-500 mg per day. Azithromycin [2] is official in demand expensive equipment and could not be available
the United States Pharmacopoeia, and it is assayed by the in many laboratories. An alternative electrochemical
high performance liquid chromatographic method. The struc- method has been described in literature [12], but it is
ture of azithromycin dihydrate is shown in Fig. (1). based on reduction of azithromycin in strongly basic me-
dia at mercury electrode. Suhagia (2006) developed a col-
*Address correspondence to this author at the School of Pharmaceutical orimetric method for the estimation of azithromycin in
Sciences, Lovely Professional University, Phagwara- 144411, Punjab, India; pharmaceutical dosage forms. In the proposed method,
Tel: +919888720835; Fax: +91 1824501900; azithromycin was oxidized with potassium permanganate
E-mail: sachin.16030@lpu.co.in (excess potassium permanganate is decolourized with ox-

1875-676X/13 $58.00+.00 © 2013 Bentham Science Publishers


Colorimetric Estimation of Azithromycin Current Pharmaceutical Analysis, 2013, Vol. 9, No. 3 311

alic acid) to liberate formaldehyde. The liberated Formal- cells at a fast scan speed by using mixture of 0.01M phos-
dehyde was determined in situ, using acetyl acetone, in phate buffer pH 7.5 and 13.5 mol/L sulphuric acid (1:9) as a
the presence of ammonium acetate, which gives a yellow blank solution as shown in Fig. (2).
coloured chromogen with absorption maxima 412 nm.
2.2.2. Analytical method Development
The procedure used was time consuming and use of resid-
ual solvents like acetyl acetone limits the use of this 2.2.2.1. Development of Colour and Preparation of Cali-
method. However, in this study potassium permanganate bration Curve
was used as an oxidizing agent which is susceptible to
One hundred milligrams of azithromycin dihydrate work-
photolytic degradation [2]. Sultana et al. (2006) reported
ing standard was weighed accurately and transferred to 100
the degradative behavior and determination of azithromy-
mL standard volumetric flask and the volume was adjusted
cin by spectrophotometry. The following research work
was based on the observation that macrolide antibiotics 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
like erythromycin undergo hydrolytic cleavage at glycosi-
was withdrawn from azithromycin dihydrate standard stock
dic linkage when reacted with 13.5 mol/L sulphuric acid
solution was diluted to obtain the known standard concentra-
and produce aglycone moiety that is erythronolide that
tions of 100 g/mL. From this, 1, 2, 3, 4 and 5 mL of ali-
exhibits strong absorption in visible region at 482 nm
quots were withdrawn in 10 mL standard volumetric flask
[13]. The same reaction formed the basis of the present
study. The method was validated as per ICH Q2 (R1) 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
guidelines [14] and applied successfully for the assay and
and 50 μg/mL, respectively. The prepared dilutions were
dissolution studies of azithromycin tablets.
heated on a water bath at 50°C for 30 minutes in order to get
2. EXPERIMENTAL yellow colour solution and finally measured at 482 nm using
UV-Visible spectrophotometer.
2.1. Materials
2.2.2.2. Sample Solutions
Azithromycin dihydrate was a gift sample from Ranbaxy
Twenty tablets of (Azipro® 500 mg) were used. The tab-
Laboratories, Gurgaon, India. Potassium dihydrogen ortho-
lets were weighed, triturated and the contents were thor-
phosphate was purchased from CDH Laboratories, Mumbai,
oughly mixed. The mass equivalent to 40 mg of azithromy-
India. Sulphuric Acid was purchased from Molychem,
cin dihydrate was weighed into a 200 mL volumetric flask,
Mumbai, India. Sodium hydroxide flakes were purchased
from Loba Chemie, Mumbai, India. All other chemicals and and phosphate buffer pH 7.4 was added to fill to volume.
Appropriate dilutions were made using 13.5 mol/L sulphuric
reagents used were of analytical grade. Triple distilled water
acid. Solutions were heated at 50°C for 30 minutes in order
was used throughout the study. Analysis was done using
to get yellow colour solution.
double beam UV – Visible spectrophotometer (Shimadzu
1800, Japan). 2.2.2.3. Stability of Azithromycin in Solution

2.2. Methods The stability of azithromycin dihydrate standard stock so-


lution in a mixture of 0.01 M phosphate buffer pH 7.5 and
2.2.1. Spectrophotometric Measurements 13.5 mol/L sulphuric acid (1:9) at a concentration of 30
g/mL was investigated at different time intervals using the
The spectra of different concentrations of reference solu-
experimental conditions.
tions of azithromycin dihydrate were recorded in 1 cm glass

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.
312 Current Pharmaceutical Analysis, 2013, Vol. 9, No. 3 Kumar et al.

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
Linearity had been accessed by calibration curve in
seven determinations at three levels, 50 %, 100 % and 150 %
which solutions of 10, 20, 30, 40 and 50 g/mL respectively
of the method concentration (30 g/mL) of the same day and
Measurements were taken on samples prepared on 3 con- under the same experimental conditions. Intermediate preci-
secutive days (n = 3). The values are reported as the mean ±
sion was evaluated by performing the analysis on three dif-
confidence interval of the calibration curves. The data was
ferent days (interday) on the same levels as mentioned above
analyzed at a wavelength of 482 nm. Evaluation parameters
and by three analysts performing the analysis in the same
such as the correlation coefficient were calculated and are
laboratory and under the same experimental conditions (be-
presented.
tween analysts). Seven replicates at a concentration of 15, 30
2.2.3.2. Specificity Studies and 45 g/mL were prepared and assayed at 482 nm. The
percentage of relative standard deviation (R.S.D.) of the ana-
Specificity was evaluated by an analysis of the visible
lytical responses was calculated.
spectrum of placebo solutions, azithromycin dihydrate work-
ing standard solution and azithromycin sample solution at 2.2.3.5. Robustness
concentration of 30 g/mL. The placebo solution was pre-
The robustness of analytical method is measure of its
pared with the same composition as the pharmaceutical for- ability to remain unaffected by small changes. In the present
mulation without the addition of azithromycin dihydrate and
study robustness has been calculated by varying the pH and
treated in the same manner as the commercial sample solu-
taking the absorbance at pH 7.2, pH 7.4 and pH 7.6 of the
tion and the reference solution Fig. (3).
sample solution (Azipro® 500 mg). Moreover, the robustness
2.2.3.3. Accuracy of the method was determined by analyzing a change of 2nm
in the wavelength of analysis. Seven replicates of the work-
The method of accuracy was determined by measuring ing standard solution and sample solution were prepared at
the reference standard recovery in triplicate at three levels,
the same concentration (30 g/mL), and the assays were car-
50 %, 100 % and 150 % of the method concentration (30
ried out at 480, 482 and 484 nm. The percentage relative
g/mL). A standard stock solution 1 mg/mL of azithromy-
standard deviation (R.S.D.) of the quantitation of azithromy-
cin dihydrate was prepared as described in section 2.2.2.1.
cin dihydrate in the tablets was calculated.
In volumetric flasks of 100 mL, aliquots of 1.5, 3.0 and 4.5
mL of this solution (which would yield concentrations of 2.2.3.6. Limit of Detection (LOD) and Limit of Quantifica-
15, 30 and 45 g/mL respectively) were combined with 15 tion (LOQ)
mL of the 200 g/mL sample solution from section 2.2.2.2.
Estimation of LOD and LOQ were determined by stan-
(this would yield a concentration of 30 g/mL). The vol-
dard deviation of response () and slope of calibration curve
ume was made upto 100mL using 13.5 mol/L sulphuric
(S). Standard deviation of Y intercepts of regression line was
acid solution. Thus the final concentrations were 45.0, 60.0
used as standard deviation. Equations 1 and 2 for LOD and
and 75 g/mL, which correspond to 50, 100 and 150 % of LOQ respectively are as follow.
the target concentration, respectively. The mean recoveries
of Azipro® 500 mg, expressed in terms of the percentage LOD= 3.3/S (1)
recovery and relative standard deviation (% RSD), were LOQ=10/S (2)
determined.

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. 3 313

2.2.4. Application of Validated Method to Assay and Disso- 3.3. Stability of Azithromycin in Solution
lution Studies of Azithromycin Dihydrate Tablet
The results from the stability study indicated that the
2.2.4.1. Assay of Azithromycin Dihydrate Tablets azithromycin dihydrate stock standard solution was stable at
room temperature for at least one week (Table 1).
The validated UV- spectrometric method was applied to
azithromycin dihydrate quantitation of the (Azipro® tablets 3.4. Method Validation
500 mg). The results were obtained by comparing the sample
spectrophotometric measurements (n = 7) with those ob- After the method development, the analytical method was
tained from the azithromycin dihydrate standard solutions (n validated according to ICH recommendations [14, 15].
= 7) at the same concentration levels. 3.4.1. Linearity and Range
2.2.4.2. Dissolution Studies The analytical curves, obtained on three consecutive days
The dissolution study of azithromycin dihydrate tablet (n = 3) by plotting the mean of absorbance at 482 nm against
(Azipro® tablets 500 mg) was carried out in 900 mL of 0.1 M the concentration, were found to be linear in the 10 to 50
phosphate buffer pH 6.0 at 75 rpm. From the dissolution g/mL range and yielded a correlation coefficient (r) of
medium 5 mL samples were withdrawn at 10, 20, 30 and 45 0.9995 (Table 2) and Fig. (4).
min respectively. From this 1 mL of solution was pipetted 3.4.2. Selectivity
out and diluted to 10 mL using 13.5 mol/L sulphuric acid.
After the dilution all the dissolution samples were heated at The spectra analysis show that the formulation excipient
50°C for 30 min and then absorbance were taken at 482 nm. of the pharmaceutical tablet product Azipro® tablets 500 mg
The study was conducted for 6 tablets and the mean % cu- did not interfere with the developed colorimetric method Fig.
mulative drug release was recorded (n = 6). (3). The spectrum showed that the placebo did not have ab-
sorbance 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
The reported methods for the determination of azithro- determining the average recoveries of samples using the
mycin dihydrate are complex, time consuming or require standard addition method. As shown in Table 3, the mean
large amount of organic solvents, some methods developed percentage recovery of Azipro® 500 mg was 99.98 % and the
on sophisticated instruments like HPLC which have higher % relative standard deviation was 0.73 %. The results were
costs. Precolumn derivatization of azithromycin dihydrate in accordance with fixed limits of 98.0 % to 102.0 %, indi-
(as it lacks chromophore) followed by HPLC analysis cating the suitability of the developed method in quantifying
makes the process tedious and expensive. In this manu- the concentration of azithromycin dihydrate in tablets. The
script, phosphate buffer pH 7.4 and sulphuric acid were accuracy value of the current method was found excellent.
chosen to obtain an inexpensive, simple and environment 3.4.4. Precision
friendly colorimetric method for the quantification of
azithromycin dihydrate in tablets. Also this method uses The precision, evaluated as the repeatability of the ana-
simple instruments. lytical method, was studied by calculating the % relative
standard deviation for the seven determination of the 15, 30,

Table 1. 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)

0h 1h 5h 6h 24 h 1 week Mean R.S.D. (%)

Responses at 0.442 0.453 0.462 0.434 0.444 0.419 0.442 0.81


482 nm
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

Table 2. 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


314 Current Pharmaceutical Analysis, 2013, Vol. 9, No. 3 Kumar et al.

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
Reference Standard Con- Total Concentration Concentration of drug Recovery R.S.D. (%) Mean Re-
(g/mL)
centration added (g/mL) of solution (g/mL) found (g/mL) (%) (n= 3) covery (%)
(Azipro®)

15 45 44.94 99.86 0.17


30 30 60 60.12 100.20 1.30 99.98
74.92
45 75 99.89 0.71

45 g/mL working standard solution performed on the same is in accordance with the Indian, British and United States
day and under the same experimental conditions. The ob- Pharmacopoeias, which permit azithromycin dihydrate in tablets
tained % R.S.D. value was less than 2.5 %. The intermediate to be in a range of 92.5 to 110.0 % of the content label claim.
precision was assessed by analyzing a sample of the pharma-
3.5.2. Dissolution Studies
ceutical formulation on 3 different days (interday precision)
and by analyzing the samples with three analysts (between – An analytical method should be developed in such a way
analysts precision). The % R.S.D. values were less than 2.5 % that it should be able to evaluate all the parameters quantita-
confirming that the method is sufficiently precise (Table 4). tively during the early product development stage till the
final product release. Dissolution studies play a major role in
3.4.5. Robustness
order to provide drug release testing during early preformu-
The robustness was found reliable, as determined by % lation studies as well as in vitro – in vivo correlation of the
R.S.D. (< 2%). It was observed that the constancy of the compound during the bioavailability studies and clinical tri-
absorbance with deliberative changes in the experimental als. Therefore dissolution studies were conducted in order to
parameter of wavelength resulted in a % R.S.D. of 0.77 and check the feasibility of this developed and validated method
change in pH resulted in a % R.S.D. of 0.68 (Table 5). The for quantification of dissolution samples. The mean % cumu-
minor changes that occurred during the analysis did not af- lative drug release was found to be 99.3 % after 45 min. The
fect the absorbance intensity of the samples. dissolution profile of Azipro® tablets (500 mg) is shown in
Fig. (5). This shows that the developed method can be suc-
3.4.6. Limits of Detection and Quantification
cessfully applied to dissolution studies.
LOD and LOQ values were found to be 0.66 ± 0.065 and
UV, UV – VIS and derivative spectrophotometry are
2.02 ± 0.061 g/mL, respectively, for Azipro® tablets (500
broadly used techniques to quantify antibiotics and other drugs
mg). These results demonstrate that the analyses were being
[16-24] because they are cost effective, simple and do not re-
performed in a region above the quantitation limit value.
quire time consuming sample preparation compared to other
3.5.1. Assay of Azithromycin Dihydrate Tablets techniques [25]. Moreover, UV- Visible spectrophotometry
produces very low amount of residues and solvents, which is
The validated method was applied to the determination of
an important ecological aspect as per regulatory point of view
azithromycin dihydrate in tablets. Three samples of Azipro®
and currently discussed in routine laboratory analysis. Because
tablets (500 mg) were analyzed. The results, expressed as the
of these reasons and the careful validation of this method, this
percentage drug as related to the content label claim, are
technique can be recommended for the routine laboratory
shown in Table 6. The results showed that the percentage drug
analysis of the antibiotic azithromycin dihydrate in tablets.
Colorimetric Estimation of Azithromycin Current Pharmaceutical Analysis, 2013, Vol. 9, No. 3 315

Table 4. Method Precision Results for Azithromycin Dihydrate Tablets

Analytical Responses

Concentration R.S.D.
1 2 3 4 5 6 7
(g/mL) (%)

15 0.243 0.234 0.238 0.247 0.238 0.240 0.245 1.88


Repeatability 30 0.433 0.443 1.11
0.438 0.440 0.435 0.434 0.446

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


Day 1 30 0.452 0.447 1.36
0.438 0.442 0.445 0.435 0.449

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


Day 2 30 0.443 0.437 1.21
0.435 0.440 0.448 0.432 0.441
Intermediate Pre-
45 0.543 0.541 0.555 0.547 0.549 0.551 0.547 0.86
cision
Day 3 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

Analyst A 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

Analyst B 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

Analyst C 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

Table 5. Robustness Test Results

Sample pH Wavelength (nm) Content (mg)a Content (%)a R.S.D. (%)

7.2 506.51 101.30

7.4 NA 498.65 99.73 0.68


Azipro 500 mg tablet
7.6 503.46 100.69

*NA 480 505.51 101.10

482 501.12 100.22 0.77

484 497.89 99.57


a
mean of seven replicates ; *NA = Not Applicable
316 Current Pharmaceutical Analysis, 2013, Vol. 9, No. 3 Kumar et al.

Table 6. Assay of Azithromycin Tablet (Azipro® 500mg Tablets) Samples A, B and C

Sample Content (mg)a Content (%)a R.S.D. (%)

1 523.16 104.63

2 518.12 103.62 1.58

3 534.36 106.87

Fig. (5). Mean % cumulative drug release profile of Azipro 500 mg Tablets (n = 6).

4. CONCLUSION [4] Carmen, L.; Rosa, C.; Ramon, C.; Mercè, G.; Dolors, P. M. Deter-
mination of macrolide antibiotics by liquid chromatography. J.
The validated analytical method for quantitative determi- Chromatogr. A., 2001, 910(2), 285-290.
nation of azithromycin dihydrate in tablets has the advantage [5] Kees, S.; Spangler, S.; Wellenhofer, M. Determination of macrol-
ides in biological matrices by high-performance liquid chromatog-
of simplicity, speed, low cost conditions and a lack of pollut- raphy with electrochemical detection. J. Chromatogr. A., 1998,
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highly satisfactory, including linearity, accuracy, precision, [6] Nigovic, B.; Imunic, B. S. Voltammetric assay of azithromycin in
selectivity, robustness and adequate detection and quantita- pharmaceutical dosage forms. J. Pharm.Biomed. Anal., 2003, 32,
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[7] Taninaka, C.; Ohtani, H.; Hanada, E.; Kotaki, H.; Sato, H.; Iga, T.
for routine quality control of azithromycin dihydrate by the Determination of erythromycin, clarithromycin, roxithromycin, and
pharmaceutical industries and quality control laboratories. azithromycin in plasma by high performance liquid chromatogra-
phy with amperometric detection. J. Chromatogr. B: Biomed. Sci.
CONFLICT OF INTEREST Appl., 2000, 738(2), 405-411.
[8] Tod, M.; Biarez, O.; Nicilas, P.; Petijean, O. Sensitive determina-
The authors confirm that this article content has no con- tion of josamycin and rokitamycin in plasma by high-performance
flict of interest. liquid chromatography with fluorescence detection. J. Chroma-
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[9] Kanfer, I.; Skinner, M. F.; Walker, R. B. Analysis of macrolide
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[10] Kamau, F. N.; Chepkwony, H. K.; Ngugi, J. K.; Debremaeker, D.;
The authors are grateful to shree Ashok Mittal, Honour- Roets, E.; Hoogmartens, J. Isocratic liquid chromatographic
able Chancellor, Lovely Professional University, Punjab, method for the analysis of azithromycin and its structurally related
India for providing facilities to carry out the aforementioned substances in bulk samples. J. Chromatogr. Sci., 2002, 40(9), 529-
research. We also owe our thanks to Ranbaxy Laboratories, 533.
Gurgaon, India, for providing the gift samples of reference [11] Horie, M.; Saito, K.; Ishii, R.; Yoshida, T.; Haramaki, Y.; Naka-
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Received: November 01, 2012 Revised: January 31, 2013 Accepted: February 06, 2013