DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC
METHOD FOR ESTIMATION OF ERYTHROMYCIN IN BULK DRUG AND
PHARMACEUTICAL FORMULATION
Anindya Bagchi*, Prosenjit Mukherjee, Harsha Bhardwaj, Jyoti gupta, Alok Semwal**
Netaji Subhas Chandra Bose Institute of pharmacy, Chakdaha,West Bengal
**Himachal institute of Pharmacy, Paonta Sahib, Himachal Pradesh
*Corrosponding author:
Anindya Bagchi
Assistant Prof.
Department of Pharmaceutical Chemistry,
Himachal Institute of Pharmacy, Paonta Sahib (H.P)
Email id: tajuanindya@gmail.com
Mobile No: +91-9318670978
Abstract: An Uv spectrophotometric method for the quantitative determination of
Erythromycin, a macrolide antibiotics in tablets was developed in present work. The
parameters linearity, accuracy, precision, limit of detection, limit of quantisation, range were
studied according to International Conference on Harmonization guidelines. UV
spectroscopic determination was carried out at an absorption maximum of 285 nm using
methanol as solvent. Results of the analyses were validated statistically and by recovery
studies. The proposed method is simple, rapid, precise and accurate and can be used for the
reliable quantisation of Erythromycin in pharmaceutical formulation. In the UV spectroscopic
method linearity over the concentration range of Erythromycin was found to be 1-9 ug/ml
with a correlation coefficient 0.9873.
Keywords: Erythromycin, UV spectrophotometric method, linearity, Macrolide Antibiotics,
ICH.
INTRODUCTION:
Erythromycin, produced by Saccharopolyspora erythreas (formerly known as Streptomyces
erythraeus) (1), is a macrolide antibiotic consisting of a 14 member ring, a ketone group, two
glycosidic bonds and a dimethyl amino group (2,3,4).
Structure of Erythromycin
It is bacteriostatic at low but bacteriocidal at high concentration. The drug targets at the
ribosome and inhibits the protein synthesis of Gram positive bacteria such as Mycoplasma
and Chlamydia. (5,6).
Erythromycin is used for treatment of several infection diseases and in patients allergic to the
penicillins. Erythromycin easily degrades in acidic conditions giving inactive compounds,
8,9-anhydro-6,9-hemiketal and erythromycin-6,9,12-spiroketal (7). To increase its acid
stability and bioavailability, erythromycin is available in several forms including estolate,
ethyl succinate and stearate. Several methods have been proposed for the analysis of
erythromycin. Dehouck et al.(8) reported the HPLC analysis of erythromycin and benzoyl
peroxide in acne gel on a Xterra RP18 column using acetonitrile, 0.2 M di- potassium
hydrogen phosphate and water (35:5:60, v/v) as a mobile phase and a detection wavelength at
215 nm.
Leal et al.(9) analyzed erythromycin and other six macrolide antibiotics by HPLC using a
C18 column, a mobile phase consisting of phosphate buffer (pH 2.5) and acetonitrile and
monitored the wavelengths in a range of 204-287 nm. HPLC-MS (mass spectrometry) was
also employed for the analysis of seven macrolide antibiotic residues in fish with a detection
limit of 0.01 μg/mL (10) Hilton and co-workers (11) used HPLC-electrospray MS in
combination with solid phase extraction (SPE) for the detection of several antibiotics
contaminated in water including erythromycin. But because of a simple Uv analysis it can be
possible to avoid such expensive experimental designing which has been tried to established
here.
MATERIALS AND METHODS:
CHEMICALS AND REAGENTS:
Methanol and dibasic phosphate buffer pH 8.0 (1:1) was used throughout UV
spectrophotometric method development and validation. Erythromycin bulk powder was
kindly gifted by Nextwave Pharma, India. The commercially available tablets of three
different of Erythromycin were procured from local market labeled content 50 mg
Erythromycin. Methanol (AR Grade, S. D. Fine Chemicals Ltd., Mumbai, India) and
Whatman filter paper no. 1 (Whatman International Ltd., England) were used in the study.
INSTRUMENTATION:
A shimadzu model 1800 (Japan) double beam UV/Visible spectrophotometer with spectral
width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cell was
used to measure absorbance of all the solutions. Spectra were automatically obtained by UVProbe system software. A Sartorius CP224S analytical balance (Gottingen, Germany), an
ultrasonic bath (Frontline FS 4, Mumbai, India).
PREPARATION OF STANDARD STOCK SOLUTION:
Erythromycin was accurately weighed (equivalent to 100 mg Erythromycin) in 100 ml
volumetric flask and diluent (100 ml) was added, shaken till dissolved and volume was made
up to the mark with diluent and mixed well (1 mg/ml).From that solution 1ml was taken and
dissolved upto 100 ml and that will give 10ug/ml concentration of solution.
PREPARATION OF WORKING STANDARD SOLUTION:
Erythromycin working standard solution was prepared by diluting standard stock solution to
produce the concentration range of (1-9) ug/ml.
VALIDATION OF THE PROPOSED METHODS:
The methods were validated with respect to linearity, accuracy, precision (method precision,
intermediate precision), limit of detection and limit of quantification according to the ICH
guidelines (12).
LINEARITY AND RANGE:
The linearity was determined by analyzing 9 independent levels of calibration curve in the
range of 1-9μg/ml. Absorbance of each solution against methanol was recorded at 285 nm
The calibration curve of absorbance vs. concentration was plotted and correlation co-efficient
and regression line equation for erythromycin were determined.
PRECISION:
METHOD PRECISION (% REPEATABILITY):
The precision of the instrument was checked by repeated scanning and measurement of the
absorbance of 8 μg/ml erythromycin standard solution (n = 6) without changing the
parameters for the method. The repeatability was expressed in terms of relative standard
deviation (% RSD).
INTERMEDIATE PRECISION (REPRODUCIBILITY):
The intraday and interday precision of the proposed methods were performed by analyzing
the corresponding responses 3 times on the same day and on 3 different days over a period of
1 week for 8different concentrations of standard solutions of erythromycin (2, 5 ,8 μg/ml).
The results were reported in terms of relative standard deviation (% RSD).
SYSTEM PRECISION:
The precision of the system was checked by repeated scanning and measurement of the
absorbance of 2 μg/ml, 5ug//ml, 8ug/ml erythromycin standard solution (n = 6) without
changing the parameters for the method. The repeatability was expressed in terms of relative
standard deviation (% RSD).
ACCURACY (% RECOVERY):
The accuracy of the methods was performed by calculating recovery of erythromycin by the
standard addition method. Known amounts of standard solutions of erythromycin were added
at 80%, 100% and 120% levels to pre quantified erythromycin sample solutions of 8 μg/ml.
At each level of the amount 3 determinations were performed. The amount of erythromycin
was estimated by applying obtained values to the respective regression equations and %
recoveries were computed.
LIMIT OF DETECTION (LOD) AND LIMIT OF QUANTIFICATION (LOQ):
The LOD and LOQ were estimated from the set of 3 calibration curves used to determine
method linearity.
LOD = 3.3 × σ/S
LOQ = 10 × σ/S
Where, σ = the standard deviation of the response and S = slope of the calibration curve.
DETERMINATION OF ERYTHROMYCIN IN PHARMACEUTICAL
FORMULATION (TABLETS):
Twenty tablets of each brand were accurately weighed and average weight was determined.
The tablets were powdered in glass mortar. A weight of the powder equivalent to 100 mg of
erythromycin was transferred to a 100 ml volumetric flask containing 50 ml methanol: buffer
(1:1) and the mixture was sonicated for 30 min then diluted to 100 ml with methanol:
Buffer(1:1) (1000 ug/ml). The solution was filtered and 1 ml of filtered solution was diluted
hundredfold to furnish a concentration of 10μg/ml. From that solution the working sample
dilute solution has been prepared.
RESULT AND DISCUSSION:
In experiment simple UV spectrophotometric instrument was used. From this the simple UV
spectrum of Erythromycin in methanol: buffer (1:1) was obtained which exhibits absorption
maxima (λ max) at 285 nm (Figure 1). The calibration curve was linear in concentration
range of 1-9 μg/ml. The proposed methods were found to be simple, sensitive, rapid,
accurate, precise and economic for the routine analysis of erythromycin in pharmaceutical
formulations. The linearity ranges was found to be 1-9 μg/ml for this method. Accuracy was
determined by calculating the recovery, and the mean was determined (Table 1). Precision
was calculated as repeatability (relative standard deviation) and intra and inter day variation
(% RSD) for erythromycin. LOD value for erythromycin was found to be 0.96 ug/ml and
LOQ value is 2.92 μg/ml respectively indicates sensitivity of the proposed method. The
method was successfully used to determine the amounts of erythromycin present in tablets.
The results obtained are in good agreement with the corresponding labelled amount (Table 2).
By observing the validation parameters, the methods were found to be sensitive, accurate and
precise. Hence the methods can be employed for the routine analysis of erythromycin in
tablet formulations. Regression analysis data and summary of validation Parameters values
for the proposed method is given on (Table 3).
Fig: 1- Uv Spectrum of Erythromycin Stearate
TABLE 1: RECOVERY DATA OF PROPOSED METHOD:
Method
employed
LEVEL
AMOUNT
TAKEN(ug/ml)
AMOUNT
ADDED
%RECOVERY±S.D
I
8
80
99.39+0.14
II
8
100
98.11+1.02
III
8
120
98.97+0.34
TABLE 2: RESULTS OF ANALYSIS OF TABLET FORMULATIONS:
Tablet
Level Claim
Parameters
Amount Found
Brand A
50 mg
Mean
98.28
S.D
0.52
Mean
98.49
S.D
0.72
Mean
98.48
S.D
0.93
Brand B
Brand C
50 mg
50 mg
TABLE 3: REGRESSION ANALYSIS DATA AND SUMMARY OF VALIDATION
PARAMETERS FOR THE PROPOSED METHODS:
Parameters
Value
λmax
285 nm
Beer’s-Lambert’s range (μg/ml )
(1-9) ug/ml
Regression equation
Y=0.0109x+0.3469
y =mx + c
Slope (m)
0. 0.0109
Intercept (c)
0.3469
Correlation coefficient (r2)
0.9873
Recovery + S. D.
99.59 + 0.13
(n = 3)
LOD (μg/ml )
0.96
LOQ (μg/ml )
2.92
Intermediate Precision(%RSD)
Interday (n = 3)
0.7661 - 1.7811
Intraday (n = 3)
0.1967 - 1.5121
CONCLUSION:
The proposed spectrophotometric method was found to be, simple, sensitive, accurate and
precise for determination of erythromycin in tablet dosage form. Unlike other researchers,
which reported the use of advanced techniques (e.g. HPLC, HPTLC and CE), we described a
simple spectrophotometric method for the determination of erythromycin. The proposed
method will not replace the methods recommended in USP or BP or other published methods.
But, it will serve as a rapid, convenient and inexpensive method. Hence it can be
conveniently adopted for routine quality analysis of the drug in tablets.
ACKNOWLEDGEMENT:
The authors are gratefully acknowledging Next wave Pharma, India for providing the gift
samples of deflazacort. Authors are also thankful to Himachal Institute of Pharmacy, Paonta
Sahib, Himachal pradesh for providing necessary facilities for the research work.
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