New
Spectrophotometric Method for Assay of Amphotericin B
in Bulk and Its Pharmaceutical Formulations
Mohan Krishna Lokireddy1*, Jayachandra
Reddy P2, M. Srinivasa Murthy1
1Vignan
Institute of Pharmaceutical Sciences, Deshmukhi, Nalgonda, Andhrapradesh, India.
2Krishna
Teja Pharmacy College, Tirupati,
India.
*Corresponding Author E-mail:
mohanlokireddy@gmail.com
ABSTRACT:
Two simple
and sensitive visible spectrophotometric methods (A and B) have been developed
for the determination of Amphotericin B (AMP) in bulk
and pharmaceutical formulations. These methods are based on the Method A was
developed based on the
formation of a colored condensation reaction with p-dimethyl amino cinnamaldehyde (PDAC) under acidic conditions (λmax 490 nm), and method B based on the oxidative
coupling reaction with DCQC (2,6-dichloroquinone N-chlorimide)
(λmax
530nm) . Beer’s law limits, precision and accuracy of these methods are
checked by the UV reference method. The results obtained are reproducible and
are statistically validated and so found to be suitable for the assay of in
bulk and pharmaceutical formulations.
INTRODUCTION:
Amphotericin B is a polyene, antifungal antibiotic produced
from a strain
of Streptomyces nodosus.
Amphotericin B is designated chemically as [1R-(1R*,
3S*, 5R*, 6R*, 9R*, 11R*, 15S*, 16R*, 17R*, 18S*, 19E, 21E, 23E, 25E, 27E, 29E,
31E, 33R*, 35S*, 36R*, 37S*)]-33-[(3-Amino-3, 6-dideoxy-β-D-mannopyranosyl)
oxy]-1,3,5,6,9,11,17,37-octahydroxy-15,16,18-trimethyl-13-oxo-14,39-dioxabicy-clo[33.3.1]
nonatriaconta-19, 21, 23, 25, 27, 29, 31-heptaene-36-carboxylic acid1.
It has a
molecular weight of 924.09 and a molecular formula of C47H73NO17.
The structural formula is:
Chemical
Structure of Amphotericin B
A number of methods such as HPLC were reported for the
estimation of AMP3-6. Literature survey revealed that few visible
spectrophotometric methods are reported for its quantitative determination in
bulk drug and pharmaceutical formulations2.
The aim of the present work is to provide simple and
sensitive visible spectrophotometric methods for the estimation of AMP, which
are basic in bulk and formulations. The efforts in this accord resulted in
developing the present methods. Hence
the authors have made
an attempt in this direction and
succeeded in developing three
visible spectrophotometric
methods by exploiting
different structural features of
the drug molecule such as the presence of an aromatic primary amino
group (condensation reaction
in method A or
oxidation followed by complex
formation in method
B).
A Systronics model` 117
UV-Visible Spectrophotometer with 1 cm matched quartz cells was used for
spectral and absorbance measurements in the UV and visible regions
respectively. All the reagents and
chemicals were of analytical grade.
A 1mg/ml solution
was prepared by dissolving 100 mg of pure AMP in 100 mL
of distilled water and this stock solution was diluted stepwise with distilled
water to obtain the working standard solutions of concentrations 100 µg/mL for method A and B respectively.
PDAC solution (BDH,
0.4%, 2.63x10-3M)
|
: |
Prepared by
dissolving 400 mg of PDAC in 100 ml of CH3OH. |
H2SO4 (Merck, Conc.)
|
: |
Used as it is. |
For method M25
|
|
|
DCQC solution (Loba,
0.2%, 9.52x10-3M)
|
: |
Prepared by
dissolving 200 mg of DCQC in 100 ml of isopropanol |
|
Buffer solution (pH 9.4) |
: |
Prepared by mixing 250 ml of 0.2M boric acid with 160
ml of 0.2M NaOH solution and diluted to 1000 ml
with distilled water and pH was adjusted to 9.4 |
For pharmaceutical formulations:
The injection powder equivalent to 100mg of (AMP) was
accurately weighed and dissolved in chloroform and filtered for methods A and
B, the filtrate was evaporated to dryness and the residue was dissolved in 100 mL of distilled water to achieve a concentration of
1mg/1mL. From which suitable dilutions
were performed for methods A and B as mentioned above.
Method A: To each one of 10 ml calibrated tubes, aliquots (0.5 –
2.5 ml, 200 mg/ml), of methanolic standard AMP solution, 2.0 ml of PDAC and 3.0 ml
of conc. sulphuric acid were added successively and the total volume in each
tube was brought to 9 ml by addition of methanol and placed in hot water bath for 25
min. Then the flasks were cooled and made up to the mark with methanol and the absorbances were measured after 5 min. at 420 nm against a
reagent blank prepared in a similar way.
The amount of drug in a sample was computed from Beer’s law plot.
Method B: In to a series of
calibrated tubes, aliquots of standard AMP solution (0.5-2.5 ml, 400mg/ml), 5.0 ml of buffer 9.4, 2.0 ml if DCQC solution were delivered. Then the tubes
were kept in a boiling water bath for 10 min. The solutions were cooled to room
temperature and the volume in each tube was made upto
10 ml with distilled water. The absorbances were
measured at 610 nm against a similar reagent blank. The amount of AMP was
estimated from its calibrations curve.
RESULTS AND
DISCUSSION:
The optical
characteristics such as Beer’s law limits, molar absorptivity
and Sandell’s sensitivity ford these methods are
given in Table-1. The precision of each method was found by measuring absorbances of six replicate samples containing known
amounts of drug and the results obtained are incorporated in Table-1. Regression analysis using the method of least
squares was made to evaluate the slope (b), intercept (a) and correlation
coefficient (R) for each system (Table-1). The relative standard deviation and
% range of error at 95% confidence level are also given in Table-1. The
accuracy of each method was ascertained by comparing the results by proposed
and reference methods (UV) statistically (Table 2). This comparison shows that there is no
significant difference between the results of proposed methods and those of the
reference ones. As an additional check
of accuracy of the proposed methods, recovery experiments were performed by
adding a fixed amount of the drug to the preanalysed
formulations. The amount of drug found
and the % recovery was calculated in
the usual way. The similarity of the results is and
obvious evidence that during the application of these methods, the excepients that are usually present in pharmaceutical
formulations do not interfere in the assay of proposed methods.
Table-1 Optical
Characteristics And Precision
|
Parameters |
Method A |
Method B |
|
λ max (nm) |
490 |
530 |
|
Beer’s law limit (μg/mL) |
10-60 |
20-120 |
|
Sandell’s sensitivity (μg/cm2/0.001
abs. unit) |
0.097 |
0.173 |
|
Molar absorptivity (L mole-1cm-1) |
9.495X103 |
5.337X103 |
|
Regression equation (Y*) |
||
|
Slope (b) |
0.0103 |
0.0057 |
|
Intercept (a) |
-0.0017 |
-0.0005 |
|
Correlation coefficient
(r) |
0.9999 |
0.9999 |
|
% RSD |
0.4118 |
0.3994 |
|
% Range of error (0.05 level confidence
limit) |
0.344 |
0.334 |
_
Y=a+bX where X is the concentration of AMP in μg/mL and Y is the
absorbance at the respective λ max.
CONCLUSION:
The proposed methods
are applicable for the assay of drug (AMP) and have an advantage of wider
range, under beer’s law limits. The proposed methods are simple, selective and
reproducible and can be used in the routine determinations of AMP in bulk samples
and formulations with reasonable precession and accuracy.
TABLE-2 Assay and
recovery of amphotericin B in pharmaceutical Formulations
|
Sample Δ (Injection Powder) |
Labeled Amount (mg) |
Amount found by reference Method |
Amount obtained (mg) |
Proposed method* |
% Recovery by proposed methods** |
|
|
A |
B |
A |
B |
|||
|
AMP A |
100 |
99.7 |
99.8 |
99.6 |
99.7 |
99.6 |
|
AMP
B |
100 |
98.6 |
98.2 |
98.4 |
99.5 |
99.6 |
|
AMP
C |
100 |
98.32 |
99.2 |
98.64 |
99.6 |
99.3 |
|
AMP
D |
100 |
99.2 |
98.86 |
99.32 |
99.43 |
99.66 |
*Average
± standard deviation of six determinations.
**After adding 3 different amounts of the pure labelled to the pharmaceutical formulation, each value is
an average of 3 determinations.
Δ Powder from different
manufactures
REFERENCES:
1.
The Merck Index,
12th Edn., Merck and Co Inc, New York, 1996.
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Nageswara Rao. L et al, A new colorimetric method for the estimation
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Loredana Elena Vijan
et al, Characterization of the
interaction of amphotericin B with cholesteryl trifluoromethylphenyl-carbamate
by UV-visible spectroscopy, Revista de Chimie (Bucharest, Romania), 59(3), 2008, 297
4.
Ilona. Gruda et al,
Application of differential spectra in
the ultraviolet-visible region to study the formation of amphotericin
B-sterol complexes, Biochimica et Biophysica Acta, Biomembranes, 602(2), 1980, 260.
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43(17),2008, 1352-1353.
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Received on 30.10.2013 Modified
on 16.12.2013
Accepted on 26.12.2013 ©AandV Publications All right reserved
Res. J. Pharm. Dosage Form. and Tech. 6(1): Jan.-Mar. 2014; Page 15-17