Visible Extractive Spectrophotometric Estimation of
Teicoplanin in Pharmaceutical Formulations
P. Janaki Pathi1*, P. Ravendra
Reddy2 and N. Appala Raju3
1Analytical Chemistry Department, Vishnu Chemicals
Limited, Jeedimetla, Hyderabad.
1,2Department of Chemistry, Sri Krishnadevaraya
University, Anantapur-515 003
3Department of
Pharmaceutical Chemistry, Sultan-Ul-Uloom College of Pharmacy, Mount Pleasant,
Road # 3, Banjara Hills, Hyderabad-500 034.
*Corresponding Author E-mail: pjp02002@yahoo.com, pjp02002@rediffmail.com
ABSTRACT:
Three simple, accurate, rapid and
sensitive methods (A, B and C) have been developed for the estimation of
Teicoplanin in pharmaceutical dosage form. The Method A is based on reaction of
Teicoplanin with ferric chloride and 1,10-phenanthroline to form a blood red
colored chromogen. The Method B is based on reaction of Teicoplanin with ferric
chloride 2,2’ bipyridyl to form blood colored chromogen. The Method C is based
on the reduction of Ferric ions of the reagent Ferric chloride to Ferrous ions
by the drug, which further in the presence of potassium ferricyanide as
oxidizing agent produces blue colored chromogen measured at 700 nm, against
reagent blank. These Methods exhibit maximum absorption at 510 nm, 520 nm and
720 nm respectively and obey the Beer’s law in the concentration range of 5-30
µg/mL, 5-50 µg/mL and 1-10 µg/mL respectively. The Methods have been
statistically evaluated and were found to be precise and accurate. The proposed
methods are economical and sensitive for the estimation of Teicoplanin in its
formulations.
KEYWORDS: UV-Visible Spectrophotometry, Teicoplanin, Ferric
chloride, 1, 10-Phenanthroline, 2, 2’ bipyridyl and Potassium ferricyanide.
INTRODUCTION:
Teicoplanin1 is actually a mixture of several compounds, five major
(named teicoplanin A2-1 through A2-5) and
four minor (named teicoplanin RS-1 through RS-4).
All teicoplanins share a same glycopeptide core, termed teicoplanin A3-1
— a fused ring structure to which two carbohydrates (mannose and N-acetylglucosamine)
are attached. The major and minor components also contain a third carbohydrate
moiety — β-D-glucos- amine and differ only by the length and conformation
of a side chain attached to it. It is chemically Ristomycin A
34-O-[2-(acetylamino)-2-deoxy-.beta.-D-glucopyranosyl]-22,31-dichloro-7-demethyl-64-O-demethyl-19-deoxy-56-O-[2-deoxy-2-[(8-methyl-1-oxononyl)
amino]-.beta.-D-glucopyranos syl]- 42-O-.alpha.-D-mannopyranosyl. The structures of the teicoplanin core and
the side chains which characterize the five major teicoplanin compounds are
shown in fig-1.
Survey of literature reveals that the drug is
determined by using HPLC2-6 and no spectrophotometric methods has
been reported. The present study describes simple, sensitive, accurate, rapid
and economical Spectrophotometric Methods A, B and C for the estimation of
Teicoplanin in its formulations.
EXPERIMENTAL:
Instrument: Elico Ultraviolet-Visible double beam spectrophotometer
SL-164 with 1 cm matched quartz cells was used for all spectral measurements.
Reagents:
All the chemicals used were of analytical reagent
grade.
1.
1,10-Phenanthroline :0.1
M in distilled water.
2.
Ferric chloride
(hexahydrate): 0.03 M in distilled water.
3.
2,2’-bipyridyl: 0.1 M in
distilled water.
4.
Ferric Chloride
hexahydrate : 0.3% w/v in distilled water
5.
Potassium ferricyanide:
0.2% w/v in distilled water.
Fig-1Chemical
structure of Teicoplanin
PROCEDURE:
Standard stock solution was prepared by dissolving 100
mg of Teicoplanin in 100 mL of distilled water to get a concentration of 1000
µg/mL. This was further diluted to get the working standard solution of 50
µg/mL, 100 µg/mL and 20 µg/mL for Method A, Method B and Method C respectively.
ASSAY PROCEDURE:
Method A: Aliquots of standard drug solution of Teicoplanin 1-6.0 mL (50 µg/mL)
were taken and transferred into series of 10 mL volumetric flasks. To each
flask 2 mL of Ferric chloride and 2 mL of 1,10-Phenanthroline (0.1M) were
added. The flask were allowed to stand in water bath at 700 c for 15
mins and cooled to room temperature and the solutions were made upto 10 mL
with distilled water. The absorbance of the red colored chromogen was measured
at 510 nm against reagent blank and a calibration curve was constructed. The
absorbance of the sample solution was measured, and the amount of Teicoplanin
was determined by referring to the calibration curve.
Method B: Aliquots of standard drug solution of Teicoplanin 0.5 – 5.0 mL (100
µg/mL) were taken and transferred into series of 10 mL volumetric flasks. To
each flask 2 mL of Ferric chloride and 2 mL of 2,2’-bipyridyl (0.1M) were
added. The flasks were allowed to stand in water bath at 700 c for
15 mins and cooled to room temperature and the solutions were made up to 10 mL
with distilled water. The absorbance of the red colored chromogen was measured
at 520 nm against reagent blank and a calibration curve was constructed. The
absorbance of the sample solution was measured, and the amount of Teicoplanin
was determined by referring to the calibration curve.
Method C: Aliquots of standard drug solution of Teicoplanin 0.5 – 5.0 mL (20
µg/mL) were taken and transferred into series of 10mL volumetric flask. To each
flask 1 mL of Ferric chloride (0.3% w/v) and 0.5 mL of Potassium ferricyanide
(0.2% w/v) were added and thoroughly shaken and set aside for 5 mins. The
volume in each flask was made upto 10 mL with distilled water. The absorbances
o the solutions were measured at 720 nm against reagent blank, within 30 mins
and the calibration curve was plotted. Similarly the absorbance of the sample
solution was measured, and the amount of Teicoplanin was determined by
referring to the calibration curve.
The methods were extended for the determination of
Teicoplanin in powder for injection (Ticocin® 400 mg Powder for injection) was
chosen. The weight of the sterile powder for injection equivalent to 100 mg of
Teicoplanin was accurately weighed and transferred to 100 mL volumetric flask.
Sterile powder for injection equivalent to 100 mg of Teicoplanin was dissolved
in 50 mL of distilled water, sonicated for 15 mins, filtered and washed with
distilled water. The filtrate and washings were combined and the final volume
was made to 100 mL with distilled water. The solution was suitably diluted and
analyzed as given under the assay procedure for bulk samples. The results are
represented in Table II. None of the excipients usually employed in the
formulation of powder for injection interfered in the analysis of Teicoplanin,
by the proposed methods.
Recovery Studies:
To ensure the accuracy and reproducibility of the
results obtained, adding known amounts of pure drug to the previously analyzed
formulated samples and these samples were reanalyzed by the proposed method and
also performed recovery experiments. The percentage recoveries thus obtained
were given in Table II.
RESULTS AND DISCUSSIONS:
In the present study, the Method A and B are based on
the reduction of Ferric chloride to ferrous form by the drug, which forms
complex with 1, 10-Phenanthroline and 2, 2’- bipyridyl to yield blood red
colored chromogen. The colored chromogens were stable for more than 3 hrs and
exhibited maximum absorption at 510 nm and 520 nm respectively. In the Method
C, the estimation is based on the reduction of ferric ions to ferrous ions by
the drug, which further in the presence of oxidizing agent like potassium
ferricyanide produces blue colored chromogen having a maximum absorbance at 720
nm. The blue colored chromogen was stable for 30 min. at room temperature.
Reaction mechanisms involved in the formation of colored species was shown in
fig—2. The conditions required for the formation of colored complexes were
optimized. Statistical analysis was carried out and the results of which were
satisfactory. The optical characteristics such as absorption maxima, Beer’s law
limits, molar absorptivity and Sand ell’s sensitivity are presented in
Table I. The regression analysis using the method of least squares was made for
slope (m), intercept (b) and correlation obtained from different concentrations
and the results are summarized in Table I.
The
reproducibility and precision of the methods are very good as shown by the low
values of coefficient of variance (CV). Recovery studies were close to 100 %
that indicates the accuracy and precision of the proposed methods, and also
indicates non-interferences from the formulation excipients. All the validated
parameters are summarized in Table II.
In conclusion, the proposed methods are simple,
sensitive, accurate and economical for the routine estimation of Teicoplanin in
bulk and in its formulations.
ACKNOWLEDGEMENT:
The authors are grateful to for the supply of
Teicoplanin as a gift sample and to the Cipla Pharmaceuticals, and to the
Management, Vishnu Chemicals, Jeedimetla, Hyderabad, for providing the
necessary facilities to carry out the research work.
Reaction Schemes involving in the formation of
Chromogens:
Proposed Scheme-1 for Mehtod-A (Fig.-2)
Step- I: When treated with known excess of Fe(III) drug undergoes oxidation
giving oxidation products of drug inclusive of reduced form of Fe(III) i.e
Fe(II) besides unreacted Fe(III). Fe(II) has a tendency to give colored complex
on treatment with 1,10-phenanthroline.
Step II: The next step concerns with the estimation of Fe (II) with 1,
10-phenanthroline which forms colored tris complex (Ferroin).
Scheme-2: Proposed Scheme for Method-B: Step-1:
(Fig.-3)
When treated with known excess of Fe(III) drug
undergoes oxidation giving oxidation products of drug inclusive of reduced
form of Fe(III) i.e Fe(II) besides unreacted Fe(III). Fe(II) has a tendency to
give colored complex on treatment with 2.2”-Bipyridyl.
Table I: ASSAY
OF TEICOPLANIN IN STERILE POWDER FOR INJECTION.
|
Sample
No.
|
Labeled Amount
in [mg]
|
% Obtained* by proposed method (mg)
|
** % Recovery
by the Proposed method
|
|
Method-A.
|
Method-B
|
Method-C
|
Method -A
|
Method- B
|
Method- C
|
|
1
|
400
|
98.7
|
98.4
|
98.6
|
97.7
|
98.4
|
98.6
|
|
2
|
400
|
99.2
|
98.8
|
98.2
|
98.8
|
98.7
|
98.6
|
|
3
|
400
|
99.1
|
98.9
|
98.8
|
99.9
|
99.2
|
99.5
|
*Average of three
determinations, ** After spiking the sample
Table: II - OPTICAL CHARACTERISTICS AND PRECISION
DATA
|
Parameters
|
Method A
|
Method B
|
Method C
|
|
l max (nm)
|
510
|
520
|
720
|
|
Beer’s law limits
mcg/ml
|
5-30
|
5-50
|
1-10
|
|
Molar
absorptivity (l/mol.cm)
|
2.043x104
|
1.515x104
|
1.872x104
|
|
Sand ell’s
sensitivity
(micrograms/cm2/0.001absorbance
unit)
|
0.092
|
0.124
|
0.1004
|
|
Regression
Equation* (Y)
Slope (m)
Intercept (c)
|
0.01087
-0.0002
|
0.00812
-0.0007
|
0.00901
0.00107
|
|
Correlation
Coefficient(r)
|
0.9999
|
0.9999
|
0.9999
|
|
Precision
(%Relative Standard Deviation)
|
0.165
|
0.248
|
0.562
|
|
Standard error of
estimate
|
0.004
|
0.0213
|
0.0345
|
*Y=mx+c, where X is
the concentration in micrograms/ml and Y is absorbance unit.
Schem-3: Proposed Scheme for Method-C:
The Chemistry involved in the color development is, the
drug reduces ferric form of Iron to ferrous form of Iron (Fe+3 to
Fe+2) which in turn couples with reagents having divalent ions like
potassium ferricyanide to form bluish green colored potassium ferro ferrous
complex.
3 Fe+2 + 2 [Fe(CN)6]-3
Fe3[ Fe(CN)6]2.
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