Download Development and Validation of Spectrophotometric Method for the

Chem Sci Trans., 2013, 2(4), 1427-1433
DOI:10.7598/cst2013.547
Chemical Science Transactions
ISSN/E-ISSN: 2278-3458/2278-3318
RESEARCH ARTICLE
Development and Validation of Spectrophotometric
Method for the Estimation of Chondroitin Sulfate in
Bulk Drug and Pharmaceutical Formulations
P. L. SOMASHEKARa*, K. P. SATHISHb, CHANDRASHEKAR JAVALIa,
A. S. TRIPATHYc and R. B. KOTANALd
a
Govt College of Pharmacy, Bangalore,Karnataka - 560 027, India
Micro Labs, Bangalore, Karnataka - 560 099, India
c
Al-Ameen College of Pharmacy, Bangalore, Karnataka - 560 027, India
d
BLDEA College of Pharmacy, Bijapur, Karnataka - 586 103, India
b
[email protected]
Received 2 February 2013 / Accepted 16 February 2013
Abstract: A simple and sensitive spectrophotometric method is described for the determination of
chondroitin sulfate in bulk drug and pharmaceuticals. The proposed method involves strong acid
hydrolysis of chondroitin sulfate into monosacchrides by concentrated hydrochloric acid and followed by
formation of furfural. The condensation of furfural with resorcinol in the presence of ferric ions yields a
yellow-orange coloured complex. This colour has absorption maxima at 435 nm against reagent blank.
The absorbance was found to increase linearly with the concentration of the drug and formed the basis for
quantification. The calibration graph was linear from 4-32 µg/mL with correlation coefficient was at
0.999. The results presented are statistically validated in accordance with the guidelines provided by ICH.
The recovery studies were carried out at three different levels. The precision was good with percentage
relative standard deviation (RSD) lower than 2.0%. The Sandell’s sensitivity was calculated and was
found 0.03782 µg/cm2. The detection limits and quantification limit are found to be 0.2166 µg/mL and
0.6563 µg/mL, respectively. Ruggedness of method was performed and the percentage relative standard
deviation (RSD) was found below 2.0%. The proposed method was successfully applied to the
determination of chondroitin sulfate in bulk drug and pharnarmaceutical tablet formulation.
Keywords: Spectrophotometric method, Chondroitin sulfate, Resorcinol
Introduction
Chondroitin sulfate belongs to a family of heteropolysaccharides called glycosaminoglycans
or GAGs. Glycosaminoglycans were formerly known as mucopolysaccharides1,2. GAGs in
the form of proteoglycans comprise the ground substance in the extracellular matrix of
connective tissue. It is made up of linear repeating units containing D-galactosamine and
D-glucuronic acid. It is found in humans in cartilage, bone, cornea, skin and the arterial wall.
Chem Sci Trans., 2013, 2(4), 1427-1433
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CHS is chemically poly- (1- 3)-N-acetyl-2-amino-2-deoxy-3-O-ß-D-glucopyranurosyl - 4 (or 6-) sulfonyl-D-galactose. It is naturally found polymer of linear repeating units
containing D-galactosamine and D-glucuronic acid. The amino group of galactosamines in
the basic unit of CHS is acetylated, yielding N-acetyl-galactosamine. The sulfate group is
esterified to the carbon 4 or 6 position in N-acetyl-galactosamine yield chondroitin sulfate A
and chondroitin sulfate C respectively. The molecular weight of chondroitin sulfate A or C
ranges from 5,000 to 50,000 Daltons and contains about 15 to 150 basic units of D-galactosamine
and D-glucuronic acid3. It is represented by the following structural formula:
Figure 1. Chemical structure of chondroitin sulfate
CHS mainly used in promotion and maintenance of the structure and function of
cartilage, pain relief of osteoarthritic joints and anti-inflammatory activity. Chondroitin
sulfate also useful in coronary artery disease, interstitial cystitis, iron absorption
enhancement, ophthalmologic uses and muscle soreness.
Literature review revealed that few analytical methods include Gel-exclusion
chromatography4,5, capillary electrophoresis6 and titration with cetyl pyridinium chloride
detecting end point with a phototrode7,8. The other methods in the literature for quantitating
chondroitin sulfate needs enzymatic digestion followed by disaccharide analyses, include
reverse phase high performance liquid chromatography9 (HPLC), anion-exchange
chromatography10, gel electrophoreses with fluorescence detection11 and colorimetric metric
method using orcinol reagent12.
Hence an attempt has been made to develop accurate, precise and reproducible
colorimetric method for estimation of chondroitin sulfate in bulk drug and tablet
formulation. The method was validated by using various parameters as per ICH guidelines13.
Experimental
Perkin Elmer double beam UV-Visible spectrophotometer was used with 1 cm matched
quartz cells.
Chemicals and reagents
Pure chondroitin sulfate was obtained as gift sample from Banner Pharmacaps (India) Pvt
Ltd, Bangalore, India, Resorcinol Qualigens fine chemicals, AR Grade), Ferric chloride
(Rasayan Laboratory), concentrated hydrochloric acid (Qualigens fine chemicals), ethanol
and double distilled water were used.
Reagents: 1. Resorcinol (0.2% w/v in concentrated hydrochloric acid and ethanol)
2. Ferric chloride (0.075% w/v in concentrated hydrochloric acid)
Reagent preparation
Dissolve 75 mg of ferric chloride in about 50 mL of concentrated hydrochloric acid. To this
solution add 5 mL ethanol containing 200 mg of resorcinol and make up to the mark with
concentrated hydrochloric acid.
Chem Sci Trans., 2013, 2(4), 1427-1433
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Preparation of standard stock solution
About 100 mg of chondroitin sulfate working reference standard of known purity (92.42%
pure) was accurately weighed into a 100 mL volumetric flask, dissolved & volume was
made up to 100 mL with water (1 mg/mL). 10 mL of above solution was diluted to 50 mL
with water (200 µg/mL).
Determination of wavelength of maximum absorbance
2 mL of working reference standard solution was pipetted into a 25 mL volumetric flask, 10 mL
of resorcinol reagent was added and solution was cooled to room. The solution mixture was
heated in a boiling water bath for 1 hour and cooled to room temperature by placing in ice-cooled
water. The volume was made up to 25 mL distilled water. The yellow-orange coloured solution
was scanned in the 400 - 800 nm range against reagent blank. The wavelength of maximum
absorbance of yellow-orange colour chromogen was found at 435 nm as shown in Figure 2.
435.0 nm 0.416 ABS
Figure 2. Absorbance maxima of chondroitin sulfate
Reaction scheme for chondroitin sulfate with resorcinol:
COOH
O
O
OR 1
O
O
O
Conc. HCL
OH
Fecl3 , Heat
OH
CH2OR1
COOH
O OH
CH2OR
OR
+
OH
OH
NHCOCH
3
OH
NHCOCH
3
N- Acetyl galactosamine
Glucuronic acid
Chondroitin sulfate
CO2 -
-3 H2O
CHO
O
Furfural
HO
HO
Resorcinol
HO
O
O
C
Yellow-orange colour
Scheme 1
O OH
OH
Chem Sci Trans., 2013, 2(4), 1427-1433
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Assay of marketed formulation
Twenty tablets were accurately weighed and crushed to obtain fine powder. An accurately
weighed tablet powder equivalent to about 100 mg of chondroitin sulfate was transferred to
100 mL volumetric flask and sonicated for 15 minutes in about 50 mL of double distilled
water and made up the volume with double distilled water. The resulting solution was
filtered through Whatman filter paper. 10 mL of filtrate solution was diluted to 50 mL with
distilled water. 2 mL of above solution was transferred into 25 mL volumetric flask, 10 mL
of resorcinol reagent solution was added and solution was cooled to room temperature. The
solution mixture was heated in a boiling water bath for 1 hour and cooled to room
temperature by placing in ice-cooled water. The volume was made up to 25 mL distilled
water. The absorbance of yellow-orange colour chromogen was measured at 435 nm against
reagent blank.
Method validation
Validation is the process of established documented evidence which provides high degree of
assurance that a specific process will consistently produce a product meeting its
predetermined specifications and quality attributes. The method was validated by various
parameters as per ICH guidelines (Table 2).
Linearity
Varying concentrations of chondroitin sulfate were treated with carbazole within the range
4 µg/mL to 32 µg/mL corresponding to 50%, 75%, 100%, 125% and 150% of chondroitin
sulfate. The linearity of chondroitin sulfate was found to be 4-32 µg/mL and linear regression
was found to be r2 = 0.9999.
Accuracy
Accuracy of the method was determined in terms of % recovery of standard chondroitin sulfate at
three different concentrations (50%, 100% and 150%). Result of the recovery study were found
to be within the acceptable criteria 100±10%, indicates sensitivity of the method towards
detection of chondroitin sulfate and non interference of excipients in the method (Table 1).
Table 1. Recovery calculation for chondroitin sulfate
Spike level
Level –I
50% -1
50% -2
50% -3
Level –II
100% -1
100% -2
100% -3
Level –III
150% -1
150% -2
150% -3
Theoretical
Value, mg/mL
Practical
Value, mg/mL
Recovery,
%
0.508
0.504
0.512
0.5085
0.5108
0.5226
100.09
101.34
102.07
1.012
1.009
1.017
1.0287
1.0358
1.0334
101.65
102.65
101.57
1.514
1.506
1.502
1.4948
1.4925
1.4948
98.73
99.01
99.52
Average
RSD,
%
101.16
0.989
101.95
0.59
99.08
0.404
1431
Chem Sci Trans., 2013, 2(4), 1427-1433
System precision
The precision of the system was determined by 6 repetitive absorbance of the same standard
solutions by using 2 mL of stock solution. As the value of % RSD of system precision study
were in within the acceptable limit (less than 2%). Hence the method provides good precision.
Method precision
The precision of the method for the assay of chondroitin sulfate was determined by the assay
of six aliquots of the homogeneous sample. As the value of %RSD of system precision study
were in within the acceptable limit (less than 2%). Hence the method provides good
precision and reproducibility.
Specificity
The study was conducted to prove that the absorbance obtained in the samples is only due to
chondroitin sulfate without any interference from other excipients. Placebo solution at
varying concentration does not show any absorbance at 435 nm. Hence the method is
specific for the determination of chondroitin sulfate.
Detection limit
The detection limit was determined by the analysis of chondroitin sulfate with known
concentrations and by establishing the minimum level at which the chondroitin sulfate was
reliably detected.
The detection limit (DL) may be expressed as; DL =3.3 σ/ S
where σ = the standard deviation of the response
S = the slope of the calibration curve
Quantitation limit
The quantitation limit was determined by the analysis of chondroitin sulfate with known
concentrations and by establishing the minimum level at which the chondroitin sulfate was
quantified with acceptable accuracy and precision.
The quantitation limit (QL) may be expressed as: QL=10 σ/ S
where σ = the standard deviation of the response
S = the slope of the calibration curve
Solution stability
The stability of the analytical solution for assay of chondroitin sulfate was determined by the
assay of sample preparation at fixed intervals of time. The % RSD for the assay values for
chondroitin sulfate up to 24 hours was found to be 0.23. This indicates that the analytical
solution is stable up to 24 hours.
Ruggedness
The ruggedness of the method for assay of chondroitin sulfate was determined by the assay
of six aliquots of the homogeneous sample on different instrument and by different analyst.
The % RSD for the assay values for chondroitin sulfate in ruggedness study was found to be
0.936. This indicates that the method has good reproducibility and very less random error.
The results of the method developed and validated for chondroitin sulfate in formulation
are depicted in the Table 2. The results developed method showed good agreement with the
labeled claim in the formulation analyzed.
Chem Sci Trans., 2013, 2(4), 1427-1433
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Table 2. Results for the validation of the analytical method for chondroitin sulfate
Parameter
1. Linearity
2. Accuracy
2. System precision
4. Method precision
5. Specificity
6. Detection Limit
7. Quantitation Limit
8. Ruggedness
9. Sandell’s sensitivity
(mg/mL/0.001 abs units)
Acceptance criteria
Regression coefficient (r2) not
less than 0.999
Beer’s Range
Regression Equation
Recovery between 98 - 102%
% RSD should be less than 2.0%
% RSD should be less than 2.0%
Non interference of placebo and
blank in analysis
% RSD should be less than 2.0%
-
Results Obtained
0.9998
4 – 32 µg/mL
y = 0.0259x+0.0014
99.08 -101.95%
0.46%
1.08%
Complies
0.2166 µg/mL
0.6563 µg/mL
0.9361 %
0.03782 µg/cm2
Results and Discussion
The proposed method for the estimation of chondroitin sulfate is based on the reaction
between resorcinol and hydrolyzed component of chondroitin sulfate in presence of ferric
ions yields a yellow-orange coloured complex. This colour has absorption maxima at 435 nm
against reagent blank. This method obeyed Beer’s concentration range 4-32 µg/mL. The
accuracy of the method was established by recovery study, as per ICH guidelines, at three
different levels viz. 50%, 100% and 150% and the % recovery ranged from 99.08 -101.95
Statistical analysis of the results shows that all the proposed procedures have good precision
and accuracy. The method was found to be sensitive with respect to Sandell’s sensitivity.
The detection limits and quantification limit are found to be 0.2166 µg/mL and 0.6563 µg/ mL,
respectively. Ruggedness of method was performed and the percentage relative standard
deviation (RSD) was found 0.9361%. Results of analysis of pharmaceutical formulations
reveal that the proposed methods are suitable for their analysis with virtually no interference
of the usual additives present in pharmaceutical formulations.
Conclusion
The proposed method is simple, sensitive and selective with reasonable precision and accuracy
for the quantification of chondroitin sulfate in bulk drug and pharmaceutical dosage forms.
Hence developed method was conveniently adopted for routine quality control analysis.
Acknowledgement
The authors are thankful to Banner Pharmacaps (India) Pvt Ltd, Bangalore, India for
providing the drug sample and facilities to carry out the present work. The authors also
thankful to Principal, Govt College of Pharmacy, Bangalore for providing the facilities to
carry out the present research work.
References
1.
Pamela C C and Richard A H, Lippincott’s Illustrated Reviews: Biochemistry. 2nd
Ed., Lippincott Williams & Wilkins Publishers, Philadephia (USA), 1994, 147-150.
1433
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Chem Sci Trans., 2013, 2(4), 1427-1433
Satyanarayana U, Biochemistry 2nd Ed., Books and Allied (P) Ltd Publishers,
Vijayawada (India), 2002, 150-155.
The Merck Index, 13th Ed., Merck & Co, INC Whitehouse station (USA), 1997, 368.
Way W, Gibson K and Breite A, J Liquid Chromatogr Relat Technol., 2000, 23(18), 2851
Choi D W, Kim M J, Kim H S, Chang S H, Jung G S, Shin K Y and Chang S Y, J
Pharma Biomed Anal, 2003, 31(6), 1229-1236.
Al-Hakim A and Linhardt R J, Anal Biochem., 1991, 195(1), 68-73.
Liang Z, Bonneville C, Senez T and Henderson T, J Pharma Biomed Anal., 2002,
28(2), 245-249.
USP, Pharmacopeial Preview, Pharmacopoeial Forum Sept-Oct, 2000, 26(5), 1432-1436.
Okamoto H, Nakajima T, Ito Y, Shimada K and Yamato S, J Chroma A, 2004,
1035(1), 137-144.
Murata K and Yokoyama Y, Anal Biochem., 1985, 146(2), 327-335.
Karousou E G, Porta G, De Luca G and Passi, J Pharma Biomed Anal., 2004, 34(4),
791-795.
Kinoshita T, Kanada K and Tsuji A, J Biochem., 1975, 77(2), 399-403.
International Conference of Harmonization (ICH) of Technical Requirement for the
Registration of Pharmaceuticals for Human Use. Validation of Analytical procedures;
Methodology. ICH-Q2B, Geneva (1996); CPMP/ICH/281/95).