Development and Validation of
Spectrophotometric Method for the Determination of Risperidone in pure and
Pharmaceutical Formulation using MBTH reagent
Mrs. Sheeja Velayudhan Kutty, Greeshma S.*,
Vidhya P.M., Mr. D. K. Sunith
Department of Pharmaceutical Analysis,
Grace College of Pharmacy, Kodunthirapully Post, Palakkad, Kerala, India, Pin
678004
Grace College of Pharmacy, Palakkad,
Kerala
Cresent College of Pharmaceutical
Sciences, Kannur, Kerala
*Corresponding Author E-mail: greeshmasivarajan@gmail.com
A simple, sensitive, precise and accurate
UV-Visible spectrophotometric method has been developed for the determination
of Risperidone in bulk drug and pharmaceutical formulation (tablet). The method
is based on the oxidative coupling reaction of the drug with
3-Methyl-2-Benzthiazolinone hydrochloride (MBTH) in the presence of Ferric
chloride Fe (III) to form green coloured chromogen exhibiting λ max at
666nm. Beer’s law was obeyed over the concentration range of 4-14µg/ml with a
linear regression value 0.993.The method was validated in accordance with the
current ICH guidelines. Interday and Intraday studies showed high degree of
repeatability, recoveries obtained do not differ significantly from 100% showed
accuracy and reliability of the method. The proposed method was applied
successfully for the determination of Risperidone in pure bulk form and in tablets
without interference from commonly encountered additives.
KEYWORDS: Risperidone, UV Visible
spectrophotometry, MBTH, Validation
INTRODUCTION:
Risperidone is a psychotropic agent used to
treat schizophrenia, action of which is mediated through a combination of
dopamine Type 2 (D 2) and serotonin Type 2 (5HT 2)
receptor antagonism. It is a selective monoaminergic antagonist with high
affinity for 5HT 2, D 2 and H1 histaminergic
receptors.[1,2]It belongs to chemical class of benzisoxazole
derivatives and is 3-[2-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)-1-piperidinyl]
ethyl]-6, 7, 8, 9-tetrahydro-2-methyl-4H-pyrido- [1, 2-a]-pyrimidin-4-one (Fig.
1) with molecular formula of C 23 H 27 FN 4 O
2 and molecular weight of 410.49. [3]
Figure 1: Chemical structure of Risperidone
Literature survey revealed that various
methods have been reported for estimation of Risperidone in biological matrices
such as plasma with help of LC / tandem mass spectrometry and by using
MEPS-LC-UV method. [4, 5] Few stability-indicating methods have been
reported for determination of Risperidone in bulk powder and tablets in presence
of its degradation products. [6, 7] However very few methods were
reported for quantitation of Risperidone in tablet dosage forms in the
literature. [8, 9] The objective of the present investigations was
to develop a simple, accurate and economical spectrophotometric method for
estimation of Risperidone in tablet formulations.
Although spectrophotometric methods are the
instrumental methods of choice commonly used in industrial laboratories, no
colorimetric method with the reagent using MBTH has been reported so far for
the determination of Risperidone. Therefore, the need for a fast, low cost and
selective method is obvious, especially for routine quality control analysis of
pharmaceutical products containing Risperidone. The spectrophotometric method
was based on the oxidative coupling reaction of reaction of Risperidone with
the reagent 3-Methyl-2-Benzthiazolinone hydrochloride (MBTH). [10-12]
MATERIALS AND METHODS:
Apparatus
A Shimadzu model Pharmaspec-1800
UV-Vis spectrophotometer with 1.0 cm matched cells was used for the electronic
spectral measurements.
Reagents
Risperidone and all other chemicals used
were analytical reagent grade. Risperidone pure drug was generously provided by
Torrent Pharmaceuticals Pvt ltd (Ahmadabad, India), as a gift sample. The
commercially available tablet “Risdone 2” (Intas Pharmaceuticals Dehradun)
containing 2mg of Risperidone was procured from the local market.
3-Methyl-2-Benzthiazolinone hydrochloride
(MBTH) 0.5%
0.5g of MBTH reagent was accurately weighed
and transferred in to a 100 ml volumetric flask, dissolved in distilled water
and made up to the mark to get a solution of 0.5%.
Ferric chloride 1%
Freshly prepared ferric chloride solution
was made by dissolving 1g in 0.5N hydrochloric acid.
Preparation of standard stock
solution
The standard Risperidone 10mg
was weighed accurately and transferred to volumetric flask (100 ml). It was
dissolved properly in methanol and made up to the mark to get a concentration
of 100µg/ml.
Determination of λmax
Aliquots of the standard stock
solution in methanol were transferred in to a series of 10 ml volumetric
flasks. To each 2.5ml of 0.5% MBTH and 2.5 ml of 1% ferric chloride were
added and the volume was made up to the mark with water. The contents of the
flask were shaken well and allowed to stand for 20 minutes to form a green
coloured complex. The solutions were scanned over visible range of 400-800 nm.
An overlay spectrum of drug was drawn out and selected the wavelength 666nm for
the analysis at which drug showed maximum absorbance (figure 2).
Procedure for calibration curve
Aliquots of the standard stock
solution in methanol, having final concentrations in the range of 4-14 µg/ml
were transferred in to a series of 10 ml volumetric flasks. To each 2.5ml of
0.5% MBTH and 2.5 ml of 1% ferric chloride were added and the volume was made
up to the mark with water. The contents of the flask were shaken well and
allowed to stand for 20 minutes to form a green coloured complex. The absorbance
of each solution was measured at 666 nm against the regent blank.(figure 3)
(table 1).
Analysis of tablet formulation
Ten tablets were accurately
weighed and triturate thoroughly to get fine powder. The powder equivalent to
10mg of Risperidone was weighed and transferred in to 100 ml volumetric flask.
The contents of the flask were dissolved in the 50 ml of the methanol with the
aid of ultrasonication for 10 minutes. The solution was filtered through
Whatmann filter paper no.41 and volume was made up to 100ml with methanol. An
aliquot of this solution was pipetted out to get a final concentration of 10
µg/ml, to that 2.5 ml of 0.5% MBTH and 2.5 ml of 1% FeCl3 was added
and made up to 10 ml with water. The contents of the flask were
shaken well and allowed to stand for 20 minutes and the absorbance of green
coloured complex produced was measured at 666nm against the reagent blank
(figure 4) (table 3 ).
RESULTS:
The method was accurate, simple,
rapid, reliable, sensitive and reproducible. The wavelength 666nm was selected
which showed good linearity between the concentrations.
Figure 2: overlay spectra of
Risperidone
Validation of the method
The validation of the developed
spectrophotometric method was carried out as per the ICH guidelines. According
to ICH guidelines the following parameters were evaluated.
Table 1 calibration curve values
|
Sl.No
|
Concentration
(µg/ml)
|
Absorbance
|
|
1
|
4
|
0.074
|
|
2
|
6
|
0.131
|
|
3
|
8
|
0.152
|
|
4
|
10
|
0.206
|
|
5
|
12
|
0.233
|
|
6
|
14
|
0.267
|
Linearity
Under the optimized experimental
conditions, the calibration curve for Risperidone was constructed by analyzing
a series of standard solutions of the drug. The regression equations for the
results were derived by using the least squares method. The Beer’s law plot was
linear (4 - 14µg/ml) with very small intercept and a correlation value of
0.993. The LOD and LOQ values were found to be 1.616 and 4.89µg/ml
respectively. The calculated Molar absorptivity indicated that the suggested
procedure is highly sensitive (Table 2).
Figure 3 calibration curve for
Risperidone
Table 2 Optical characteristics
and statistical data of the regression equation for the reaction of the
proposed method
|
Parameters
|
Optical characteristics
|
|
Colour
|
Green
|
|
λmax
|
666 nm
|
|
Linearity range
|
4 - 14µg/ml
|
|
Molar absorptivity value
|
197.367l/mol/cm
|
|
Limit of Detection
|
1.616µg/ml
|
|
Limit of Quantification
|
4.89µg/ml
|
|
Regression equation
|
|
|
Slope
|
0.019
|
|
Intercept
|
0.002
|
|
Correlation Coefficient
|
0.993
|
Table 3 Analysis of marketed
formulation
|
Marketed formulation
|
Label claim (mg)
|
Amount of drug estimated (mg)
|
% mean amount estimated
|
|
Risdone 2
|
2
|
1.9894
|
99.47
|
Accuracy
The accuracy of the proposed
method was also confirmed through recovery studies using the method of standard
addition. Results indicated good recoveries which reflect the selectivity of
Risperidone from the commonly encountered excipients and additives. Therefore
the proposed method can be considered specific for the determination of
Risperidone in commercial dosage forms (Table 4 ).
Precision:
Precision of the method was
determined by performing Interday variation, intraday variation and
repeatability studies and expressed in the forms of %RSD. In Interday
variation, the absorbances of working standard solutions of Risperidone (4 - 14
µg/ml) were measured on three consecutive days. In intraday variation the
absorbance were measured three times a day. In repeatability study, six
determinations of the fixed concentration of the drug were analyzed separately
(table 5 ).
Figure 4 spectrum of Risperidone
from tablet formulation
Table 4 Recovery studies of
Risperidone
|
Standard drug added (%)
|
Amount of pure drug added (µg/ml)
|
Concentration of sample present(µg/ml)
|
%Recovery
|
%RSD
|
|
80
|
3.2
|
4
|
98.68
|
1.8496
|
|
100
|
4
|
4
|
101.31
|
1.6129
|
|
120
|
4.8
|
4
|
100.15
|
0.0121
|
Table 5: precision data
|
Particulars
|
Fortified
amount (µg/ml)
|
Amount
found (µg/ml)
|
%RSD
|
|
Repeatability
(n=6)
|
4
|
3.93
|
1.547
|
|
Reproducibility
(n=6)
|
4
|
4.06
|
1.5
|
DISCUSSION:
In the present method, the drug
reacts with MBTH in the presence of FeCl3 to give a green coloured
product. Actually, this is an iron catalyzed oxidative coupling reaction of
MBTH with the drug. Under the reaction conditions, MBTH on oxidation loses two
electrons and one proton forming an electrophilic intermediate, which is the
active coupling species. This intermediate undergoes electrophilic substitution
with the drug to form the coloured product. Experiments were carried out to
optimize the reaction condition for complete colour formation. The optimum
concentration and volume were selected on the basis of their ability to give
maximum absorbance. Different concentrations and different volumes were tried
for all the reagents, by varying the parameters at a time. In this method it
was found that optimum concentration of MBTH reagent was 0.5%w/v and optimum
concentration of FeCl3 was 1%w/v. The optimum volume was found to be
2.5 ml for MBTH and 2.5 ml for Ferric chloride.
We have proposed a new
spectrophotometric method for the determination of Risperidone, which is fairly
sensitive, simple, and economical with reasonable precision and accuracy. The
optical parameters and statistical comparison justify this method for
application in routine drug estimation in pure and dosage forms. Also, the
procedures do not involve any critical reaction conditions or tedious sample
preparation steps. The proposed method is of great value in quality control
determinations of Risperidone because of its adequate accuracy, reliability for
the determination of this drug in the pharmaceutical dosage forms without
interference from commonly encountered excipients. So, the recommended method
is well suited for the assay and evaluation of drugs in pharmaceutical
preparation.
ACKNOWLEDGEMENTS:
We express our sincere thanks to
Torrent pharmaceuticals private limited, Ahmadabad, India for providing the
gift sample of Risperidone, and also we are grateful to the Principal and
staffs of Grace College of Pharmacy, Kodunthirapully, Kerala, India for
providing the facilities to carry out the present work.
REFERENCES:
1. Martindale the complete drug reference,
thirty fifth edition volume 1 page no.921 – 922.
2. K D Tripathi Essentials of medical
pharmacology, sixth edition page no. 167 – 429.
3.
The Merck Index,
thirteenth edition compound no.8316, page no 1478.
4.
Manickam
Aravagiri and Stephen R. Marder. Simultaneous determination of Risperidone and
9- hydroxyrisperidone in plasma by liquid chromatography/electro spray tandem
mass spectrometry. J.
Mass Spectrum. 35,
718–724 (2000)
5.
Roberto
Mandrioli, Laura Mercolini, Domenica Lateana, Giancarlo Boncompagni, Maria
Augusta raggi. Analysis of Risperidone and 9-hydroxy Risperidone in human
plasma, urine and saliva by MEPS-LC uv.JCHROMB.2010;11.033
6.
A. P.
Suthar1, S. A. Dubey2, S. R. Patel1, A. M. Shah1. Determination of Risperidone
and forced degradation behaviour by HPLC in tablet dosage form IJPRIF Vol.1,
No.3, pp 568-574.
7.
Zarna R
Dedania, Ronak R Dedania, Navin R sheth, jigar B Patel and bhavana Patel. Stability Indicating HPLC
Determination of Risperidone in Bulk Drug and Pharmaceutical Formulations. International journal of
analytical chemistry. 2011
8.
Kulkarni, S,
Chhabra, G, Shivani. Method Development and validation of UV spectrophotometric method for the
determination of Risperidone in bulk and tablets formulation. IJPCR 1 (1);
2012
9.
Moynul
Hasan, Abdullh Al Masud and Jamiuddin Ahmed. Development and validation of
spectrophotometric method for the determination of Risperidone in bulk drug and
pharmaceutical formulation. IJPSR, 2011; Vol. 2(2): 378-382.
10.
V.Siva
Ramakrishna, P.B.Sudha Lakshmi, D.Ravi kumar, C. Ram babu . Spectrophotometric determination of
Valacyclovir HCl through oxidative coupling reaction in bulk and its
pharmaceutical preparations. International
Journal of ChemTech Research..4(1); 138-142.
11.
A Bose, P P
Dash, M K Sahoo. Simple spectrophotometric methods for estimation of
aceclofenac from bulk and formulation. Pharmaceutical methods, 1( 1); oct – dec 2010.
12.
Sheeja
Velayudhan kutty, Susamma cicy Eapen, Mohammed shameer, faizal p p.
Validated UV – Visible spectrophotometric method for the estimation of
Fenofibrate in pure and pharmaceutical formulation using MBTH reagent, Intenational journal of pharmaceutical
sciences and drug research 4(1);
2012; 74 -76.
13.
K.
Raghubabu, L. Santhi Swarup, B. Kalyana Ramu, G. Rupakumari, M. Narayanarao and
C. Ramdas. Development and Validated Visible Spectrophotometric Methods for the
Assay of Donepezil hydrochloride in Pharmaceutical preparations. Research J.
Pharm. and Tech. 5 (2): Feb. 2012; 228-232.