Development and Validated Visible Spectrophotometric Methods for the Assay of Donepezil hydrochloride in Pharmaceutical preparations

 

K. Raghubabu¹*, L. Santhi Swarup¹, B. Kalyana Ramu², G. Rupakumari, M. Narayanarao³ and C. Ramdas³

¹Department of Engineering Chemistry, AU College of Engineering (A), Andhra University, Visakhapatnam -530003 Andhra Pradesh (India)

²Department of Chemistry, Maharajah’s College (Aided and Autonomous), Vizianagaram-535002 (AP) India.

³M/S Tychy Industries, R &D Division, Hyderabad-AP (India)

Corresponding author: drraghualways@yahoo.co.in

ABSTRACT:

Two simple and sensitive visible spectrophotometric methods (M₁ and M₂) have been developed for the estimation of Donepezil hydrochloride (DH) in bulk and dosage forms. Method M₁ involves Internal salt formation of aconitic anhydride, dehydration product of citric acid [CIA] with acetic anhydride [Ac₂O] to form colored chromogen with an absorption maximum of 580 nm and the method M₂ is based on the formation of green colored coordination complex by the drug with cobalt thiocyanate which is quantitatively extractable into nitro benzene with an absorption maximum of 620 nm. Beer’s law obeyed in the concentration range of 8-24µg/ml for method M₁and 16-48 µg/ml for method M₂. Commercially available tablets were analyzed and the results are statistically compared with those obtained by the reference method and validated by recovery studies. The results are found satisfactory and reproducible. These methods are applied successfully for the estimation of the Donepezil hydrochloride in the presence of other ingredients that are usually present in dosage forms. These methods offer the advantages of rapidity, simplicity and sensitivity and normal cost and can be easily applied to resource-poor settings without the need for expensive instrumentation and reagents.

 

 

INTRODUCTION:

Donepezil hydrochloride(DH), (Fig.1) (±)-2,3-dihydro-5,6-dimethoxy 2-[[1-(phenyl methyl)-4-piperidinyl] methyl]-1H-inden-1-one hydrochloride, is a specific and reversible inhibitor of acetylcholine esterase (AChE) of the second generation drug approved by the US FDA for the symptomatic treatment of mild to moderate dementia of the Alzheimer’s type disease (AD). It is a new class of acetylcholine esterase (AChE) inhibitor having an N-benzyl piperidine and an indanone moiety which shows longer and more selective action. DH was found in vitro to be over 1000 times more potent an inhibitor of this enzyme than that of butyryl cholinesterase (BuChE), an enzyme which is present mainly in the periphery, outside the central nervous system. The drug is also known as E2020or Aricept ¹ and it exerts its therapeutic effect by increasing acetylcholine concentrations and enhancing cholinergic functions. The drug is not yet official in any pharmacopoeia.

 

Some analytical methods which include HPLC^2-19 including stability indicating, HPTLC²⁰, LC-MS-MS ^21-27, voltametry²⁸, Capillary electrophoresis ²⁹, UV and visible spectrophotometric ^30-33 have been reported in the literature for the determination of DH in biological fluids and pharmaceutical preparations. The main purpose of the present study was to establish a relatively simple, sensitive, validated and inexpensive visible spectrophotometric method for the determination of DH in pure form and in pharmaceutical dosage forms, since most of the previous methods have been found to be relatively complicated, expensive and less selectivity and sensitivity. So the authors have made some attempts in this direction and succeeded in developing these methods based on the reaction between the drug and citric acid –acetic anhydride³⁴ (CIA/AC₂O- M₁) or cobalt thiocyanate³⁵ (CTC-M₂) reagents . These methods can be extended for the routine assay of DH formulations.

 

MATERIALS AND METHODS:

Apparatus and chemicals

A Systronics UV/Visible spectrophotometer model-2203 with 10mm matched quartz cells was used for all spectral measurements. Systronics model-362 pH meter was used for all the pH measurements. A pure drug sample of DH was provided as a gift sample by Tychy industries, Hyderabad AP (India). Donep10mg tablets of Alkem Laboratories and Aricept10mg Tablets of Eisai pharmaceuticals, purchased from local market. All the chemicals used were of analytical grade. Citric acid monohydrate (Prepared by dissolving 1.2 grams of (1.2%, 6.245X10^-2M) Citric acid in 5 ml methanol initially followed by dilution up to 100ml with acetic anhydride) and Acetic anhydride (SD Fine chemicals), CTC (2.50x10^-1M, solution prepared by dissolving 7.25 g of cobalt nitrate and 3.8 g of ammonium thiocyanate in 100ml distilled water), Citrate buffer pH(2.0) (prepared by mixing 306ml of 0.1M tri sodium citrate with 694ml of 0.1M HCl and pH was adjusted to 2.0) were prepared .

 

Fig.1: Chemical structure of Donepezil hydrochloride

 

Preparation of Standard and sample drug stock solution:

An accurately weighed quantity of DH (pure or tablet powder) equivalent to 100mg was mixed with 5ml of 10% Na₂CO₃ solution and transferred into 125ml separating funnel. The freebase released was extracted with 3x15ml portion of chloroform and the combined chloroform layer was brought up to 100ml with the same solvent to get 1mg/ml DH drug stock solution in free base form. This free base stock solution was further diluted step wise with the same solvent to get the working standard solution concentrations [M₁-200 µg/ml, M₂-400 µg/ml].

 

Fig.2: Absorption spectra of DH-CIA-AC₂O

 

Procedure / Assay:

Method M₁:

Aliquots of standard DH drug solution [1.0-3.0ml;200µg/ml in free base form] in chloroform were taken into a series of 25ml graduated tubes and gently evaporated in a boiling water bath to dryness. To this, 10ml of citric acid- Acetic anhydride reagent was added and the tubes were immersed in a boiling water bath for 30 minutes then the tubes were cooled to room temperature and made up to the mark with acetic anhydride. The absorbance of the colored solutions was measured after 15minutes at 580 nm against the reagent blank (Fig-2 showing absorption spectra) within the stability period of 15-60min.The amount of DH was computed from its calibration graph (Fig-3 showing Beer’s law plot).

 

Fig.3: Beer’s law plot of DH-CIA/AC₂O

 

Method M₂

Aliquots of standard DH solution (1.0ml - 3.0ml, 400µg/ml in free base form) were delivered into a series of 125ml separating funnels. Then 2.0ml of buffer solution (pH 2.0) and 5.0ml CTC solution were added. The total volume of aqueous phase in each separating funnel was adjusted to 15.0ml with distilled water. To each separating funnel 10.0ml of nitrobenzene was added and contents were shaken for 2 minutes. The two phases were allowed to separate and absorbance of nitrobenzene layer was measured at 620nm against a similar reagent blank (Fig-4 showing absorption spectra).The colored product was stable for 1 hour. The amount of DH in the sample solution was com puted from its calibration graph (Fig-5 showing Beer’s law plot).

 

RESULTS AND DISCUSSION:

In developing these methods, systematic studies of the effects of various parameters were undertaken by varying one parameter at a time and controlling all others fixed (OVAT method). The effect of various parameters such as time, volume and strength of reagents, pH buffer solution and order of addition of reagents, stability period and solvent for final dilution of the colored species were studied and the optimum conditions were established. Among the various water immiscible organic solvents (C₆H₆, CHCl₃, dichloro methane, nitro benzene, chloro benzene and CCl₄) tested for the extraction of colored coordinate complex into organic layer, nitrobenzene was preferred for selective extraction of colored complex from organic phase in method M₂.

Table - 1 Optical characteristics, precision and accuracy of the proposed methods

Parameters	Method A	Method B
λ max(nm)	580	620
Beer’s law limit (µg/ml)	8- 24	16-48
Sandell’s sensitivity (µg/cm2/0.001 abs. unit)	0.002633745	0.004129032
Molar absorptivity (Litre/mole/cm)	157934.8125	100740.3125
Regression equation (Y) *= a +b x
Intercept (a)	-0.081	-0.105
Slope(b)	0.02	0.013
%RSD	1.684	0.9501
% Range of errors(95% Confidence limits) 0.05 significance level 0.01 significance level	1.767	0.9997
	2.77	1.56

*Y = a + b x, where Y is the absorbance and x is the concentration of DH in µg/ml

 

 

Table-2 Analysis of DH in pharmaceutical formulations

Method	*Formulations	Labeled Amount (mg)	Found by Proposed Methods			Found by Reference Method ± SD	#% Recovery by Proposed Method ± SD
			**Amount found ± SD	t	F
A	Tablet-1	5	4.98±0.033	0.896	1.93	4.97±0.024	99.51±0.65
	Tablet-2	10	9.86±0.091	1.36	2.901	9.89±0.54	98.58±0.91
B	Tablet-1	5	4.975±0.021	1.55	1.31	4.97±0.024	99.51 ± 0.412
	Tablet-2	10	9.81 ± 0.044	1.99	1.45	9.89±0.54	98.95 ± 0.76

* Tablet- 1 and Tablet-2 from two different companies.

**Average ± Standard deviation of six determinations, the t- and f-values refer to comparison of the proposed method with UV reference method. Theoretical values at 95% confidence limits t =2.57 and F = 5.05.

# Recovery of 10mg added to the pre analyzed sample (average of three determinations).

Reference method (reported UV method) using methanol (λ _max=231nm).

 

 

Different solvents like acetic anhydride, acetic acid, methanol, ethanol and isopropanol were also used as diluents but acetic anhydride was found to be ideal for final dilution in method M₁. The ratio of organic to aqueous phase was found to be 1:1.5 by slope ratio method for method M₂. The optical characteristics such as Beer’s law limit, Sandell‘s sensitivity, molar absorptivity, percent relative standard deviation, (calculated from the six measurements containing 3/4^th of the amount of the upper Beer’s law limits ) were calculated and the results are summarized in Table-1.

 

Fig.4: Absorption spectra of DH-CTC

 

Fig.5: Beer’s Law plot of DH-CTC

 

Commercial formulations containing DH were successfully analyzed by the proposed methods. The values obtained by the proposed and reference methods for formulations were compared statistically by the t-and F-test and found not to differ significantly. As an additional demonstration of accuracy, recovery experiments were performed by adding a fixed amount of the drug to the pre analyzed formulations at three different concentration levels. These results are summarized in Table-2.

 

Recovery experiments indicated the absence of interference from the commonly encountered pharmaceutical excipients present in formulations. The proposed methods are found to be simple, sensitive and accurate and can be used for the routine quality control analysis of DH in bulk and dosage forms.

 

Fig.6: Scheme of reactions

 

Chemistry of colored species

In method M₂ the green color species formation is the coordination complex of the drug (electron donor) and the central metal of cobalt thiocyanate, which is extractable into nitro benzene from aqueous solution and in method M₁ red-violet color internal salt of aconitic anhydride is formed when DH was treated with CTC or CIA/Ac₂O reagents. The formations of colored species are due to the presence of the tertiary amino group in it. It is based on the analogy of tertiary amine as given in scheme (Fig-6).

 

CONCLUSION:

The reagents utilized in the proposed methods are cheap, readily available and the procedures do not involve any critical reaction conditions or tedious sample preparation. The proposed visible spectrophotometric methods are validated as per ICH guide lines and possess reasonable precision, accuracy, simple, sensitive and can be used as alternative methods to the reported ones for the routine determination of DH depending on the need and situation.

 

ACKNOWLEDGEMENT:

The authors are very much thankful to the M/S Tychy Industries for providing gift sample of the drug and also thanks to University authorities for providing necessary facilities in this work.

 

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Received on 26.11.2011          Modified on 20.12.2011

Accepted on 29.12.2011         © RJPT All right reserved