Bio-analytical Method for the Pre-clinical Estimation of Donepezil Hydrochloride in Rabbit Plasma by RP-HPLC

Venna R Surya Anusha¹*, Koppala RVS Chaitanya², Rajeswari Aleti¹,

Kolapalli Venkata Ramana Murthy¹

¹AU College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India, 530003.

²Department of Pharmacology, Vignan Institute of Pharmaceutical Technology, Kapujaggrajupeta, Duvvada, Visakhapatnam, India, 530049.

*Corresponding Author E-mail: rajeswarianusha@gmail.com

ABSTRACT:

A simple, reliable and sensitive RP-HPLC bioanalytical method with PDA as detector was developed for the estimation of donepezil HCl in the rabbit plasma and validated. The biological samples were extracted and analysed using loratadine as internal standard. The sample analysed with Inertsil ODS 150mm x 4.6mm, 5.0µm particle size column using 70:30 v/v of 0.1% trifluoroacetic acid and acetonitrile mobile phase at flow rate of 1mL/min. All the parameters validated were within the limits proposed in bioanalytical method validation guidelines. The linearity was observed in the range of 5-200ng/mL with LOD of 0.9ng/mL. The method was observed to have short analysis time of 6 min and also found to be successful in studying the pharmacokinetic parameters of donepezil HCl.

KEYWORDS: HPLC, Donepezil HCl, Biological estimation, Rabbit plasma, RP-HPLC.

INTRODUCTION: 

Donepezil hydrochloride is a (±)-2, 3-dihydro-5, 6-dimethoxy-2-[[1- (phenylmethyl)-4-piperidinyl]methyl]-1H-inden-1- one hydrochloride with a molecular formula of C₂₄H₂₉NO₃ · HCl. It is an official drug in United States Pharmacopoeia and Indian Pharmacopoeia.^1,2 It is used to treat the Alzheimer’s associated dementia. In Alzheimer’s disease, the concentration and activity of acetylcholine, a neurotransmitter needed for memory and learning is reduced. Donepezil HCl exerts reversible inhibition of the cholinesterase enzyme, which hydrolyses the acetylcholine thereby increasing acetylcholine function^3,4. It may not cure the disease but improves the memory and awareness by returning the balance of neurotransmitters in brain. Depending on the severity of condition, 5 mg, 10 mg tablets and sustained release 23 mg tablets are administered once daily^5,6

Figure 1: Chemical Structure of Donepezil HCl

Bioanalytical methods of drug estimation were important in pre-clinical and clinical studies of the efficacies of newly developed dosage forms. In an attempt to meet the requirement, different methods were developed and reported for the bioanalytical estimation of donepezil HCl viz., HPLC/UV method⁷, electrophoresis/UV⁸ method, LC/MS⁹ method and HPLC/Fluorescence method¹⁰ are reported for human plasma sample analysis of donepezil HCl. However, very few methods are available for estimation of donepezil HCl in animal models including HPLC/fluorescence in rabbit plasma¹⁰ and HPLC/PDA in rat plasma¹¹. Though rabbit is the mostly used animal model of pre-clinical analysis, Fluorescence detector technique has limitations of loss of recognition capability, photoinstability, interference of pH, oxygen levels of sample and short lifespan of fluorophores. HPLC/PDA detector has proved to be successful in estimation of many drugs^12-19. Hence our aim is to develop a simple, sensitive, cost-effective, validated RP-HPLC method for quantification of donepezil HCl in rabbit plasma using PDA detector using liquid-liquid microextraction process.

CHEMICALS AND REAGENTS:

Donepezil Hydrochloride was supplied as gift sample by Dr. Reddy’s Laboratories. Donepezil HCl 23mg tablets (ARICEP) manufactured by Eisai Pharmaceuticals India Pvt. Ltd (Ramky Pharma City, Visakhapatnam, India) were purchased from the local registered medical store. Acetonitrile (HPLC grade) was procured from Merck Limited (Mumbai, India). Other chemicals used were purchased from Sigma Aldrich and were of analytical grade. Water was produced by Millipore Milli-Q Plus water treatment system (Millipore Bedford Corp., Bedford, MA, USA).

EXPERIMENTAL ANIMALS:

The study was conducted in accordance with institutional guidelines of animal ethics committee with approved protocol no.83/1074/PO/Re/S/05/CPCSEA. All the experimental animals (Newzealand rabbit; weighing 2-3kgs) were housed in cages at temperature of 20-22C with RH of 65% maintained on light/dark cycle. The feed and water were supplied ad libitum throughout the acclimatization and study period.

INSTRUMENTATION:

Method development and analysis was carried out in Waters Alliance HPLC (e2695) equipped with Waters PDA detector (2998) with Inertsil ODS 150mm x 4.6 mm, 5.0µm particle size column operated in an isocratic mode at flow rate of 1.0mL/minute, coupled to PDA detector. The analysis was carried at ambient temperature with Loratadine as internal standard. 10µL injection volume was fixed for all the samples with the mobile phase of 70:30v/v of 0.1% trifluoroacetic acid and acetonitrile. The determination was performed in range of 200-400nm with specific wavelengths detection at 261.9nm and 257.2nm respectively for donepezil HCl and loratadine as shown in figure 2.

Figure 2: Detection wavelength of donepezil HCl and loratadine HCl

Method Development:

Preparation of standard stock and working standards:

20mg of the drug (donepezil HCl/loratadine) was weighed and taken in a 100ml volumetric flask. The drug was dissolved and made up to volume with triple distilled water to form a 200µg/mL primary stock solution. The secondary stock solution of 40µg/mL was prepared by diluting 20mL of the primary stock to 100 mL. 1mL and 2mL of secondary stock solution was further diluted separately to 100mL in a volumetric flask to 400ng/mL and 800ng/mL concentration solution respectively. All the dilutions were made with triple distilled water. The final stock solution of donepezil HCl (400ng/mL) was added in volume of 25, 50, 125, 250, 375, 500, 625 and 750 to 200µL rabbit plasma along with 500µL loratadine (100ng/mL concentration) and 500µL acetonitrile. And further diluted to 2mL volume with triple distilled water to get the working standard solution of donepezil HCl with concentrations: 5, 10, 25, 50, 75, 100, 125, 150ng/mL respectively.  200ng/mL concentration solution was prepared in similar procedure with final stock solution of (800ng/mL).

Extraction procedure:

The mixtures were vortexed for 2minutes and then centrifuged at 10,000rpm for 10minutes. Supernatant plasma was collected using a micropipette and 10µL was injected into the column by Hamilton microsyringe. The data analysis was performed with Masslynx™ Sofware. The chromatogram was quantified in terms of the peak area ratio of donepezil HCl to an internal standard. All the determinations are performed in triplicate and the average values are reported.

METHOD VALIDATION:^20-22

Specificity/selectivity:

Different plasma lots were analysed to make sure that no interferences were present in the chromatographic peak at the retention time of donepezil HCl The acceptance criteria were that the interferences if produced should have <20% area response.

Accuracy and precision:

The accuracy of the analytical method was estimated by analysing three replicates of donepezil HCl samples at four different QC level concentrations (lower limit of quantification (LLOQ), low quality control (LQC), medium quality control (MQC), High quality control (HQC)). 5, 25, 100, 150ng/mL were used as LLOQ, LQC, MQC, HQC respectively. The acceptance criteria of accuracy for the standard concentration were 90-110% from nominal value except for LLOQ which can be 80-120%. The deviation of the actual value from the mean was expressed as the relative error (%RE) as a measure for accuracy. %RE was calculated from Eq. 1.

The four-level quality control samples were analysed on different runs to assess the intra and inter-assay precision and the relative standard deviation of the result was calculated. According to the USP monograph of the donepezil HCl, the %RSD should not be more than 2%.

                     Mean conc. – Actual conc.

% RE = -------------------------------------- x 10…… Eq.-1

                            Actual conc.

Recovery:

The efficiency of donepezil HCl and internal standard extraction (recovery) from human plasma was determined by comparing responses of actual samples and plasma spiked samples at equivalent concentrations. Recovery was determined at three quality control levels (LQC, MQC, HQC) with the same concentration as used for precision using Eq.2. The peak area ratio of donepezil HCl in plasma and peak area ratio of donepezil HCl in the mobile phase at the same concentration were compared and expressed as percentage values. The recovery values should be reproducible and consistent.

Percentage recovery

Peak area of drug extracted from plasma

=--------------------------------------- X 100 …. Eq.-2

            Peak area of drug in Mobile plasma

Pharmacokinetic Study:

The developed method was applied to investigate the pharmacokinetic studies of donepezil HCl in rabbit after oral administration of pure drug. After dosing, the blood samples were collected from marginal ear vein with 24 gauge surgical needle at regular time intervals of 0 (pre-dose), 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, and 80hours. The blood was collected into EDTA coated microcentrifuge rubes and later centrifuged at 2000rpm for 10 minutes to separate plasma and stored at -20^ᵒC. 200µL mL of the plasma sample collected was taken in centrifuge tube along with 500µL of internal standard solution (Loratadine solutionwith 100ng/mL concentration), 500µL acetonitrile and 800µL distilled water to get a final volume of 2mL. The mixture was vortexed for 2 minutes and then centrifuged at 10,000rpm for 10minutes to separate the precipitated proteins. The clear supernatant was collected and 10µL was injected.

Pharmacokinetic Model:^23,24

The pharmacokinetic parameters were calculated based on standard non-compartmental technique using an excel spreadsheet (MS Office 2013).  Various pharmacokinetic parameters such as Peak plasma concentration (C_max), Time of peak plasma concentration (T_max), Area under plasma concentration -time curve (AUC), Elimination rate constant (k_el), Absorption rate constant (k_a)_, Apparent elimination half-life (t_1/2), Mean residence time (MRT), Apparent total body clearance (Cl/F), Apparent volume of distribution (V_d/F) were estimated.

RESULTS AND DISCUSSION:

A sensitive method for estimation of donepezil HCl in rabbit plasma was developed by modifying the earlier reported method. The change of mobile phase composition from 50% methanol to 30% acetonitrile has shown good results along with a reduction in organic solvent usage and cost. An acceptable retention time with a good peak shape was obtained from the Inertsil ODS column which is evident from the tailing factor. All the chromatogram has shown tailing factor less than 1.5 which meets the requirement specified in donepezil HCl USP monograph. Loratadine has similar chromatographic behaviour as donepezil HCl without interference producing nearer R_t values and peak symmetry and is hence selected as the internal standard. Among the different aqueous phases studied, 0.1% TFA showed good sensitivity and peak symmetry (figure 3). Very low TFA concentrations produced unaccepted peak symmetry. Hence 0.1% TFA (pH 3) at a ratio of 70:30 v/v with acetonitrile was used for better separation of donepezil HCL and loratadine bands. Donepezil HCL and loratadine have shown retention times of 2.9 and 5.9 min respectively under the specified chromatographic conditions as shown in the Figure. 3. Total analysis time of test and internal standard closes in 6 minutes allowing short and quick analysis.

Calibration curve linearity:

The linearity was established in the range of 5-200 ng/mL. The data of peak area ratio calculation is shown in Table 1. A good linear relation was found between the peak area ratio of donepezil HCl to loratadine and the concentration of donepezil HCl as determined by linear regression. It has shown a correlation coefficient (r) of 0.99995 and the regression line equation as y = 0.0075x - 0.00051 as shown in Figure 4. All the standard concentrations in the linearity range have shown an accuracy between 97-101% which lies between the accepted range of 90-110%. The LOD was observed to be 0.9ng/mL.

Figure 3: HPLC chromatogram of donepezil HCl and loratadine in rabbit plasma

Table 1: Concentration vs. peak area ratios for donepezil HCl in rabbit plasma

Concentration of donepezil HCL (ng/mL)	Peak area		Ratio	% cv
	Donepezil HCL	Loratidine HCl (IS)
5	2548	68789	0.037	0.013
10	5115	68598	0.075	0.009
25	12578	68745	0.183	0.014
50	25575	68985	0.371	0.005
75	37895	68154	0.556	0.005
100	51154	68240	0.75	0.003
125	63987	68226	0.938	0.009
150	76985	68357	1.126	0.16
200	102874	68974	1.491	0.13

Figure. 4: Calibration curve of donepezil HCl in rabbit plasma

Accuracy and precision:

Accuracy and precision estimation for the four levels were evaluated in triplicate and are expressed as %RE and %RSD respectively as shown in Table 2. The %RSD values at all the four quality control levels are <2% and the %RE was not more than 5% suggesting an accuracy of 95%. Hence, both theparameters are within the range of the suitability requirements specified in the donepezil HCl USP monograph. Hence, the analytical method was accurate and precise.

Specificity:

In all the plasma blanks, no interferences were seen in the chromatogram near or at the retention time and hence the response at the retention time of donepezil HCl was less than 20% of LLOQ response as shown in Table 3 and the retention time was 2.9minutes in all the cases.

Recovery:

The percentage recovery of donepezil HCl in plasma samples of concentrations 25, 100, 150ng/mL was found to be 99.79, 100.04 and 98.46% respectively as shown in Table 4.

Table 2: Intra-assay and inter-assay accuracy and precision of donepezil HCl in rabbit plasma (n=3)

Assay type	Level	Actual conc. (ng/mL)	Mean measured conc. (ng/mL)	s.d.	Precision (%RSD)	Accuracy (%RE)
Intra assay	LLOQ	5	5.05	0.06	1.10	1.00
	LQC	25	24.62	0.35	1.42	1.50
	MQC	100	99.87	0.16	0.16	0.13
	HQC	150	152.26	2.14	1.41	1.51
Inter assay	LLOQ	5	5.10	0.08	1.58	2.10
	LQC	25	25.20	0.02	0.07	0.80
	MQC	100	99.65	0.43	0.44	0.35
	HQC	150	156.64	0.41	0.26	4.43

Table 3: Specificity of donepezil HCl in rabbit plasma (n=3)

S. No	LLOQ		% interference
	Peak Area	RT
1	2598	2.97	Nil
2	2545	2.97	Nil
3	2558	2.96	Nil
4	2564	2.96	Nil
5	2587	2.92	Nil
6	2649	2.93	Nil

Table 4: Recovery of donepezil HCl from pre-analyzed rabbit plasma (n=3)

Drug	QC level	Conc. of drug added (ng/mL)	% Recovery
Donepezil HCl	Low	25	99.79
	Medium	100	100.04
	High	150	98.46

Pharmacokinetic study in rabbits:

The proposed method was successfully implemented for the pharmacokinetic study of donepezil HCl after oral administration in rabbits. The calibration curve range fitted the range of estimated samples. The various pharmacokinetic parameters calculated were presented in the Table. 5. Mean± s.d plasma concentration versus time profile is shown in Figure. 5

a

b

Figure 5. a) plasma concentration-time profile of donepezil HCl (Mean ± s.d, n=3) b) representative chromatogram of plasma sample obtained after 1 hour of post oral drug administration.

Table 5: Pharmacokinetic parameters of pure donepezil HCl and RDT (mean±s.d., n=4)

Pharmacokinetic parameters	Units	Pure drug
Cmax	ng/mL	78.89
Tmax	h	3.0
kel	1/h	0.0433
ka	1/h	1.44
t1/2	h	16.01
AUC0-60	ng/mL.h	1317.45
AUC0-∞	ng/mL.h	1412.56
AUMC0-t	ng/mL.h^2	24073.7
AUMC0-∞	ng/mL.h^2	30111.9
MRT	h	21.32
Cl	L/h	2.12
Vd	L	49.06

CONCLUSION:

The present study was aimed to develop easy and accurate method for donepezil HCl drug estimation in bioanalytical samples using simple extraction method. The proposed method of HPLC/PDA with 70:30 v/v of 0.1% trifluoroacetic acid and acetonitrilehas shown a linearity range of 5-200 ng/mL. The composition of mobile phase has very low ratio of organic phase ensuring an economical and eco-friendly procedure. The applicability of developed method was also confirmed by the pharmacokinetic study of pure drug administration in rabbit as oral suspension. Our study provides the information to evaluate pharmacokinetic parameters of donepezil HCl in the rabbits.

CONFLICT OF INTEREST:

The authors have no conflict of interest regarding this investigation.

REFERENCES:

1. Indian Pharmacopeia 2007, Volume -2. The Indian Pharmacopoeia Commission, Ghaziabad, Government of India, Ministry of Health and Welfare; 2007.

2. US Pharmacopeia and National Formulary. United States Pharmacopeial Convention, Inc.; 2006. Available at: https://www.uspnf.com/sites/default/files/usp_pdf/EN/USPNF/revisions/donepezilhydrochloride.pdf.

3. Rogers SL and Friedhoff LT. The efficacy and safety of donepezil in patients with Alzheimer’s disease: results of a US multicentre, randomized, double-blind, placebo-controlled trial. The Donepezil Study Group. Dementia, 1996; 7: 293-303.

4. Burt T. Donepezil and related cholinesterase inhibitors as mood and behavioral controlling agents. Current psychiatry reports. 2000; 2: 473-478.

5. US Pharmacopeia and National Formulary. United States Pharmacopeial Convention, Inc.; 2006. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/nda/97/020690Orig1s000rev.pdf.

6. United States Food and Drug Administration. Aricept prescribing information. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2012/020690s035,021720s008,022568s005lbl.pdf.

7. Yasui-Furukori N, Furuya R, Takahata T, Tateishi T. Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection. Journal of Chromatography B. 2002; 768(2): 261-265.

8. Bhateria M, Ramakrishna R, Pakala DB, Bhatta RS. Development of an LC–MS/MS method for simultaneous determination of memantine and donepezil in rat plasma and its application to pharmacokinetic study. Journal of Chromatography B. 2015; 1001: 131-139.

9. Noetzli M, Ansermot N, Dobrinas M, Eap CB. Simultaneous determination of antidementia drugs in human plasma: Procedure transfer from HPLC–MS to UPLC–MS/MS. Journal of Pharmaceutical and Biomedical Analysis. 2012; 64:16-25.

10. Mohamed FA, Khashaba PY, Shahin RY, El-Wekil MM. Determination of donepezil in spiked rabbit plasma by high-performance liquid chromatography with fluorescence detection. Royal Society Open Science. 2019;6(1):181476.

11. Krishna KV, Saha RN, Singhvi G, Dubey SK. Pre-clinical pharmacokinetic-pharmacodynamic modelling and biodistribution studies of donepezil hydrochloride by a validated HPLC method. RSC Advances. 2018;8(44):24740-24749.

12. Bagyalakshmi J, VijayarajS, Sindhu, Ravi TK. Method Development and Validation of Erythrosine (E127) Using RP-HPLC Coupled With PDA Detector. Asian J. Research Chem. 2008; 1(2): 95-96.

13. TentuNageswara Rao, Muralidhar Reddy Avuthu, Venkata Reddy B, Murthy SNVS. Forced degradation study of Terbinafine HCl by HPLC with PDA detector. Asian J. Research Chem. 2016; 9(11): 561-565.

14. TentuNageswara Rao, Vijayalakshmi A, Karri Apparao, Krishnarao N. A New Analytical Method Validation and Quantification of OlmesartanMedoxomil and its related impurities in bulk drug product by HPLC. Asian J. Pharm. Tech. 2017; 7(3): 147-152.

15. Satya Raga Devi A, Ashutosh Kumar S, Saravanan J, ManidipaDebnath, Greeshma V, Sai Krishna N, Naga Madhusudhan Rao Ch. A New RP-HPLC Method Development for Simultaneous Estimation of Metformin and Gliclazide in Bulk as well as in Pharmaceutical Formulation by using PDA Detector. Research J. Pharm. and Tech. 2014; 7(2): 142-150.

16. Ashutosh Kumar S, ManidipaDebnath, Seshagiri Rao J.V.L.N., GowriSankar D. Development and Validation of a Sensitive RP-HPLC method for Simultaneous Estimation of Rosuvastatin and Fenofibrate in Tablet Dosage form by using PDA Detector in Gradient Mode. Research J. Pharm. and Tech. 2016; 9(5): 549-554.

17. MeruguManasa, VijeyAanandhi M. Stability Indicating Method Development and Validation of Semaglutide by RP-HPLC in Pharmaceutical substance and Pharmaceutical Product. Research J. Pharm. and Tech 2021;14(3):1385-1389

18. Prashanthi Y, TentuNageswara Rao, Yellapu Srinivas. Method Development and Validation of Alectinib Drug by RP-HPLC in Bulk and Pharmaceutical Dosage Form. Asian J. Pharm. Ana. 2018; 8(4): 186-190.

19. Balaswami B, Ramana PV, Rao BS, Sanjeeva P. A New Simple Stability-Indicating RP-HPLC-PDA Method for Simultaneous Estimation of Triplicate Mixture of Sofosbuvir, Velpatasvir and Voxilaprevir in Tablet Dosage Form. Research Journal of Pharmacy and Technology. 2018;11(9):4147-56.

20. US Pharmacopeia and National Formulary. (USP31-NF26) <621>Chromatography. United States Pharmacopeial Convention, Inc. 2006; 29(5): 152.

21. Amruta S. Kadam, Nayana V. Pimpodkar, Puja S.Gaikwad, Sushila D. Chavan. Bioanalytical Method Validation. Asian J. Pharm. Ana. 2015; 5(4): 219-225.

22. R. B. Saudagar, P. G. Thete. Bioanalytical Method Validation: A Concise Review. Asian J. Res. Pharm. Sci. 2018; 8(2):107-114. Advaita B. Patel. Analytical Method Validation: Collation between International uidelines. Asian J. Research Chem. 2017; 10(6): 857-866.

23. Chung M. Computation of model-independent pharmacokinetic parameters during multiple dosing. Journal of Pharmaceutical Science, 1984; 73: 570-571.

24. Yamaoka K, Nakagawa T and Uno T. Statistical moments in pharmacokinetics. Journal of Pharmacokinetics and Biopharmaceutics 1978; 6: 547-558.

Received on 05.11.2021            Modified on 08.08.2022

Accepted on 10.06.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(11):5207-5212.

DOI: 10.52711/0974-360X.2023.00844