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Donepezil hydrochloride, 2-[((1-benzylpiperidin-4-yl)methyl)]-5,6-dimethoxy-2,3-di-
hydoinden-1-one monohydrochloride (DP) (Fig. 1) is a centrally and selectively acting
acetylcholinesterase inhibitor. Donepezil has been reported to be effective in the treatment
of cognitive impairment and memory loss in patients with Alzheimer’s disease. It is well
tolerated when 5 mg of the drug is prescribed daily (1).
In clinical trials, significant correlations were found between the plasma concentra-
tion of donepezil and percentage of acetylcholinesterase inhibition. A 50 % inhibition of
acetylcholinesterase activity was obtained at a plasma drug concentration of 15.6 ng mL–1
and inhibition plateaus at the plasma concentration of donepezil higher than 50 ng mL–1
(2). Therefore, plasma drug concentration can be a useful tool to predict the clinical out-
come of donepezil in the treatment of Alzheimer’s disease.
403
Acta Pharm. 61 (2011) 403–413 Original research paper
DOI: 10.2478/V10007-011-0035-1
Determination of donepezil hydrochloride in human
plasma and pharmaceutical formulations by HPLC
with fluorescence detection
MOHAMMED A. ABONASSIF
MOHAMMED M. HEFNAWY
MOHAMED G. KASSEM
GAMAL A. E. MOSTAFA*
Pharmaceutical Chemistry Department
College of Pharmacy
King Saud University, P.O. Box 2457
Riyadh 11451, Saudi Arabia
Accepted September 7, 2011
A sensitive, isocratic reversed-phase high performance
liquid chromatographic method involving fluorescence
detection was developed for the determination of done-
pezil hydrochloride in tablets and in human plasma. Pin-
dolol was used as an internal standard. Good chromato-
graphic separation was achieved by using an analytical
column C18. The system operated at room temperature us-
ing a mobile phase consisting of methanol, phosphate buf-
fer (0.02 mol L–1) and triethyl amine (pH 3.5) (55 : 45 : 0.5,
V/V/V) at a flow rate 0.9 mL–1 min. The analyte and in-
ternal standard were extracted from human plasma via
liquid-liquid extraction. The proposed method was vali-
dated for sensitivity, selectivity, linearity, accuracy and pre-
cision. The calibration curve was linear over the range of
5–2000 ng mL–1 of donepezil with detection limit of
1.5 ng mL–1. Intra- and inter-day relative standard devia-
tions were less than 2.5 %. The method was found to be
suitable for quality control of donepezil hydrochloride
in bulk drug as well as in human plasma.
Keywords: donepezil hydrochloride, RP-HPLC, fluorescen-
ce detection, dosage form, plasma
* Correspondence; e-mail: gamal_most@yahoo.com
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Previous studies have reported quantification of donepezil in pharmaceutical pre-
parations (3–5) and in plasma (6, 7) by HPLC with UV detection. However, most of the
above mentioned methods have a common limitation of low sensitivity and long chro-
matographic run time. Further, LC-MS (8, 9) and capillary electrophoresis (CE) (10) were
presented for the determination of DP. Although LC-MS is selective and sensitive and
has been successfully applied to analysis of DP, both LC-MS and CE required expensive
instrumentation.
To the best of our knowledge, only one HPLC method with fluorescence detection
was developed (11). Fluorescence detection was employed because it can provide excellent
selectivity and sensitivity. The reported HPLC-FL method for the assay of DP in plasma
and microdialysate samples was developed using micellar liquid chromatography with
a short C30 column (11). Micellar liquid chromatographic technique (MLC) is used mainly
to enhance retention and selectivity of various solutes, which would otherwise be inse-
parable or poorly resolved. One of the main drawbacks of this technique is the reduced
efficiency of separation caused by the micelles (12, 13).
The main purpose of this study was to develop a sensitive, simple, and reliable me-
thod to quantitate donepezil hydrochloride in a relatively short time with high linearity.
Therefore, this study was focused on the development of a simple and rapid isocratic
RP-HPLC-FL method that can be employed for the routine analysis of donepezil hydro-
chloride in bulk drug formulations and in human plasma.
EXPERIMENTAL
Reagents and materials
Donepezil and pindolol were obtained from Sigma Chemical Co. (USA). HPLC-gra-
de methanol, analytical grade triethylamine and phosphoric acid were purchased from
BDH Chemicals (UK). Bidistilled water was purified using a Milli-Q plus cartridge puri-
fication system (Millipore, Waters, USA) to get ultra pure water of 18 mW. Aricept®tab-
lets, 5 and 10 mg, products of Eisai Co., Ltd (Japan) were obtained from the local market.
Human blood was obtained from male adult healthy volunteers and whole blood was
received from the blood blank unit of the King Khalid University Hospital (Riyadh, KSA).
It was kept frozen until use. This work was approved by the Deanship of Scientific Re-
search at the King Saud University.
Instrumentation and chromatographic conditions
The LC analysis was carried out on a Water HPLC system (USA) equipped with a
1500 series HPLC pump, operated in isocratic mode to deliver the mobile at a flow rate
of 0.9 mL min–1. A dual wavelength fluorescence detector (2475) and an autosampler
(717 plus) were used. The data was collected with an Empower pro Chromatography
Manager Data collection system. Chromatographic separations were performed on an
analytical column Phenyl Hypersil C18 (125 mm ´4.6 mm i.d. ´3mm particle diameter)
manufactured by Phenomenex (USA). All solutions were degassed by ultrasonication
(Technal, Brazil) and filtered through a 0.45-mm Millex filter (Millipore).
404
M. A. Abonassif et al.: Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with
fluorescence detection, Acta Pharm. 61 (2011) 403–413.
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The mobile phase consisted of methanol, 0.02 mol L–1 (pH 3.5) buffer phosphate
and triethylamine (55: 45: 0.5, V/V/V). It was filtered and degassed. Buffer phosphate
was prepared from monobasic sodium phosphate, triethylamine and phosphoric acid.
The samples (15 mL each) were injected with the aid of an auto-sampler. The fluores-
cence detector was set at 290 nm as the excitation wavelength and 315 nm as the emis-
sion wavelength.
Preparation of standard solutions
Stock solutions of both donepezil and pindolol as internal standard (IS) were prepa-
red by dissolving the appropriate amount of each compound in methanol to yield a con-
centration of 1 mg mL–1. Stock solutions were stable for at least two months when stored
in refrigerator, and no evidence of degradation of the analyte was observed on the chro-
matograms during that period. Working solutions of DP (10.0, 1.0 and 0.1 mgmL
–1) and
100 mgmL
–1 of IS were obtained by suitable dilutions of stock solutions with methanol.
Determination of donepezil in the pharmaceutical dosage forms
Commercially available formulations (Aricept®tablets) labeled to contain either 5
or 10 mg donepezil hydrochloride were analyzed. Ten tablets of each formulation were
weighed and then powdered. Powder samples, equivalent to 5 or 10 mg of DP, were pla-
ced in a 100-mL volumetric flask with the aid of methanol. The content of the flask was
vortexed for 3 min and sonicated for 15 min. The content in the flask was made up to the
volume with methanol. Aliquot was filtered and further diluted with the mobile phase
to obtained the final sample solution of 5 mgmL
–1 of DP. Donepezil was determined by
using external standard working solutions from pure reference DP run simultaneously.
Extraction of plasma sample and preparation of plasma quality control samples
Hundred microliters of human plasma were spiked with 15, 500 and 1000 ng of DP
in 2.0-mL disposable polypropylene microcentrifuge tubes. Each was vortexed for 30 s.
The solution was mixed with 600 mL of acetonitrile, vortexed at high speed for 1 min and
centrifuged at 10,000 rpm for 30 min. The supernatant was transferred to a 2.0-mL dis-
posable microcentrifuge tube and evaporated to dryness under a nitrogen stream. The
residue was reconstituted in 100 mL of mobile phase and 15 mL was injected into the
HPLC system for DP determination. Blank human plasma samples were processed in
the same manner using methanol instead of DP.
The quality control (QC) samples at three concentration levels (15, 500 and 1000 ng
mL–1 were prepared by spiking drug-free plasma with appropriate volumes of DP and
IS. Before spiking, the drug-free plasma was tested to make sure that there were no en-
dogenous interferences at the retention time of DP and IS.
Validation
Validation was preformed according to the criteria set in references 14–17.
Linearity range. – Calibration plots for the DP were prepared by diluting stock solu-
tions to yield seven concentration levels (5, 20, 100, 200, 500, 1000 and 2000 ng mL–1).
405
M. A. Abonassif et al.: Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with
fluorescence detection, Acta Pharm. 61 (2011) 403–413.
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Calibration curves were constructed using the observed analyte peak area ratio versus
the nominal concentration of analyte.
Precision and accuracy. – Intra-day precision was expressed through the relative stan-
dard deviation of five replicate assays of samples at three concentration levels. Inter-day
precision was determined by analyzing the same set of samples on five different days.
Accuracy was determined (using the data from precision assessment) as the close-
ness of spiked samples to the nominal value. The recovery was evaluated by comparing
the peak area of spiked analyte samples to pure analyte from the stock solution that was
injected directly into the HPLC system.
Accuracy and precision of the method were determined for DP according to the FDA
guidance for bioanalytical method validation (14).
Selectivity. – The selectivity of the assay was checked by analyzing six independent
blank plasma samples. Chromatograms obtained by analyzing human plasma samples
were compared with chromatograms obtained by analyzing human plasma samples spi-
ked with the analyte. Further, the selectivity of the assay was checked by analyzing 10
Ariecpt®Tablets samples. Chromatograms were compared with the chromatograms ob-
tained by analyzing the standard solution containing the drug.
Limit of detection and limit of quantification. – The limit of detection (LOD) and the
limit of quantification (LOQ) (14) were determined at 3.3 and 10 times the base-line noise,
respectively.
RESULTS AND DISCUSSION
Method development
Determination of DP in human plasma using HPLC with fluorescence detection us-
ing micellar liquid chromatography has been reported (11). The main drawback of this
technique is the reduced efficiency of separation caused by the micelles (12, 13). The pre-
sent work represents an HPLC method for the determination of DP in human plasma at
nano level. Table I. summarizes the general characteristics of the proposed HPLC method.
Chromatographic conditions were aimed to achieve efficient separation and resolu-
tion. Also, the response should be adequate with a sharp peak shape and a short run
time per analysis for both the analyte and IS. This includes selection of the mobile-phase,
406
M. A. Abonassif et al.: Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with
fluorescence detection, Acta Pharm. 61 (2011) 403–413.
Fig. 1. Chemical structure of: a) donepezil hydrochloride and b) pindolol.
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flow rate, column type and injection volume. Different ratios of methanol/water and
acetonitrile/water combinations were tried as the mobile phase, along with phosphate
buffer, on m-bonded silica C18. Use of Phenyl Hypersil C18 (150 ´3.9 mm ´4mm) chro-
matographic column helped fast separation and elution of the drug and IS. An optimum
mobile phase was very critical for their separation as they had similar retention mecha-
nisms and retention times. It was observed that a mobile phase consisting of methanol/
0.02 mol L–1 phosphate buffer (pH = 3.5) /triethylamine (55:45:0.5) was most appropriate
for good resolution, elution, and peak shape. Pindolol as an internal standard was easily
separated and eluted along with the analyte. Total chromatographic run time was 15 min
using 0.9 mL min–1 flow rate. There was no effect of IS on analyte recovery and sen-
sitivity of detection. Typical chromatograms of DP spiked in human plasma are shown in
Fig. 2. Retention times were 11.4 and 8.1 min, for DP and IS, respectively.
Peak characteristics
In order to determine adequate resolution and repeatability of the proposed method,
suitability parameters including the retention factor, selectivity, and resolution and peak
asymmetry were investigated and the results are abridged in Table I. The peak retention
factor (k') for both the drug and IS was calculated from k' = (tR–to)/to, where tRand to
are retention times of the peak of interest and the solvent front, respectively. The separa-
tion factor (a) was estimated from a=k'2/k'1where k'1and k'2are retention factors of
the drug and IS, respectively. Useful and practical measurement of peak shape, the peak
asymmetry factor, As, was calculated at 10 % of peak height. The resolution factor (Rs)
was calculated by Rs= 1/4 (a–1)(N1/2)[(k'/(1 + k')], where Nis the column plate num-
407
M. A. Abonassif et al.: Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with
fluorescence detection, Acta Pharm. 61 (2011) 403–413.
Table I. Analytical parameters for the determination of donepezil using the proposed methoda
Parameter
Calibration line
Slope ±SD 0.004 ±0.0003
Intercept ±SD 0.0179 ±0.0024
R0.998
Linearity range (ng mL–1) 5.0–2000.0
LOD (ng mL–1)a5.0
LOQ (ng mL–1)a1.5
Retention time for DP (min) 11.4
Retention time for IS (min) 8.1
Capacity factor (k') 5.01
Separation factor (a)1.5
Resolution factor (Rs) 3.02
Peak asymmetry factor at 10 % peak height 1.05
aValues are the mean of six measurements.
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ber and k' is the average retention factor for the two bands. The column plate number
was determined using the formula, N= 5.54 (tR/wh)2, where whis the bandwidth at 50 %
of peak height.
Validation of the method
The method was extensively validated as per the United States Food and Drug Ad-
ministration (FDA) guidelines.
Linearity, LOD and LOQ. – Excellent linear relationship was demonstrated between
the peak area ratio of DP and IS vs. plasma DP concentration over a range of 5.0–2000.0
ng mL–1. The mean linear regression equation of the peak ratio (y) versus drug concen-
tration (ng mL–1) in spiked plasma samples (g) showed the correlation coefficient R>
0.998 (y= 0.004g+ 0.0179). Good linearity of the calibration graphs and negligible scatter
of experimental points are evident from the values of the correlation coefficient and
standard deviation of the obtained data (18). Table I shows the statistical analysis of ex-
perimental data obtained by the least-squares treatment of the results. The LOD was 1.5
ng mL–1. The LOQ of the calibration graph was 5.0 ng mL–1(18).
Selectivity. – Analytical figures of merit for this method are shown in Fig. 2. Done-
pezil and IS (pindolol), were well separated under the HPLC conditions applied. Reten-
tion times were 11.4 ±0.1 and 8.1 ±0.1 min for donepezil and IS, respectively. This has
indicated appropriate selectivity of the elaborated procedure. Excipients commonly co-
-formulated with DP such as magnesium stearate, cellulose, starch, calcium hydrogen-
phosphate, colloidal silicon dioxide and coloring agents did not interfere with donepezil
determination, indicating high selectivity of the method. Also, no interfering peaks were
co-eluted with the compounds of interest (Figs. 3a and b), which could originate from
endogenous substances in human plasma.
Precision and accuracy. – The precision of the method was evaluated in terms of re-
peatability (intra-day) and intermediate precision (inter-day) (15–17). Three different con-
centrations of QC samples were analyzed in six independent series during the same day
and in different days; each sample was injected in triplicate. The RSD values of intra-
and inter-day studies for DP showed that the precision of the method was satisfactory
(Table II) with intra- and inter day RSDs less than 1.5–1.8 %. The results obtained for rela-
tive error (er) (15–17) at three concentrations are shown in Table II and were (±0.2–2.4 %).
408
M. A. Abonassif et al.: Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with
fluorescence detection, Acta Pharm. 61 (2011) 403–413.
EU
10
0.0
5
15
20
2
5
Time
(
min
)
0.0 2 4 6 8 10 12 14 16 18 20 22
IS
DP
Fig. 2. Chromatogram
of 500 ng mL–1 donepe-
zil (DP) from the formu-
lation with 15 mgmL
1of
pindolol (IS). First peak
is solvent fronting.
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Ruggedness. – Ruggedness of the HPLC method was evaluated by carrying out the
analysis using two different analysts and different instruments on different days. RSD
values of less than 2 % were obtained for repetitive measurements and operators. The
results indicated that the method is rugged enough.
Application of the method to pharmaceutical formulations and plasma
Reliability of the proposed method for donepezil quantification was assessed first
for its determination in water. Determination of donepezil in solutions (five replicates)
by direct HPLC or standard addition method gave average recovery values of 97.6–99.8 %
with relative standard deviation of 1.5–1.8 (Table II). This indicates high model accuracy
of the proposed method.
Results obtained for the analysis of DP in each formulation by the proposed and the
HPLC USP methods (19) are given in Table III.
409
M. A. Abonassif et al.: Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with
fluorescence detection, Acta Pharm. 61 (2011) 403–413.
EU
18
16
14
12
10
8
6
4
2
0
Time
(
min
)
0 2 4 6 8 10 12 14 16 18 20 22 24
b)
EU
0
1
2
3
4
5
6
7
Time ( min )
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
a
)
IS
DP
Fig. 3. Chromatogram of: a) blank human plasma (drug-free plasma), and (b) reference spiked with
5ngmL
–1 donepezil and 15 mg mL–1 pindolol (IS). First three peaks in Fig. 3b is the plasma back-
ground.
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Comparison the experimental means for the two methods was carried out using the
null hypothesis of |t|2at p= 0.05 and n= 5. It was found that |t|2was 1.7 and 1.5, which
was less than the tabulated value (|t|2= 3.36) (18). This indicate comparable accuracy of
the proposed method to that of as USP method (19). Comparison of the precision of the
proposed method with that of the USP method was also carried out using the two-tailed
410
M. A. Abonassif et al.: Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with
fluorescence detection, Acta Pharm. 61 (2011) 403–413.
Table II. Accuracy and precision data for donepezil in standard solution
Actual concentration
(ng mL–1)
Found concentration
(ng mL–1)a
RSD
(%)
er
(%)
5.00 4.88 ±0.0011.8 2.4
10.00 9.80 ±0.00 1.8 2.0
20.00 19.60 ±0.00 1.8 2.0
100.00 99.60 ±0.0021.7 0.4
500.00 499.00 ±0.01 1.6 0.2
1000.00 998.00 ±0.02 1.5 0.2
2000.00 1996.00 ±0.03 1.5 0.2
aMean ±SD, n=6.
Table III. Determination of DP means in pharmaceutical dosage forms
Dosage form Proposed method
found (mg)a
HPLC metod (19)
found (mg)a|t|2F-testb
Aricept®5mg 4.88 ±0.02 4.90 ±0.01 1.70 3.1
10 mg 9.80 ±0.02 9.79 ±0.02 1.60 2.8
aMean ±SD, n=6.
bThe tabulated value of tis 3.36 and of Fis 6.38.
Table IV. Accuracy and precision data for donepezil in spiked human plasma
DP Added concentration
(ng mL–1)
Found concentration
(ng mL–1)Recovery (%)aRSD (%)a
Within-a-day
15.0 13.8 92.2 6.5
500.0 472.5 94.5 5.0
1000.0 950.0 95.0 5.0
Between-days
15.0 13.7 91.4 6.5
500.0 470.0 94.0 6.0
1000.0 945.0 94.5 5.0
n=6
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F-test (18). It is clear that experimental F4,4 values were 3.1 and 2.8, which are obviously
lower than the tabulated value of F4,4 for p= 0.05 and n= 5 (6.38) (18). This proves com-
parable precision of both methods.
Recovery of donepezil was also determined by analysis of spiked plasma. As shown
in Table IV recovery ranged from 92.3 to 94.5 % in the linearity range of 5.0–2000.0 ng
mL–1 of DP. In human plasma samples with DP concentration 15–1000 ng mL–1 RSD was
less than 7 %.
Table V summarizes the characteristics of the reported HPLC methods with the de-
veloped one.
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M. A. Abonassif et al.: Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with
fluorescence detection, Acta Pharm. 61 (2011) 403–413.
Table V. A summary of the characteristics of the earlier reported HPLC methods and the improved
method
Stationary
phase
Analytical
range LOD Precision
(RSD, %)
Correlation
coefficient
(R)
Application Refe-
rence
C18
(Microsorb-MV) 10–60 mgmL
–1 10 mgmL
–1 0.5 0.9995
Pharmaceuti-
cal formula-
tions
4
C18
Uptispher ODB
(250´4.6 mm,
5mm)
0.35–0.64 mg mL–1 0.06 mgmL
–1 <0.31 0.9980
Pharmaceuti-
cal formula-
tions and
impurities
3
C18
STR ODS-II
(250´4.6 mm,
5mm)
3–90 ng mL–1 3ngmL
–1 7.3–7.6 0.9987 Human
plasma 6
Chiralcel OD
(chiral column) 0.05–2 mgmL
–1 20 ng mL–1 £10 0.994
Pharmaceuti-
cal formula-
tions and
human
plasma
7
C30
Develosil
Combi-RP-5
(250´4.6 mm,
5mm)
5–500 nmol L–1
10–500 nmol L–1
1–50 nmol L–1
2.5 nmol L–1
5.0 nmol L–1
0.5 nmol L–1
£9.3
0.999
1.0
0.999
Rat plasma
microdialysa-
te human
plasma
11
C18
Phenyl Hypersil
(125´4.6 mm,
3mm)
5–2000 ng mL–1 2.0 ng mL–1 £6.5 0.998
Pharmaceuti-
cal formula-
tions and
human
plasma
This
paper
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CONCLUSIONS
In a conclusion, a simple, sensitive and reliable RP-HPLC-FL method for measuring
DP in human plasma and pharmaceutical formulations has been developed and valida-
ted. The low volume of plasma needed, the simplicity of separation procedure, and the
short run time make this method suitable for quick and routine analyses. Hence, it can
be recommended for routine quality control and for pharmacokinetic studies.
Acknowledgments. – The authors extend their appreciation to the Deanship of Scientific Research
at the King Saud University for funding the work through the research group project No. RGP-
-VPP-037.
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SA@ETAK
Odre|ivanje donepezil hidroklorida u humanoj plazmi i ljekovitim oblicima
pomo}u HPLC s detekcijom fluorescencije
MOHAMMED A. ABONASSIF, MOHAMMED M. HEFNAWY, MOHAMED G. KASSEM i GAMAL A. E. MOSTAFA
Ovaj rad opisuje HPLC metodu odre|ivanja donepezil hidroklorida (DP) u tableta-
ma i u ljudskoj plazmi u nano podru~ju. Postavljena je osjetljiva metoda izokrati~ne HPLC
s fluorescencijskom detekcijom. Kao unutarnji standard upotrebljen je pindolol. Dobro
kromatografsko odjeljivanje postignuto je primjenom analiti~ke kolone C18. Radna tem-
peratura bila je sobna, a kao mobilna faza upotrebljena je smjesa metanola, fosfatnog
pufera (0,02 mol L–1) i trietilamina (pH 3,5) (55 : 45 : 0.5, V/V/V). Analit i unutarnji stan-
dard su ekstrahirani iz ljudske plazme ekstrakcijom teku}e-teku}e. Predlo`ena metoda
je validirana s obzirom na selektivnost, podru~je linearnosti, ispravnost i preciznost. Ka-
libracijska funkcija bila je linearna u podru~ju od 5-2000 ng mL–1 donepezila, a granica
detekcije iznosila je 2 ng mL–1. Relativna standardna devijacija za repetabilnost i interme-
dijarnu preciznost bila je manja od 2,5 %. Metoda je primjenljliva u kontroli kvalitete
ljekovitih formulacija s DP-om i u pra}enju DP-a u ljudskoj plazmi.
Klju~ne rije~i: donepezil, RP-HPLC, dozirani oblik, plazma
Pharmaceutical Chemistry Department, College of Pharmacy, King Saud University, P.O. Box 2457
Riyadh11451, Saudi Arabia
413
M. A. Abonassif et al.: Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with
fluorescence detection, Acta Pharm. 61 (2011) 403–413.
Unauthenticated
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