RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom.

2006; 20: 31933198 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.2718

Rapid and sensitive determination of donepezil in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study
Zhiyong Xie1, Qiongfeng Liao2*, Xinjun Xu1, Meicun Yao1, Jinzhi Wan1 and Dan Liu1
1 2

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China College of Chinese Traditional Medicine, Guangzhou University of Traditional Chinese Medicine, Guangzhou, China

Received 2 June 2006; Revised 28 August 2006; Accepted 30 August 2006

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of donepezil in human plasma samples. Diphenhydramine was used as the internal standard. The collision-induced transition m/z 380 ! 91 was used to analyze donepezil in selected reaction monitoring mode. The signal intensity of the m/z 380 ! 91 transition was found to relate linearly with donepezil concentrations in plasma from 0.120.0 ng/mL. The lower limit of quantication of the LC/MS/MS method was 0.1 ng/mL. The intra- and inter-day precisions were below 10.2% and the accuracy was between S2.3% and R2.8%. The validated LC/MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 5 mg donepezil hydrochloride. The non-compartmental pharmacokinetic model was used to t the donepezil plasma concentration-time curve. Maximum plasma concentration was 12.3 W 2.73 ng/mL which occurred at 3.50 W 1.61 h post-dosing. The apparent elimination half-life and the area under the curve were, respectively, 60.86 W 12.05 h and 609.3 W 122.2 ng h/mL. LC/MS/MS is a rapid, sensitive and specic method for determining donepezil in human plasma samples. Copyright # 2006 John Wiley & Sons, Ltd.

Alzheimers disease accounts for about 50% of clinically diagnosed dementia.1 Alzheizmers dementia is a neurodegenerative disease characterized by progressive cognitive impairment. Mild to moderate Alzheimers dementia is usually treated with donepezil hydrochloride, {2,3-dihydro5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]methyl]1H-inden-1-one} hydrochloride (Fig. 1), to slow down the degenerative progression of the disease and the patients cognitive impairment.2 Donepezil is an acetylcholinesterase inhibitor which enhances the acetylcholine level of the brain and its cholinergic function. In humans, donepezil is metabolized mainly by the hepatic cytochrome P-450 2D6 and 3A4 isozymes. Elimination of donepezil from the blood is characterized by a doseindependent elimination half-life of about 70 h.3,4 Because plasma donepezil concentrations are related linearly to acetylcholinesterase inhibition,5 plasma donepezil concentration is a useful tool to predict donepezil efcacy.

*Correspondence to: Q. Liao, Department of Pharmaceutical Analysis, College of Chinese Traditional Medicine, Guangzhou University of Traditional Chinese Medicine, 12 Jichang Road, Guangzhou 510405, China. E-mail: liaoqf2075@yahoo.com Contract/grant sponsor: Laboratory Open Fund of the Sun Yat-sen University; contract/grant number: KF2005009.

Previous investigations have shown that donepezil in biological uids can be detected using high-performance liquid chromatography (HPLC) equipped with a ultraviolet (UV),612 uorescence (FL),13,14 or mass spectrometric (MS)1519 detector. The HPLC/UV method has been used to determine donepezil in human plasma,12,13,15,19 but the method is insensitive (!1.0 ng/mL) and requires a large volume of blood sample (!1.0 mL).612 Radwan et al.12 have reported a stereoselective HPLC/UV assay for determining donepezil enantiomers. The assay has a low limit of quantication (LLOQ) of about 50 ng/mL and requires at least 20 min to complete the run. LC/MS1518 also has been used to analyze donepezil in different biological uids. The LLOQ of LC/MS is about 0.1 ng/mL and the run time for each sample is longer than 7 min. Matsui et al.19 have developed a sensitive and enantioselective LC/MS/MS assay for donepezil enantiomers in human plasma using a deuterium-labeled internal standard (IS). The LLOQ is 0.0206 ng/mL. The purposes of the present study were to develop and validate a LC/MS/MS method for determining donepezil in human plasma using diphenhydramine (Fig. 1) as the IS.2023

Copyright # 2006 John Wiley & Sons, Ltd.

3194 Z. Xie et al.

O N
CH3 N CH3

A
100 Relative Abundance 80 60 40 20 0

F: + c Full ms2 380.10@-30.00 [ 30.00-500.00] 91.0

O N m/z 91

H3CO OCH3

H3CO OCH3

II

Figure 1. Structures of donepezil (I) and diphenhydramine (II), the internal standard.

105.0 100

151.0 200

243.0 m/z 300

380.1 380.7 400 500

EXPERIMENTAL Chemicals and reagents

Donepezil hydrochloride (purity !99.9%) and diphenhydramine hydrochloride (purity !99.8%) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC-grade methanol, acetonitrile and formic acid were purchased from Tedia Company Inc. (Beijing, China). All other reagents were of analytical grade. Blank human plasma was obtained from the Guangzhou Blood Donor Service (Guangzhou, China). Ultrapure water was obtained from a Milli Q-plus system (Billerica, MA, USA).

B
Relative Abundance

100 80 60 40 20 0

F: + c Full ms2 256.00@-10.00 [ 30.00-300.00] 167.00

CH3 N CH3 O m/z 167

164.96 152.08 60 100 140 180 m/z 220

256.13 260 300

Figure 2. Product ion mass spectra of [MH] ions of (A) donepezil and (B) diphenhydramine (internal standard). pressure of approximately 1.2 mTorr (1 Torr 133.3 Pa). The optimized collision energies chosen for donepezil and IS were 33 and 10 eV, respectively. Figure 2 shows the product ion mass spectra of the [MH] ion of donepezil and IS.

Equipment
The LC/MS/MS system consisted of a Surveyor MS pump, a Surveyor autosampler (ThermoFinnigan, USA) and a ThermoFinnigan TSQ Quantum triple quadrupole mass spectrometer (San Jose, CA, USA) equipped with an electrospray ionization (ESI) source. Data acquisition was performed with Xcalibur 1.3 software (ThermoFinnigan, USA). Peak integration and calibration were performed using LCQuan software (ThermoFinnigan, USA).

Preparation of stock and working solutions

The stock solutions of donepezil (400 mg/mL) and diphenhydramine (400 mg/mL) were prepared in methanol and serially diluted to produce a 4 mg/mL stock solution in methanol/water (50:50, v/v). The donepezil stock solution was then diluted to give working solutions of 1, 2, 8, 20, 50, 100 and 200 ng/mL in methanol/water (50:50, v/v). Diphenhydramine working solution (200 ng/mL) was also prepared by diluting the 400 mg/mL stock solution of diphenhydramine with methanol/water (50:50, v/v). All stock solutions and working solutions were stored at 48C.

Chromatographic conditions
Chromatographic separation was achieved using an Aquasil C18 column (150 mm 2.1 mm i.d., 5 mm; Thermo Finnigan, USA) with a 4.0 mm 2.0 mm i.d. Security Guard C18 (5 mm) guard column (Phenomenex, Torrance, CA, USA). The mobile phase consisted of methanol/acetonitrile/1% formic acid (70:10:20, v/v/v), delivered at a ow rate of 0.3 mL/ min. The column temperature was maintained at 258C.

Preparation of calibration standards and quality control (QC) samples

Calibration curves were prepared by spiking 50 mL of the appropriate working solution to 500 mL blank human plasma. Effective donepezil concentrations in plasma samples were 0.1, 0.2, 0.8, 2.0, 5.0, 10.0, and 20.0 ng/mL. The QC samples used in the validation and during the pharmacokinetic (PK) study were prepared in the same way as the calibration standards. The nominal donepezil plasma concentrations of QC samples were 0.2, 2.0 and 20.0 ng/mL. The spiked plasma samples (standards and QC samples) were extracted in each analytical batch along with the unknown samples.

Mass spectrometric conditions

The mass spectrometer was operated in the positive mode. Quantication was performed using selected reaction monitoring (SRM) of the transitions of m/z 380 ! 91 for donepezil and m/z 256 ! 167 for diphenhydramine (IS), respectively, with a scan time of 0.3 s per transition. The tuning parameters were optimized for donepezil and IS by infusing a solution, containing 1 mg/mL of each analyte, at a ow rate of 10 mL/min into the mobile phase (0.3 mL/min) using a post-column T connection. The optimal MS parameters obtained were as follows: the spray voltage was 3500 V with a source collision-induced dissociation (CID) voltage of 10 V, the heated capillary temperature was 3508C. Nitrogen was used as the sheath gas (50 psi) and auxiliary gas (12 psi). Argon was used as the collision gas at a
Copyright # 2006 John Wiley & Sons, Ltd.

Sample preparation
To a 500-mL aliquot of plasma sample, 50 mL of IS (200 ng/mL diphenhydramine), 50 mL of methanol/water (50:50, v/v) and 0.5 mL of 10 mmol/L phosphate buffer (pH 14) were
Rapid Commun. Mass Spectrom. 2006; 20: 31933198 DOI: 10.1002/rcm

LC/MS/MS determination of plasma donepezil

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added. The sample was briey mixed and ethyl acetate (3 mL) was added. The mixture was vortex-mixed for approximately 1 min, then shaken on a mechanical shaker for 15 min. After centrifugation at 3000 rpm for 10 min, the upper organic layer was removed and evaporated to dryness at 408C under a gentle stream of nitrogen. The dry residue was reconstituted in 200 mL of the mobile phase, then vortexmixed. A 10-mL aliquot of the resulting solution was injected onto the LC/MS/MS system.

compared with those QC samples freshly prepared, and the percentage concentration deviation was calculated.

Application to PK study
The method was applied to determine the plasma concentrations of donepezil from a clinical trial study in which 20 healthy male volunteers each received orally a tablet containing 5 mg donepezil hydrochloride. Venous blood samples (5 mL) were withdrawn from each volunteer and placed into heparinized tubes according to the following time schedule: 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, 24.0, 48.0, 96.0, 144.0 and 192.0 h post-dosing. Blood samples were centrifuged immediately at 3500 rpm for 10 min to obtain the plasma. The plasma samples were labeled and kept frozen at 208C until analysis. The volunteers were all adult Chinese who were selected after completing a thorough medical, biochemical and physical examination. Informed consent was obtained from all subjects after explaining the aims and risks of the study. The study protocol was approved by the Human Investigation Ethical Committee at the School of Pharmaceutical Sciences at Central South University, Changsha, China. The plasma concentration-time proles obtained from the experimental subjects were analyzed by non-compartmental analysis using the TopFit 2.0 software package (Thomae GmbH, Germany). Maximum plasma concentration (Cmax) and the time-to-maximum concentration (Tmax) were estimated by visual inspection of semi-logarithmic plots of the concentration-time curves. The area under the curve (AUC0 t) was calculated using the linear-trapezoidal rule, with extrapolation to innity (AUC0 1) from the last detectable concentration using the terminal elimination rate constant (ke) calculated by linear regression of the nal loglinear part of the drug concentration-time curve. Apparent elimination half-life (t1/2) was calculated as t1/2 0.693/ke.

Method validation
The method was validated by linearity, LLOQ, accuracy and precision. To evaluate linearity, plasma calibration curves were prepared and assayed in duplicate on three separate days. Accuracy and precision were also assessed by determination of QC samples using six replicate preparations of plasma samples at three concentration levels (Table 1) for donepezil on three validation days. Accuracy was expressed by relative error (RE) and precision by relative standard deviation (RSD). The LLOQs, dened as the lowest concentration at which both precision and accuracy were less than or equal to 20%, were evaluated by analyzing samples which were prepared in six replicates as follows: spiking 50 mL of the standard solution, containing 1 ng/mL donepezil, to 500 mL of blank human plasma. For the determination of extraction recovery, blank human plasma was processed according to the sample preparation procedure as described above. The organic layer was evaporated to dryness, and dry extracts were reconstituted in the mobile phase adding appropriate standards at concentrations corresponding to the nal concentration of the extracted plasma samples. These spike-after-extraction samples represented 100% recovery. The extraction recoveries of donepezil were determined by comparing the mean peak areas of six extracted low, medium and high QC samples to mean peak areas of six spike-after-extract samples at the same concentrations. Recovery of IS was also evaluated by comparing the mean peak areas of six extracted medium QC samples to mean peak areas of six reference solutions spiked in extracted plasma samples of the same concentrations. Stability of processing (three freeze/thaw cycles, benchtop for 4 h), chromatography (re-injection) and sample storage (208C for 45 days) was assessed by analyzing triplicates (n 3) of QC samples (at the concentrations of 0.2, 2.0 and 20.0 mg/mL for donepezil), which were exposed to different time and temperature conditions. The results were Table 1. Accuracy and precision for donepezil at the plasma concentrations of the LLOQ
Nominal plasma conc. (ng/mL) 0.10 Mean measured conc. (ng/mL) 0.09 0.10 0.12 0.10 0.10 0.10 RE (%) 2.05 Intra-day RSD (%) 6.90

RESULTS AND DISCUSSION Mass spectrometry

Electrospray ionization (ESI) was used in the present study because ESI mass spectra of donepezil are more sensitive than atmospheric pressure chemical ionization (APCI) spectra in detecting donepezil (data not shown). Following the selection of the ESI source, donepezil and diphenhydramine, respectively, were found to form protonated molecules [MH] at m/z 380 and 256 that could be used as precursor ions for the analysis of donepezil. Figure 2 shows the product ion mass spectra of the [MH] ion of donepezil and diphenhydramine; fragment ions at m/z 91 and 167 were observed for donepezil and diphenhydramine, respectively. Based on the results of these studies, the transitions of m/z 380 ! 91 for donepezil and m/z 256 ! 167 for diphenhydramine were used to analyze the human plasma donepezil samples in selected reaction monitoring (SRM) mode. Several LC/MS/MS parameters were optimized to obtain the maximum sensitivity for the m/z 380 ! 91 transition. The collision energy in this study was set at 30 eV because the collision behavior of the [MH] ion of donepezil was found to depend strongly on this parameter. In contrast, the capillary temperature and the spray voltage
Rapid Commun. Mass Spectrom. 2006; 20: 31933198 DOI: 10.1002/rcm

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3196 Z. Xie et al.

Relative Abundance

were left unchanged at the recommended values of 3508C and 3.5 kV, respectively, because they were not found to affect the detection sensitivity of the analytes signicantly. Other LC/MS/MS parameters including the source CID and the collision pressure also were maintained at the auto-tuned values since they did not signicantly affect the spectral behavior of the analytes.

A
100 50 m/z 380 m/z 91 NL: 1.00E4

0 100 m/z 256 m/z 167 50 0 0.0 0.2

NL: 1.00E5

HPLC mobile phase

The retention times of donepezil and diphenhydramine in the HPLC chromatogram were 2.0 and 2.2 min, respectively. The HPLC mobile phase consisted of a solution of methanol, acetonitrile and 1% formic acid because: (1) the composition of the solvents could reduce the run time of each sample to about 2.8 min which was much shorter than most methods reported for donepezil analysis;19 (2) the use of 70% methanol provided a low background chromatogram and a rapid separation of donepezil and diphenhydramine; (3) the addition of a small amount of formic acid in the mobile phase improved analyte detection and sensitivity; and (4) a symmetrical peak was achieved only after adding 10% acetonitrile to the mobile phase.

0.6

1.0

1.4

1.8

2.2

2.6

Time (min)

B
100 m/z 380 m/z 91 I, 1.98 NL: 2.01E3

Relative Abundance

50

0 100 m/z 256 m/z 167 50 0 0.0 0.2

II, 2.17 NL: 3.07E5

0.6

1.0

1.4

1.8

2.2

2.6

Time (min)

Sample preparation
Attempts were made to prepare the plasma samples using either protein precipitation or liquid-liquid extraction (LLE). Protein precipitants such as methanol, acetonitrile, ethanol and acetone produced sample extracts that reduced the sensitivity of the mass spectral detector (data not shown). Different LLE extraction conditions also were evaluated in order to reduce extraction time and improve donepezil recovery. These included the use of different organic solvents and aqueous pH modiers such as NH3 buffer (10 mmol/L, pH 8), 0.5 mol/L Na2CO3, 1.0 mol/L NaOH, and 10 mmol/L phosphate buffer (pH 14) in the extraction. Phosphate buffer (pH 14) was found to be the best aqueous pH modier for the LC/MS/MS method; plasma analyte and IS in phosphate buffer could be extracted by ethyl acetate with a high recovery (!65% for donepezil and IS) and a small standard deviation (SD, 7.4%).

C
100 m/z 380 m/z 91 I, 1.97 NL: 4.08E4

Relative Abundance

50

0 100 m/z 256 m/z 167 50 0 0.0 0.2

II, 2.17 NL: 3.02E5

0.6

1.0

1.4

1.8

2.2

2.6

Time (min)

Figure 3. Representative SRM chromatograms for (A) drugfree plasma; (B) plasma (500 mL) spiked with 0.1 ng/mL of donepezil and 20 ng/mL of internal standard (IS); and (C) a volunteers 96 h plasma sample after an oral dose of 5 mg donepezil hydrochloride. I: donepezil; II: diphenhydramine (IS).

Selectivity
Selectivity was assessed by comparing the chromatograms of six different batches of blank plasma with the chromatograms from the same plasma samples but that had been spiked with 0.1 ng/mL of donepezil (LLOQ concentration). Figure 3 shows a typical chromatogram of the plasma blank, the spiked plasma sample, and the 96 h plasma sample from a healthy volunteer after receiving donepezil. As shown in Fig. 3, no endogenous chemical peak was found to interfere with the analysis of the analytes. comparing the peak areas of donepezil (0.2, 2.0, and 20 ng/ mL) and the IS (20 ng/mL) in ve QC samples spiked postextraction with standard solutions that had been prepared in the same way as the QC samples except that water was substituted for drug-free plasma. For donepezil, the absolute MEs were found to be between 96.8% and 103.8%. For the IS, the absolute ME was 3.7%. These observations indicated that no endogenous substances signicantly affect the ionization of these analytes. The relative standard deviations (RSDs) of absolute MEs at different concentrations of donepezil in the ve batches of human plasma were below 8.2%. For the IS, the RSD of absolute ME was below 6.2%. These data also indicate that the relative MEs for donepezil and IS are small in this assay.

Matrix effects
Although matrix-matched calibration standards were used in the analytical procedure, matrix effects due to co-eluted endogenous substances might reduce the selectivity, sensitivity, accuracy or precision of the assay. Therefore, an assessment of matrix effects (MEs) was conducted according to the procedure of Matuszewski et al.:24 the absolute MEs of ve different batches of blank plasma were evaluated by
Copyright # 2006 John Wiley & Sons, Ltd.

Linearity of calibration curve and LLOQ

Peak area ratio versus donepezil concentration plots were analyzed by the least-squares linear regression with a
Rapid Commun. Mass Spectrom. 2006; 20: 31933198 DOI: 10.1002/rcm

LC/MS/MS determination of plasma donepezil

Concentration (ng/mL)

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Table 2. Accuracy and precision results for donepezil in human plasma (3 days, 6 replicates per day)
Nominal plasma Mean measured Intra-day Inter-day conc. (ng/mL) conc. (ng/mL) RE (%) RSD (%) RSD (%) 0.20 2.00 20.00 0.20 2.02 20.56 2.25 0.83 2.79 8.58 3.55 3.84 10.18 7.51 4.33

15

10

0 0 48 96 144 192

Time (h)

weighting of 1/x . The straight line could be described by the equation, y 1.187 103 4.749 102x with r2 0.9937, where y represents the ratios of the donepezil peak area to that of diphenhydramine and x represents the plasma concentrations of donepezil. Based on the results of this analysis, peak area ratio is found to be linearly related to plasma donepezil concentrations ranging from 0.1 to 20.0 ng/mL. The LLOQ was established at 0.1 ng/mL for donepezil, which was sufcient for clinical PK studies in humans following oral administration of donepezil. The precision and accuracy values corresponding to the LLOQ are shown in Table 1.

Figure 4. Mean plasma concentration-time prole of donepezil after an oral dose of 5 mg donepezil hydrochloride to 20 healthy volunteers (each point represents mean SD).

Table 3. Pharmocokinetic parameters of donepezil in humans after receiving a single oral dose of 5 mg donepezil hydrochloride
Parameter Cmax (ng/mL) Tmax (h) t1/2 (h) ke (L/h) AUC0 t (ng h/mL) AUC0 1 (ng h/mL) Oral of 5 mg 12.3 2.73 3.50 1.61 60.86 12.05 0.11 0.002 609.3 122.2 989.6 233.7

Precision and accuracy

Precision and accuracy of the assay were determined by performing the complete analytical runs of replicate QC samples (n 6) at three concentrations on the same day and on the three consecutive days. The data from these QC samples were examined by a one-way analysis of variance (ANOVA). The intra-day and inter-day precisions were less than 10.2% for each QC level of donepezil. The accuracy, determined from QC samples, was within 2.8% for each QC level. The results are summarized in Table 2. an oral dose of 5 mg donepezil hydrochloride. Figure 4 shows the mean donepezil plasma concentration versus time curve. Table 3 lists the pharmacokinetic parameters of donepezil. The Cmax, Tmax, t1/2, ke, AUC0 192 h, and AUC01 of donepezil were, respectively, 12.3 2.73 ng/mL, 3.50 1.61 h, 60.86 12.05 h, 0.11 0.002 L/h, 609.3 122.2 ng h/ mL, and 989.6 233.7 ng h/mL. The pharmacokinetic parameters in this study are very similar to those reported in previous studies.1518,2527

Extraction recovery and stability

Extraction recoveries for donepezil at 0.2, 2.0 and 20.0 ng/mL were 66.9 7.4%, 65.5 3.1% and 66.7 2.5% (n 3), respectively. The RSDs of extraction recoveries were better than 7.4% for the entire standard concentration ranges, indicating consistent recoveries of donepezil from plasma samples. Extraction recovery for the IS (20 ng/mL) was 72.0 2.6% (n 3). The stability of donepezil in human plasma was also investigated under a variety of storage and process conditions. The analyte was found to be stable (RE < 8.6%) in human plasma after three freeze (208C)/thaw (room temperature) cycles. The analyte also was stable after being left on the laboratory bench for 4 h at room temperature (RE < 8.8%) and in reconstitution solutions at room temperature for 24 h (RE < 7.3%). Therefore, frozen plasma samples could be kept at 208C for at least 45 days (RE < 5.8%) without signicant donepezil degradation.

CONCLUSIONS
An LC/MS/MS method with an ESI interface was developed and validated for determining donepezil in human plasma. The method had several advantages over the previously reported methods; it was rapid, sensitive, robust and highly selective. Also, this method has a short run time (about 2.8 min) and requires only a simple sample preparation procedure. In our hands, more than 150 samples were analyzed in one day. The method was successfully applied to determine plasma donepezil concentrations in a pharmacokinetic study involving healthy Chinese volunteers. The pharmacokinetic prole in this study was similar to those reported in previous studies.

Acknowledgements
The authors thank the Laboratory Open Fund (KF2005009) of the Sun Yat-sen University for providing nancial support. We also thank Dr. F. Law of the Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada, for editing the manuscript.
Rapid Commun. Mass Spectrom. 2006; 20: 31933198 DOI: 10.1002/rcm

Application of the method to a PK study in healthy volunteers

The validated LC/MS/MS method was used to obtain the plasma kinetic prole of donepezil in humans after receiving
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Rapid Commun. Mass Spectrom. 2006; 20: 31933198 DOI: 10.1002/rcm