Hydrophilic Interaction Chromatography-Tandem Mass ... - Springer Link

Arch Pharm Res Vol 31, No 9, 1205-1211, 2008 DOI 10.1007/s12272-001-1290-6

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Hydrophilic Interaction Chromatography-Tandem Mass Spectrometry of Donepezil in Human Plasma: Application to a Pharmacokinetic Study of Donepezil in Volunteers Eun Jeong Park, Hye Won Lee, Hye Young Ji, Hoe Yoon Kim1, Mann Hyung Lee2, Eun-Seok Park1, Kang Choon Lee1, and Hye Suk Lee Drug Metabolism and Bioanalysis Laboratory, College of Pharmacy, Wonkwang University, Iksan 570-749, Korea, 1College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea, and 2College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Korea (Received March 18, 2008/Revised June 5, 2008/Accetped August 13, 2008)

A selective, sensitive and rapid hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry was developed for the determination of donepezil in human plasma. Donepezil was twice extracted from human plasma using methyl tert-butyl ether at basic pH. The analytes were separated on an Atlantis HILIC Silica column with the mobile phase of acetonitrile: ammonium formate (50 mM, pH 4.0) (85:15, v/v) and detected by tandem mass spectrometry in the selective reaction monitoring mode. The calibration curve was linear (r = 0.9994) over the concentration range of 0.10-50.0 ng/mL and the lower limit of quantification was 0.1 ng/mL using 200 µL plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 2.7 to 10.5% and -10.0 to 0.0%, respectively. There was no matrix effect for donepezil and cisapride. The present method was successfully applied to the pharmacokinetic study of donepezil after oral dose of donepezil hydrochloride (10 mg tablet) to male healthy volunteers. Key words: Donepezil, HILIC-MS/MS, Human plasma

INTRODUCTION Donepezil [2-(1-Benzyl-piperidin-3-ylmethyl)-5,6-dimethoxy-indan-1-one] (Fig. 1) is used for the treatment of mild to moderate Alzheimer’s disease to slow down the degenerative progression. Donepezil is a selective and reversible acetylcholinesterase inhibitor which enhances the acetylcholine level of the brain and its cholinergic function. For the determination of donepezil in human plasma or urine samples, reversed-phase high-performance liquid chromatography (RP-HPLC) with UV (Tiseo et al., 1998; Yasui-Furukori et al., 2002), fluorescence (Haginaka and Seyama, 1992; Nakashima et al., 2006), and tandem mass spectrometry (MS/MS) detection (Thevis et al., 2006; Xie et al., 2006; Apostolou et al., 2007; Lee et al., 2007) were Correspondence to: Hye Suk Lee, Drug Metabolism & Bioanalysis Lab., College of Pharmacy, Wonkwang University, Shinyongdong, Iksan 570749, Korea Tel: 82-63-850-6817, Fax: 82-63-851-2013 E-mail: [email protected]

reported. The simultaneous determination of donepezil enantiomers were performed by chiral HPLC with MS (Matsui et al., 1995), MS/MS (Matsui et al., 1999), and fluorescence detection (Haginaka and Seyama, 1992). The sample preparation procedures for the extraction of donepezil from plasma and urine samples consist of liquidliquid extraction (Matsui et al., 1999; Yasui-Furukori et al., 2002; Nakashima et al., 2006; Xie et al., 2006; Apostolou et al., 2007; Lee et al., 2007). Those methods used a large amount of plasma samples (500-1000 µL plasma) in order to obtain the high sensitivity (Matsui et al., 1995, 1999; Yasui-Furukori et al., 2002; Nakashima et al., 2006; Xie et al., 2006; Lee et al., 2007) and the matrix effect in RP-HPLC with MS/MS detection was observed (Apostolou et al., 2007). Hydrophilic interaction liquid chromatography (HILIC) technique using bare silica or polar bonded phase and low aqueous/high organic mobile phase has been shown to be a valuable tool for MS analysis of the polar compounds in biological samples (Naidong, 2003; Paek et al., 2004; Hemstrom and Irgum, 2006; Ji et al., 2006, 2008;

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Jeong et al., 2007; McCalley, 2007; Dejaegher et al., 2008; Hao et al., 2008; Hsieh, 2008). There are some advantages of HILIC are compared to RP-HPLC: MS sensitivity improvement due to higher organic content in the mobile phase, higher flow rates and less matrix effect (Naidong, 2003; Hemstrom and Irgum, 2006; McCalley, 2007; Hao et al., 2008; Hsieh, 2008). To develop the rapid, robust and sensitive method for the quantification of donepezil in human plasma, HILICMS/MS method was described and validated using small volume of human plasma (200 µL). The present method has been successfully applied to the pharmacokinetic study of donepezil after oral administration of donepezil hydrochloride (10 mg) to humans.

MATERIALS AND METHODS Materials and reagents Donepezil hydrochloride (purity, 99.35%) and cisapride (purity, 99.48%; internal standard) were the gifts from Dong-A Pharm. Co. Ltd. (Yongin, Korea). Acetonitrile and methyl tert-butyl ether were HPLC grade and obtained from Burdick & Jackson Inc. (Muskegon, MI, USA). The other chemicals were of the highest quality available. Drug-free human plasma containing sodium heparin as the anticoagulant was obtained from healthy volunteers. Preparation of calibration standards and quality control samples Primary stock solutions of donepezil and cisapride (1 mg/mL) were prepared in acetonitrile. Working standard solutions of donepezil were prepared by diluting primary stock solution with acetonitrile. The internal standard working solution (25 ng/mL cisapride) was prepared by diluting an aliquot of primary stock solution with acetonitrile. All standard solutions were stored at ca 4oC in 20 mL scintillation vial in the dark when not in use. Human plasma calibration standards of donepezil, i.e., 0.1, 0.2, 0.5, 1.0, 5.0, 25.0, and 50.0 ng/mL, were prepared by spiking the working standard solutions into a pool of drug-free human plasma. Quality control (QC) samples at 0.1, 0.3, 20.0, and 40.0 ng/mL were prepared in bulk by adding 250 µL of the appropriate working standard solutions (4.0, 12.0, 800, and 1600 ng/mL) to drug-free human plasma (9.75 mL). The bulk samples were aliquoted (200 µL) into polypropylene tubes and stored at -80oC until analysis. Sample preparation 200 µL of human blank plasma, calibration standards and QC samples were mixed with 10 µL of internal standard working solution (25 ng/mL cispride) and 50 µL of 0.5 M NaOH. The mixtures were extracted twice with

E. J. Park et al.

1000 µL of methyl tert-butyl ether in 2.0 mL-polypropylene tubes by vortex-mixing for 2 min at high speed and centrifuged at 5000 × g for 5 min. 1700 µL of the organic layer was evaporated to dryness at 35oC for 20 min using vacuum concentrator (EZ-plus, Genevac, UK). The residues were dissolved in 100 µL of the mobile phase by sonicating for 2 min, centrifuged at 3000 × g for 5 min and transferred to injection vials. The aliquot (7 µL) was injected into the LC-MS/MS system.

HILIC-MS/MS analysis The LC-MS/MS system consisted of a Nanospace SI-2 pump, SI-2 column oven, an SI-2 autosampler (Shiseido, Tokyo, Japan) and a tandem quadrupole mass spectrometer (TSQ Quantum Access, ThermoFisher Scientific, CA, USA). The separation was performed on an Atlantis HILIC Silica column (5 µm, 3 mm i.d. × 50 mm, Waters, Milford, MA, USA) using a mixture of acetonitrile: ammonium formate (50 mM, pH 4.0) (85:15, v/v) at a flow rate of 0.5 mL/min. The column and autosampler tray were maintained at 50oC and 4oC, respectively. The analytical run time was 2.0 min. The electrospray ionization source settings for the analysis of donepezil and cisapride were as follows: spray voltage 4.0 kV; vaporizer temperature 250oC; capillary temperature 330oC; sheath gas pressure 35 psi; auxiliary gas pressure 10 psi. The tube lens offsets for donepezil and cisapride were 77 V and 74 V, respectively, in order to produce a strong protonated molecular ion [MH+] without formation of adduct and fragment ions. Fragmentation of MH+ for donepezil and cisapride was performed at collision energy of 34 V and 28 V by collision-activated dissociation with argon as the collision gas at a pressure setting of 1.5 on the instrument. Selective reaction monitoring (SRM) mode was employed for the quantification: m/z 380.0→91.1 for donepezil and m/z 466.2→184.0 for cisapride. The LCMS/MS system control and data processing were performed by the Xcalibar® software (ThermoFisher Scientific). Method validation Batches, consisting of triplicate calibration standards at each concentration, were analyzed on five different days to complete the method validation. In each batch, QC samples at 0.1, 0.3, 20.0, and 40.0 ng/mL were assayed in sets of five replicates to evaluate the intra- and inter-day precision and accuracy. The percentage deviation of the mean from true values, expressed as relative error (RE), and the coefficient of variation (CV) serve as the measure of accuracy and precision, respectively. The absolute and relative matrix effects and recoveries of donepezil and cisapride were assessed by analyzing three sets of standards at four concentrations (0.1, 0.3, 20.0, and 40.0 ng/mL) according to the approach of

HILIC-MS/MS Analysis of Donepezil in Human Plasma

Matuszewski et al.(2003). The absolute matrix effect for donepezil and cisapride was assessed by comparing mean peak areas of the analyte spiked at four concentrations into the extracts originating from five different lots of human blank plasma samples (set 2) to mean peak areas for neat solutions of the analyte in 85% acetonitrile in ammonium formate (50 mM, pH 4.0) (set 1). A value of 100% indicates no absolute matrix effect because the response in the solvent and in the plasma extracts were the same. A value of 100% indicates an ionization enhancement. The relative matrix effect was assessed by the variability in the peak areas of the analyte spiked post-extraction into five different plasma extracts (set 2) expressed as CVs (%). Recoveries of donepezil and cisapride were determined by comparing mean peak areas of analytes spiked before extraction into the same five different sources as set 2 (set 3) with those of the analytes spiked post-extraction into different blank plasma extracts at four concentrations (set 2). Five replicates of QC samples at each of low and high concentration (0.3 and 40.0 ng/mL, respectively) were subjected to three freeze/thaw cycles or the storage at room temperature for 2 h before processing to evaluate the freeze/thaw stability and room temperature storage stability. Post-extraction batch integrity was determined by batch reinjection.

Clinical application This method was applied to a pharmacokinetic study of donepezil after an oral administration of donepezil hydrochloride to male volunteers. The protocol was approved by an institutional review board at Research Institute for Pharmaceutical Sciences, Sungkyunkwan University (Suwon, Korea) and the informed consent was obtained from the subjects after explaining the nature and purpose details of the study. Thirty healthy male volunteers, fasted for 12 hours, received a single oral dose of donepezil hydrochloride (10 mg, Aricept®, Daewoong Pharm. Co., Hwasung, Korea) with 240 mL of water. Blood samples (5 mL) were withdrawn from the forearm vein at 0, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 12, 24, 48, 96, 144, 192, and 240 h post dosing, transferred to VacutainerTM tubes (sodium heparin, BD, NJ, U.S.A.) and centrifuged at 10000 × g for 5 min. The plasma samples were transferred to polypropylene tubes and stored at -70oC prior to analysis. The maximum concentration (Cmax) and the time to maximum concentration (Tmax) were determined by visual inspection from each volunteer’s plasma concentrationtime curve for donepezil. Area under the plasma concentration-time curve (AUC) was calculated by the linear trapezoidal method from 0 to 240 h.

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RESULTS AND DISCUSSION HILIC-MS/MS The positive electrospray ionization of donepezil and cisapride (internal standard) produced the protonated molecular ions (MH+) at m/z 380.0 and m/z 466.2, respectively, without any evidence of fragmentation and adduct formation. MH+ ions from donepezil and cisapride were selected as the precursor ion and subsequently fragmented in MS/MS mode to obtain the product ion spectra yielding useful structural information (Fig. 1). The prominent product ion for donepezil was m/z 91.1 (benzyl group) and the prominent product ion for cisapride was m/z 184.0 (5-chloro-4-amino-2-methoxy-phenylketone). The quantification of the analytes was performed using the SRM mode due to the high selectivity and sensitivity: m/z 380.0→91.1 for donepezil and m/z 466.2 →184.0 for cisapride. The selectivity of HILIC separations can be drastically changed by the judicious selection of column and the nature of buffer and mobile phase pH (Naidong, 2003; Hemstrom and Irgum, 2006; McCalley, 2007; Dejaegher et al., 2008; Hao et al., 2008; Ji et al., 2008). Donepezil showed good retention and peak shape using ammonium formate at pH 4.0. The higher organic content in the mobile

Fig. 1. Product ion mass spectra of (a) donepezil and (b) cisapride (internal standard)

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Fig. 2. SRM HILIC-MS/MS chromatograms of (a) a human blank plasma, (b) human plasma sample spiked with 0.1 ng/mL of donepezil and (c) a human plasma sample obtained 24 h after oral administration of donepezil hydrochloride (10 mg) to a male volunteers

phase of HILIC resulted in the sensitivity improvement due to the high efficiency of spraying and desolvation techniques. The use of 85% acetonitrile as mobile phase in HILIC-MS/MS analysis of donepezil resulted in the sensitivity enhancement of donepezil in MS/MS detection, and therefore, the plasma sample volume (200 µL) used in this study was smaller than that (500-1000 µL) of RPLC-MS/MS (Lee et al., 2007; Xie et al., 2006) to obtain the same limit of quantification (LOQ, 0.1 ng/mL). The typical HILIC-MS/MS SRM chromatograms of a blank human plasma and a human plasma sample spiked with donepezil at LOQ (0.1 ng/mL) were shown in Fig. 2. There was no interference peak at the retention times of donepezil (1.3 min) and cisapride (1.1 min) in the analysis of blank plasma samples obtained from forty humans, confirming the selectivity of the present method.

No column deterioration was observed after analysis of 800 human plasma samples. Sample carryover effect was not observed.

Linearity Calibration curves were obtained over the concentration range of 0.1 to 50.0 ng/mL for donepezil in human plasma. Linear regression analysis with a weighting of 1/concentration gave the optimum accuracy (RE: -10~5.8%) and precision (CV: ≤6.1%) of the corresponding calculated concentrations at each level (Table I). The low CV value (5.0%) for the slope indicated the repeatability of the method (Table I). Precision, accuracy and LOQ Table II shows a summary of intra- and inter-assay

Table I. Calculated concentrations of donepezil in calibration standards prepared in human plasma (n = 5) Statistical variable Mean (ng/mL) CV (%) RE (%)

Theoretical concentration (ng/mL) 0.10

0.20

0.50

1.00

5.00

25.0

50.00

0.09 6.10 -10.00

0.20 5.70 0.00

0.52 5.50 4.00

1.01 2.30 1.00

5.29 2.20 5.80

24.4 1.4 -2.4

50.40 0.50 0.80

slope

Intercept

r

0.5269 5.0000

0.0114

0.9994 0.0500

Table II. Precision and accuracy of donepezil in human plasma quality control samples Statistical variable Mean CV (%) RE (%)

Intra-batch (ng/mL, n = 5)

Inter-batch (ng/mL, n = 5)

0.10

0.30

20.0

40.0

0.10

0.30

20.0

40.0

0.09 7.90 -10.00

0.29 2.90 -3.30

19.3 3.1 -3.5

37.0 2.7 -7.5

0.09 10.5 -10.0

0.30 6.50 0.00

19.8 3.0 -1.0

37.2 4.1 -7.0


precision and accuracy data for donepezil QC samples. Both intra- and inter-assay CV values ranged from 2.7 to 10.5% at four QC levels. The intra-and inter-assay RE values for donepezil were -10.0 to 0.0% at four QC levels. These results indicated that the present method has the acceptable accuracy and precision. The LOQ was set at 0.1 ng/mL for donepezil using 200 µL of human plasma. Representative chromatogram at the LOQ is shown in Fig. 2b and the signal-to-noise ratio for donepezil are higher than 20.

Recovery The liquid-liquid extraction of donepezil from plasma and urine was performed by the use of hexane, mixture of hexane and isopropanol, methyl tert-butyl ether or ethyl acetate (Matsui et al., 1999; Yasui-Furukori et al., 2002; Nakashima et al., 2006; Thevis et al., 2006; Xie et al., 2006; Apostolou et al., 2007; Lee et al., 2007). Liquidliquid extraction of donepezil and cisapride twice using methyl tert-butyl ether at basic pH resulted in the selectivity and good recovery. The extraction recoveries of donepezil and cisapride (internal standard) were 86.4% and 93.0%, respectively (Table III). Matrix effect The absolute matrix effects, the ratio of the mean peak area of an analyte spiked post-extraction to the mean peak area of same analyte standards multiplied by 100, for donepezil and cisapride were 100.5 and 101.7%, respectively (Table III). This HILIC method resulted in less absolute matrix effect for donepezil although the matrix effect due to ion suppression was observed in RPLC-MS/ MS method (Apostolou et al., 2007). CVs of the peak

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areas of donepezil and cisapride spiked into each plasma extract originated from five human sources (set 2) were 5.6 to 8.3% for donepezil and 5.4 to 7.6% for cisapride (internal standard), suggesting that there was little relative matrix effect.

Stability The stability of processing (freeze–thaw and short-term storage at room temperature) and chromatography (reinjection) was assessed and shown to be of insignificant effect (Table IV). Three freeze-thaw cycles and a 2 hstorage at room temperature of QC samples at the low and high concentrations before analysis had little effect on the quantification. Extracted QCs and calibration standards were allowed to stand at 4oC for 24 hours prior to injection without affecting the quantification. Clinical application This method has been successfully applied to the anTable IV. Stability of samples (n = 5) Statistical variable

Three freeze/ 2 h at room thaw cycles temperature

Post-preparative stability (24 h at 4oC)

QC (ng/mL) Mean (ng/mL) CV (%) RE (%)

0.30 0.30 10.10 0.00

0.30 0.34 0.00 13.30

0.30 0.30 0.00 0.00

QC (ng/mL) Mean (ng/mL) CV (%) RE (%)

40.00 40.60 3.60 1.50

40.00 40.20 3.70 0.50

40.00 35.30 5.80 -11.80

Table III. Absolute matrix effect and recovery of donepezil and cisapride (internal standard) in five different lots of human plasma effect Nominal Absolute matrix (%)a concentration (ng/mL) donepezil cisapride 0.10 0.30 20.0 40.0 Mean

101.2 197.8 100.7 100.2 100.5

102.0 102.2 102.5 100.1 101.7

Recovery (%)b donepezil cisapride 87.3 87.9 86.8 83.6 86.4

91.0 94.7 94.6 91.9 93.0

a

Absolute matrix effect was expressed as the ratio of the mean peak area of an analyte spiked into plasma extract after liquidliquid extraction to the mean peak area of the same analyte standards in mobile phase multiplied by 100. b Recovery was calculated as the ratio of the mean peak area of an analyte spiked into plasma before liquid-liquid extraction to the mean peak area of an analyte spiked into plasma extract postextraction multiplied by 100.

Fig. 3. Mean plasma concentration-time plot of donepezil after an oral dose of donepezil hydrochloride (10 mg) to thirty male volunteers. Each point represents the mean ± S.D. (n = 30).

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alysis of 1200 plasma samples in pharmacokinetic study of donepezil. Representative chromatograms of the extract of a plasma sample obtained 24 h after oral administration of donepezil hydrochloride at a dose of 10 mg to human are shown in Fig. 2c. Mean plasma concentrationtime plot of donepezil obtained after a single oral dose of donepezil hydrochloride (10 mg) to thirty healthy male volunteers is shown in Fig. 3. Cmax, Tmax, AUC, and halflife of donepezil were 23.3 ± 5.2 ng/mL, 2.8 ± 1.2 h, 892.2 ± 254.6 ng · h/mL, and 73.2 ± 25.1 h, respectively. In conclusion, a selective, sensitive and reliable HILICMS/MS method has been successfully developed for the determination of donepezil in human plasma. Donepezil and cisapride were twice extracted from plasma samples using methyl tert-butyl ether at basic pH as sample preparation procedure. The present method demonstrated the acceptable selectivity, sensitivity (LOQ: 0.1 ng/mL), precision, accuracy and stability, and less matrix effect. This method was successfully applied to the determination of donepezil in human plasma samples obtained after an oral administration of donepezil hydrochloride (10 mg).

ACKNOWLEDGEMENTS This work was supported by the Korea Science and Engineering Foundation (KOSEF) NRL Program grant funded by the Korea Government (MEST) (R0A-2008000-20074-0).

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