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Hydrophilic interaction liquid chromatography–tandem mass spectrometry for the determination of adefovir in human plasma and its application to a pharmacokinetic study
Journal of Chromatography B, 878 (2010) 2111–2116
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Hydrophilic interaction liquid chromatography–tandem mass spectrometry for the determination of adefovir in human plasma and its application to a pharmacokinetic study Zhili Xiong, Yi Zhang, Feng Qin, Ting Qin, Shuyan Yang, Famei Li ∗ Department of Analytical Chemistry, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
a r t i c l e
i n f o
Article history: Received 18 March 2010 Accepted 18 June 2010 Available online 26 June 2010 Keywords: Adefovir HILIC–MS/MS Human plasma Pharmacokinetic study
a b s t r a c t A selective, rapid and sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for the first time to determine adefovir in human plasma and applied to a pharmacokinetic study. Plasma samples were prepared by protein precipitation with methanol followed by a further cleaning using dichloromethane. The chromatographic separation was carried out on an ACQUITY UPLCTM BEH HILIC column with the mobile phase of methanol–water–formic acid (85:15:0.2, v/v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The method was rapid with a run time of 3 min per sample. The linear calibration curves were obtained in the concentration range of 1.02–102 ng/mL (r2 ≥ 0.99) with the lower limit of quantification (LLOQ) of 1.02 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 12% and the accuracy (relative error, R.E.) was from 0.6% to 3.2% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of adefovir in healthy volunteers after oral administration of adefovir dipivoxil tablet. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Adefovir, 9-(2-phosphonylmethoxyethyl) adenine (Fig. 1a) has broad-spectrum activity against human immunodeficiency virus, herpes viruses, hepatitis B viruses and adenoviruses [1]. Unfortunately, adefovir exhibited low oral bioavailability in both animals [2] and humans [3]. The bioavailability of adefovir has been substantially improved by using the bis-pivaloyloxymethyl ester of adefovir (adefovir dipivoxil) as a prodrug with enhanced lipophilicity and in vitro activity [4]. However, dose-limiting nephrotoxicity such as proximal renal tubular dysfunction in approximately 17% of patients has been associated with the use of adefovir dipivoxil [5]. Therefore, therapeutic drug monitoring is always necessary to maximize the antiviral efficacy and minimize the toxicity during adefovir dipivoxil chemotherapy. Adefovir is the primary circulating metabolite identified after oral administration of adefovir dipivoxil [6]. The development of a sensitive and specific method to determine adefovir in human plasma is necessary and valuable for pharmacokinetic study and therapeutic drug monitoring of adefovir dipivoxil. Analysis of adefovir in preparations by high-performance liquid chromatography
∗ Corresponding author. Tel.: +86 24 2398 6289; fax: +86 24 2398 6289. E-mail address: [email protected] (F. Li). 1570-0232/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2010.06.024
(HPLC) with ultraviolet detection has been reported [7]. Methods using pre-column derivatization with chloroacetaldehyde to form highly fluorescent derivatives followed by separation on reservedphase ion-pairing HPLC [3,6,8] were reported for the determination of adefovir in human plasma. The pretreatment of samples was tedious and time-consuming, and those methods were not sensitive enough for evaluating the pharmacokinetics of adefovir dipivoxil with LLOQ higher than 10 ng/mL. Several sensitive highperformance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) methods [9–12] have been developed using electrospray ionization (ESI) for the measurement of adefovir in human plasma or serum. Although those methods were sensitive for the pharmacokinetic study of adefovir, long analysis time (longer than 8 min) precluded their use in therapeutic drug monitoring. Hydrophilic interaction chromatography (HILIC) is a method where polar stationary phase (bare silica or derivatized silica) is used in conjunction with a low aqueous/high organic mobile phase. It has been proved to be a powerful way to separate polar compounds in biological samples with reversed retention compared to the traditional reversed-phase liquid chromatography (RPLC) [13–17]. In view of LC–MS analysis, the higher organic content in the mobile of HILIC resulted in higher sensitivity, flow rate and less matrix effect. In this study, a HILIC–MS/MS method was described for the first time to determine adefovir in human plasma, which facilitates the determination with the total run time of 3.0 min. The method was
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Fig. 1. Product ion spectra of adefovir (a) and pidotimod (I.S.) (b).
fully validated and applied to the pharmacokinetic study in healthy volunteers after oral administration of 10 mg adefovir dipivoxil in tablets. 2. Materials and methods 2.1. Reagents and chemicals Reference standard of adefovir (99.6% purity) was kindly provided by the Medicinal Chemistry Department of Shenyang Pharmaceutical University. Pidotimod (internal standard (I.S.), 99.5% purity, Fig. 1b) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, PR China). Methanol of HPLC grade was obtained from Tedia (Fairfield, OH, USA). Formic acid (HPLC grade) was purchased from Dikma (Richmond Hill, NY, USA). Water was purified by redistillation and filtered through a 0.22 m membrane filter before use. 2.2. Apparatus and operation conditions 2.2.1. Liquid chromatographic conditions The chromatography was performed on an ACQUITY UPLCTM system (Waters Corp., Milford, MA, USA). An ACQUITY UPLCTM BEH HILIC column (2.1 × 50 mm I.D., 1.7 m) was employed for the separation at 40 ◦ C. The mobile phase was composed of methanol–water–formic acid (85:15:0.2, v/v/v). The flow rate was set at 0.30 mL/min. The autosampler was conditioned at 4 ◦ C and the injection volume was 20 L using partial loop mode for sample injection. 2.2.2. Mass spectrometric conditions The mass spectrometric detection was carried out on a Micromass® Quattro microTM API triple-quadrupole tandem mass spectrometer (Waters Corp.) equipped with ESI interface. The ESI source was set in positive ionization mode. The quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z 274.4 → 136.0, 274.4 → 162.0, 274.4 → 226.1 for adefovir and m/z 245.0 → 133.8 for I.S. respectively, with scan time of 0.20 s per transition. The optimal MS parameters were as follows: capillary voltage 3.5 kV, cone voltage 25 V, source temperature 110 ◦ C and desolvation temperature 450 ◦ C. Nitrogen was used as the desolvation and cone gas with a flow rate of 550 and 30 L/h, respectively. Argon was used as the collision gas at a pressure of approximately 0.255 Pa. The optimized collision energy values for adefovir and I.S. were 25 and 13 eV, respectively. All data collected in centroid mode was acquired and processed using MassLynxTM NT 4.1 software with QuanLynxTM program (Waters Corp., Milford, MA, USA).
2.3. Preparation of standards and quality control samples Stock standard solutions of adefovir and I.S. were prepared in water at the concentration of 102 and 101 g/mL, respectively. The I.S. solution was prepared by dilution with methanol to 202 ng/mL. Adefovir stock solution was serially diluted with methanol to provide working standard solutions at desired concentrations. Another stock solution of adefovir for the preparation of quality control (QC) samples was prepared independently with a concentration of 101 g/mL. All the solutions were stored at 4 ◦ C and brought to room temperature before use. The calibration standards were prepared by evaporating 50 L of working standard solutions to dryness and then fully mixing with 500 L of blank plasma. The effective concentrations in standard plasma samples were 1.02, 2.04, 5.10, 10.2, 20.4, 51.0, 102 ng/mL. One calibration curve was constructed on each analysis day using freshly prepared calibration standard samples. The QC samples were prepared in bulk with a similar procedure as that for standard samples and aliquots were stored at −20 ◦ C. The LLOQ, low, mid and high concentrations of QC samples were 1.02, 2.53, 7.07 and 85.8 ng/mL. The standard samples and QC samples were extracted on each analysis day with the same procedures for plasma samples as described below. 2.4. Plasma sample preparation The I.S. solution (50 L) was pipetted into 2.0-mL polypropylene micro-centrifuge tube and evaporated to dryness under a gentle stream of nitrogen at 40 ◦ C. The residue was vortex-mixed with 500 L of plasma for 30 s followed by addition of 1.0 mL of methanol to each tube. The mixture was vortex-mixed for 60 s and centrifuged at 11,200 × g for 15 min. The supernatant was transferred into another tube and dichloromethane was added to remove the possible impurity. After vortex-mixing for 60 s and centrifugation at 2000 × g for 10 min the upper layer was evaporated to dryness under a gentle stream of nitrogen at 40 ◦ C. The residue was reconstituted in 50 L of methanol–water (85:15, v/v) and filtered through a 0.22 m membrane filter. An aliquot of 20 L was injected into the HILIC–MS/MS system for analysis. 3. Method validation The method was validated for selectivity, linearity, precision, accuracy, extraction recovery, matrix effect and stability according to FDA guidance for validation of bioanalytical methods [18]. Validation runs were conducted on three consecutive days. The peak area ratios of adefovir to I.S. from QC samples were interpolated
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from the calibration curve on the same day to give the concentration of adefovir. 3.1. Selectivity The selectivity was investigated by comparing chromatograms of six different batches of blank plasma from six subjects to those of corresponding standard plasma samples spiked with adefovir and I.S. and plasma sample after oral dose of adefovir dipivoxil tablets. 3.2. Linearity and lower limit of quantification Calibration curves were constructed by assaying standard plasma samples at seven concentrations in the range of 1.02–102 ng/mL with weighted (1/x2 ) least squares linear regression. According to FDA guidance, the LLOQ is defined as the lowest amount of an analyte in a sample that can be quantitatively determined with acceptable precision and accuracy, which are a relative standard deviation (R.S.D.) of 20% and a relative error (R.E.) within ±20%. 3.3. Precision and accuracy The intra-day precision and accuracy were evaluated by a replicate analysis of QC samples of adefovir on the same day. The validation run consisted of two sets of calibration standard samples and six replicates of QC samples at three concentrations. For determining the inter-day accuracy and precision, analysis of three batches of QC samples was performed on three consecutive days. The precision was expressed as R.S.D. and the accuracy as R.E. 3.4. Extraction recovery and matrix effect The extraction recovery of adefovir was determined by comparing the peak areas obtained from blank plasma samples spiked with the analyte before extraction with those from blank plasma samples to which the analyte was added after extraction. This procedure was repeated for five replicates at three QC concentration levels of 2.53, 7.07 and 85.8 ng/mL. The matrix effect was measured by comparing the peak response of sample spiked post-extraction with that of pure standard solution dried directly and reconstituted with the same mobile phase. The extraction recovery and matrix effect of I.S. were also evaluated using the same procedure. 3.5. Stability The stability of dilution solution of adefovir was evaluated at room temperature for 6 h by comparing the peak areas with those of freshly prepared dilution solutions. The stability of stock solution was evaluated for 30 days by comparing the peak area per unit mass of analyte in the stock solution stored at 4 ◦ C with that in freshly prepared stock solution. The stability of adefovir in human plasma was assessed by analyzing three replicates of low, mid and high QC samples under various temperature and time conditions. The freeze-thaw stability study was performed by subjecting unextracted QC samples to three freeze (−20 ◦ C)-thaw (room temperature) cycles. The QC samples were stored at −20 ◦ C for 30 days and at ambient temperature for 4 h to determine long-term and short-term stability, respectively. The post-preparative stability was studied by analyzing the extracted QC samples kept in the autosampler at 4 ◦ C for 12 h. All QC samples for stability test were determined by using calibration curve of freshly prepared standard samples. The concentrations obtained were compared with the nominal values.
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3.6. Application to pharmacokinetic study The method was applied to the determination of the plasma concentrations of adefovir from a clinical trial in which 20 healthy male volunteers received one adefovir dipivoxil tablet (containing 10 mg adefovir dipivoxil). The pharmacokinetic study was approved by the local Ethics Committee and carried out in the hospital. All volunteers gave their signed informed consent to participate in the study according to the principles of the Declaration of Helsinki. The blood samples were collected into sodium heparin-containing tubes before dosing and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 12, 24, 36, 48 h post-dosing. The plasma was separated by centrifugation and stored at −20 ◦ C until analyzed. TopFit v 2 0 software is used to calculate the pharmacokinetic parameters. The pharmacokinetic model is non-compartmental. The maximum plasma concentrations (Cmax ) and their times (Tmax ) were noted directly from the measured data. The elimination rate constant (ke ) was calculated by linear regression of the terminal points of the semi-log plot of plasma concentration against time. Elimination half-life (t1/2 ) was calculated using the formula t1/2 = 0.693/ke . The area under the plasma concentration–time curve (AUC0−t ) to the last measurable plasma concentration (Ct ) was calculated by the linear trapezoidal rule. The area under the plasma concentration–time curve to time infinity (AUC0−∞ ) was calculated as AUC0−∞ = AUC0−t + Ct /ke . 4. Results and discussion 4.1. Selection of internal standard An internal standard in the analysis of biological sample could be a structurally similar analog of analyte or a stable labeled compound [18]. Deuterated standard would be a preferred internal standard in HPLC–MS assay, however, it was not always commercially available in our case. Therefore pidotimod, diphenhydramine, and phenacetin having amino group in their structure as adefovir were tested as the I.S. They all had satisfactory detection response in positive ionization mode of ESI. Finally, pidotimod was chosen as the I.S. due to its similarity to adefovir in retention, ionization and extraction efficiency. 4.2. Development of mass spectrometry MS/MS operation parameters were carefully optimized for the determination of adefovir. A standard solution (1 g/mL) of adefovir and the I.S. was directly infused alongwith the mobile phase into the mass spectrometer with ESI ionization source. The mass spectrometer was tuned in both positive and negative ionization modes for adefovir contains both amino and phosphoric acid groups. The response observed in positive ionization mode was higher than that in negative ionization mode. In the precursor ion full-scan spectra, the most abundant ions were the protonated molecules [M+H]+ at m/z 274.4 and 245.0 for adefovir and the I.S., respectively. Parameters such as desolvation temperature, ESI source temperature, capillary and cone voltage, flow rate of desolvation gas and cone gas were optimized to obtain highest intensity of protonated molecule of adefovir. The product ion scan spectra of [M+H]+ for adefovir showed several major fragment ions at m/z 94.6, 136.0, 162.0, 192.1, 226.1, 256.4 (Fig. 1a). The formation pathways of these fragment ions have been reported in the literature [19]. The fragment ion at m/z 162.0 formed by breaking the single carbon–oxygen bond in the side chain was present in the highest abundance. The fragment ion at m/z 136.0 and 226.1 showed a relative intensity above 80% and 50% of the base peak respectively and other fragments not above 60%. To improve the detection sensitiv-
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Fig. 2. The retention and MS response of adefovir (10 ng/mL) on C18 column and HILIC column. (a) Column: ACQUITY UPLCTM BEH C18 (2.1 mm × 50 mm, 1.7 m); mobile phase: methanol–water containing 0.2% formic acid (15:85, v/v); flow rate: 0.3 mL/min; (b) Column: ACQUITY UPLC BEH HILIC (2.1 mm × 50 mm, 1.7 (m); mobile phase: methanol–water containing 0.2% formic acid (85:15, v/v); flow rate: 0.3 mL/min.
ity the three major diagnostic fragment ions, m/z 136.0, 162.0 and 226.1, were monitored in the MRM for adefovir. The most abundant fragment ion at m/z 133.8 (Fig. 1b) was chosen for the MRM acquisition for the I.S. 4.3. Development of chromatography RPLC is a powerful and versatile technique for the separation of a wide range of compounds. However, the retention of polar analytes in RPLC usually requires a high proportion of aqueous phase in the mobile phase, which results in poor ionization efficiency and the response in MS due to difficulty in desolvating mobile phase. In the literatures [10–12], in order to retain adefovir on reversed phase, the percentage of aqueous phase was up to 80%. In this study a reversed-phase column was also attempted. From Fig. 2 it can be seen that adefovir eluted from an ACQUITY UPLCTM BEH C18 column (2.1 mm × 50 mm, 1.7 m) unretained even with mobile phase of methanol–water containing 0.2% formic acid (15:85, v/v). The retention time is 0.52 min, shorter than the dead time (0.67 min) on the column with mobile phase at a flow rate of 0.3 mL/min. When a UPLC HILIC column of same dimensions and particle size was employed adefovir retained well (tR = 1.19 min) on the column with mobile phase of methanol–water containing 0.2% formic acid (85:15, v/v) at the same flow rate. The relatively high organic solvent concentration in the mobile phase increased ESI efficiency due to a better desolvation and reduced surface tension, in effect enhancing signal response in MS [20]. About ten times higher response of adefovir in MS after the UPLC HILIC separation than that after the UPLC C18 (1.7 m) column separation was observed (Fig. 2). Furthermore, low column back pressure originated from the use of high organic content mobile phase in the HILIC method makes it possible for the implementation of high flow rate, which can significantly shorten the analysis time. A chromatographic run time as short as 3.0 min per sample was about half of those reported in the literatures (6–8 min) [9–12]. In this study, a 1.7 m un-derivatized ethylene bridged hybrid (BEH) particle was employed under HILIC condition. The reduction of particle diameter of the packing material also contributed to the enhanced chromatographic efficiency resolution and sensitivity [17,21,22]. The mobile phase systems of acetonitrile–water and methanol–water in various proportions were tested. The signal to noise (S/N) ratio of adefovir was obviously higher
with methanol–water as the mobile phase than that with acetonitrile–water. Methanol proportion in the mobile phase from 60% to 90% was considered in the followed experiment. In view of the response of adefovir, retention times and peak shapes of both adefovir and I.S., 85% methanol was chosen as the mobile phase. Although in HILIC–MS the sensitivity is mainly affected by the proportion of the organic solution in the mobile phase, buffers typically acetic acid, formic acid and their ammonium salts are also attempted to improve the ionization of analyte due to their well volatility and solubility at such high percentages of organic solvent. In this study the response of adefovir was distinctly increased by adding formic acid into the mobile phase. The effect of formic acid of 0.1%, 0.2% and 0.3% in aqueous phase on the response and peak shape of adefovir was investigated and 0.2% formic acid was found to give the best results. 4.4. Selection of extraction method The highly polar character of adefovir makes it difficult to be extracted from plasma with organic solvents. In the literature [9] a solid phase extraction (SPE) was applied to purify and enrich adefovir from human plasma, which would be expensive and not suitable for sample preparation in large numbers. Therefore, a procedure of protein precipitation combined with interference elimination using dichloromethane extraction and concentration by evaporation was chosen as the sample preparation method, which provided the same purification and concentration effect as SPE. Several protein precipitants such as ethanol, methanol and acetonitrile were investigated. Methanol was chosen as the precipitant due to its satisfactory efficiency in analyte extraction and less matrix effect compared to that observed with other precipitants. 4.5. Method validation 4.5.1. Selectivity Comparing the chromatograms of six batches of blank plasma with those of the spiked plasma demonstrated a good selectivity of the method. As shown in Fig. 3a, no significant interferences from endogenous substances were observed at the retention times of adefovir and the I.S. Carry-over was eliminated by rinsing system, which was demonstrated by analyzing blank samples immediately following the samples at highest concentration.
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Fig. 3. Representative MRM chromatograms of adefovir (peak 1, channel 1) and pidotimod (I.S., peak 2, channel 2) in human plasma samples. (a) A blank plasma sample; (b) a blank plasma sample spiked with adefovir at the LLOQ of 1.02 ng/mL and the I.S. (202 ng/mL); (c) a plasma sample from a volunteer 0.5 h after oral administration of adefovir dipivoxil.
4.5.2. Linearity and LLOQ The standard calibration curves for adefovir were linear over the concentration range of 1.02–102 ng/mL (r2 ≥ 0.99). A typical regression equation for the calibration curves is y = 1.19 × 10−1 x − 3.84 × 10−2 , r = 0.9947, where y is the peak area ratio of adefovir to the I.S., and x is the concentration of adefovir in plasma. The data of the linearity parameters of the method during the method validation are given in Table 1. The lower limit of quantification (LLOQ) for adefovir was 1.02 ng/mL in plasma with precision (R.S.D.) below 20% and accuracy (R.E.) within ±20% (Table 2) and a corresponding chromatogram is given in Fig. 3b. With the LLOQ of 1.02 ng/mL, the present method can determine adefovir concentration in plasma samples until 48 h after a single oral dose of 10 mg adefovir
Table 1 Linearity parameters of the method in the range of 1.02–102 ng/mL during method validation. Run
Intercept (×10−2 )
Slope (×10−1 )
Regression
1 2 3 Mean S.D.
3.84 4.70 4.04 4.19 0.45
1.19 1.28 1.15 1.21 0.07
0.9947 0.9973 0.9961 0.9960 0.0013
dipivoxil. The method is sensitive enough to investigate the pharmacokinetic behavior of adefovir in human. 4.5.3. Precision and accuracy The data of intra- and inter-day precision and accuracy of the method are given in Table 2. The intra- and inter-day R.S.D.s were not more than 6.0% and 11.6%, respectively, and R.E.s were from 0.6% to 3.2% at three QC levels, indicating an acceptable precision and accuracy of the present method. 4.5.4. Extraction recovery and matrix effect The extraction recoveries of adefovir from human plasma were 84.9 ± 5.7%, 79.0 ± 6.5%, 71.9 ± 5.6% at concentrations of 2.53, 7.07 and 85.8 ng/mL, respectively. The mean extraction recovery of the I.S. was 79.8 ± 5.2%. The consistency in recoveries of adefovir and I.S. supported the procedure for its application to routine analysis. Matrix effect is due to co-elution of some components present in biological samples. These co-eluted components may not give a signal in MRM of target analyte but can certainly decrease or increase the analyte response dramatically to affect the sensitivity, accuracy and precision of a method. Thus, the evaluation of matrix effect from the co-eluting components on analyte ionization is necessary for an HPLC–MS/MS method. The matrix effect on both adefovir and
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Table 2 Precision and accuracy for the determination of adefovir in human plasma (intra-day: n = 6; inter-day: n = 6 series per day, 3 days).
LLOQ Low QC Mid QC High QC
Added C (ng/mL)
Found C (ng/mL)
1.02 2.53 7.07 85.8
1.05 2.61 7.12 87.8
± ± ± ±
0.07 0.15 0.43 4.2
Table 3 Stability of adefovir in human plasma at three QC levels (n = 3). Stability
Mean ± S.D. 2.53 (ng/mL)
Short-term stability Long-term stability Freeze-thaw stability Post-preparative stability
2.58 2.55 2.62 2.78
± ± ± ±
0.08 0.30 0.23 0.03
7.07 (ng/mL) 7.07 6.71 7.15 6.90
± ± ± ±
0.39 0.78 0.10 0.33
85.8 (ng/mL) 90.0 87.8 92.3 86.0
± ± ± ±
2.2 5.3 5.0 3.8
Intra-run R.S.D. (%)
Inter-run R.S.D. (%)
Accuracy R.E. (%)
5.3 6.0 5.5 4.2
11.6 4.2 8.7 7.8
3.2 3.0 0.6 2.3
declined with a t1/2 of 10.5 ± 0.9 h. The AUC0−t and AUC0−∞ values obtained were 302 ± 45 and 318 ± 43 ng h/mL, respectively. These pharmacokinetic parameters were in accordance with those reported in the literatures [10–12], indicating the applicability of this method to the pharmacokinetic study of adefovir. 5. Conclusion A HILIC–MS/MS method for the determination of adefovir in human plasma is described for the first time. The method was highly efficient with a short run time of 3.0 min. The validation demonstrated that the developed method had high sensitive, selectivity and satisfactory precision and accuracy. The method has been successfully applied to the pharmacokinetic study of adefovir dipivoxil given in tablet to healthy volunteers. Acknowledgement
Fig. 4. Mean plasma concentration–time curve of adefovir in male volunteers after a single oral dose of adefovir dipivoxil.
This work was supported by Key Project for Drug Innovation (2009ZX09301-012) from the Ministry of Science and Technology of China. References
the I.S. was between 85% and 115%, indicating that no co-eluting substance influenced the ionization of the analytes and I.S. 4.5.5. Stability The stability evaluation for adefovir stock solution at 4 ◦ C for 30 days gave REs from 3.8% to 4.2% and that for dilution solutions at room temperature for 6 h gave REs from −3.8% to 4.0%, which indicated a good stability of adefovir in stock solution and dilution solutions. The stock solution stored for over 30 days was discarded and another was prepared. The results from all stability tests of adefovir in plasma samples are presented in Table 3, which indicated a good stability of adefovir in plasma stored at room temperature for 4 h, at −20 ◦ C for 30 days and during three freeze-thaw cycles, and in prepared samples at 4 ◦ C for 12 h. The method is therefore proved to be applicable to routine analysis. 4.5.6. Pharmacokinetic application It is reported that adefovir dipivoxil and its monoester were not observed in plasma, suggesting rapid hydrolysis of the prodrug to adefovir before reaching the systemic circulation [23]. Therefore, pharmacokinetics of adefovir was evaluated following oral dosing of the prodrug. This validated HILIC–MS/MS method was successfully applied to the pharmacokinetic study of adefovir in healthy male volunteers after oral administration of adefovir dipivoxil. The mean plasma concentration–time curve of adefovir in single dose study is shown in Fig. 4. After administration of a single dose of 10 mg adefovir dipivoxil, the Cmax and Tmax of adefovir were 24.7 ± 3.6 ng/mL and 1.14 ± 0.25 h, respectively. The plasma concentration of adefovir
[1] M. Buti, R. Esteban, Drugs Today (Barc) 39 (2003) 127. [2] K.C. Cundy, J.P. Shaw, W.A. Lee, Antimicrob. Agents Chemother. 38 (1994) 365. [3] K.C. Cundy, P. Barditch-Crovo, R.E. Walker, A.C. Collier, D. Ebeling, J. Toole, H.S. Jaffe, Antimicrob. Agents Chemother. 39 (1995) 2401. [4] R.B. Qaqish, K.A. Mattes, D.J. Ritchie, Clin. Ther. 25 (2003) 3084. [5] E.J. Fisher, K. Chaloner, D.L. Cohn, L.B. Grant, B. Alston, C.L. Brosgart, B. Schmetter, W.M. El-Sadr, J. Sampson, AIDS 15 (2001) 1695. [6] R.W. Sparidans, A. Veldkamp, R.M. Hoetelmans, J.H. Beijnen, J. Chromatogr. B 736 (1999) 115. [7] P. Augustijins, P. Annaert, S. Adriaens, E. De Clercq, R. Kinget, J. Liquid Chromatogr. Relat. Technol. 19 (1996) 2271. [8] L. Naesens, J. Balzarini, E. De Clercq, Clin. Chem. 38 (1992) 480. [9] Y.F. Liu, G.F. Xu, C. Xu, L. Garcia, C.C. Lin, L.T. Yeh, J. Chromatogr. B 803 (2004) 293. [10] X.Y. Chen, D. Liu, L. Zhu, D.F. Zhong, Rapid Commun. Mass Spectrom. 19 (2005) 1893. [11] H.C. Bi, G.P. Zhong, S.F. Zhuo, X. Chen, M. Huang, Rapid Commun. Mass Spectrom. 19 (2005) 2911. [12] D.Q. Sun, H.S. Wang, B.J. Wang, R.C. Guo, J. Pharm. Biomed. Anal. 42 (2006) 372. [13] H.W. Lee, H.Y. Ji, H.Y. Kim, E.S. Park, K.C. Lee, H.S. Lee, J. Chromatogr. B 877 (2009) 1716. [14] A. Liu, S.P. Coleman, J. Chromatogr. B 877 (2009) 3695. [15] L.S. Huang, F. Marzan, A.L. Jayewardene, P.S. Lizak, X.H. Li, F.T. Aweeka, J. Chromatogr. B 877 (2009) 285. [16] Y. Li, X.Z. Zhang, X.Y. Wang, S. Li, J.X. Ruan, Z.Q. Zhang, J. Chromatogr. B 877 (2009) 933. [17] X.M. Cai, L.J. Zou, J. Dong, L.L. Zhao, Y.Y. Wang, Q. Xu, X.Y. Xue, X.L. Zhang, X.M. Liang, Anal. Chim. Acta 650 (2009) 10. [18] USFDA (2001) http://www.fda.gov/downloads/Drugs/GuidanceCompliance RegulatoryInformation/Guidances/ucm070107.pdf. [19] X.Y. Chen, J. Xing, D.F. Zhong, J. Mass Spectrom. 39 (2004) 145. [20] D. McCalley, J. Chromatogr. A 1171 (2007) 46. [21] E. Grumbach, D. Diehl, U. Neue, J. Sep. Sci. 31 (2008) 1511. [22] H. Gika, G. Theodoridis, I. Wilson, J. Sep. Sci. 31 (2008) 1598. [23] W.T. Hughes, J.L. Shenep, J.H. Rodman, A. Fridland, R. Willoughby, S. Blanchard, L. Purdue, D.F. Coakley, K.C. Cundy, M. Culnane, B. Zimmer, S. Burchett, J.S. Read, Antimicrob. Agents Chemother. 44 (2000) 1041.
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Hydrophilic interaction liquid chromatography–tandem mass spectrometry for the determination of adefovir in human plasma and its application to a pharmacokinetic study Zhili Xiong, Yi Zhang, Feng Qin, Ting Qin, Shuyan Yang, Famei Li ∗ Department of Analytical Chemistry, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
a r t i c l e
i n f o
Article history: Received 18 March 2010 Accepted 18 June 2010 Available online 26 June 2010 Keywords: Adefovir HILIC–MS/MS Human plasma Pharmacokinetic study
a b s t r a c t A selective, rapid and sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for the first time to determine adefovir in human plasma and applied to a pharmacokinetic study. Plasma samples were prepared by protein precipitation with methanol followed by a further cleaning using dichloromethane. The chromatographic separation was carried out on an ACQUITY UPLCTM BEH HILIC column with the mobile phase of methanol–water–formic acid (85:15:0.2, v/v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The method was rapid with a run time of 3 min per sample. The linear calibration curves were obtained in the concentration range of 1.02–102 ng/mL (r2 ≥ 0.99) with the lower limit of quantification (LLOQ) of 1.02 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 12% and the accuracy (relative error, R.E.) was from 0.6% to 3.2% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of adefovir in healthy volunteers after oral administration of adefovir dipivoxil tablet. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Adefovir, 9-(2-phosphonylmethoxyethyl) adenine (Fig. 1a) has broad-spectrum activity against human immunodeficiency virus, herpes viruses, hepatitis B viruses and adenoviruses [1]. Unfortunately, adefovir exhibited low oral bioavailability in both animals [2] and humans [3]. The bioavailability of adefovir has been substantially improved by using the bis-pivaloyloxymethyl ester of adefovir (adefovir dipivoxil) as a prodrug with enhanced lipophilicity and in vitro activity [4]. However, dose-limiting nephrotoxicity such as proximal renal tubular dysfunction in approximately 17% of patients has been associated with the use of adefovir dipivoxil [5]. Therefore, therapeutic drug monitoring is always necessary to maximize the antiviral efficacy and minimize the toxicity during adefovir dipivoxil chemotherapy. Adefovir is the primary circulating metabolite identified after oral administration of adefovir dipivoxil [6]. The development of a sensitive and specific method to determine adefovir in human plasma is necessary and valuable for pharmacokinetic study and therapeutic drug monitoring of adefovir dipivoxil. Analysis of adefovir in preparations by high-performance liquid chromatography
∗ Corresponding author. Tel.: +86 24 2398 6289; fax: +86 24 2398 6289. E-mail address: [email protected] (F. Li). 1570-0232/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2010.06.024
(HPLC) with ultraviolet detection has been reported [7]. Methods using pre-column derivatization with chloroacetaldehyde to form highly fluorescent derivatives followed by separation on reservedphase ion-pairing HPLC [3,6,8] were reported for the determination of adefovir in human plasma. The pretreatment of samples was tedious and time-consuming, and those methods were not sensitive enough for evaluating the pharmacokinetics of adefovir dipivoxil with LLOQ higher than 10 ng/mL. Several sensitive highperformance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) methods [9–12] have been developed using electrospray ionization (ESI) for the measurement of adefovir in human plasma or serum. Although those methods were sensitive for the pharmacokinetic study of adefovir, long analysis time (longer than 8 min) precluded their use in therapeutic drug monitoring. Hydrophilic interaction chromatography (HILIC) is a method where polar stationary phase (bare silica or derivatized silica) is used in conjunction with a low aqueous/high organic mobile phase. It has been proved to be a powerful way to separate polar compounds in biological samples with reversed retention compared to the traditional reversed-phase liquid chromatography (RPLC) [13–17]. In view of LC–MS analysis, the higher organic content in the mobile of HILIC resulted in higher sensitivity, flow rate and less matrix effect. In this study, a HILIC–MS/MS method was described for the first time to determine adefovir in human plasma, which facilitates the determination with the total run time of 3.0 min. The method was
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Fig. 1. Product ion spectra of adefovir (a) and pidotimod (I.S.) (b).
fully validated and applied to the pharmacokinetic study in healthy volunteers after oral administration of 10 mg adefovir dipivoxil in tablets. 2. Materials and methods 2.1. Reagents and chemicals Reference standard of adefovir (99.6% purity) was kindly provided by the Medicinal Chemistry Department of Shenyang Pharmaceutical University. Pidotimod (internal standard (I.S.), 99.5% purity, Fig. 1b) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, PR China). Methanol of HPLC grade was obtained from Tedia (Fairfield, OH, USA). Formic acid (HPLC grade) was purchased from Dikma (Richmond Hill, NY, USA). Water was purified by redistillation and filtered through a 0.22 m membrane filter before use. 2.2. Apparatus and operation conditions 2.2.1. Liquid chromatographic conditions The chromatography was performed on an ACQUITY UPLCTM system (Waters Corp., Milford, MA, USA). An ACQUITY UPLCTM BEH HILIC column (2.1 × 50 mm I.D., 1.7 m) was employed for the separation at 40 ◦ C. The mobile phase was composed of methanol–water–formic acid (85:15:0.2, v/v/v). The flow rate was set at 0.30 mL/min. The autosampler was conditioned at 4 ◦ C and the injection volume was 20 L using partial loop mode for sample injection. 2.2.2. Mass spectrometric conditions The mass spectrometric detection was carried out on a Micromass® Quattro microTM API triple-quadrupole tandem mass spectrometer (Waters Corp.) equipped with ESI interface. The ESI source was set in positive ionization mode. The quantification was performed using multiple reaction monitoring (MRM) of the transitions of m/z 274.4 → 136.0, 274.4 → 162.0, 274.4 → 226.1 for adefovir and m/z 245.0 → 133.8 for I.S. respectively, with scan time of 0.20 s per transition. The optimal MS parameters were as follows: capillary voltage 3.5 kV, cone voltage 25 V, source temperature 110 ◦ C and desolvation temperature 450 ◦ C. Nitrogen was used as the desolvation and cone gas with a flow rate of 550 and 30 L/h, respectively. Argon was used as the collision gas at a pressure of approximately 0.255 Pa. The optimized collision energy values for adefovir and I.S. were 25 and 13 eV, respectively. All data collected in centroid mode was acquired and processed using MassLynxTM NT 4.1 software with QuanLynxTM program (Waters Corp., Milford, MA, USA).
2.3. Preparation of standards and quality control samples Stock standard solutions of adefovir and I.S. were prepared in water at the concentration of 102 and 101 g/mL, respectively. The I.S. solution was prepared by dilution with methanol to 202 ng/mL. Adefovir stock solution was serially diluted with methanol to provide working standard solutions at desired concentrations. Another stock solution of adefovir for the preparation of quality control (QC) samples was prepared independently with a concentration of 101 g/mL. All the solutions were stored at 4 ◦ C and brought to room temperature before use. The calibration standards were prepared by evaporating 50 L of working standard solutions to dryness and then fully mixing with 500 L of blank plasma. The effective concentrations in standard plasma samples were 1.02, 2.04, 5.10, 10.2, 20.4, 51.0, 102 ng/mL. One calibration curve was constructed on each analysis day using freshly prepared calibration standard samples. The QC samples were prepared in bulk with a similar procedure as that for standard samples and aliquots were stored at −20 ◦ C. The LLOQ, low, mid and high concentrations of QC samples were 1.02, 2.53, 7.07 and 85.8 ng/mL. The standard samples and QC samples were extracted on each analysis day with the same procedures for plasma samples as described below. 2.4. Plasma sample preparation The I.S. solution (50 L) was pipetted into 2.0-mL polypropylene micro-centrifuge tube and evaporated to dryness under a gentle stream of nitrogen at 40 ◦ C. The residue was vortex-mixed with 500 L of plasma for 30 s followed by addition of 1.0 mL of methanol to each tube. The mixture was vortex-mixed for 60 s and centrifuged at 11,200 × g for 15 min. The supernatant was transferred into another tube and dichloromethane was added to remove the possible impurity. After vortex-mixing for 60 s and centrifugation at 2000 × g for 10 min the upper layer was evaporated to dryness under a gentle stream of nitrogen at 40 ◦ C. The residue was reconstituted in 50 L of methanol–water (85:15, v/v) and filtered through a 0.22 m membrane filter. An aliquot of 20 L was injected into the HILIC–MS/MS system for analysis. 3. Method validation The method was validated for selectivity, linearity, precision, accuracy, extraction recovery, matrix effect and stability according to FDA guidance for validation of bioanalytical methods [18]. Validation runs were conducted on three consecutive days. The peak area ratios of adefovir to I.S. from QC samples were interpolated
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from the calibration curve on the same day to give the concentration of adefovir. 3.1. Selectivity The selectivity was investigated by comparing chromatograms of six different batches of blank plasma from six subjects to those of corresponding standard plasma samples spiked with adefovir and I.S. and plasma sample after oral dose of adefovir dipivoxil tablets. 3.2. Linearity and lower limit of quantification Calibration curves were constructed by assaying standard plasma samples at seven concentrations in the range of 1.02–102 ng/mL with weighted (1/x2 ) least squares linear regression. According to FDA guidance, the LLOQ is defined as the lowest amount of an analyte in a sample that can be quantitatively determined with acceptable precision and accuracy, which are a relative standard deviation (R.S.D.) of 20% and a relative error (R.E.) within ±20%. 3.3. Precision and accuracy The intra-day precision and accuracy were evaluated by a replicate analysis of QC samples of adefovir on the same day. The validation run consisted of two sets of calibration standard samples and six replicates of QC samples at three concentrations. For determining the inter-day accuracy and precision, analysis of three batches of QC samples was performed on three consecutive days. The precision was expressed as R.S.D. and the accuracy as R.E. 3.4. Extraction recovery and matrix effect The extraction recovery of adefovir was determined by comparing the peak areas obtained from blank plasma samples spiked with the analyte before extraction with those from blank plasma samples to which the analyte was added after extraction. This procedure was repeated for five replicates at three QC concentration levels of 2.53, 7.07 and 85.8 ng/mL. The matrix effect was measured by comparing the peak response of sample spiked post-extraction with that of pure standard solution dried directly and reconstituted with the same mobile phase. The extraction recovery and matrix effect of I.S. were also evaluated using the same procedure. 3.5. Stability The stability of dilution solution of adefovir was evaluated at room temperature for 6 h by comparing the peak areas with those of freshly prepared dilution solutions. The stability of stock solution was evaluated for 30 days by comparing the peak area per unit mass of analyte in the stock solution stored at 4 ◦ C with that in freshly prepared stock solution. The stability of adefovir in human plasma was assessed by analyzing three replicates of low, mid and high QC samples under various temperature and time conditions. The freeze-thaw stability study was performed by subjecting unextracted QC samples to three freeze (−20 ◦ C)-thaw (room temperature) cycles. The QC samples were stored at −20 ◦ C for 30 days and at ambient temperature for 4 h to determine long-term and short-term stability, respectively. The post-preparative stability was studied by analyzing the extracted QC samples kept in the autosampler at 4 ◦ C for 12 h. All QC samples for stability test were determined by using calibration curve of freshly prepared standard samples. The concentrations obtained were compared with the nominal values.
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3.6. Application to pharmacokinetic study The method was applied to the determination of the plasma concentrations of adefovir from a clinical trial in which 20 healthy male volunteers received one adefovir dipivoxil tablet (containing 10 mg adefovir dipivoxil). The pharmacokinetic study was approved by the local Ethics Committee and carried out in the hospital. All volunteers gave their signed informed consent to participate in the study according to the principles of the Declaration of Helsinki. The blood samples were collected into sodium heparin-containing tubes before dosing and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 12, 24, 36, 48 h post-dosing. The plasma was separated by centrifugation and stored at −20 ◦ C until analyzed. TopFit v 2 0 software is used to calculate the pharmacokinetic parameters. The pharmacokinetic model is non-compartmental. The maximum plasma concentrations (Cmax ) and their times (Tmax ) were noted directly from the measured data. The elimination rate constant (ke ) was calculated by linear regression of the terminal points of the semi-log plot of plasma concentration against time. Elimination half-life (t1/2 ) was calculated using the formula t1/2 = 0.693/ke . The area under the plasma concentration–time curve (AUC0−t ) to the last measurable plasma concentration (Ct ) was calculated by the linear trapezoidal rule. The area under the plasma concentration–time curve to time infinity (AUC0−∞ ) was calculated as AUC0−∞ = AUC0−t + Ct /ke . 4. Results and discussion 4.1. Selection of internal standard An internal standard in the analysis of biological sample could be a structurally similar analog of analyte or a stable labeled compound [18]. Deuterated standard would be a preferred internal standard in HPLC–MS assay, however, it was not always commercially available in our case. Therefore pidotimod, diphenhydramine, and phenacetin having amino group in their structure as adefovir were tested as the I.S. They all had satisfactory detection response in positive ionization mode of ESI. Finally, pidotimod was chosen as the I.S. due to its similarity to adefovir in retention, ionization and extraction efficiency. 4.2. Development of mass spectrometry MS/MS operation parameters were carefully optimized for the determination of adefovir. A standard solution (1 g/mL) of adefovir and the I.S. was directly infused alongwith the mobile phase into the mass spectrometer with ESI ionization source. The mass spectrometer was tuned in both positive and negative ionization modes for adefovir contains both amino and phosphoric acid groups. The response observed in positive ionization mode was higher than that in negative ionization mode. In the precursor ion full-scan spectra, the most abundant ions were the protonated molecules [M+H]+ at m/z 274.4 and 245.0 for adefovir and the I.S., respectively. Parameters such as desolvation temperature, ESI source temperature, capillary and cone voltage, flow rate of desolvation gas and cone gas were optimized to obtain highest intensity of protonated molecule of adefovir. The product ion scan spectra of [M+H]+ for adefovir showed several major fragment ions at m/z 94.6, 136.0, 162.0, 192.1, 226.1, 256.4 (Fig. 1a). The formation pathways of these fragment ions have been reported in the literature [19]. The fragment ion at m/z 162.0 formed by breaking the single carbon–oxygen bond in the side chain was present in the highest abundance. The fragment ion at m/z 136.0 and 226.1 showed a relative intensity above 80% and 50% of the base peak respectively and other fragments not above 60%. To improve the detection sensitiv-
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Fig. 2. The retention and MS response of adefovir (10 ng/mL) on C18 column and HILIC column. (a) Column: ACQUITY UPLCTM BEH C18 (2.1 mm × 50 mm, 1.7 m); mobile phase: methanol–water containing 0.2% formic acid (15:85, v/v); flow rate: 0.3 mL/min; (b) Column: ACQUITY UPLC BEH HILIC (2.1 mm × 50 mm, 1.7 (m); mobile phase: methanol–water containing 0.2% formic acid (85:15, v/v); flow rate: 0.3 mL/min.
ity the three major diagnostic fragment ions, m/z 136.0, 162.0 and 226.1, were monitored in the MRM for adefovir. The most abundant fragment ion at m/z 133.8 (Fig. 1b) was chosen for the MRM acquisition for the I.S. 4.3. Development of chromatography RPLC is a powerful and versatile technique for the separation of a wide range of compounds. However, the retention of polar analytes in RPLC usually requires a high proportion of aqueous phase in the mobile phase, which results in poor ionization efficiency and the response in MS due to difficulty in desolvating mobile phase. In the literatures [10–12], in order to retain adefovir on reversed phase, the percentage of aqueous phase was up to 80%. In this study a reversed-phase column was also attempted. From Fig. 2 it can be seen that adefovir eluted from an ACQUITY UPLCTM BEH C18 column (2.1 mm × 50 mm, 1.7 m) unretained even with mobile phase of methanol–water containing 0.2% formic acid (15:85, v/v). The retention time is 0.52 min, shorter than the dead time (0.67 min) on the column with mobile phase at a flow rate of 0.3 mL/min. When a UPLC HILIC column of same dimensions and particle size was employed adefovir retained well (tR = 1.19 min) on the column with mobile phase of methanol–water containing 0.2% formic acid (85:15, v/v) at the same flow rate. The relatively high organic solvent concentration in the mobile phase increased ESI efficiency due to a better desolvation and reduced surface tension, in effect enhancing signal response in MS [20]. About ten times higher response of adefovir in MS after the UPLC HILIC separation than that after the UPLC C18 (1.7 m) column separation was observed (Fig. 2). Furthermore, low column back pressure originated from the use of high organic content mobile phase in the HILIC method makes it possible for the implementation of high flow rate, which can significantly shorten the analysis time. A chromatographic run time as short as 3.0 min per sample was about half of those reported in the literatures (6–8 min) [9–12]. In this study, a 1.7 m un-derivatized ethylene bridged hybrid (BEH) particle was employed under HILIC condition. The reduction of particle diameter of the packing material also contributed to the enhanced chromatographic efficiency resolution and sensitivity [17,21,22]. The mobile phase systems of acetonitrile–water and methanol–water in various proportions were tested. The signal to noise (S/N) ratio of adefovir was obviously higher
with methanol–water as the mobile phase than that with acetonitrile–water. Methanol proportion in the mobile phase from 60% to 90% was considered in the followed experiment. In view of the response of adefovir, retention times and peak shapes of both adefovir and I.S., 85% methanol was chosen as the mobile phase. Although in HILIC–MS the sensitivity is mainly affected by the proportion of the organic solution in the mobile phase, buffers typically acetic acid, formic acid and their ammonium salts are also attempted to improve the ionization of analyte due to their well volatility and solubility at such high percentages of organic solvent. In this study the response of adefovir was distinctly increased by adding formic acid into the mobile phase. The effect of formic acid of 0.1%, 0.2% and 0.3% in aqueous phase on the response and peak shape of adefovir was investigated and 0.2% formic acid was found to give the best results. 4.4. Selection of extraction method The highly polar character of adefovir makes it difficult to be extracted from plasma with organic solvents. In the literature [9] a solid phase extraction (SPE) was applied to purify and enrich adefovir from human plasma, which would be expensive and not suitable for sample preparation in large numbers. Therefore, a procedure of protein precipitation combined with interference elimination using dichloromethane extraction and concentration by evaporation was chosen as the sample preparation method, which provided the same purification and concentration effect as SPE. Several protein precipitants such as ethanol, methanol and acetonitrile were investigated. Methanol was chosen as the precipitant due to its satisfactory efficiency in analyte extraction and less matrix effect compared to that observed with other precipitants. 4.5. Method validation 4.5.1. Selectivity Comparing the chromatograms of six batches of blank plasma with those of the spiked plasma demonstrated a good selectivity of the method. As shown in Fig. 3a, no significant interferences from endogenous substances were observed at the retention times of adefovir and the I.S. Carry-over was eliminated by rinsing system, which was demonstrated by analyzing blank samples immediately following the samples at highest concentration.
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Fig. 3. Representative MRM chromatograms of adefovir (peak 1, channel 1) and pidotimod (I.S., peak 2, channel 2) in human plasma samples. (a) A blank plasma sample; (b) a blank plasma sample spiked with adefovir at the LLOQ of 1.02 ng/mL and the I.S. (202 ng/mL); (c) a plasma sample from a volunteer 0.5 h after oral administration of adefovir dipivoxil.
4.5.2. Linearity and LLOQ The standard calibration curves for adefovir were linear over the concentration range of 1.02–102 ng/mL (r2 ≥ 0.99). A typical regression equation for the calibration curves is y = 1.19 × 10−1 x − 3.84 × 10−2 , r = 0.9947, where y is the peak area ratio of adefovir to the I.S., and x is the concentration of adefovir in plasma. The data of the linearity parameters of the method during the method validation are given in Table 1. The lower limit of quantification (LLOQ) for adefovir was 1.02 ng/mL in plasma with precision (R.S.D.) below 20% and accuracy (R.E.) within ±20% (Table 2) and a corresponding chromatogram is given in Fig. 3b. With the LLOQ of 1.02 ng/mL, the present method can determine adefovir concentration in plasma samples until 48 h after a single oral dose of 10 mg adefovir
Table 1 Linearity parameters of the method in the range of 1.02–102 ng/mL during method validation. Run
Intercept (×10−2 )
Slope (×10−1 )
Regression
1 2 3 Mean S.D.
3.84 4.70 4.04 4.19 0.45
1.19 1.28 1.15 1.21 0.07
0.9947 0.9973 0.9961 0.9960 0.0013
dipivoxil. The method is sensitive enough to investigate the pharmacokinetic behavior of adefovir in human. 4.5.3. Precision and accuracy The data of intra- and inter-day precision and accuracy of the method are given in Table 2. The intra- and inter-day R.S.D.s were not more than 6.0% and 11.6%, respectively, and R.E.s were from 0.6% to 3.2% at three QC levels, indicating an acceptable precision and accuracy of the present method. 4.5.4. Extraction recovery and matrix effect The extraction recoveries of adefovir from human plasma were 84.9 ± 5.7%, 79.0 ± 6.5%, 71.9 ± 5.6% at concentrations of 2.53, 7.07 and 85.8 ng/mL, respectively. The mean extraction recovery of the I.S. was 79.8 ± 5.2%. The consistency in recoveries of adefovir and I.S. supported the procedure for its application to routine analysis. Matrix effect is due to co-elution of some components present in biological samples. These co-eluted components may not give a signal in MRM of target analyte but can certainly decrease or increase the analyte response dramatically to affect the sensitivity, accuracy and precision of a method. Thus, the evaluation of matrix effect from the co-eluting components on analyte ionization is necessary for an HPLC–MS/MS method. The matrix effect on both adefovir and
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Table 2 Precision and accuracy for the determination of adefovir in human plasma (intra-day: n = 6; inter-day: n = 6 series per day, 3 days).
LLOQ Low QC Mid QC High QC
Added C (ng/mL)
Found C (ng/mL)
1.02 2.53 7.07 85.8
1.05 2.61 7.12 87.8
± ± ± ±
0.07 0.15 0.43 4.2
Table 3 Stability of adefovir in human plasma at three QC levels (n = 3). Stability
Mean ± S.D. 2.53 (ng/mL)
Short-term stability Long-term stability Freeze-thaw stability Post-preparative stability
2.58 2.55 2.62 2.78
± ± ± ±
0.08 0.30 0.23 0.03
7.07 (ng/mL) 7.07 6.71 7.15 6.90
± ± ± ±
0.39 0.78 0.10 0.33
85.8 (ng/mL) 90.0 87.8 92.3 86.0
± ± ± ±
2.2 5.3 5.0 3.8
Intra-run R.S.D. (%)
Inter-run R.S.D. (%)
Accuracy R.E. (%)
5.3 6.0 5.5 4.2
11.6 4.2 8.7 7.8
3.2 3.0 0.6 2.3
declined with a t1/2 of 10.5 ± 0.9 h. The AUC0−t and AUC0−∞ values obtained were 302 ± 45 and 318 ± 43 ng h/mL, respectively. These pharmacokinetic parameters were in accordance with those reported in the literatures [10–12], indicating the applicability of this method to the pharmacokinetic study of adefovir. 5. Conclusion A HILIC–MS/MS method for the determination of adefovir in human plasma is described for the first time. The method was highly efficient with a short run time of 3.0 min. The validation demonstrated that the developed method had high sensitive, selectivity and satisfactory precision and accuracy. The method has been successfully applied to the pharmacokinetic study of adefovir dipivoxil given in tablet to healthy volunteers. Acknowledgement
Fig. 4. Mean plasma concentration–time curve of adefovir in male volunteers after a single oral dose of adefovir dipivoxil.
This work was supported by Key Project for Drug Innovation (2009ZX09301-012) from the Ministry of Science and Technology of China. References
the I.S. was between 85% and 115%, indicating that no co-eluting substance influenced the ionization of the analytes and I.S. 4.5.5. Stability The stability evaluation for adefovir stock solution at 4 ◦ C for 30 days gave REs from 3.8% to 4.2% and that for dilution solutions at room temperature for 6 h gave REs from −3.8% to 4.0%, which indicated a good stability of adefovir in stock solution and dilution solutions. The stock solution stored for over 30 days was discarded and another was prepared. The results from all stability tests of adefovir in plasma samples are presented in Table 3, which indicated a good stability of adefovir in plasma stored at room temperature for 4 h, at −20 ◦ C for 30 days and during three freeze-thaw cycles, and in prepared samples at 4 ◦ C for 12 h. The method is therefore proved to be applicable to routine analysis. 4.5.6. Pharmacokinetic application It is reported that adefovir dipivoxil and its monoester were not observed in plasma, suggesting rapid hydrolysis of the prodrug to adefovir before reaching the systemic circulation [23]. Therefore, pharmacokinetics of adefovir was evaluated following oral dosing of the prodrug. This validated HILIC–MS/MS method was successfully applied to the pharmacokinetic study of adefovir in healthy male volunteers after oral administration of adefovir dipivoxil. The mean plasma concentration–time curve of adefovir in single dose study is shown in Fig. 4. After administration of a single dose of 10 mg adefovir dipivoxil, the Cmax and Tmax of adefovir were 24.7 ± 3.6 ng/mL and 1.14 ± 0.25 h, respectively. The plasma concentration of adefovir
[1] M. Buti, R. Esteban, Drugs Today (Barc) 39 (2003) 127. [2] K.C. Cundy, J.P. Shaw, W.A. Lee, Antimicrob. Agents Chemother. 38 (1994) 365. [3] K.C. Cundy, P. Barditch-Crovo, R.E. Walker, A.C. Collier, D. Ebeling, J. Toole, H.S. Jaffe, Antimicrob. Agents Chemother. 39 (1995) 2401. [4] R.B. Qaqish, K.A. Mattes, D.J. Ritchie, Clin. Ther. 25 (2003) 3084. [5] E.J. Fisher, K. Chaloner, D.L. Cohn, L.B. Grant, B. Alston, C.L. Brosgart, B. Schmetter, W.M. El-Sadr, J. Sampson, AIDS 15 (2001) 1695. [6] R.W. Sparidans, A. Veldkamp, R.M. Hoetelmans, J.H. Beijnen, J. Chromatogr. B 736 (1999) 115. [7] P. Augustijins, P. Annaert, S. Adriaens, E. De Clercq, R. Kinget, J. Liquid Chromatogr. Relat. Technol. 19 (1996) 2271. [8] L. Naesens, J. Balzarini, E. De Clercq, Clin. Chem. 38 (1992) 480. [9] Y.F. Liu, G.F. Xu, C. Xu, L. Garcia, C.C. Lin, L.T. Yeh, J. Chromatogr. B 803 (2004) 293. [10] X.Y. Chen, D. Liu, L. Zhu, D.F. Zhong, Rapid Commun. Mass Spectrom. 19 (2005) 1893. [11] H.C. Bi, G.P. Zhong, S.F. Zhuo, X. Chen, M. Huang, Rapid Commun. Mass Spectrom. 19 (2005) 2911. [12] D.Q. Sun, H.S. Wang, B.J. Wang, R.C. Guo, J. Pharm. Biomed. Anal. 42 (2006) 372. [13] H.W. Lee, H.Y. Ji, H.Y. Kim, E.S. Park, K.C. Lee, H.S. Lee, J. Chromatogr. B 877 (2009) 1716. [14] A. Liu, S.P. Coleman, J. Chromatogr. B 877 (2009) 3695. [15] L.S. Huang, F. Marzan, A.L. Jayewardene, P.S. Lizak, X.H. Li, F.T. Aweeka, J. Chromatogr. B 877 (2009) 285. [16] Y. Li, X.Z. Zhang, X.Y. Wang, S. Li, J.X. Ruan, Z.Q. Zhang, J. Chromatogr. B 877 (2009) 933. [17] X.M. Cai, L.J. Zou, J. Dong, L.L. Zhao, Y.Y. Wang, Q. Xu, X.Y. Xue, X.L. Zhang, X.M. Liang, Anal. Chim. Acta 650 (2009) 10. [18] USFDA (2001) http://www.fda.gov/downloads/Drugs/GuidanceCompliance RegulatoryInformation/Guidances/ucm070107.pdf. [19] X.Y. Chen, J. Xing, D.F. Zhong, J. Mass Spectrom. 39 (2004) 145. [20] D. McCalley, J. Chromatogr. A 1171 (2007) 46. [21] E. Grumbach, D. Diehl, U. Neue, J. Sep. Sci. 31 (2008) 1511. [22] H. Gika, G. Theodoridis, I. Wilson, J. Sep. Sci. 31 (2008) 1598. [23] W.T. Hughes, J.L. Shenep, J.H. Rodman, A. Fridland, R. Willoughby, S. Blanchard, L. Purdue, D.F. Coakley, K.C. Cundy, M. Culnane, B. Zimmer, S. Burchett, J.S. Read, Antimicrob. Agents Chemother. 44 (2000) 1041.