Journal of Chromatography B, 985 (2015) 172–179
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Quantitation of pilsicainide in microscale samples of human biological fluids using liquid chromatography–tandem mass spectrometry Mikiko Shimizu a , Masayuki Hashiguchi b,∗ , Tsuyoshi Shiga c , Koichi Nakamura d , Hiro-omi Tamura a , Mayumi Mochizuki b a
Department of Hygienic Chemistry, Faculty of Pharmacy, Keio University, Tokyo, Japan Division for Evaluation and Analysis of Drug Information, Faculty of Pharmacy, Keio University, Tokyo, Japan c Department of Cardiology, Tokyo Women’s Medical University, Tokyo, Japan d Clinical Research Tokyo Hospital, Tokyo, Japan b
a r t i c l e
i n f o
Article history: Received 4 June 2014 Accepted 11 January 2015 Available online 22 January 2015 Keywords: Pilsicainide LC–MS/MS Plasma Urine Pharmacokinetics Therapeutic drug monitoring
a b s t r a c t This paper describes a sensitive, reliable method to determine pilsicainide (PLC) levels in microscale sample volumes of human biological fluids using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI). PLC and quinidine as an internal standard were extracted with diethylether from 0.1 mL of alkalinized biological fluids. The extract was injected into an analytical column (l-column 2 ODS, 75 mm × 2.1 mm i.d.). The mobile phase for separation consisted of 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v) and was delivered at a flow rate of 0.2 mL/min. The drift voltage was 100 V. The sampling aperture was heated at 120 ◦ C and the shield temperature was 260 ◦ C. The ion transitions used to monitor analytes were m/z 273 → m/z 110 for PLC and m/z 325 → m/z 79 for quinidine. The total time for chromatographic separation was less than 8 min. The validated concentration ranges of this method for PLC were 5–2000 ng/mL in plasma, 5–500 ng/mL in ultrafiltered plasma solution, and 25–2000 ng/mL in urine. Mean recoveries of PLC in plasma, ultrafiltered plasma solution, and urine were 93.2–99.7%, 91.4–100.6%, and 93.9–104.7%, respectively. Intra- and interday coefficients of variation for PLC were less than 6.0% and 4.3% in plasma, 6.1% and 3.7% in ultrafiltered plasma solution, and 5.4% and 2.5% in urine at the above concentration ranges, respectively. The lower limit of quantification for PLC in plasma, ultrafiltered plasma solution, and urine were 5 ng/mL, 5 ng/mL, and 25 ng/mL, respectively. This method can be applied to pharmacokinetic study and therapeutic drug monitoring in special populations such as neonates, infants, and the elderly by making effective use of residual samples used for general clinical laboratory testing. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Pilsicainide (PLC) is a class Ic antiarrhythmic agent derived from lidocaine. It was developed in Japan and is widely used for the treatment of supraventricular and ventricular arrhythmia. An overdose of PLC results in adverse drug reactions including circulatory disorders, mental disorders, neuropathy, ventricular fibrillation, ventricular tachycardia, and syncope [1,2]. As the therapeutic range of PLC (0.2–0.9 g/mL) in plasma [3] is narrow, therapeutic drug monitoring (TDM) is recommended in the clinical practice when PLC is administered to patients with arrhythmia.
∗ Corresponding author. Tel.: +81 3 5400 2120; fax: +81 3 5400 2120. E-mail address: [email protected] (M. Hashiguchi). http://dx.doi.org/10.1016/j.jchromb.2015.01.013 1570-0232/© 2015 Elsevier B.V. All rights reserved.
In terms of the pharmacokinetics of PLC in humans, about 90% of orally administered PLC is recovered in urine; 75–86% is recovered as the unchanged form, and about 5% is recovered as 2hydroxymethylate and minor amounts of other metabolites [4]. The elimination half-life (t1/2 ) of PLC is 4.5–4.9 h. As the prolongation of the t1/2 of PLC is related to decreased creatinine clearance in the kidneys [5,6], it is necessary to select the dose of PLC depending on the degree of renal dysfunction. Therefore, because the renal clearance of PLC may need to be estimated in those patients, the urinary concentration of PLC must be determined at first. PLC also binds to ␣1 -acid glycoprotein, which is an acute-phase reaction protein, and albumin in plasma [7]. Therefore, the unbound plasma concentration of PLC, which is related to its pharmacological effects, will change with differences in the fractions of plasma proteins in various pathophysiological conditions such as inflammatory disease and kidney disease.
M. Shimizu et al. / J. Chromatogr. B 985 (2015) 172–179
In patients with such conditions, TDM to detect the unbound plasma concentration of PLC is recommended to ensure the administration of an effective dose and/or avoid adverse drug effects. Generally, however, it is not possible to obtain a sufficient volume of ultrafiltered plasma solution for the measurement of the unbound plasma concentration of PLC because the centrifugal separation of plasma usually must be a volume of less than 10%, reflecting the equilibrium between the unbound and bound fractions of the compound. In addition, in some clinical situations, frequent sampling of large volumes of blood is not possible, as in neonates, infants, and the elderly, when TDM must be performed routinely. In such situations, a sensitive method to determine the plasma concentration of PLC which reuses the residual material for general biochemistry laboratory testing would be both efficient and economical as well as minimally invasive. The mechanism underlying PLC elimination has yet to be clearly understood because the results of some studies on the mechanism of renal elimination of PLC yielded controversial results, and PLC pharmacokinetics show wide interindividual variation, including hepatic/gastrointestinal metabolism and renal excretion. We previously reported that PLC is excreted via the organic cationic transport system in the renal proximal tubule in humans [8] and the elimination of PLC was not inhibited by verapamil, a potent P-glycoprotein inhibitor, in human and experimental studies [9]. In contrast, Tsuruoka et al. [10] suggested that the excretion of PLC in the kidney is mediated by human multidrug resistance protein 1 based on the results of a pharmacokinetic drug–drug interaction study of PLC. Moreover, a case report suggested that the metabolic rate of PLC was increased by cytochrome p450 induction [11]. Therefore, a quantitative method to investigate the detailed elimination mechanism of PLC is needed. Currently, high-performance liquid chromatography (HPLC) is the main method for determining the concentration of PLC in biological fluids. To the best of our knowledge, only five previous reports described the determination of PLC plasma and/or urine concentrations in humans [4,5,7,8,12]. The limit of detection (LOD) of PLC using solid-phase extraction was reported to be 50 ng/mL in 1-mL sample of both human plasma and urine [4,5], and that using the liquid–liquid extraction method was 10 ng/mL [8] and 50 ng/mL [7] in 0.5-mL sample, respectively. However, while assay methods using a solid-phase extraction column are convenient, they are not economical. Kim et al. [12] used 50 L of plasma obtained using the deproteination method to assay PLC, although that method cannot measure the unbound PLC concentration. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay has several advantages over HPLC, including high sensitivity and no interference from background peaks due to specific m/z ion monitoring. The high sensitivity of this assay is suitable for TDM in special populations such as neonates and infants from whom only 100 L of whole blood can be collected, and/or for measurement of the unbound plasma concentration of PLC since only a microscale volume of sample is required. Recently, Qui et al. [13] have reported a human pharmacokinetic study of PLC using LC–MS/MS, but this was not a quantitative methodology study. There has been no paper reporting on the quantitative methodology of PLC using LC–MS/MS. This paper describes a sensitive, reliable method for the determination of PLC in microscale volumes of human plasma, ultrafiltered plasma solution, and urine using LC–MS/MS with an electrospray ionization (ESI) interface, which was validated according to the US Food and Drug Administration (FDA) guidelines [14]. Additionally, this assay is suitable for TDM in hospital and in pharmacokinetic studies in humans following oral administration of PLC hydrochloride (50 mg) because it is more convenient and less time-consuming than other methods.
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2. Experimental 2.1. Chemicals PLC (pKa 10.2) hydrochloride was kindly provided by DaiichiSankyo Pharmaceutical Industry (Tokyo, Japan). Quinidine (pKa 4.3, 8.4), used as the internal standard (I.S.), was purchased from Wako Pure Chemical Industries (Osaka, Japan). The purity of these materials was greater than 98%. Ammonium acetate, methanol, diethylether, sodium bicarbonate, and water of LC/MS grade were also from Wako Pure Chemical Industries.
2.2. LC–MS/MS conditions The assay was developed using a 3200 QTRAP LC/MS/MS system (AB SCIEX, Tokyo, Japan), including a LC-20AD prominence liquid chromatograph, DGU-20A3 prominence degasser, CTO-20A prominence column oven, SIL-20AC HT prominence autosampler, and CBM-20A prominence communications bus module (Shimadzu, Kyoto, Japan). The module was controlled by Analyst Software on Windows NT 4.0 and connected to a 3200 QTRAP LC/MS/MS-based mass spectrometer equipped with an ESI source (AB SCIEX). The analytes were separated on an l-column (75 mm × 2.1 mm i.d., particle size 2 m, CERI, Saitama, Japan), preceded by an l-column precolumn filter. The temperature of the column was maintained at 25 ◦ C. Samples were eluted isocratically using a mobile phase composed of 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v) at a flow rate of 0.2 mL/min. The MS conditions were as follows: curtain gas 40, collision gas 4, ion-spray voltage 5500 V, temperature 700 ◦ C, ion source gas 1–70 psi, ion source gas 2–70 psi, declustering potential 70 V, entrance potential 10 V, collision energy 50 V, and collision cell exit potential 3 V. The positive-ion mode was used, and selected-ion monitoring was performed at m/z 273 → m/z 110 for PLC and m/z 325 → m/z 79 for quinidine as the I.S.
2.3. Preparation of stock and working solutions Stock solutions of PLC and the I.S. were prepared by dissolving an appropriate amount of each compound in methanol to yield concentrations of 1 mg/mL for generating standard curves. Working standard solutions of PLC were prepared by serial dilution with 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v). The working standard solution of the I.S. (1 g/mL) was obtained by 1000-fold dilution of the stock solution (1 mg/mL) with 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v). Stock solutions were stable at −20 ◦ C for at least 10 months for the purpose of analysis.
2.4. Extraction procedure A 0.1 mL of I.S. solution (1 g/mL) and 0.1 mL of Na2 CO3 (0.1 M) were added to 0.1 mL of human biological fluids (plasma, ultrafiltered plasma solution, or urine) in a 1.5-mL plastic tube. The tubes were vortex-mixed for 10 s, and 1 mL of diethylether was added as an extraction solution. After 30 s of vortex-mixing, the mixture was centrifuged at 9000 × g for 5 min at 4 ◦ C, and the organic phase (1 mL) was evaporated to dryness at 40 ◦ C under a stream of nitrogen gas. The residue was dissolved in 250 L of 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v) and vortex-mixed. A 2-L portion from each sample was injected into the LC–MS/MS system.
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M. Shimizu et al. / J. Chromatogr. B 985 (2015) 172–179
2.5. Preparation of calibration standards and quality control samples The human drug-free plasma, ultrafiltered plasma solution, and urine (so-called blank plasma, ultrafiltered blank plasma solution, and blank urine) were used for the validation studies of the analytical method. Calibration curves were prepared by spiking 0.1 mL of the working solutions in 0.1 mL of drug-free plasma, ultrafiltered plasma solution, or urine to yield final PLC concentrations of 5, 50, 250, 500, and 2000 ng/mL for plasma; 5, 50, and 500 ng/mL for ultrafiltered plasma solution; and 25, 250, 500, and 2000 ng/mL for urine. Standard curves were prepared daily and constructed by linear-regression analysis of the compound to the I.S. peak-area ratio versus the respective concentrations of PLC. Stock solutions of PLC were separately prepared for quality control samples in the same manner as for the standard curves. Quality control samples were obtained by spiking 0.1 mL of the working solutions in 0.1 mL of drug-free plasma, ultrafiltered plasma solution, or urine to yield final PLC concentrations of 25, 100, and 1000 ng/mL for plasma; 25, 100, and 250 ng/mL for ultrafiltered plasma solution; and 50, 100, and 1000 ng/mL for urine. Quality control samples were kept at −20 ◦ C until analysis.
2.6. Precision and accuracy Intra- and interday precision and accuracy were evaluated by assaying quality control samples with three different concentrations of PLC. Intra- and interday precision was assessed by analyzing five quality control samples at each concentration on the same day and mean values of a quality control sample for 5 days, respectively. The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the lower limit of quantification (LLOQ), at which it should not exceed 20% of the CV [13]. Accuracy was calculated as percent error (relative error) [(measured concentration − spiked concentration)/spiked concentration] × 100 (%), the precision of which was quantified by calculating intra- and inter-CV values. The matrix effect was evaluated by comparing the area of pilsicainide dissolved with blank matrix extract with the areas dissolved with the mobile phase at concentrations of 25 ng/mL, 100 ng/mL, and 1000 ng/mL for plasma, 25 ng/mL, 100 ng/mL, and 250 ng/mL for ultrafiltered plasma solution, and 50 ng/mL, 100 ng/mL, and 1000 ng/mL for urine, respectively. For the I.S., the matrix effect was evaluated by comparing the area of quinidine dissolved with blank matrix extract with the areas dissolved with the mobile phase at concentrations of 1 g/mL for plasma, ultrafiltered plasma solution, and urine. The stability of PLC in plasma, ultrafiltered plasma solution, and urine was assessed by analyzing QC samples at three concentrations through three freeze–thaw cycles (−20 ◦ C to room temperature as one cycle), on the bench at room temperature for 72 h, at −20 ◦ C after being stored in a freezer for 1 and 12 months, and on the autosampler rack at 4 ◦ C in the autosampler for 48 h (extracted sample/autosampler stability). All the samples were analyzed with the calibration curves that were freshly prepared. The analytes were considered stable when percentage deviation was ±15%. Chromatographic performance was evaluated by the retention factor (k): k=
tR t0
=
tR − t0 t0
where tR and tR are the relative retention time and the retention time of the analyte, respectively, and t0 is the elution time of an unretained solute.
2.7. Pharmacokinetic study design and sample collection Three healthy male Japanese volunteers were enrolled in this study after giving written informed consent. The mean (±standard deviation) age and body weight of the volunteers were 28.3 (±7.5) years and 63.7 (±6.5) kg, respectively. This study was approved by the Institutional Review Boards of Tokyo Women’s Medical University, Clinical Research Hospital Tokyo, and Ethics Committee of the Faculty of Pharmacy, Keio University. Each volunteer received an oral dose of 50 mg of PLC hydrochloride with 150 mL of water after an overnight fast. All volunteers were allowed to drink freely during the study. Blood was drawn into heparinized Vacutainer tubes (Becton Dickinson & Co., Rutherford, NJ) from a forearm vein before and 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 6.0, 12.0, and 24.0 h after drug administration. The blood samples were centrifuged (1450 × g for 10 min at 4 ◦ C) immediately, and the plasma was stored in plastic vials at −20 ◦ C until analysis. The degree of PLC binding to the plasma components was determined in the plasma samples 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 6.0, 12.0, and 24.0 h after drug administration. The PLC plasma protein binding experiment was conducted after subjecting the samples to centrifugation at 1450 × g for 5 min at 25 ◦ C in a micro ultrafiltration system with Centricut V-10 (Kurabou Industries Ltd., Osaka, Japan). Urine samples were collected and stored in plastic bottles at the following time intervals: 0–1, 1–2, 2–3, 3–4, 4–8, 8–16, and 16–32 h after PLC administration. The urinary volume was recorded, and a portion of each sample was stored at −20 ◦ C until analysis.
2.8. Pharmacokinetic data and statistical analyses The maximum plasma concentration (Cmax ) and time to reach Cmax (tmax ) of PLC were determined directly from the individual concentration–time data. The elimination rate constant (ke ) was obtained by linear-regression analysis using at least three sampling points of the terminal log-linear declining phase to the final measurable concentration. The elimination half-life (t1/2 ) was determined from the equation: t1/2 = ln 2/ke . The area under the plasma concentration–time curve from time zero to the final sampling time (tn ) of the plasma level (AUC0–tn ) was determined following the trapezoidal rule. The AUC from time zero to infinity (AUC0–∞ ) was calculated according to the following equation: AUC0–∞ = AUC0–tn +
Cpt ke
where Cpt is the observed plasma concentration at tn . The apparent oral clearance (CLoral ) was determined by the following equation: CLoral =
Dose AUC0–∞
The fraction of the absorbed dose excreted unchanged in the urine (Ae) was calculated as the amount of PLC recovered in the urine divided by the amount of PLC administered. The unbound fraction in plasma (fu) was expressed as the quotient of the ultrafiltered concentration divided by the prefiltered plasma concentration. The fractional renal clearance (CLr) (0–1, 1–2, 2–3, 3–4, 4–8, 8–16 h) was expressed as the quotient of the amount recovered in urine divided by the midpoint of the plasma concentration of PLC for the corresponding periods. The CLr over the period from 0 to 32 h was expressed as the quotient of the amount recovered in the urine divided by the AUC0–∞ .
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Fig. 1. Product ion spectra of (A) PLC and (B) the I.S. quinidine.
3. Results and discussion
3.2. Linearity
3.1. Chromatographic optimization
Calibration curves were linear over the concentration range from 5 to 2000 ng/mL for PLC in plasma (r2 = 0.9993 and F = 5239.156, p < 0.001, n = 5), in ultrafiltered plasma solution (r2 = 0.9991 and F = 18,831.428, p < 0.001, n = 5), and in urine (r2 = 0.9997 and F = 2008.547, p < 0.001, n = 5), respectively (Table 1).
This paper describes a new method for the determination of PLC in human biological fluids (plasma, ultrafiltered plasma solution, and urine) using LC–MS/MS with an ESI interface. Our goal was to develop a more sensitive, reliable method requiring only a microscale volume of human biological fluids to perform TDM in clinical practice or to evaluate precisely the human pharmacokinetics of orally administered PLC with LC–MS/MS. In general, the LC–MS/MS method is superior to HPLC–UV, especially in its sensitivity and selectivity in detecting PLC in plasma and urine. In this study, we selected the MS conditions with the ESI interface described in Section 2.2. Mass spectra of PLC and quinidine are shown in Fig. 1, and ions at 273 → 110 and 325 → 79, respectively, were selected for subsequent quantitative analysis. The calibration curve for PLC ranged from 5 to 2000 ng/mL in human plasma, from 5 to 500 ng/mL in ultrafiltered plasma solution, and from 25 to 2000 ng/mL in urine, which were the concentration ranges of PLC in biological fluids in previous reports [7–10]. In addition, a LLOQ was obtained by simple extraction procedure using diethylether. Moreover, there were no interfering peaks of endogenous substances during the retention times of PLC and the I.S. (Fig. 2) when this method was applied in a human pharmacokinetic study in three healthy volunteers. To the best of our knowledge, this is the first quantitative methodology report on the determination of PLC which enables the determination of orally administered PLC using a microscale volume (
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Quantitation of pilsicainide in microscale samples of human biological fluids using liquid chromatography–tandem mass spectrometry Mikiko Shimizu a , Masayuki Hashiguchi b,∗ , Tsuyoshi Shiga c , Koichi Nakamura d , Hiro-omi Tamura a , Mayumi Mochizuki b a
Department of Hygienic Chemistry, Faculty of Pharmacy, Keio University, Tokyo, Japan Division for Evaluation and Analysis of Drug Information, Faculty of Pharmacy, Keio University, Tokyo, Japan c Department of Cardiology, Tokyo Women’s Medical University, Tokyo, Japan d Clinical Research Tokyo Hospital, Tokyo, Japan b
a r t i c l e
i n f o
Article history: Received 4 June 2014 Accepted 11 January 2015 Available online 22 January 2015 Keywords: Pilsicainide LC–MS/MS Plasma Urine Pharmacokinetics Therapeutic drug monitoring
a b s t r a c t This paper describes a sensitive, reliable method to determine pilsicainide (PLC) levels in microscale sample volumes of human biological fluids using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI). PLC and quinidine as an internal standard were extracted with diethylether from 0.1 mL of alkalinized biological fluids. The extract was injected into an analytical column (l-column 2 ODS, 75 mm × 2.1 mm i.d.). The mobile phase for separation consisted of 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v) and was delivered at a flow rate of 0.2 mL/min. The drift voltage was 100 V. The sampling aperture was heated at 120 ◦ C and the shield temperature was 260 ◦ C. The ion transitions used to monitor analytes were m/z 273 → m/z 110 for PLC and m/z 325 → m/z 79 for quinidine. The total time for chromatographic separation was less than 8 min. The validated concentration ranges of this method for PLC were 5–2000 ng/mL in plasma, 5–500 ng/mL in ultrafiltered plasma solution, and 25–2000 ng/mL in urine. Mean recoveries of PLC in plasma, ultrafiltered plasma solution, and urine were 93.2–99.7%, 91.4–100.6%, and 93.9–104.7%, respectively. Intra- and interday coefficients of variation for PLC were less than 6.0% and 4.3% in plasma, 6.1% and 3.7% in ultrafiltered plasma solution, and 5.4% and 2.5% in urine at the above concentration ranges, respectively. The lower limit of quantification for PLC in plasma, ultrafiltered plasma solution, and urine were 5 ng/mL, 5 ng/mL, and 25 ng/mL, respectively. This method can be applied to pharmacokinetic study and therapeutic drug monitoring in special populations such as neonates, infants, and the elderly by making effective use of residual samples used for general clinical laboratory testing. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Pilsicainide (PLC) is a class Ic antiarrhythmic agent derived from lidocaine. It was developed in Japan and is widely used for the treatment of supraventricular and ventricular arrhythmia. An overdose of PLC results in adverse drug reactions including circulatory disorders, mental disorders, neuropathy, ventricular fibrillation, ventricular tachycardia, and syncope [1,2]. As the therapeutic range of PLC (0.2–0.9 g/mL) in plasma [3] is narrow, therapeutic drug monitoring (TDM) is recommended in the clinical practice when PLC is administered to patients with arrhythmia.
∗ Corresponding author. Tel.: +81 3 5400 2120; fax: +81 3 5400 2120. E-mail address: [email protected] (M. Hashiguchi). http://dx.doi.org/10.1016/j.jchromb.2015.01.013 1570-0232/© 2015 Elsevier B.V. All rights reserved.
In terms of the pharmacokinetics of PLC in humans, about 90% of orally administered PLC is recovered in urine; 75–86% is recovered as the unchanged form, and about 5% is recovered as 2hydroxymethylate and minor amounts of other metabolites [4]. The elimination half-life (t1/2 ) of PLC is 4.5–4.9 h. As the prolongation of the t1/2 of PLC is related to decreased creatinine clearance in the kidneys [5,6], it is necessary to select the dose of PLC depending on the degree of renal dysfunction. Therefore, because the renal clearance of PLC may need to be estimated in those patients, the urinary concentration of PLC must be determined at first. PLC also binds to ␣1 -acid glycoprotein, which is an acute-phase reaction protein, and albumin in plasma [7]. Therefore, the unbound plasma concentration of PLC, which is related to its pharmacological effects, will change with differences in the fractions of plasma proteins in various pathophysiological conditions such as inflammatory disease and kidney disease.
M. Shimizu et al. / J. Chromatogr. B 985 (2015) 172–179
In patients with such conditions, TDM to detect the unbound plasma concentration of PLC is recommended to ensure the administration of an effective dose and/or avoid adverse drug effects. Generally, however, it is not possible to obtain a sufficient volume of ultrafiltered plasma solution for the measurement of the unbound plasma concentration of PLC because the centrifugal separation of plasma usually must be a volume of less than 10%, reflecting the equilibrium between the unbound and bound fractions of the compound. In addition, in some clinical situations, frequent sampling of large volumes of blood is not possible, as in neonates, infants, and the elderly, when TDM must be performed routinely. In such situations, a sensitive method to determine the plasma concentration of PLC which reuses the residual material for general biochemistry laboratory testing would be both efficient and economical as well as minimally invasive. The mechanism underlying PLC elimination has yet to be clearly understood because the results of some studies on the mechanism of renal elimination of PLC yielded controversial results, and PLC pharmacokinetics show wide interindividual variation, including hepatic/gastrointestinal metabolism and renal excretion. We previously reported that PLC is excreted via the organic cationic transport system in the renal proximal tubule in humans [8] and the elimination of PLC was not inhibited by verapamil, a potent P-glycoprotein inhibitor, in human and experimental studies [9]. In contrast, Tsuruoka et al. [10] suggested that the excretion of PLC in the kidney is mediated by human multidrug resistance protein 1 based on the results of a pharmacokinetic drug–drug interaction study of PLC. Moreover, a case report suggested that the metabolic rate of PLC was increased by cytochrome p450 induction [11]. Therefore, a quantitative method to investigate the detailed elimination mechanism of PLC is needed. Currently, high-performance liquid chromatography (HPLC) is the main method for determining the concentration of PLC in biological fluids. To the best of our knowledge, only five previous reports described the determination of PLC plasma and/or urine concentrations in humans [4,5,7,8,12]. The limit of detection (LOD) of PLC using solid-phase extraction was reported to be 50 ng/mL in 1-mL sample of both human plasma and urine [4,5], and that using the liquid–liquid extraction method was 10 ng/mL [8] and 50 ng/mL [7] in 0.5-mL sample, respectively. However, while assay methods using a solid-phase extraction column are convenient, they are not economical. Kim et al. [12] used 50 L of plasma obtained using the deproteination method to assay PLC, although that method cannot measure the unbound PLC concentration. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay has several advantages over HPLC, including high sensitivity and no interference from background peaks due to specific m/z ion monitoring. The high sensitivity of this assay is suitable for TDM in special populations such as neonates and infants from whom only 100 L of whole blood can be collected, and/or for measurement of the unbound plasma concentration of PLC since only a microscale volume of sample is required. Recently, Qui et al. [13] have reported a human pharmacokinetic study of PLC using LC–MS/MS, but this was not a quantitative methodology study. There has been no paper reporting on the quantitative methodology of PLC using LC–MS/MS. This paper describes a sensitive, reliable method for the determination of PLC in microscale volumes of human plasma, ultrafiltered plasma solution, and urine using LC–MS/MS with an electrospray ionization (ESI) interface, which was validated according to the US Food and Drug Administration (FDA) guidelines [14]. Additionally, this assay is suitable for TDM in hospital and in pharmacokinetic studies in humans following oral administration of PLC hydrochloride (50 mg) because it is more convenient and less time-consuming than other methods.
173
2. Experimental 2.1. Chemicals PLC (pKa 10.2) hydrochloride was kindly provided by DaiichiSankyo Pharmaceutical Industry (Tokyo, Japan). Quinidine (pKa 4.3, 8.4), used as the internal standard (I.S.), was purchased from Wako Pure Chemical Industries (Osaka, Japan). The purity of these materials was greater than 98%. Ammonium acetate, methanol, diethylether, sodium bicarbonate, and water of LC/MS grade were also from Wako Pure Chemical Industries.
2.2. LC–MS/MS conditions The assay was developed using a 3200 QTRAP LC/MS/MS system (AB SCIEX, Tokyo, Japan), including a LC-20AD prominence liquid chromatograph, DGU-20A3 prominence degasser, CTO-20A prominence column oven, SIL-20AC HT prominence autosampler, and CBM-20A prominence communications bus module (Shimadzu, Kyoto, Japan). The module was controlled by Analyst Software on Windows NT 4.0 and connected to a 3200 QTRAP LC/MS/MS-based mass spectrometer equipped with an ESI source (AB SCIEX). The analytes were separated on an l-column (75 mm × 2.1 mm i.d., particle size 2 m, CERI, Saitama, Japan), preceded by an l-column precolumn filter. The temperature of the column was maintained at 25 ◦ C. Samples were eluted isocratically using a mobile phase composed of 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v) at a flow rate of 0.2 mL/min. The MS conditions were as follows: curtain gas 40, collision gas 4, ion-spray voltage 5500 V, temperature 700 ◦ C, ion source gas 1–70 psi, ion source gas 2–70 psi, declustering potential 70 V, entrance potential 10 V, collision energy 50 V, and collision cell exit potential 3 V. The positive-ion mode was used, and selected-ion monitoring was performed at m/z 273 → m/z 110 for PLC and m/z 325 → m/z 79 for quinidine as the I.S.
2.3. Preparation of stock and working solutions Stock solutions of PLC and the I.S. were prepared by dissolving an appropriate amount of each compound in methanol to yield concentrations of 1 mg/mL for generating standard curves. Working standard solutions of PLC were prepared by serial dilution with 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v). The working standard solution of the I.S. (1 g/mL) was obtained by 1000-fold dilution of the stock solution (1 mg/mL) with 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v). Stock solutions were stable at −20 ◦ C for at least 10 months for the purpose of analysis.
2.4. Extraction procedure A 0.1 mL of I.S. solution (1 g/mL) and 0.1 mL of Na2 CO3 (0.1 M) were added to 0.1 mL of human biological fluids (plasma, ultrafiltered plasma solution, or urine) in a 1.5-mL plastic tube. The tubes were vortex-mixed for 10 s, and 1 mL of diethylether was added as an extraction solution. After 30 s of vortex-mixing, the mixture was centrifuged at 9000 × g for 5 min at 4 ◦ C, and the organic phase (1 mL) was evaporated to dryness at 40 ◦ C under a stream of nitrogen gas. The residue was dissolved in 250 L of 5 mM ammonium acetate (pH 4.5)/methanol (4:1, v/v) and vortex-mixed. A 2-L portion from each sample was injected into the LC–MS/MS system.
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M. Shimizu et al. / J. Chromatogr. B 985 (2015) 172–179
2.5. Preparation of calibration standards and quality control samples The human drug-free plasma, ultrafiltered plasma solution, and urine (so-called blank plasma, ultrafiltered blank plasma solution, and blank urine) were used for the validation studies of the analytical method. Calibration curves were prepared by spiking 0.1 mL of the working solutions in 0.1 mL of drug-free plasma, ultrafiltered plasma solution, or urine to yield final PLC concentrations of 5, 50, 250, 500, and 2000 ng/mL for plasma; 5, 50, and 500 ng/mL for ultrafiltered plasma solution; and 25, 250, 500, and 2000 ng/mL for urine. Standard curves were prepared daily and constructed by linear-regression analysis of the compound to the I.S. peak-area ratio versus the respective concentrations of PLC. Stock solutions of PLC were separately prepared for quality control samples in the same manner as for the standard curves. Quality control samples were obtained by spiking 0.1 mL of the working solutions in 0.1 mL of drug-free plasma, ultrafiltered plasma solution, or urine to yield final PLC concentrations of 25, 100, and 1000 ng/mL for plasma; 25, 100, and 250 ng/mL for ultrafiltered plasma solution; and 50, 100, and 1000 ng/mL for urine. Quality control samples were kept at −20 ◦ C until analysis.
2.6. Precision and accuracy Intra- and interday precision and accuracy were evaluated by assaying quality control samples with three different concentrations of PLC. Intra- and interday precision was assessed by analyzing five quality control samples at each concentration on the same day and mean values of a quality control sample for 5 days, respectively. The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the lower limit of quantification (LLOQ), at which it should not exceed 20% of the CV [13]. Accuracy was calculated as percent error (relative error) [(measured concentration − spiked concentration)/spiked concentration] × 100 (%), the precision of which was quantified by calculating intra- and inter-CV values. The matrix effect was evaluated by comparing the area of pilsicainide dissolved with blank matrix extract with the areas dissolved with the mobile phase at concentrations of 25 ng/mL, 100 ng/mL, and 1000 ng/mL for plasma, 25 ng/mL, 100 ng/mL, and 250 ng/mL for ultrafiltered plasma solution, and 50 ng/mL, 100 ng/mL, and 1000 ng/mL for urine, respectively. For the I.S., the matrix effect was evaluated by comparing the area of quinidine dissolved with blank matrix extract with the areas dissolved with the mobile phase at concentrations of 1 g/mL for plasma, ultrafiltered plasma solution, and urine. The stability of PLC in plasma, ultrafiltered plasma solution, and urine was assessed by analyzing QC samples at three concentrations through three freeze–thaw cycles (−20 ◦ C to room temperature as one cycle), on the bench at room temperature for 72 h, at −20 ◦ C after being stored in a freezer for 1 and 12 months, and on the autosampler rack at 4 ◦ C in the autosampler for 48 h (extracted sample/autosampler stability). All the samples were analyzed with the calibration curves that were freshly prepared. The analytes were considered stable when percentage deviation was ±15%. Chromatographic performance was evaluated by the retention factor (k): k=
tR t0
=
tR − t0 t0
where tR and tR are the relative retention time and the retention time of the analyte, respectively, and t0 is the elution time of an unretained solute.
2.7. Pharmacokinetic study design and sample collection Three healthy male Japanese volunteers were enrolled in this study after giving written informed consent. The mean (±standard deviation) age and body weight of the volunteers were 28.3 (±7.5) years and 63.7 (±6.5) kg, respectively. This study was approved by the Institutional Review Boards of Tokyo Women’s Medical University, Clinical Research Hospital Tokyo, and Ethics Committee of the Faculty of Pharmacy, Keio University. Each volunteer received an oral dose of 50 mg of PLC hydrochloride with 150 mL of water after an overnight fast. All volunteers were allowed to drink freely during the study. Blood was drawn into heparinized Vacutainer tubes (Becton Dickinson & Co., Rutherford, NJ) from a forearm vein before and 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 6.0, 12.0, and 24.0 h after drug administration. The blood samples were centrifuged (1450 × g for 10 min at 4 ◦ C) immediately, and the plasma was stored in plastic vials at −20 ◦ C until analysis. The degree of PLC binding to the plasma components was determined in the plasma samples 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 6.0, 12.0, and 24.0 h after drug administration. The PLC plasma protein binding experiment was conducted after subjecting the samples to centrifugation at 1450 × g for 5 min at 25 ◦ C in a micro ultrafiltration system with Centricut V-10 (Kurabou Industries Ltd., Osaka, Japan). Urine samples were collected and stored in plastic bottles at the following time intervals: 0–1, 1–2, 2–3, 3–4, 4–8, 8–16, and 16–32 h after PLC administration. The urinary volume was recorded, and a portion of each sample was stored at −20 ◦ C until analysis.
2.8. Pharmacokinetic data and statistical analyses The maximum plasma concentration (Cmax ) and time to reach Cmax (tmax ) of PLC were determined directly from the individual concentration–time data. The elimination rate constant (ke ) was obtained by linear-regression analysis using at least three sampling points of the terminal log-linear declining phase to the final measurable concentration. The elimination half-life (t1/2 ) was determined from the equation: t1/2 = ln 2/ke . The area under the plasma concentration–time curve from time zero to the final sampling time (tn ) of the plasma level (AUC0–tn ) was determined following the trapezoidal rule. The AUC from time zero to infinity (AUC0–∞ ) was calculated according to the following equation: AUC0–∞ = AUC0–tn +
Cpt ke
where Cpt is the observed plasma concentration at tn . The apparent oral clearance (CLoral ) was determined by the following equation: CLoral =
Dose AUC0–∞
The fraction of the absorbed dose excreted unchanged in the urine (Ae) was calculated as the amount of PLC recovered in the urine divided by the amount of PLC administered. The unbound fraction in plasma (fu) was expressed as the quotient of the ultrafiltered concentration divided by the prefiltered plasma concentration. The fractional renal clearance (CLr) (0–1, 1–2, 2–3, 3–4, 4–8, 8–16 h) was expressed as the quotient of the amount recovered in urine divided by the midpoint of the plasma concentration of PLC for the corresponding periods. The CLr over the period from 0 to 32 h was expressed as the quotient of the amount recovered in the urine divided by the AUC0–∞ .
M. Shimizu et al. / J. Chromatogr. B 985 (2015) 172–179
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Fig. 1. Product ion spectra of (A) PLC and (B) the I.S. quinidine.
3. Results and discussion
3.2. Linearity
3.1. Chromatographic optimization
Calibration curves were linear over the concentration range from 5 to 2000 ng/mL for PLC in plasma (r2 = 0.9993 and F = 5239.156, p < 0.001, n = 5), in ultrafiltered plasma solution (r2 = 0.9991 and F = 18,831.428, p < 0.001, n = 5), and in urine (r2 = 0.9997 and F = 2008.547, p < 0.001, n = 5), respectively (Table 1).
This paper describes a new method for the determination of PLC in human biological fluids (plasma, ultrafiltered plasma solution, and urine) using LC–MS/MS with an ESI interface. Our goal was to develop a more sensitive, reliable method requiring only a microscale volume of human biological fluids to perform TDM in clinical practice or to evaluate precisely the human pharmacokinetics of orally administered PLC with LC–MS/MS. In general, the LC–MS/MS method is superior to HPLC–UV, especially in its sensitivity and selectivity in detecting PLC in plasma and urine. In this study, we selected the MS conditions with the ESI interface described in Section 2.2. Mass spectra of PLC and quinidine are shown in Fig. 1, and ions at 273 → 110 and 325 → 79, respectively, were selected for subsequent quantitative analysis. The calibration curve for PLC ranged from 5 to 2000 ng/mL in human plasma, from 5 to 500 ng/mL in ultrafiltered plasma solution, and from 25 to 2000 ng/mL in urine, which were the concentration ranges of PLC in biological fluids in previous reports [7–10]. In addition, a LLOQ was obtained by simple extraction procedure using diethylether. Moreover, there were no interfering peaks of endogenous substances during the retention times of PLC and the I.S. (Fig. 2) when this method was applied in a human pharmacokinetic study in three healthy volunteers. To the best of our knowledge, this is the first quantitative methodology report on the determination of PLC which enables the determination of orally administered PLC using a microscale volume (
