Journal of Chromatography B, 958 (2014) 117–123

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Determination of mesoridazine by liquid chromatography–tandem mass spectrometry and its application to pharmacokinetic study in rats So Hee Im a,b , Myoung Joo Park a , Hyewon Seo a , Sung Heum Choi a , Sang Kyum Kim b , Sung-Hoon Ahn a,∗ a b

Department of Drug Discovery Platform Technology, Korea Research Institute of Chemical Technology (KRICT), Daejeon, Republic of Korea College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea

a r t i c l e

i n f o

Article history: Received 3 September 2013 Received in revised form 7 March 2014 Accepted 16 March 2014 Available online 24 March 2014 Keywords: LC–MS/MS Mesoridazine Method validation Rat plasma Pharmacokinetics Toxicokinetics

a b s t r a c t The object of the present study was to develop and validate an assay method of mesoridazine in rat plasma using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Plasma samples from rats were prepared by simple protein precipitation and injected onto the LC–MS/MS system for quantification. Mesoridazine and chlorpromazine as an internal standard (IS) were separated by a reversed phase C18 column. A mobile phase was composed of 10 mM ammonium formate in water and acetonitrile (ACN) (v/v) by a linear gradient system, increasing the percentage of ACN from 2% at 0.4 min to 98% at 2.5 min with 4 min total run time. The ion transitions monitored in positive-ion mode [M + H]+ of multiplereaction monitoring (MRM) were m/z 387 > 126 for mesoridazine and m/z 319 > 86 for IS. The detector response was specific and linear for mesoridazine at concentrations within the range 0.001–4 ␮g/ml and the correlation coefficient (R2 ) was greater than 0.999 and the signal-to-noise ratios for the samples were ≥10. The intra- and inter-day precision and accuracy of the method were determined to be within the acceptance criteria for assay validation guidelines. The matrix effects were approximately 101 and 99.5% from rat plasma for mesoridazine and chlorpromazine, respectively. Mesoridazine was stable under various processing and/or handling conditions. Mesoridazine concentrations were readily measured in rat plasma samples after intravenous and oral administration. This assay method can be practically useful to the pharmacokinetic and/or toxicokinetic studies of mesoridazine. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Mesoridazine, (10-[2-(1-methyl-2-piperidyl)ethyl]-2-(methylsufinyl)phenothiazine), is an antipsychotic agent as phenothiazines class antipsychotics [1–5] and a metabolite of thioridazine by a side-chain sulfoxidation [6–10]. Although mesoridazine has also been used for the treatment of psychotic disease such as schizophrenia similar to thioridazine during a half century, this drug has its severe cardiac toxicity including irregular heartbeats and QT-prolongation. Because of its side effects, mesoridazine is under the situation of withdrawal in some market. One of the reasons for cardiac toxicity of mesoridazine was involved with QT interval prolongation by inhibition of potassium hERG (human ether-a-go-go-related gene) channels [11–14]. There have been

∗ Corresponding author. Tel.: +82 42 860 7265; fax: +82 42 860 7459. E-mail address: [email protected] (S.-H. Ahn). http://dx.doi.org/10.1016/j.jchromb.2014.03.020 1570-0232/© 2014 Elsevier B.V. All rights reserved.

several reports about the relationship between exposure level and toxicity of mesoridazine [15–18]. However, the pharmacokinetic and/or toxicokinetic analysis including the mechanisms of absorption, distribution, metabolism, and excretion (ADME) of mesoridazine has not been clear yet. The validated and advanced assay method in biological fluids can be helpful for these studies. Mesoridazine was determined by the various assay methods including gas–liquid chromatography (GLC) [19–22], gas chromatography (GC) [23], and radioimmunoassay (RIA) [24], high-performance liquid chromatography (HPLC) [25–29]. Most of these methods are not good application to analyze mesoridazine levels in biological samples because of their low sensitivity and poor selectivity. Even though the determination of mesoridazine was also applied using liquid chromatography-tandem mass spectrometry (LC–MS/MS) methods with other Cytochrome P450 substrates or antipsychotics [30,31], these analytical methods were not enough for rapid and sensitive analytical method for further advanced pharmacokinetic studies using animal models for further

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pharmacological and toxicological studies because these methods were not focused on mesoridazine. An LC–MS/MS method has relatively short run times and higher sensitivity and selectivity in assays of various biological samples [32,33]. Multiple reaction monitoring (MRM) offers the selection of certain ions chosen by a specific precursor ion and collision-induced fragmental ion and a very powerful system for representative methods available for the determination of drug levels. There has been reported for LC–MS/MS method for mesoridazine including other antipsychotics. The method in the literature was not focused on mesoridazine, therefore, the analytical method showed timeconsuming liquid–liquid extraction (LLE) method and less sensitive (∼10 ng/ml) compared to this method. The aims of this study, therefore, were to develop and validate a rapid, simple, selective and sensitive method to assay mesoridazine in rat plasma using one-step protein precipitation that is applicable to pharmacokinetic studies. Furthermore, this sensitive validated method can be applicable to further pharmacokinetic, toxicokinetic, and pharmacological studies for the safety usage of mesoridazine using LC–MS/MS equipped with an electro-spray ionization (ESI) mode. 2. Experimental 2.1. Chemicals Mesoridazine, chlorpromazine, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG) 400 and ammonium formate were purchased at high purity over HPLC, TLC or Bioreagent grade from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN) of HPLC grade was from J.T. Baker (Phillipsburg, NJ, USA). Distilled water was obtained from a Milli-Q system (Millipore, Bedford, MA, USA) in our laboratory. 2.2. Calibration standard and quality control samples A stock solution of mesoridazine was prepared in DMSO at the concentration of 1 mg/ml. Working standard solutions were made by dilution of the standard stock solution in acetonitrile. Internal standard (IS) working solution (100 ng/ml chlorpromazine in ACN) was prepared from an IS stock solution (1 mg/ml). All solutions were stored at −20 ◦ C before sample preparation. A standard calibration curve for mesoridazine was prepared by spiking working standard solution in rat plasma at the concentration ranges of 0.001, 0.003, 0.1, 1, 4 and 5 ␮g/ml. Quality control (QC) samples were prepared for mesoridazine concentrations of 0.003, 0.1 and 4 ␮g/ml in blank plasma. 2.3. Instrumentation and chromatographic conditions The concentration of mesoridazine in rat plasma was quantified using Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) with an API 4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer (AB Sciex, Foster City, CA, USA) equipped with a turbo electrospray interface. The chromatographic separation of analyte was performed on a Hypersil GOLD C18 column (50 mm × 2.1 mm i.d., 3 ␮m; Thermo, Waltham, MA, USA) with a SecurityGuard C18 guard column (4 mm × 20 mm i.d., Phenomenex, USA) maintained at 35 ◦ C. The separation was carried out a linear gradient of 10 mM ammonium formate in water and ACN (v/v), increasing the percentage of ACN from 2% at 0.4 min to 98% at 2.5 min. The flow rate was 0.4 ml/min and overall chromatographic run time was 4 min. The mass spectrometer was set in MRM mode using target ions at m/z 387 → 126 of mesoridazine and m/z 319 → 86 for chlorpromazine (IS). The optimized instrument conditions were as follow: ion spray voltage,

5500 V; source temperature, 550 ◦ C; curtain gas (CUR), 10 psi; nebulizing gas (GS1), 50 psi; heating gas (GS2), 50 psi; collision energy (CE), 35 V for mesoridazine and 29 V for IS, respectively. Analytical data were performed with Analyst software Version 1.4.2. (AB Sciex). 2.4. Sample preparation Before analysis, plasma samples were thawed at room temperature together with calibration standards and QC samples. A rat plasma sample (30 ␮l) was placed in a 1.5 ml microfuge tube and mixed with 270 ␮l of IS solution (100 ng/ml chlorpromazine in acetonitrile) to precipitate the plasma proteins. The mixture was vigorously mixed for 10 min using vortex mixer and followed by centrifugation at 10,000g for 10 min at 4 ◦ C using temperature controlled microcentrifuge (Eppendorf, Hamburg, Germany). An aliquot of 5 ␮l supernatant was directly injected into the LC–MS/MS system for analysis after transferring to a fresh vial. 2.5. Validation The analytical method for mesoridazine was determined with regard to selectivity, sensitivity, linearity, precision and accuracy. For selectivity validation, interference by endogenous compounds was assessed by comparison of the chromatograms for blank plasma samples. For sensitivity of mesoridazine, the limit of quantification (LOQ) was determined as the concentration with sufficient precision within 20% of the relative standard deviation (RSD) and acceptable accuracy between 80 and 120% of the theoretical value. In this study, the limit of detection (LOD) and LOQ values were 0.5 and 1 ng/ml with the signal-to-noise ratios over 3 and 10, respectively. To evaluate the linearity, using standard plasma solution containing mesoridazine in the concentration range of 0.001–4 ␮g/ml. Calibration curves were constructed by weighted (1/x) least squares linear regression of the peak area ratios (y) of mesoridazine to internal standard, versus the concentration (x) in ␮g/ml. Precision and accuracy of the method were estimated using replicate samples (n = 5). The intra- and inter-day precisions were estimated by analyzing the spiked samples at four different concentrations (0.001, 0.003, 0.1 and 4 ␮g/ml) in a single day and for five days, respectively. Precision was expressed as RSD at each concentration level. The percentage accuracy of deviation of the calculated concentration from theoretical concentration was expressed as the relative error (RE). 2.6. Matrix effect The matrix effect for mesoridazine was assessed by analyzing 2 sets of standards at three concentrations (0.003, 0.1 and 4 ␮g/ml). The matrix effect was performed by comparing the peak areas of the analytes that was spiked into the post-precipitation matrix (set 1) with those of the reference standards prepared by spiking with the same concentration of mesoridazine in the mobile phase (set 2): matrix effect = mean peak area of an analyte added postprecipitation (set 1)/mean peak area of the same analyte standards (set 2) × 100. Each sample set was performed in triplicate. 2.7. Stability The stability of mesoridazine was analyzed to evaluate the analyte stability in plasma samples under various storage or handling conditions. Assessment of mesoridazine stability in rat plasma included short- and long-term tests at the QC levels. To analyze short-term stability included: (a) freeze–thaw cycle stability; (b) exposure of samples to room temperature for 1 day; (c) exposure to 4 ◦ C for 1 day; (d) exposure to −20 ◦ C for 1 day; (e) exposure to

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−80 ◦ C for 1 day; (f) exposure in mobile phase at 4 ◦ C for 1 day; (g) exposure in mobile phase at 4 ◦ C for 1 week after preparation, and (h) exposure in mobile phase at room temperature for 1 day after preparation. To examine long-term stability included: (a) exposure of samples to −20 ◦ C for 30 days, and (b) exposure to −80 ◦ C for 30 days. 2.8. Pharmacokinetic study Male Sprague–Dawley rats (8-week of age, 250 g) were used for pharmacokinetic analysis of mesoridazine. Animals were kept plastic cages with free access to standard rat diet and water. The room was maintained at a temperature of 23 ± 3 ◦ C, relative humidity of 50 ± 10% with an approximately 12-h dark/light cycle. The dosing solutions were formulated by the concentrations, 2.5 mg/ml and 12.5 mg/ml in PEG 400:distilled water:DMSO

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at a ratio of 40:55:5 for intravenous and oral administrations, respectively. Blood samples (about 0.2 ml) were collected from the femoral vein into heparinized collection tubes at pre-dose, 0.033, 0.167, 0.5, 1, 2, 4, 8, 12 and 24 h after intravenous administration (5 mg/kg dose, n = 5) and pre-dose, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h after oral administration (25 mg/kg dose, n = 5). The dosage was administrated at a final volume of 2 ml/kg (e.g., 500 ␮l of dosage volume in case of 250 g rat). Blood samples were centrifuged promptly and stored at −70 ◦ C until analysis. For the in vivo study of mesoridazine, pharmacokinetic parameters were calculated by non-compartmental analysis using the nonlinear least squares regression program WinNonlin 5.3 (Pharsight, Mountain View, CA, USA). The area under the plasma concentration–time curve from time zero to the last time point measured concentration (AUCh ) and to infinite time (AUC∞ ) by adding extrapolated area were calculated using the trapezoidal method. The extent of

Fig. 1. The structures and product-ion scan spectra of (A) mesoridazine and (B) chlorpromazine (IS).

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Table 1 Reproducibility and accuracy for mesoridazine in rat plasma (n = 5). Theoretical concentration (␮g/ml) 0.001 0.003 0.1 4 a b

Intra-day

Inter-day

Concentration found (␮g/ml)

RSDa (%)

REb (%)

Concentration found (␮g/ml)

RSD (%)

RE (%)

0.00110 0.00290 0.106 3.86

0.590 2.59 1.44 1.88

6.32 1.72 6.03 3.49

0.00120 0.00300 0.107 3.92

8.31 2.41 5.27 3.63

16.9 1.64 6.59 1.98

RSD (%) = standard deviation of concentration/mean concentration × 100. RE (%) = (calculated concentration − theoretical concentration)/theoretical concentration × 100.

absolute oral bioavailability (F) was calculated as AUC∞,po /AUC∞ , iv × 100%. The maximum peak concentration (Cmax ), time to reach Cmax (Tmax ), the terminal elimination half-life (t1/2 ), total clearance (CL), apparent volume of distribution at steady state (Vss ), and mean residence time (MRT) for mesoridazine were obtained using individual plasma concentration–time profiles. All data are presented as mean ± S.D. 3. Results and discussion 3.1. Mass spectra and chromatography The product ion mass spectra of mesoridazine and internal standard are shown in Fig. 1. Under scan mode, mesoridazine and internal standard yielded mostly ions [M + H]+ at m/z 387 and 319, respectively. The most abundant fragmentation ion was identified in the mass spectra, in which the product ion for mesoridazine was detected at m/z 126. Quantification of mesoridazine and IS were performed using the MRM for high selectivity and sensitivity of acquisition data in positive electrospray ionization (ESI) mode; following precursor to product ion transitions was used: m/z 387 → 126 for mesoridazine, m/z 319 → 86 for chlorpromazine (IS). Transitions from the specific precursor to product ion fragment were optimized. After appropriate adjusting the chromatographic separation conditions, a reproducible separation of mesoridazine and IS was determined. A chromatogram obtained by using a blank plasma sample did not contain any interfering peaks at the retention times for mesoridazine and IS. Consequentially, the chromatographic condition with retention times of 1.56 min for mesoridazine and 1.58 min for IS with apparently symmetric peaks, respectively. Typical peak shape and retention times of MRM chromatograms are shown in Fig. 2. The retention times for mesoridazine and IS in rats following a single intravenous administration of the drug were consistent with the values obtained from standard plasma samples, indicating that the specificity of the assay was adequate. Therefore, these analytical conditions were used in subsequent studies. 3.2. Sample preparation The sample was processed using simple protein precipitation method with acetonitrile. Protein precipitation method is significantly easier, convenient and non-time consuming than the liquid–liquid extraction method and equally effective in determining the concentrations of analyte, which is suitable for pharmacokinetics analysis [34–36]. Furthermore, no interference peaks were observed and symmetrical peak shape and stable baseline was generated using the MRM mode. Compared with previous liquid–liquid extraction method for the detection of mesoridazine in blood using LC–MS/MS [31], this method based on simple protein precipitation technique was applicable to easy sample preparation with less time. Thus, protein precipitation method using acetonitrile was employed in this study for sample preparation.

3.3. Validation and matrix effect Under the analytical conditions using LC–MS/MS, chromatograms of double blank and blank rat plasma spiked with internal standard are presented in Fig. 2. There were no significantly interfering peaks from the constituents of the drug-free rat plasma with the retention time of the mesoridazine or IS. The calibration curve for mesoridazine in rat plasma was linear over the concentration range of 0.001–4 ␮g/ml. The best linear fit and least squares regression analysis of the data indicated that the correlation coefficient (R2 ) for analyte was excess of 0.999 of calibration curve. A summary of the intra- and inter-day precision and accuracy is shown in Table 1. In general, the precision and accuracy were within 8.31% except the inter-day accuracy of LOD (16.9%). The intra- and inter-day precision in RSD for mesoridazine were