Quetiapine fumarate (QTP), whose chemical name is bis[2-(2-[4-(dibenzo[b
][1,4]thiazepin-11-yl)]ethoxy)ethanol]fumarate, Figure 1
A, is a dibenzothiazepine derivative with an atypical neuropharmacological profile. Studies [1
] have showed that quetiapine is very effective for negative and positive schizophrenia, as well as cognitive impairment, with slightly choline resistant and rarely granulocytopenia. Due to the highest serotonin/dopamine binding ratio, quetiapine makes the serotonin type 2 (5-HT2
)-receptor blocking effect about twice as strong as the dopamine D2
-receptor blocking effect. As a result of this binding pattern, the extrapyramidal side effects of quetiapine are minimal [3
]. Those characteristics make quetiapine well tolerated and effective in patients who have Alzheimer’s disease or Parkinson’s disease [3
]. In addition, quetiapine is metabolized by the CYP450 system, primarily by CYP3A4 enzyme [5
Chemical structures of quetiapine (A) and internal standard mirtazapine (B).
Chemical structures of quetiapine (A) and internal standard mirtazapine (B).
To the best of our knowledge, many methods had been described so far in the literature for the determination of quetiapine, including high-performance liquid chromatography with ultraviolet detection (HPLC-UV) [10
], UPLC-UV [13
], and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) [14
]. The detection limit of UV generally can only reach μg level. HPLC-MS/MS, which is expensive for most laboratories, has been the most common choice for this purpose to date. While the obvious advantages of CE are simplicity, economies, high efficiency, good selectivity, small sample volume, and short analysis time. Moreover, various separation modes can be selected for different analytes. Vincenzo Pucci et al.
] used a spectrophotometric and CZE method to determinate quetiapine in commercial tablets for the quality control. However, when we used ultraviolet detection, the sensitivity of CE was always limited because of the little injection volume and small detection window. To resolve this problem, some stacking CE methods were developed to enhance sensitivity [18
], such as field amplified sample stacking (FASS), field enhanced/amplified sample injection (FESI), dynamic pH junction or large volume sample stacking (LVSS), and using ionic liquid during pretreatment. In addition, FESI was the easiest sample stacking method by electrokinetic injection that the conductivity of the BGE was at least ten folds than sample due to the simple requirement. However, there was no literature about the quantitative analysis of quetiapine using any stacking CE method until now.
Therefore, the aim of this paper was to develop and validate a simple and rapid CZE method for the determination of quetiapine in beagle dog plasma. For this purpose, a new FESI-CZE method was developed, optimized and validated in terms of precision, linearity, accuracy, robustness, and detection and quantitation limits for determination of quetiapine.
3. Experimental Section
3.1. Chemicals and Reagents
Quetiapine fumarate standard reference material (99.9% purity) was purchased from National Institutes for Food and Drug Control (Beijing, China). The immediate release tablets formulation of quetiapine fumarate (200 mg, lot: KA187) were kindly supplied by AstraZeneca S. p. A. (Basiglio, Milan, Italy). The sustained release tablets formulation of quetiapine fumarate (200 mg, lot: C10125A) also obtained from AstraZeneca S. p. A. Mirtazapine (99.9% purity) was used as the internal standard (IS, Figure 1
B) obtained from Dalian Melon Biological Technology Co. Ltd. (Dalian, China). NaH2
was obtained from China Medicine (Group) Shanghai Chemical Reagent Corporation (Shanghai, PR China). Phosphoric acid was from Sigma (St. Louis, MO, USA). Methyl alcohol and acetonitrile were from MERK KGaA (Darmstadt, Germany). Methyl tert-butyl ether (chromatographic grade) was from J.T. Baker (Phillipsburg, NJ, USA). All chemicals were analytical grade or better. Beagle dog plasma (sodium heparin as an anticoagulant) was obtained from Shanghai Xingang experiment animal field. Water was deionized and purified by using a Milli-Q system (Millipore, Milford, MA, USA) and was used to prepare all aqueous solutions.
3.2. CE Instrumentation
Analyses were performed on a CE system consisted of a Beckman P/ACE MDQ instrument (Beckman Coulter, Brea, CA, USA) equipped with a photodiode array detection detector (PDA) and P/ACE System MDQ Software. The system of 32-Karat software (Beckman) was used for data acquisition, processing, and analysis. Detection was performed on-column at 210 nm, where quetiapine had the maximum absorption. An uncoated fused-silica capillary (31.2 cm × 75 μm i.d., effective length 21 cm) was acquired from Hebei Yongnian Optical Fiber Factory (Hebei, China).
3.3. Capillary Electrophoretic Conditions
The phosphate buffer (50 mM, pH 2.5) was used in this study as the background electrolyte (BGE). It was prepared by accurately weighing 0.78 g of sodium dihydrogen phosphate and thoroughly mixed it with 100 mL deionized water. Then, the pH of this buffer was adjusted to 2.5 with phosphoric acid. BGE was prepared freshly every day and filtered through a 0.45 μm hydrophilic cellulose membrane filter and degassed by sonication prior to use. The capillary temperature of the optimal separation was maintained at 25 °C by immersion of the capillary in the cartridge around of circulating cooling liquid. The separation voltage was set at 13 kV with the current of about 98 μA. The sample was injected by electrokinetic injection modes (5 kV × 10 s). A new capillary was conditioned by rinsed with 1 M NaOH for 30 min, and then flushed with 0.1 M NaOH, H2O, 0.1 M HCl and H2O (15 min each), sequentially. At the beginning of each working day the capillary was rinsed with H2O (2 min), 0.1 M NaOH (10 min), H2O (2 min), and running buffer (10 min) in regular sequence. At routine condition between runs, the capillary was rinsed in turn with 0.1 M NaOH, 0.1 M HCl, H2O and then BGE, each for 2 min.
3.4. Preparation of Stock Solutions, Calibration Samples and Quality Control Samples
Stock solutions of quetiapine fumarate and the IS were prepared in methanol at concentration of 1 mg/mL, respectively. The quetiapine fumarate working standard solutions were prepared daily by diluting the stock solution with distilled water to desired concentration of the range from 0.01 to 10 μg/mL. The stock solution of IS was diluted with distilled water to working solution (1 μg/mL). All described solutions were stored at 4 °C.
Calibration samples were obtained by diluting standard working solutions (10 μL) with drug-free beagle dog control plasma (90 μL), to span a calibration standard range of 1–1000 ng/mL (1, 2, 10, 50, 200, 500, and 1000 ng/mL). Quality control (QC) samples (2, 50, 800 ng/mL) were independently prepared by spiking appropriate amount of the working standard solutions in drug-free beagle dog control plasma.
3.5. Extraction Procedure
Samples were prepared by LLE in 96-well deep format plate (2 mL, Corning Life Sciences-Axygen Inc, CA USA) to increase throughput. We used an automatic multichannel electronic pipette (INTEGRA Bioscience AG, Switzerland) to complete liquid transfer steps. Plasma samples were thawed at room temperature after taken out from −80 °C freezer. 100 μL aliquot of plasma samples were added into 96-well deep format plate. Aliquots of 10 μL IS working solution (1 μg/mL) were added and vortexed homogenized for 30 s. The extraction consisted in addition of 500 μL methyl tert-butyl ether. Then the mixture was vortex-mixed for 3 min in a platform and centrifuged at 3000 g for 10 min. 400 μL of the supernatant organic layer was transferred from the original sample plate into a new 96-well deep format plate. And then the plate was evaporated under a gentle nitrogen flow at 30 °C. All dry residues were reconstituted by addition of 20 μL acetonitrile–water (65:35, v/v). Finally, the plate was vortex-mixed for 5 min, then centrifuged for 10 min at 4000 g. The supernatant was injected into the CZE system.
3.6. Pharmacokinetic Study in Beagle Dogs
This method was applied to determine plasma concentrations of quetiapine fumarate from a preclinical trial in beagle dogs. Beagle dogs were respectively received a single oral dose of 200 mg of quetiapine fumarate immediate release tablet and sustained release tablet. About 1 mL of blood samples were collected in heparinized centrifuge tubes at before administration (0 min) and at the time of 10, 20, 30, 45 min, and 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24 h after dosing and centrifuged at 1370 g for 10 min to separate the plasma fraction. The obtained plasma samples were stored at −20 °C until analysis. The study was approved by a local ethics committee.