Analysis and pharmacokinetics of quetiapine and two metabolites in human plasma using reversed-phase HPLC with ultraviolet and electrochemical detection
Introduction
Quetiapine fumarate (‘Seroquel’; bis[2-(2-[4-(dibenzo[b,f][1,4]thiazepin-11-yl)piperazin-1-yl] ethoxy) ethanol] fumarate) is a dibenzothiazepine derivative developed by Zeneca Pharmaceuticals and approved for the management of manifestation of psychotic disorders. The preclinical profile of quetiapine predicts that it would have the pharmacologic properties of atypical antipsychotics [1]. This is supported by the results of clinical studies which indicate that, in the therapeutic dose range of 150 to 750 mg day−1, quetiapine is effective in improving the positive and negative symptoms of schizophrenia, and that quetiapine treatment is not associated with sustained elevation of plasma prolactin concentrations and induction of extrapyramidal symptoms (EPS) across the clinical dose range [2]. To investigate the pharmacokinetics of quetiapine in humans, a sensitive and specific assay for quetiapine and two of its active metabolites was required. A previously developed HPLC method [3] performed well for quetiapine, but lacked sensitivity to support low-dose studies and was non-reproducible for the 7-hydroxylated metabolite. A GC/MSD method was developed [3] which afforded sensitivity, but lacked ruggedness. This HPLC procedure has been successfully developed and validated, with a quantitation limit of 2.50 ng ml−1 for each analyte in human plasma. The assay employs a three-step liquid–liquid extraction of quetiapine and its 7-hydroxylated (ICI 214,227) and 7-hydroxylated, N-dealkylated (M 236,303) metabolites from human plasma, and utilizes ultraviolet (UV) detection of quetiapine and electrochemical detection of the metabolites (Fig. 1). An example from our laboratory of the application of this methodology to the determination of the pharmacokinetics of quetiapine and its metabolites in human plasma with the time course of dopamine D2 and serotinin 5-HT2 receptor occupancy in the brain using positron emission tomography (PET) is presented. The PET method and results will be presented in a separate paper.
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Materials
Quetiapine (ICI 204,636), internal standard (M 214,652) and metabolite standards (ICI 214,227 and M 236,303) were synthesized at Zeneca Pharmaceuticals (Wilmington, DE). High purity methanol, ethyl acetate and acetonitrile were purchased from Burdick & Jackson (Muskegon, MI). Ammonium hydroxide, hydrochloric acid, phosphoric acid, sodium phosphate and potassium chloride were from J. T. Baker (Phillipsburg, NJ). HPLC-grade water was prepared with a Nano-Pure water purification system.
Preparation of reagents and solutions
Stock
Blood sample collection and processing
Venous blood samples (7 ml each) were collected in tubes containing sodium heparin (green-top Vacutainer BD-6483, Becton–Dickinson, Cockeysville, MD). Immediately after collection, each blood sample was gently inverted a few times for complete mixing with the anticoagulant and then centrifuged at room temperature within 30 min of collection to separate the plasma. The resulting plasma samples were transferred, using glass transfer pipettes, to polypropylene tubes, then frozen immediately. The
Chromatography
A chromatogram of extracted control human plasma and that of control human plasma spiked with 20.0 ng ml−1 quetiapine and M 214,652 are compared in Fig. 2. Detection was by UV absorption at 225 nm. While the two hydroxylated metabolites also absorbed at this wavelength, they eluted in regions of the chromatogram which contained endogenous plasma components, which interfered with their quantitation at their expected plasma concentrations. An electrochemical detector was placed in series with the
Discussion
A sensitive and specific HPLC method, with both UV absorption and electrochemical detection, was developed and validated for the determination of quetiapine and its metabolites in human plasma. This method offers a significant improvement in sensitivity, specificity and reproducibility over the previous HPLC method [3], and enhanced robustness compared to the previous GC/MS procedure [3].
The use of narrow-bore chromatography necessitates the use of small injection volumes, and the sensitivity
Conclusions
Improvements in sensitivity, specificity and reproducibility were achieved using an HPLC procedure with UV and electrochemical detection for the quantification of quetiapine and two of its active metabolites. An experienced operator can comfortably prepare 80 plasma extracts a day for automated chromatographic analysis. The wide linear range of the assay can support both low-dose studies in normal volunteers and higher dose studies in subjects with schizophrenia and other psychotic disorders.
Acknowledgements
The authors would like to thank S. D. Brooks and K. M. Palermo for providing bioanalytical expertise during the conduct of these studies. ‘Seroquel’ is a trademark, the property of Zeneca Limited.
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