Akademik Veri Yönetim Sistemi
13th International Drug Chemistry Conference (13th DCC), Antalya, Türkiye, 6 - 09 Şubat 2025, ss.253
Schizophrenia is a complex and chronic mental disorder that generally causes disturbances in cognition, perception, emotions, and behavior [1]. Pharmaceutical interventions are critical for its management, with clozapine being one of the most effective treatments [2]. However, there are several studies suggesting an alteration in clozapine metabolism associated with coffee intake or cigarette smoking, highlighting possible interactions between clozapine and caffeine (metabolized also by CYP1A2 enzyme) and clozapine and smoking (CYP1A2 enzyme inducer) [3, 4]. However, the mechanism of alteration still is not well understood and needs further investigations. In this study, a novel analytical method which is based on extraction of clozapine, caffeine, nicotine and their primary metabolites from plasma using thin film microextraction (TFME) followed by high-performance liquid chromatographic (HPLC) separation was proposed for investigation of the abovementioned interactions in plasma samples taken from schizophrenia patients. For this purpose, as the first step of the study, a chromatographic method was developed using Kinetex EVO C18 Core-Shell Column (250 mm × 4.6 mm, 5 µm) for which several parameters critical in separation of the analytes were optimized. Caffeine, nicotine, clozapine, and their respective metabolites were selected as target analytes during method optimization to assess system performance and achieve efficient separation. As the second step, TFME blades were prepared by two coating methods, namely, spraying and dip coating, using hydrophilic lipophilic balanced (HLB) particles as extractive phase for coverage of both polar and nonpolar compounds. Finally, before validation of the method, TFME protocol was optimized in terms of desorption time, extraction time and sample dilution to provide the best sensitivity at shortest time. Based on these evaluations, in the final chromatographic method a 35 min three phase gradient (with pH 4.0 ammonium acetate buffer, methanol, and acetonitrile) was used. During the chromatographic run, the flow rate, column temperature, and monitored wavelength were set as 1 mL/min, 30 ˚C, and 260 nm, respectively. Among the investigated TFME sampler preparation methods, dip coating was found to produce more reproducible TFME samplers which provided 7.0% and 10.9% relative standard deviations (N= 22) in extraction of clozapine and caffeine from water, respectively. Desorption time optimization studies showed that following the extraction all analytes can be quantitatively desorbed from the TFME samplers in 15 minutes, as no significant changes were observed in the results beyond this duration, when methanol/acetonitrile/water (40/40/20) was used as an eluent. Moreover, several plasma dilution ratios were evaluated (undiluted, 1:1 1:2, v/v) which indicated that as dilution of plasma increases the free concentration of protein bound analytes increases, resulting in higher recoveries during the TFME process. Finally, the extraction time optimization carried out in diluted plasma (1:2, v/v) showed that 2 hours of extraction is optimal. The preliminary results obtained in the study showed that the novel method developed here can be a suitable candidate for quantitative determination of clozapine, caffeine, nicotine, and their metabolites in plasma samples.