Quetiapine

Standard solutions of chlorPromazine, haloperidol, and clozapine were purchased from LGC Promochem (Barcelona, Spain). Promazine, levomePromazine, and cyamemazine were purchased from Sigma Aldrich (Lisbon, Portugal). Quetiapine was kindly offered by AstraZeneca PLC (London, UK). All standards were acquired at 1 mg/mL. N-metil-N-(trimetilsilil) trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS) were acquired from Macherey-Nagel (Düren, Germany). Deionized water was obtained from a Milli-Q System (Millipore, Billerica, MA, USA). Ascorbic acid was purchased from Fisher Scientific (UK), while sodium azide was purchased from Panreac Química SA (Barcelona, Spain), and sodium fluoride was provided by Sigma Aldrich (Sintra, Portugal). Whatman™ 903 protein saver cards were acquired from Solítica (Lisbon, Portugal).
A work solution with all target analytes was prepared by proper dilution of standard solutions with methanol to a final concentration of 10 µg/mL, except for haloperidol, which was diluted to 2 µg/mL. The Internal Standard (IS) Promazine was also diluted, with methanol, to a final concentration of 0.5 µg/mL. Promazine is an antipsychotic drug that is not commercially available in Portugal, so it is unlikely to be found in real patient samples. All the solutions were stored in the absence of light at 4 °C.

All reagents were obtained from Sigma-Aldrich, Missouri, USA unless stipulated otherwise. Quetiapine was donated by AstraZeneca, Stockholm, Sweden; aripiprazole by Bristol-Myers Squibb, New Jersey, USA and AG1478 (EGFR inhibitor) purchased from A.G. Scientific, Inc., California, USA. Primary antibodies, including phospho-p44/42 MAPK, p44/42 MAP kinase, phospho-p90RSK, RSK1/RSK2/RSK3 and β-Actin were from Cell Signaling Technology, Massachusetts, USA and c-Fos from Assay Designs, Michigan, USA. Secondary antibodies, including goat anti-mouse and goat anti-rabbit horseradish peroxidase (HRP)-conjugated immunoglobulins (IgGs) were supplied by DAKO, NSW, Australia.

CPZ (Sigma-Aldrich, St. Louis, MO, USA) was added to the rodent chow with a final concentration of 0.2% (w/w). As a positive control, additional mice were treated with quetiapine (AstraZeneca, Wilmington, DE, USA, 10 mg·kg⁻¹·d⁻¹, QTP), a widely used antipsychotic [18 (link)]. SZASD is a traditional Chinese herb formula, which is composed of Chrysanthemum (菊花), Rehmannia glutinosa (干地黄), and Polygoni Multiflori Radix (何首乌) and other components [5 ]. All herbs were purchased from Beijing Tong Ren Tang (Group, Co., Ltd., Beijing, China). The mixed herbs were boiled at 100°C in an appropriate volume for 1 h, and the extraction procedure was repeated twice. The aqueous extracts were filtered, combined, and further concentrated using rotary evaporation under vacuum in a 60°C water bath. The concentrated extract was lyophilized to create a SZASD powder (yield: 32.0%) and stored under desiccation at room temperature.

MK-801 [(+)-MK-801 hydrogen maleate] purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and quetiapine obtained from AstraZeneca Pharmaceuticals (Macclesfield, UK) were both freshly dissolved in saline. Mice were treated with chronic quetiapine (0 or 10 mg/kg/day, intraperitoneally) for 28 days. From day 22 to 28, 1 h after the administration of quetiapine, the mice were administered MK-801 (0 or 2 mg/kg/day, subcutaneously). No mortality was observed in the MK-801-treated mice. The volume of all injected solutions was 10 ml/kg. Totally 40 mice were randomly assigned into four groups (n=10 in each group): saline + saline (CON), quetiapine + saline (Que), MK-801 + saline (MK), MK-801 + quetiapine (MK+Que).