Sterile water for injection

For the preparation of APO solutions at different concentrations, the commercially available Apofin solution for subcutaneous injection (Chiesi Farmaceutici, Parma, Italy) was used. The commercial drug (3 mL) contains 30 mg of apomorphine hydrochloride (M_w = 303.78332 g/mol) as the active ingredient, corresponding to a molar concentration of 3.3 × 10⁻² M of R-apomorphine, plus sodium metabisulfite and hydrochloric acid as excipients.
The APO solutions were obtained by suitable dilution of the commercial drug with sterile water for injection (B. Braun) to reach the final concentrations of 100, 10, 1 and 0.1 µg/mL (i.e., from 3.3 × 10⁻⁴ M to 3.3 × 10⁻⁷ M). The pH values of all solutions were in the range between 5.5 and 6.5, as determined by indicator paper.

Blood samples were collected from the treated rats, at pre-determined time points (1, 4, 7, 28 and 56 days), into heparinised tubes via the lateral tail vein. Plasma was separated from the whole blood and prepared as described, prior to HPLC analysis.
Vaginal washes were collected by washing the vaginal cavity with 200 μl sterile water for injection (B. Braun Medical Ltd., Sheffield, UK) at pre-determined time points (1, 4, 7, 28 and 56 days). The samples were centrifuged at 14,000× g for 10 min at 4 °C to remove any debris. A 35 μl volume of sample was then mixed with 65 μl acetonitrile (ACN), and a sample of this was analysed by HPLC. For data analysis purposes, arithmetic means were determined and plasma samples with RPV concentrations below the LOQ of the system were included in data tables as half the LOQ, namely 25 ng/ml.