Whatman 903 filter paper cards

Initial ifosfamide and cyclophosphamide 1mg/mL solutions were individually prepared in 100% MeOH. From the initial ifosfamide solution, work solutions were prepared (20X) in 70% MeOH in order to obtain solutions for calibrators at 2, 10, 20, 50, 100, 150 and 200 μg/mL concentrations. Solutions for low, medium, and high quality control points were prepared at 6, 80, and 160 μg/mL respectively. Independent calibration curves (100, 500, 1000, 2500, 5000, 7500 and 10000 ng/mL) were prepared by adding 475 μL of total blood (haematocrit 30% and 45%) and 25 μL of ifosfamide solution at the different concentrations (20X). 40 μL of the mix were poured onto the Whatman 903 filter paper cards in each circle, separately. Cards were allowed to dry at room temperature for 6 hours, then were labelled and stored in low gas-permeability plastic bags including desiccant packs at 4°C and -80°C until analysed. Quality control points at low, medium and high levels (300, 4000 and 8000 ng/mL, respectively) were prepared with the same procedure as the calibration curves, using the same initial solutions (20X).

This study used serum samples collected during a pilot excretion study performed by Lamon et al.,¹⁸ in which six healthy volunteers received a single intravenous injection of 200 μg CERA (MIRCERA®, Roche Pharma AG) and which was approved by the local ethical committee (protocol #05/08). The volunteers were Caucasian men aged 20–28 years, with a mean body mass index of 23.3 kg/m² (SD 1.48 kg/m²). None of the volunteers was involved in elite sport. Blood samples were collected every morning over the first 4 post‐administration days. Thereafter, blood sampling was performed on days 6, 8, 10, 13, 16, 20, and 24. For four of the subjects, blood was also collected on day 27 post‐injection (Figure 1). Venous blood was collected from the elbow crease into EDTA tubes from heathy volunteers. After centrifugation at 300g for 10 min, the erythrocytes were isolated and washed twice with hypotonic solution (0.9% NaCl) and then mixed gently with an appropriate serum (50:50 ratio) as described by Reverter‐Branchat et al.¹⁷ Subsequently, 20‐μl aliquots of this modeled blood were spotted onto the DBS card (Whatman 903™ filter paper cards). The cards were left to dry for a minimum of 1 hour, and then the cards were stored at room temperature in sealed plastic bags until analysis. Same procedure was used for reconstituted volumetric absorptive microsampling (VAMS) (Neoteryx™).