Qtrap 5500 system

Blood serum samples were diluted 1000-fold with IS solution and centrifuged at 18,000×g for 5 min. The supernatant was transferred into a clean vial and 5 μL was injected into an LC-MS/MS system. LC analysis was performed using a Nexera LC system (Shimadzu, Kyoto, Japan). An L-column 2 ODS column (150 mm × 1.5 mm i.d.; 5 μm particle size; CERI, Tokyo, Japan), equipped with a guard column (OPTI-GUARD 1 mm C18; Optimize Technologies, Inc., Oregon City, OR, USA), was used for chromatographic separation. The mobile phase consisted of 10 mmol/L ammonium formate (95%) and methanol (5%) (solvent A), and 10 mmol/L ammonium formate (5%) and methanol (95%) (solvent B). The solvent gradient was increased linearly from 0 to 100% solvent B in 15 min and was maintained at this composition for 5 min. Subsequently, the gradient was changed to 0% solvent B and maintained for 10 min to re-equilibrate the column. The flow rate of the mobile phase was set at 0.1 mL/min and the column temperature was maintained at 40 °C. MS/MS detection was performed using a QTRAP 5500 system (SCIEX, Framingham, MA, USA). Quantitation was performed in SRM mode. The SRM transition 152 > 110 was used for quantitation, and the transition 152 > 65 was used as qualifier ions. The SRM transition 156 > 114 was used for IS (acetaminophen-D₄). All experiments were conducted in positive ion mode.

Blood samples were centrifuged (2000 × g, 10 min, at 4°C) to extract plasma, which was snap frozen at −20°C within 60 min of the respective times of collection. Microdialysis samples were snap frozen at −20°C within 15 min of their respective times of collection. Blood and microdialysis samples were subsequently stored at −80°C until analysis. Doxycycline concentrations in microdialysate and plasma samples were analysed using a validated liquid chromatography–tandem mass spectrometry method, consisting of a Symbiosis™ ALIAS chromatographic system (Spark, The Netherlands) and a Qtrap 5500 system (Sciex, Framingham, MA, USA). Gradient elution was performed at a flow rate of 0.4 ml min⁻¹. The mass spectrometer was operated in positive electrospray ionization mode. The analytical method was validated according to the European Medicines Agency guideline on bioanalytical method validation 12. Quantification was performed by multiple reaction monitoring, with chlortetracycline (Fluka 46 133, Sigma‐Aldrich, St. Louis, MO, USA) used as internal standard. Two calibration ranges were applied for microdialysate samples – a lower range at 1–100 ng ml⁻¹, and a higher range at 50–600 ng ml⁻¹. For plasma, the calibration range used was 100–750 ng ml⁻¹.

Blood samples were taken from arterial catheters at 2, 4, 8 and 12 hours after the start of clonidine infusion. Subsequently, a sample was taken once daily until the termination of treatment. After stopping the infusion, blood samples were taken at 0, 8, 16, 24 and 48 h. Blood samples were stored at −20°C and transferred to Amsterdam University Medical Centre AUMC for analysis. Plasma concentrations were measured using a validated high‐perfomance liquid chromatography–mass spectrometry system (liquid chromatography: LC30 UPLC, Shimadzu, Kyoto, Japan; mass spectrometry: QTRAP 5500 system, Sciex, Framingham, MA, USA), as described previously by Kleiber et al.¹⁴ The lower limit of quantification (LLOQ) was 0.1 μg/L and the upper limit of quantification was 20 μg/L. The accuracy of the assay was between 99–114%.