Development of a sensitive and quantitative UHPLC-MS/MS method to study the whole-body uptake of pharmaceuticals in...

An analytical procedure to measure the whole-body uptake of pharmaceuticals in zebrafish has been developed using state-of-the-art methodologies. A sample preparation procedure for 9 pharmaceuticals displaying a variety in physicochemical properties was developed using 10-day old zebrafish (TG898). For an efficient homogenization of the samples and subsequent recovery of the compounds of interest, different amounts of organic solvents in combination with acidic modifiers were added to zebrafish samples. Samples were subsequently processed using a powerful bath sonicator and centrifuged. Supernatant was then removed and evaporated in a vacuum oven before being reconstituted in a mobile phase-like solvent. Samples were analyzed using ultra-high performance liquid chromatography (UHPLC) on an Acquity BEH C₁₈ column (100 × 2.1mm, d_p=1.7µm) coupled to a Waters Xevo TQ-S mass spectrometer. For this purpose, a generic gradient was run, wherein the percentage of acetonitrile was varied from 3% to 82% in 10.5min at a flow rate of 0.41mL/min. Linearity of the method was demonstrated for all compounds (R² > 0.997) in a practically relevant concentration range. Matrix effects were between 81% and 106%, except for amitriptyline (51%). Using this method, it was demonstrated that a sample pretreatment using 1:2 (v/v) water:methanol in combination with 0.1% formic acid resulted in acceptable recoveries between 74% and 100% for all compounds. Together with the obtained lower limits of quantification of the analytical method (between 0.005 and 1.5ng/mL), this allowed the use of a single zebrafish to study the whole-body uptake of a particular drug, after incubating zebrafish at the maximum tolerated concentration for this drug.