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Centrifuge the calibration standards, QC, and ISTD mix vials before opening at 10 000 x g (rcf) for 2 min. Dissolve calibration standards (red caps) in 100 µL water. Dissolve QCs (green, blue and yellow caps) in 100 µL water. Dissolve ISTD mix (orange cap) in 1200 µL water. Shake all vials (Cal, QCs, ISTD) for 15 min at 1200 rpm and vortex several times. Centrifuge the QCs and your plasma samples for 5 min at 2750 x g (rcf) and 4 °C. Add 10 µL of the ISTD mix to each well of the Kit plate except A1. Pipette 10 µL PBS / Cal / QC / sample according to your plate layout generated by MetIDQTM. Dry for 30 min under nitrogen (using a Nitrogen Evaporator or a Pressure Manifold). Prepare the pre-mix for derivatization in the plastic tube of the Kit box (1900 µL of each: ethanol, water and pyridine). Add 300 µL phenylisothiocyanate (PITC) and vortex rigorously (at least 10 sec) until the derivatization solution is clear. Make half of the solution for the starter kit. Add 50 µL of the derivatization solution to each well using a repeater at maximum dispensing speed. Cover the plate with the plastic lid and incubate for 25 min, then dry for 60 min under nitrogen. Prepare extraction solvent and add 300 µL to each well. You can use the repeater at low dispensing speed or an 8-channel pipette. Shake for 30 min at 450 rpm (on the scale of min-max 0-1400 rpm). Centrifuge for 2 min at 500 x g (rcf) or use a Pressure Manifold. Visually check that the fill level is the same in all wells of the capture plate. If not, repeat and apply higher g or higher pressure, respectively. Carefully remove the upper filter plate and transfer the volume specified for your instrument (p. 77-80) to another empty 96-deep well plate that you find in the Kit box (label “LC plate”). Finish the LC plate and dilute. Prepare the FIA plate and dilute. Seal both plates with the silicon mat. Firmly press the mat’s naps into the wells.

Please refer the optional items for chromatography protocol. Additional information is decribed in the manual provided by Biocrates.

Please refer the optional items for mass spectrometry protocol. Additional information is decribed in the manual provided by Biocrates.