On-chip electromembrane extraction for monitoring drug metabolism in real time by electrospray ionization mass spectrometry.
A temperature controlled (37 °C) metabolic reaction chamber with a volume of 1 mL was coupled directly to electrospray ionization mass spectrometry (ESI-MS) by the use of a 50 μm deep counter flow micro-chip electromembrane extraction (EME) system. The EME/ESI-MS system was used to study the in vitro metabolism of amitriptyline in real time. There was no need to stop the metabolisms by protein precipitation as in conventional metabolic studies, since the EME selectively extracted the drug and metabolites from the reaction solution comprised of rat liver microsomes in buffer. Compositional changes in the reaction chamber were continuously detected 9 seconds later in the MS. Most of this time delay was due to transport of the purified extract towards the ESI source. The EME step effectively removed the enzymatic material, buffer and salts from the reaction mixture, and prevented these species from being introduced into the ESI-MS system. The on-chip EME/ESI-MS system provided repeatability for the amitriptyline signal intensity within 3.1% relative standard deviation (RSD) (n = 6), gave a linear response for amitriptyline in the tested concentration range of 0.25 to 15 μM, and was found not to be prone to ion-suppression from major metabolites introduced simultaneously into the EME/ESI-MS system. The setup allowed the study of fast reactions kinetics. The half-life, t(1/2), for the metabolism of 10 μM amitriptyline was 1.4 minutes with a 12.6% RSD (n = 6).
School of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark. njp@farma.ku.dk
Nickolaj Jacob
NJ
Petersen
Jacob Sønderby
JS
Pedersen
Nicklas Nørgård
NN
Poulsen
Henrik
H
Jensen
Christian
C
Skonberg
Steen Honoré
SH
Hansen
Stig
S
Pedersen-Bjergaard
D000639:Amitriptyline / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D005591:Chemical Fractionation / Q000295:instrumentation / Q000379:methods*; D056656:Lab-On-A-Chip Devices; D008297:Male; D008567:Membranes, Artificial; D046208:Microchip Analytical Procedures / Q000379:methods*; D004364:Pharmaceutical Preparations / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013696:Temperature; D013997:Time Factors
D008567:Membranes, Artificial; D004364:Pharmaceutical Preparations; D000639:Amitriptyline