Oxidative metabolism

ROXY EC/MS for mimicing natures redox reactions.

ROXY EC/LC System upfront MS
^{mimicing natures redox reactions}

• Fast synthesis of metabolites (µ-preparative)
• Rapid risk assessments of drug-protein binding
• Signal enhancement in MS
• Electrochemical cleavage of proteins/peptides
• Oxidative stress/damage of proteins, DNA, lipids, etc 

The on-line coupling of Electrochemistry (EC) with LC/MS has shown great potential for the investigation of drug metabolism. It offers fast electrochemical synthesis of drug metabolites for reliable quantification by mass spectrometry (MS) to be operated as a high throughput screening system. The system is suitable to mimic (synthesize) phase I metabolites within minutes, that otherwise are obtained by time consuming in vivo (laboratory animals) or in vitro experiments (isolated liver cells, microsomes).

• fast: within minutes vs. days using traditional methods
• zero matrix effect, no isolation steps
• direct identification of oxidative labile sites in drug molecule
• zero risk of adduct formation with cell matrix
• high throughput screening of metabolites

A pre-injection on-line electrochemical cell has been used to mimic liver metabolism. Metabolites are instantaneously generated and analysed. Briefly, a syringe pump is used to flush xenobiotics through an electrochemical cell to fill an injection loop. The oxidation products are injected and analysed using HPLC-MS.

Comparing Fig. 1 and 2 clearly indicates that the electrochemical simulation leads to all metabolites also found in conventional method making it a perfect tool for Metabolism.

Electrochemical simulation

Mass traces of Tetrazepam metabolites formed in the electrochemical reaction cell of the ROXY™ EC/LC system.

Microsomal approach

Mass traces of Tetrazepam metabolites formed by the microsomal approach. Experiments were performed using pooled rat liver microsomes from male Sprague Dawley rats. Courtesy: A. Baumann, B. Schubert, H. Oberacher and U. Karst, University Münster, Münster, Germany.

Sysem configuration

For the simulation of oxidative metabolism Antec provides a number of electrode materials that are useful for the synthesis of metabolites. A platinum, and the new Magic Diamond™ electrodes are supplied. The system further comprises of the following parts:

• AS 110 autosampler, cool, micro
• LC 110S HPLC pump, 2 x
• OR 110 organiser rack, dual channel
• Clarity data acquisition software, incl LC, AS modules
• ROXY potentiostat DCC
• Reactor cell with Pt, GC, Au, MD WE, and HyREF 

Notes

• 210_001A_04 - ROXY Event programming and Settings
• 210_001_07 - Oxidative Metabolism of Acetaminophen
• 210_002A_01 - ROXY ECLC system userdefined programs for AS 110
• 210_002_01 - Automated screening with ROXY ECLC system
• 210_003_01 - uPrepCell for Metabolite Synthesis
• 210_004_04 - Oxidative Metabolism of Amodiaquine

Posters

• 221_020_02 - Disulfides with Magic Diamond Electrode