Determination of a deuterohemin–peptide conjugate in rat plasma by liquid chromatography–tandem mass spectrometry and application to a preclinical pharmacokinetic study

• A LC–MS/MS method was first established for DhHP-6, a deuterohemin–peptide.
• Acidic environment was maintained during sample preparation to obtain high recovery.
• Good recovery, minimal matrix effect was achieved using a simple sample preparation.
• The method was applied to support a preclinical pharmacokinetic study in rat.

The deuterohemin–peptide conjugate (DhHP-6) is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species. This paper reports the development of a sensitive and rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the determination of DhHP-6 in rat plasma using triptorelin as an internal standard (IS). 50 μL plasma was used in sample preparation, and a simple protein precipitation procedure with acetonitrile was involved. Satisfactory peak shapes of analyte and IS were obtained on an Agilent HC-C18 column by using a gradient elution with 10 mM ammonium acetate–0.5% formic acid (v:v) and acetonitrile, there was no significant interference impacting the determination. A calibration curve obtained from this method was linear within the concentration range 10–3000 ng/mL with intra- and inter-day precisions of 4.2–6.8% and 3.2–8.9%, respectively and accuracy of −1.3% to 2.1%. The recovery was above 80% with low matrix effects. The method was successfully applied to support a preclinical pharmacokinetic study in rat.

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