title: Simultaneous determination of a novel antitrypanosomal compound (OSU-36) and its ester derivative (OSU-40) in plasma by HPLC: application to first pharmacokinetic study in rats.
abstract: OBJECTIVE
  To develop an HPLC-UV method for determination of a novel antitrypanosomal compound (OSU-36) and its ester prodrug (OSU-40) in rat plasma and to apply the method for pharmacokinetic evaluation of both compounds in rats.
  
  
  METHODS
  Since an attempt to assay for OSU-36 and OSU-40 in non-stabilized plasma resulted in highly non-linear calibration curves and poor sensitivity due to instability of the compounds, the plasma was stabilized using paraoxon and ascorbic acid. The sample treatment included protein precipitation by acetonitrile; evaporation; reconstitution with acetonitrile and filtration. The chromatography conditions included Xterra RP18 3.5 µm 4.6X100 mm column and gradient mobile phase system of acetonitrile-water.
  
  
  RESULTS
  The limits of quantification (LOQ) were 50 ng/mL and 40 ng/mL for OSU-36 and OSU-40, respectively. The intra- and interday precision and accuracies were below 13% for low, medium and high quality control samples for both compounds. While OSU-40 has been stable in all tested handling conditions, OSU-36 was unstable in plasma after 20 days storage in -80 °C or 4h 28 °C storage. The developed method has been applied for a pharmacokinetic study in rats which revealed that an ester prodrug OSU-40 is rapidly converted to OSU-36 within the plasma compartment by plasma esterases. OSU-36, in turn, relatively quickly undergoes oxidative metabolism, including within the plasma compartment.
  
  
  CONCLUSIONS
  A supplementation of rat plasma with an esterase inhibitor to prevent degradation of ester prodrug (OSU-40), and with antioxidant to prevent oxidation of OSU-36, is necessary for reliable determination of both compounds. Due to limited stability of OSU-36 in stabilized rat plasma, long-term storage of samples or prolonged handling in room temperature conditions is not recommended.
authors.affiliation: Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, BC, Canada. pgershko@interchange.ubc.ca
authors.forename: Pavel
authors.initials: P
authors.lastname: Gershkovich
authors.forename: Kishor M
authors.initials: KM
authors.lastname: Wasan
authors.forename: Olena
authors.initials: O
authors.lastname: Sivak
authors.forename: Summer
authors.initials: S
authors.lastname: Lysakowski
authors.forename: Carolyn
authors.initials: C
authors.lastname: Reid
authors.forename: Kokku
authors.initials: K
authors.lastname: Premalatha
authors.forename: Karl A
authors.initials: KA
authors.lastname: Werbovetz
mesh_terms: D000085:Acetates / Q000493:pharmacokinetics*; D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D004356:Drug Storage; D008297:Male; D010261:Paraoxon / Q000737:chemistry; D011355:Prodrugs; D011806:Quinolinium Compounds / Q000493:pharmacokinetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013696:Temperature; D014344:Trypanocidal Agents / Q000493:pharmacokinetics*
chemical_list: C560079:1-benzyl-1,2-dihydro-2,2,4 -trimethylquinolin-6-ol; C560080:1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate; D000085:Acetates; D011355:Prodrugs; D011806:Quinolinium Compounds; D014344:Trypanocidal Agents; D001205:Ascorbic Acid; D010261:Paraoxon