title: Detection of tetrahydrobiopterin by LC-MS/MS in plasma from multiple species.
abstract: BACKGROUND
  Tetrahydrobiopterin (BH4) is a naturally occurring pteridine and cofactor for a variety of enzymes, including phenylalanine-4-hydroxylase, nitric oxide synthetase and glyceryl ether monooxygenase. BH4 is readily oxidized to dihydrobiopterin and biopterin (B), however only BH4 can provide proper cofactor functions. BH4 is the active ingredient in Kuvan™ for the treatment of phenylketonuria. In order to measure BH4 in plasma from nonclinical and clinical samples with good accuracy, precision, sensitivity and robustness, an LC-MS/MS method was validated. To overcome the oxidation of BH4 in postcollection plasma, the approach was to measure the concentration of BH4 indirectly by measuring B concentration and applying an oxidation conversion ratio. Different endogenous levels of BH4 are determined in human, monkey, dog, rabbit, rat and mouse plasma. Furthermore, the conversion ratio of BH4 to B for each species is different and determined empirically. Plasma is transferred into cryogenic vials containing 0.1% dithioerythritol to prevent oxidation of BH4. The samples are then extracted and oxidized under basic conditions. B is measured with LC-MS/MS using negative ion mode.
  
  
  RESULTS
  The method is accurate, and precise to within 15%. The lower limit of quantitation in matrix is 5, 50 or 100 ng/ml, depending on the species endogenous levels of BH4. The pharmacokinetics of a single oral dose at three concentrations of BH4 administered to C57BL/6 mice is presented. In this mouse study, the T(1/2) of BH4 in plasma was approximately 1.2 h.
  
  
  CONCLUSIONS
  The validated LC-MS/MS method to determine plasma BH4 concentration described herein has been used to support many nonclinical and clinical toxicokinetic and pharmacokinetic studies. BH4 is sensitive to oxidation and has a complicated biology. The method successfully supported the approval of Kuvan for the treatment of phenylketonuria.
authors.affiliation: Quest Pharmaceutical Services LLC, Newark, DE, USA.
authors.forename: Yuwen
authors.initials: Y
authors.lastname: Zhao
authors.forename: Jerry
authors.initials: J
authors.lastname: Cao
authors.forename: Yuan-Shek
authors.initials: YS
authors.lastname: Chen
authors.forename: Yongdong
authors.initials: Y
authors.lastname: Zhu
authors.forename: Colin
authors.initials: C
authors.lastname: Patrick
authors.forename: Benjamin
authors.initials: B
authors.lastname: Chien
authors.forename: Alphonsus
authors.initials: A
authors.lastname: Cheng
authors.forename: Erik D
authors.initials: ED
authors.lastname: Foehr
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000097:blood* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D016018:Least-Squares Analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D010661:Phenylketonurias / Q000188:drug therapy; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001708:Biopterins; C003402:sapropterin