title: Breaking the sensitivity limitations of cytochrome P450 oxidation product: dansyl chloride derivatisation of 4-OH mephenytoin, a CYP2C19 metabolite and its application to in vitro CYP inhibition assay.
abstract: A rapid selective and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the quantitative determination of derivatised cytochrome P450-2C19 oxidation product (dansyl-4-OH mephenytoin) and its underivatised form (4-OH mephenytoin). Samples were anaysed on C18 column (Waters Xbridge, 50 mm×4.6 mm, 3.5 μm particle size) with the mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. A gradient method with a short run time of 2.5 min and 3.5 min was developed for the analysis of dansyl-4-OH mephenytoin and 4-OH mephenytoin, respectively. The standard curve was linear (r(2)=0.9972 for 4-OH mephenytoin; r(2)=0.9946 for dansyl-4-OH mephenytoin) over the concentration range of 0.16 to 40 ng/mL for both derivatised and underivatised forms. The CV (%) and relative error (RE) for inter and intraassay at three QC levels for dansyl-4-OH mephenytoin was 0.97-5.85% and -9.80 to 2.51%, respectively. Whereas, for 4-OH mephenytoin the CV (%) and RE (%) at three QC levels was 0.82-3.47% and -6.69 to -0.01%, respectively. The developed method was validated for various parameters such as linearity, precision & accuracy, extraction recovery, matrix effect, autosampler stability and was proved to be consistent across three QC levels with overall CV (%) less than 15. Dansylation helped in increasing the sensitivity of hydroxy mephenytoin by 100-200 fold. Given the simplicity involved in derivatisation process, we believe that this novel methodology will change the current approaches used for the enhancing the detection sensitivity of 4-OH mephenytoin.
authors: DMPK Laboratory (Biology Division), GVK BIO, Nacharam, Hyderabad 500076, Telangana, India. Electronic address: vijayabhaskar.v@gvkbio.com.
authors: V
authors: V
authors: Vijayabhaskar
authors: DMPK Laboratory (Biology Division), GVK BIO, Nacharam, Hyderabad 500076, Telangana, India.
authors: Pratima
authors: P
authors: Srivastava
authors: DMPK Laboratory (Biology Division), GVK BIO, Nacharam, Hyderabad 500076, Telangana, India.
authors: Sriram
authors: S
authors: Rajagopal
mesh_terms: D065731:Cytochrome P-450 CYP2C19 / Q000737:chemistry*; D003619:Dansyl Compounds / Q000737:chemistry*; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D007700:Kinetics; D008617:Mephenytoin / Q000737:chemistry*; D010084:Oxidation-Reduction
chemical_list: D003619:Dansyl Compounds; D004791:Enzyme Inhibitors; D065731:Cytochrome P-450 CYP2C19; C029038:dansyl chloride; D008617:Mephenytoin
keywords: 4-OH mephenytoin; CYP2C19; Dansyl chloride; IC50; LC–MS/MS; Method validation