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.
J Chromatogr B Analyt Technol Biomed Life Sci
; 989: 27-36, 2015 May 01.
Article
in En
| MEDLINE
| ID: mdl-25797720
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Dansyl Compounds
/
Enzyme Inhibitors
/
Enzyme Assays
/
Cytochrome P-450 CYP2C19
/
Mephenytoin
Type of study:
Diagnostic_studies
/
Evaluation_studies
Language:
En
Journal:
J Chromatogr B Analyt Technol Biomed Life Sci
Journal subject:
ENGENHARIA BIOMEDICA
Year:
2015
Document type:
Article