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

• Dansylchloride derivatisation of 4-OH mephenytoin was developed and validated.
• Derivatisation helped in increasing the sensitivity by 100–200 fold.
• Higher sensitivity enabled the analysis of samples on lower sensitive mass spec.
• Vmax, Km values of substrate were similar (derivatised vs underivatised metabolite).
• IC50 value of inhibitor was similar (derivatised vs underivatised metabolite).

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 (r2 = 0.9972 for 4-OH mephenytoin; r2 = 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.