Quantification of mevalonate-5-phosphate using UPLC-MS/MS for determination of mevalonate kinase activity

Abstract Objectives Mevalonate kinase deficiency, a rare autosomal recessive autoinflammatory disease, is caused by mutations in the MVK gene encoding mevalonate kinase (MK). MK catalyzes the phosphorylation of mevalonic acid to mevalonate-5-phosphate (MVAP) in the pathway of isoprenoid and sterol synthesis. The disease phenotype correlates with residual activity ranging from Design and methods Wild-type MK and the variant V261A, which is associated with HIDS, were recombinantly expressed in Escherichia coli . Enzyme activity was determined by formation of MVAP over time quantified by isotope dilution UPLC-MS/MS. The method was validated according to the FDA Guidance for Bioanalytical Method Validation. Results Sensitivity for detection of MAVP by UPLC-MS/MS was improved by derivatization with butanol–HCl (LLOQ, 5.0 fmol) and the method was linear from 0.5 to 250 μmol/L ( R 2  > 0.99) with a precision of ≥ 89% and an accuracy of ± 2.7%. The imprecision of the activity assay, including the enzymatic reaction and the UPLC-MS/MS quantification, was 8.3%. The variant V261A showed a significantly decreased activity of 53.1%. Conclusion Accurate determination of MK activity was enabled by sensitive and reproducible detection of MVAP using UPLC-MS/MS. The novel method may improve molecular characterization of MVK mutations, provide robust genotype–phenotype correlations, and accelerate compound screening for drug candidates restoring variant MK activity.