An Improved, Rapid, and Sensitive LC‐MS/MS Method for Quantification of Nitisinone From Human Plasma

ABSTRACT

Nitisinone (NTBC) has been the standard therapy for hereditary tyrosinemia type 1 (HT‐1) since the early 1990s and remains the only effective treatment. To ascertain its therapeutic levels, a rapid and sensitive liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for NTBC quantification in plasma is crucial. Existing methods often employ gradient elution, longer run time with limited sensitivity and selectivity, highlighting the need for improvement and to develop an isocratic method with sensitive quantification of NTBC. A rapid and highly sensitive LC‐MS/MS method was developed with direct protein precipitation for NTBC in plasma. Isocratic separation was achieved on a Purospher Star C‐18 column (30 × 4 mm, 3 µm) using ultrapure water and acetonitrile (48:52; v/v) with 0.2% formic acid. Acquisition was performed in multiple reaction monitoring mode with the transitions: m/z 328.0→281.0 and 328.0→239.0 for NTBC and m/z 284.1→141.1 for probenecid (internal standard) in negative ion mode. The selectivity, linearity, sensitivity, accuracy, precision, and recovery of the developed method were validated as per US‐FDA guidelines for bioanalytical method validation. The developed method demonstrated linearity over a concentration range of 1.56–400 ng/mL (4.73–1210 nM), with a coefficient of determination r² = 0.9991. For NTBC validation, the intra‐day precision (%CV) ranged from 1.82% to 11.07%, with accuracy between 93.20% and 107.81%, while the inter‐day precision (%CV) varied from 1.91% to 12.65%, with accuracy spanning from 89.19% to 112.83%. The method exhibited high sensitivity, with a limit of detection (LOD) of 0.76 nM (0.25 ng/mL) and a lower limit of quantification (LLOQ) of 1.18 nM (0.39 ng/mL). A highly sensitive LC‐MS/MS method for NTBC quantification from human plasma was developed and validated, incorporating significant improvements in analytical performance, including reduced run time with enhanced sensitivity and selectivity.