Isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedures for the quantification of total and free phenytoin in human serum and plasma.

Objectives: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed and validated to accurately measure serum and plasma concentrations of total and free phenytoin.

Methods: Quantitative nuclear magnetic resonance spectroscopy (qNMR) was used to determine the absolute content of the reference material, ensuring its traceability to SI units. The separation of phenytoin from potential unknown interferences was achieved with reversed-phase chromatography, utilizing a C8 column. A protein precipitation protocol was established for preparation of total phenytoin samples, while free phenytoin samples were prepared by membrane separation utilizing a commercially available ultrafiltration device. Assay validation and determination of measurement uncertainties was performed according to the guidelines of the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement.

Results: These RMPs demonstrated high selectivity and specificity, with no evidence of matrix effects, allowing quantification of total and free phenytoin in ranges of 0.640-48.0 μg/mL and 0.0800-4.80 μg/mL, respectively. Intermediate precision was <3.8 %, and repeatability was 1.4-3.8 %, over all concentration levels, for both forms of phenytoin. For total phenytoin, relative mean bias ranged from -2.7-0.3 % in native serum and from 0.0-1.1 % in lithium heparin plasma. Relative mean biases for free phenytoin were 3.5-4.1 % for both native serum and ultrafiltrates. Measurement uncertainties for single measurements and target value assignment were 1.8-2.5 % and 0.9-1.7 %, respectively, for total phenytoin. For free phenytoin, these measurement uncertainties were 2.0-3.9 % and 0.9-1.4 % for single measurements and target value assignment, respectively.

Conclusions: We present a novel LC-MS/MS-based RMP for phenytoin in human serum and plasma that provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.