Objectives Topiramate is an antiepileptic drug (AED) used for the monotherapy or adjunctive treatment of epilepsy and for the prophylaxis of migraine. It has several pharmacodynamic properties that contribute to both its clinically useful properties and observed adverse effects. Accurate measurement of its concentration is therefore essential for dose adjustment/optimisation of AED therapy. Our aim was to develop and validate a novel reference measurement procedure (RMP) for the quantification of topiramate in human serum and plasma. Methods An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method in combination with a protein-precipitation-based sample preparation allows for quantification of topiramate in human serum and plasma. To assure traceability to SI units, quantitative nuclear magnetic resonance (qNMR) was applied to characterize the reference material used as primary calibrator for this RMP. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Accuracy and precision was evaluated performing an extensive five day precision experiment and measurement uncertainty was evaluated according Guide to the Expression of Uncertainty in Measurement (GUM). Results The method enabled topiramate quantification within the range of 1.20–36.0 μg/mL without interference from structurally related compounds and no evidence of a matrix effect. Intermediate precision was ≤3.2 % and repeatability was 1.4–2.5 % across all concentration levels. The relative mean bias was −0.3 to 3.5 %. Expanded measurement uncertainties for target value assignment (n=6) were found to be ≤2.9 % (k=2) independent of the concentration level and the nature of the sample. Conclusions In human serum and plasma, the RMP demonstrated high analytical performance for topiramate quantification and fulfilled the requirements on measurement uncertainty. Traceability to SI units was established by qNMR content determination of the topiramate, which was used for direct calibration of the RMP. This RMP is, therefore, fit for purpose for routine assay standardization and clinical sample evaluation.
Objectives To develop an isotope dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for levetiracetam quantification in human serum and plasma. Methods Quantitative nuclear magnetic resonance spectroscopy (qNMR) was used to characterize the RMP material to ensure traceability to SI units. To quantify levetiracetam, an LC-MS/MS method was optimized using a C8 column for chromatographic separation following protein-precipitation-based sample preparation. Spiked matrix samples of serum and plasma were used to test selectivity and specificity. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Precision and accuracy were evaluated over 5 days. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM). Results The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of levetiracetam within the range of 1.53–90.0 μg/mL. Intermediate precision was <2.2% and repeatability was 1.1–1.7% across all concentrations. The relative mean bias ranged from −2.5% to −0.3% across all levels and matrices within the measuring range. Diluted samples were found with a mean bias ranging from −0.1 to 2.9%. The predefined acceptance criterion for measurement uncertainty was met and determined for individual measurements independently of the concentration level and sample type to be ≤4.0% (k=2). Conclusions We present a novel LC-MS/MS)-based candidate RMP for levetiracetam in human serum and plasma. Its expanded measurement uncertainty of ≤4.0% meets the clinical needs in levetiracetam monitoring. Utilizing qNMR to characterize levetiracetam reference materials allowed metrological traceability to SI units.
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