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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., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Phenobarbital serves as an antiepileptic drug (AED) and finds application in the treatment of epilepsy either as monotherapy or adjunctive therapy. This drug exhibits various pharmacodynamic properties that account for its beneficial effects as well as potential side effects. Accurate measurement of its concentration is critical for optimizing AED therapy through appropriate dose adjustments. Therefore, our objective was to develop and validate a new reference measurement procedure (RMP) for the accurate quantification of phenobarbital levels in human serum and plasma. A sample preparation protocol based on protein precipitation followed by a high dilution step was established in combination with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a C8 column to separate target analytes from known and unknown interferences. Assay validation and determination of measurement uncertainty were performed based on current guidelines. Selectivity and Specificity were assessed using spiked serum and plasma samples; to investigate possible matrix effects (MEs) a post-column infusion experiment and a comparison of standard line slopes was performed. Precision and accuracy were determined within a multiday precision experiment. The RMP was shown to be highly selective and specific, with no evidence of matrix interferences. It can be used to quantify phenobarbital in the range of 1.92 to 72.0 μg/mL. Intermediate precision was less than 3.2 %, and repeatability coefficient of variation (CV) ranged from 1.3 to 2.0 % across all concentration levels. The relative mean bias ranged from −3.0 to −0.7 % for native serum levels, and from −2.8 to 0.8 % for Li-heparin plasma levels. The measurement uncertainties (k=1) for single measurements and target value assignment were 1.9 to 3.3 % and 0.9 to 1.6 %, respectively. A novel LC-MS/MS-based candidate RMP for the quantification of phenobarbital in human serum and plasma is presented which can be used for the standardization of routine assays and the evaluation of clinically relevant samples. [ABSTRACT FROM AUTHOR]

A reference measurement procedure (RMP) using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated with the aim of accurately measuring carbamazepine-10,11-epoxide concentrations in human serum and plasma. To establish traceability to SI units, the absolute content of the reference material was determined using quantitative nuclear magnetic resonance (qNMR) spectroscopy. As sample preparation a protein precipitation protocol followed by a high dilution step was established. Chromatographic separation from carbamazepine and potential metabolites was achieved using a C18 stationary phase. Selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement were evaluated based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. The RMP demonstrated very good selectivity and specificity, showing no evidence of a matrix effect. This enabled accurate quantification of carbamazepine-epoxide in the concentration range of 0.0400–12.0 μg/mL. The intermediate precision was found to be less than 2.1 %, and the repeatability coefficient of variation (CV) ranged from 1.2 to 1.8 % across all concentration levels. Regarding accuracy, the relative mean bias varied from 1.4 to 2.5 % for native serum levels and from 1.4 to 3.5 % for Li-heparin plasma levels. The measurement uncertainty for single measurements ranged from 1.6 to 2.1 %. In this study, we introduce a new LC-MS/MS-based candidate RMP for accurately measuring carbamazepine-10,11-epoxide in human serum and plasma. This novel method offers a traceable and dependable platform, making it suitable for standardizing routine assays and assessing clinically relevant samples. [ABSTRACT FROM AUTHOR]

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 carbamazepine. Quantitative nuclear magnetic resonance (qNMR) spectroscopy was used to determine the absolute content of the reference material, ensuring its traceability to SI units. The separation of carbamazepine from potential interferences, whether known or unknown, was achieved using a C18 column. A protein precipitation protocol followed by a high dilution step was established for sample preparation. Assay validation and determination of measurement uncertainty were performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute, the International Conference on Harmonization (ICH), and the Guide to the Expression of Uncertainty in Measurement (GUM). In order to demonstrate equivalence to the already existing RMP a method comparison study was performed. The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of carbamazepine within the range of 0.800–18.0 μg/mL. Intermediate precision and repeatability (n=60 measurements) was found to be <1.6 % and <1.3 % over all concentration levels and independent from the matrix. The relative mean bias ranged from −0.1 to 0.6 % for native serum and from −0.3 to −0.1 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were found to be <1.8 % and <1.3 %, respectively. Method comparison showed a good agreement between the Joint Committee of Traceability in Laboratory Medicine (JCTLM) listed RMP and the candidate RMP resulting in a Passing–Bablok regression equation with a slope of 1.01 and an intercept of −0.01. The bias in the patient cohort was found to be 0.9 %. We present a novel LC-MS/MS-based candidate RMP for carbamazepine in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples. [ABSTRACT FROM AUTHOR]

To describe and validate an isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure (RMP) for zonisamide to accurately measure serum and plasma concentrations. Quantitative nuclear magnetic resonance (qNMR) spectroscopy was employed to determine the absolute content of the reference material used in order to establish traceability to SI units. Separation of zonisamide from known or unknown interferences was performed on a C8 column. For sample preparation a protocol based on protein precipitation in combination with a high dilution step was established. Assay validation and determination of measurement uncertainty were performed based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of zonisamide within the range of 1.50–60.0 μg/mL. Intermediate precision was <1.4 % and repeatability CV ranged from 0.7 to 1.2 % over all concentration levels. The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment ranged from 1.1 to 1.4 % and 0.8–1.0 %, respectively. We present a novel LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples. [ABSTRACT FROM AUTHOR]

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. 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). 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. 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. [ABSTRACT FROM AUTHOR]

We developed an isotope dilution (ID)-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for lamotrigine in human serum and plasma, using quantitative nuclear magnetic resonance-characterized reference standards to ensure traceability to the International System of Units. A sample preparation protocol based on protein precipitation combined with LC-MS/MS analysis using a C18 column for chromatographic separation was established for the quantification of lamotrigine in human serum and plasma. Assay validation was performed according to current guidelines. Spiked serum and plasma samples were used to assess selectivity and specificity; a post-column infusion experiment and comparison of standard line slopes were performed to ascertain possible matrix effects. Precision and accuracy were determined in a 5 days validation experiment. Measurement uncertainty was determined per the Guide to the Expression of Uncertainty in Measurement. The method allowed the quantification of lamotrigine in serum and plasma in a range of 0.600–24.0 μg/mL without any observable matrix effects. The relative mean bias (n=6) ranged from 1.7 to 3.7%; intermediate precision, including variances in between-day, -calibration, and -injection, was ≤2.4%, independent of the level and matrix. Total measurement uncertainty for a single measurement was ≤2.6%; expanded uncertainty was ≤5.2% (coverage factor k=2). This candidate RMP based on ID-LC-MS/MS provides a traceable and reliable platform for the standardization of routine assays and the evaluation of clinical samples. [ABSTRACT FROM AUTHOR]