Dr. Risch Ostschweiz AG, Buchs, Switzerland.
111618 Roche Diagnostics GmbH , Penzberg, Germany.
Clin Chem Lab Med. 2024 Feb 27;62(7):1314-1326. doi: 10.1515/cclm-2023-1104. Print 2024 Jun 25.
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.
苯巴比妥作为一种抗癫痫药物（AED），无论是作为单药治疗还是辅助治疗，都可用于治疗癫痫。该药具有多种药效学特性，这些特性既带来了有益的效果，也带来了潜在的副作用。准确测量其浓度对于通过适当调整剂量来优化 AED 治疗至关重要。因此，我们的目标是开发和验证一种新的参考测量程序（RMP），以准确量化人血清和血浆中的苯巴比妥水平。
建立了一种基于蛋白沉淀的样品制备方案，结合使用 C8 柱的液相色谱-串联质谱（LC-MS/MS）方法进行高稀释步骤，以将目标分析物与已知和未知的干扰物分离。根据现行指南进行了分析验证和测量不确定度的测定。使用加标血清和血浆样本评估选择性和特异性；为了研究可能的基质效应（ME），进行了柱后输注实验和标准曲线斜率的比较。在多日精密度实验中确定了精密度和准确度。
该 RMP 被证明具有高度的选择性和特异性，没有证据表明存在基质干扰。它可用于在 1.92 至 72.0μg/mL 的范围内定量苯巴比妥。中间精密度小于 3.2%，在所有浓度水平下，重复性变异系数（CV）范围为 1.3%至 2.0%。对于天然血清水平，相对平均偏差范围为-3.0%至-0.7%，对于 Li-肝素血浆水平，相对平均偏差范围为-2.8%至 0.8%。单次测量和目标值赋值的测量不确定度（k=1）分别为 1.9%至 3.3%和 0.9%至 1.6%。
提出了一种新的基于 LC-MS/MS 的人血清和血浆中苯巴比妥定量候选 RMP，可用于常规分析的标准化和临床相关样本的评估。
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