Khromov Tatjana, Dihazi Gry Helene, Brockmeyer Phillipp, Fischer Andreas, Streit Frank
Department of Clinical Chemistry, University Medical Center Goettingen, Goettingen, Germany.
Department of Oral and Maxillofacial Surgery, University Medical Center Goettingen, Goettingen, Germany.
Front Neurol. 2025 Mar 14;16:1493201. doi: 10.3389/fneur.2025.1493201. eCollection 2025.
Epilepsy is a common neurological disorder associated with seizures that impact patients' quality of life. Treatment includes antiepileptic drugs (AEDs), each effective only at a specific dose, making continuous therapeutic drug monitoring (TDM) useful in clinical cases under inpatient conditions. Conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) lacks automation for 24/7 operation, limiting clinical applicability. This study validates a fully automated 24/7 AED monitoring system using the Clinical Laboratory Automated Sample Preparation Module 2030 (CLAM-2030).
The method was validated according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines by evaluating linearity, precision, accuracy, carry over, matrix effects, and calibration stability. Twenty-six AEDs were quantified in plasma using multiple reaction monitoring (MRM) transitions in positive and negative electrospray ionization modes. Sample preparation was fully automated: 20 μL methanol was used to wet the column, followed by 20 μL internal standard and 100 μL acetonitrile for protein precipitation. The supernatant was filtered and injected directly into the LC system. Chromatographic separation was achieved within 4.5 min using a C18 column (2.1 × 50 mm, 2.7 μm) under gradient conditions with a mobile phase of 0.2 mM ammonium formate and 0.002% formic acid.
The method demonstrated excellent linearity over the validated concentration ranges ( > 0.99 for all analytes). Within-run imprecision was <15% at the lower limit of quantitation (LLOQ), while between-run imprecision was <10% for most AEDs. Accuracy was within ±10% of nominal concentrations at all quality control (QC) levels. Matrix effects were within acceptable limits (<30% variation) for 23 of 26 analytes, with compensatory corrections applied for carbamazepine-D, felbamate-D, and levetiracetam-D. Carry over was negligible [<2% for all AEDs except retigabine and N-desmethylselegiline (NDMS), which remained below 6.5%]. Calibration stability was maintained over 5 days with concentration and peak area variation <10%. An interlaboratory comparison (ring test) showed a relative standard deviation <20% for all analytes.
This study establishes a robust, fully automated, high-throughput method for continuous AED monitoring in the clinical setting. The CLAM-2030-LCMS-8060NX system enables reliable 24/7 TDM with minimal technical expertise, ensuring optimized AED therapy and improved patient outcomes.
癫痫是一种常见的神经系统疾病，伴有发作，会影响患者的生活质量。治疗方法包括抗癫痫药物（AEDs），每种药物仅在特定剂量下有效，这使得在住院条件下的临床病例中进行持续的治疗药物监测（TDM）很有用。传统的液相色谱 - 串联质谱法（LC-MS/MS）缺乏用于全天候运行的自动化功能，限制了其临床适用性。本研究验证了一种使用临床实验室自动样品制备模块2030（CLAM - 2030）的全自动24/7 AED监测系统。
根据美国食品药品监督管理局（FDA）和欧洲药品管理局（EMA）的指南，通过评估线性、精密度、准确度、残留、基质效应和校准稳定性来验证该方法。在正离子和负离子电喷雾电离模式下，使用多反应监测（MRM）转换对血浆中的26种AEDs进行定量。样品制备完全自动化：用20μL甲醇湿润柱子，然后加入20μL内标和100μL乙腈进行蛋白沉淀。将上清液过滤后直接注入液相色谱系统。使用C18柱（2.1×50mm，2.7μm）在梯度条件下，以0.2mM甲酸铵和0.002%甲酸为流动相，在4.5分钟内实现色谱分离。
该方法在验证的浓度范围内显示出优异的线性（所有分析物的相关系数>0.99）。在定量下限（LLOQ）处，批内精密度<15%，而对于大多数AEDs，批间精密度<10%。在所有质量控制（QC）水平下，准确度在标称浓度的±10%以内。26种分析物中的23种的基质效应在可接受范围内（变化<30%），对卡马西平 - D、非氨酯 - D和左乙拉西坦 - D进行了补偿校正。残留可忽略不计[除瑞替加滨和N - 去甲基司来吉兰（NDMS）外，所有AEDs的残留<2%，后者保持在6.5%以下]。校准稳定性在5天内保持，浓度和峰面积变化<10%。实验室间比较（环形试验）显示所有分析物的相对标准偏差<20%。
本研究建立了一种用于临床环境中持续AED监测的强大、全自动、高通量方法。CLAM - 2030 - LCMS - 8060NX系统能够以最少的技术专业知识实现可靠的24/7 TDM，确保优化AED治疗并改善患者预后。
