Simultaneous Quantification of 11 Antiseizure Medications and Metabolites in Serum for Therapeutic Drug Monitoring Using High-Performance Liquid Chromatography with Ultraviolet Detection: A Short Communication

Abstract

Background: Therapeutic drug monitoring (TDM) of antiseizure medications (ASMs) is widely used to guide therapy, avoid toxicity, and assess patient compliance. Commercial immunologic quantification methods are common practice; however, as they are only applicable to one specific drug and prone to cross-reacting metabolites, their practical applicability is limited. In this article, the authors proposed a high-performance liquid chromatography method using ultraviolet detection (HPLC-UV) for simultaneous quantification of 11 ASMs and active metabolites (carbamazepine, felbamate, lacosamide, lamotrigine, levetiracetam, phenobarbital, phenytoin, primidone, zonisamide, carbamazepine-10,11-epoxide, and licarbazepine) in serum.

Methods: Chromatographic separation was performed on a Phenomenex Luna PFP(2) (3-µm particle size; 150 × 4.6 mm i.d.) analytical column. The mobile phase comprised phosphate buffer (20 mM; pH 3), acetonitrile (ACN), and methanol using gradient elution. Analyses were conducted at 35°C and a 1.3-mL/min flow rate. The detection wavelength for all analytes was 210 nm. The samples were prepared by protein precipitation using ACN.

Results: The HPLC-UV method was validated according to the FDA guidelines and applied to measure patient samples in TDM. Calibration curves showed excellent linearity (r2 > 0.99) and covered the entire reference range for each analyte. Intraday and interday imprecisions and inaccuracies were <10% for all samples. Extensive stability testing showed no significant degradation (<15%), and interference measurements additionally ensured clinical applicability. Furthermore, the sensitivity was comparable with that of previously published HPLC methods using mass spectrometry.

Conclusions: The authors developed an HPLC-UV method for the simultaneous quantification of 11 ASMs in the human serum and demonstrated its practical applicability in TDM. The method requires only standard laboratory equipment and simple sample preparation, making TDM available in less specialized laboratories.