We validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for quantifying levetiracetam (LEV) and lamotrigine (LAM) in serum specimens. We developed an UHPLC-MS/MS method using Triple Quad 4500MD (SCIEX, Singapore) with 10 μM LEV-²H₃ (LEV-internal standard [IS]) and 10 μM LAM ¹³C₃, d3 (LAM-IS) as internal standards. The mass spectrometer detected the transitions from the precursor to product ions (mass-to-charge ratio [m/z] 171.0→126.0 for LEV, m/z 177.1→132.1 for LEV-IS, m/z 256.0→210.9 for LAM, and m/z 262.1→216.8 for LAM-IS, respectively). We used a Kinetex (2.6 μm C18 100Å, 100×3.0 mm; Phenomenex, USA) column on an ExionLC AD UHPLC. The UHPLC-MS/MS method yielded a linear response from 0.60 to 340.13 μg/mL for LEV (R²=0.9997) and from 0.17 to 99.98 μg/mL for LAM (R²=0.9995). The lower limits of quantification using this method were 0.60 and 0.17 μg/mL for LEV and LAM, respectively. The recovery of LEV and LAM UHPLC-MS/MS method measurements were within ±6% of the targeted values. The intra-day and inter-day coefficients of variation were all acceptable with values of less than 7% in both cases. Carry-over was not observed in any of the results. Ion suppression or enhancement was not observed in the blank and six samples for both LEV and LAM test results. The UHPLC-MS/MS LEV and LAM assay showed adequate recovery, precision, and sensitivity, and an adequate AMR, and thus is suitable for routine clinical work.

Keywords: Levetiracetam, Lamotrigine, Liquid chromatography-tandem mass spectrometry, Method validation