Abstracts

Rationale: Pregnancy induces various physiological changes that can significantly alter the pharmacokinetics of antiseizure medications (ASM), potentially increasing the risk of seizures. To facilitate convenient self-collection of pharmacokinetic study samples and reduce the necessity for in-person study, we developed and validated a robust microsampling assay to measure the concentrations of three ASMs - levetiracetam (LEV), lamotrigine (LTG), and brivaracetam (BRV)—simultaneously.

Methods: Calibrators were prepared by spiking 20 μL of whole blood (WB) with purified standards of LEV, LTG, and BRV. Deuterated internal standards were used for each analyte. Quality control (QC) samples were made by spiking WB and then splitting each QC level into 2 groups, one of which was absorbed onto a Volumetric Absorptive Microsampling (VAMS®, Neoteryx) collection device, to target the lower limit of quantification (LLOQ) as well as low, medium, and high points on the calibration curve. A protein precipitation was used for extraction and analyzed via LCMS. The coefficients of variation (CV) for QC values were calculated for both WB and VAMS tips to assess accuracy and precision. Analyses were conducted at three different QC levels, both within a single day (within-run) and (between-run) across three consecutive days. Matrix effects and extraction recovery were evaluated in accordance with FDA guidance for bioanalytical methods.¹ Stability of VAMS samples was tested at room temperature (RT) for overnight and at three days, and -80°C at three days.

Results: The final calibration curve was best represented by a quadratic fit and 1/C2 weighting, with concentration ranges LEV (0.5-100 μg/mL), LTG (0.1-30 μg/mL), and BRV (0.1-10 μg/mL). Precision (< 10%CV at low, middle, and high concentration QCs and < 15% at lower limit of quantification QC-LLOQ) and accuracy (range 85-115%) were acceptable within- and between-runs. Recovery rates for LEV, LTG, and BRV in VAMS were 90.5%, 80.9%, and 82.3%, respectively, compared to 104.9%, 89.3%, and 98.9%, in WB at medium
QC levels (LEV 20 μg/mL, LTG 5 μg/mL, BRV 1.5 μg/mL). Stability tests showed no significant degradation of VAMS samples under three tested conditions.

Conclusions: We developed and validated a sensitive and reliable LCMS assay for the quantification of LEV, LTG, and BRV using a microsampling approach from whole blood and VAMS. The assay demonstrated robust performance, indicating its potential to facilitate pharmacokinetic studies in patients through self-collection. The utilization of VAMS for self-collection enhances the feasibility of more frequent sampling, thereby minimizing inconvenience and associated costs. Additionally, this method increases the potential for research participation from a more geographically diverse population. Future research is needed to incorporate additional ASMs and explore their long-term stability.

References:
1) Bioanalytical Method Validation Guidance for Industry. May 2018.

Funding: This work was funded by the NIH NICHD R01HD105305 grant.