Simultaneous Determination of Lamotrigine, Oxcarbazepine, Lacosamide, and Topiramate in Rat Plasma by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

This study established an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to study the pharmacokinetics of four antiepileptic drugs, lamotrigine, oxcarbazepine, lacosamide, and topiramate, in rats after oral administration. The gradient elution was performed on a UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column with acetonitrile-0.1% formic acid as the mobile phase at a flow rate of 0.4 mL/min. Protein precipitation by acetonitrile was adopted for plasma sample pretreatment. Electrospray- (ESI-) positive/negative ion switching and multiple reaction monitoring (MRM) modes were adopted for ion quantitative determination of antiepileptic drugs. UPLC-MS/MS detection and Drug and Statistics (DAS) software fitting were performed to blood samples collected from rats after oral administration of lamotrigine, oxcarbazepine, lacosamide, and topiramate (5 mg/kg). All drugs examined showed linearity within 5–5000 ng/ml (R2 > 0.9987), the intraday accuracy was within 92%–108%, and the interday accuracy was within 93%–109%. The relative standard deviations (RSD) of intraday and interday were less than 15%. The matrix effect was within 91%–105%, and the recovery was better than 88%. The established UPLC-MS/MS method was successfully applied to the pharmacokinetic study of lamotrigine, oxcarbazepine, lacosamide, and topiramate in rats.


Introduction
Epilepsy is a common nervous system disease [1][2][3]. Epilepsy treatment usually relies on antiepileptic drugs that belong to the symptom control drugs [4,5]. Most epilepsy patients require long-term medication; however, patients show individual differences in drug response [6][7][8][9][10]. To simultaneously improve the effectiveness and safety of clinical drugs and provide a reliable scientific basis for diagnosing and treating drug overdose poisoning, a rapid and accurate method is necessary to determine drug concentration in plasma.
e methods for the determination of antiepileptic drugs mainly include high-performance liquid chromatography (HPLC) [11][12][13][14], immunoassay [15,16], and gas chromatography-mass spectrometer (GC-MS) [17,18]. Liquid chromatography with tandem mass spectrometry (LC-MS/ MS) can obtain strong adduct ion peaks of the compounds under first-order mass spectrometry [19][20][21]. Although methods for measuring individual antiepileptic drug concentration have been widely reported, some patients with severe conditions require multiple drugs simultaneously. It is urgent to establish a rapid and quantitative screening method for several antiepileptic drugs to meet the clinical needs.
In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established to simultaneously determine plasma concentrations of four antiepileptic drugs (lamotrigine, oxcarbazepine, lacosamide, and topiramate) in rats, using midazolam as an internal standard. e proposed method could be helpful for blood concentration monitoring, individual administration plan formulation, drug abuse monitoring, and pharmacokinetic study of these drugs.

Standard Curve
Preparation. An appropriate amount of standard working solution of lamotrigine, oxcarbazepine, lacosamide, and topiramate was added to the rat plasma to prepare standard curves (5, 20, 100, 500, 1000, 2000, and 5000 ng/mL of the standard solution). e quality control (QC) samples were prepared in the same way as the plasma standard curve to obtain low, medium, and high concentrations (10, 900, and 4500 ng/mL) solutions.

Method Verification
e verification method was established following the US Food and Drug Administration (FDA) Bioanalytical Method Verification Guidelines. e verification items included selectivity, matrix effect, linearity, precision, accuracy, recovery, and stability [24]. e method's selectivity was evaluated by analyzing blank rat plasma, blank plasma-spiked lamotrigine, oxcarbazepine, lacosamide, topiramate, and internal standard.
Calibration curves of lamotrigine, oxcarbazepine, lacosamide, and topiramate were constructed by analyzing spiked calibration samples on three separate days. e lower limit of quantification (LLOQ) was defined as the lowest concentration on the calibration curves, and the deviation should be within ±20%.
Blank rat plasma was extracted and spiked with the analyte at 10, 900, and 4500 ng/mL to evaluate the matrix effect. e corresponding peak areas were then compared to neat standard solutions at equivalent concentrations.
Accuracy and precision were assessed by determining QC in six replicates (10, 900, and 4500 ng/mL) over three days of validation testing. e precision is expressed as RSD.
e recovery of lamotrigine, oxcarbazepine, lacosamide, and topiramate was evaluated by comparing the peak area of extracted QC samples with those of reference QC solutions reconstituted in blank plasma extracts (n � 6). e stability of lamotrigine, oxcarbazepine, lacosamide, and topiramate in rat plasma was evaluated by analyzing three plasma sample replicates at 10, 900, and 4500 ng/mL, exposed to different conditions. ese results were compared with the freshly prepared plasma samples.  Figure 3 shows the UPLC-MS/MS chromatograms of rat blank plasma samples and plasma samples spiked with lamotrigine, oxcarbazepine, lacosamide, and topiramate. e results showed that the endogenous substances in rat plasma samples had almost no effect on the determination of the four compounds.

Standard Curve
Line. Lamotrigine, oxcarbazepine, lacosamide, and topiramate concentrations in rat plasma showed a linear relationship within the range of 5-5000 ng/ mL. e standard curve equations are given in Table 1     International Journal of Analytical Chemistry y represents the plasma concentration of lamotrigine, oxcarbazepine, lacosamide, and topiramate, and x represents the peak area of the drugs. e LLOQ of lamotrigine, oxcarbazepine, lacosamide, and topiramate in rat plasma was 5 ng/mL. e detection limit was 2 ng/mL with a signalto-noise of 3. Table 2, the intraday accuracy of lamotrigine, oxcarbazepine, lacosamide, and topiramate is within 92%-108%, and the interday accuracy is within 93%-109%; the RSDs of intraday and interday are less than 15%; the matrix effect is within 91%-105%, and the recovery is above 88%. It is concluded that the established UPLC-MS/MS method was suitable for pharmacokinetic studies of lamotrigine, oxcarbazepine, lacosamide, and topiramate.

Stability.
e rat plasma stability tests were conducted at room temperature for 2 h, −20°C for 30 days, and three freeze-thaw cycles. e results in Table 3 show an accuracy within 92-108% and an RSD lower than 13%, indicating that lamotrigine, oxcarbazepine, lacosamide, and topiramate have good stability.

Pharmacokinetic Studies.
e blood drug concentration over time curve is shown in Figure 4. e pharmacokinetic parameters peak drug concentration (C max ), time to peak (T max ), terminal elimination half-life (t 1/2 ), area under the drug-time curve (AUC), clearance rate (CL), apparent volume of distribution (Vd), and mean residence time (MRT) were calculated according to the noncompartment model (Table 4). When the sample concentration was higher than 5000 ng/mL, blank rat plasma was diluted 10 times prior to processing.

Discussion
ESI-positive/negative electrode selection is often adopted in methodological studies. Lamotrigine, oxcarbazepine, and lacosamide are alkaloids more suitable for ESI-positive detection. However, topiramate is more suitable for ESInegative electrode detection. erefore, positive and negative ion switching modes were adopted to simultaneously detect lamotrigine, oxcarbazepine, lacosamide, and topiramate.
For LC conditions, the retention time of endogenous interferences should be as far away as possible from the examined compounds and internal standards [25,26]. For this purpose, the relative chromatographic behavior of the column and mobile phase plays a decisive role; therefore, multiple columns and mobile phases were examined. First, BEH C18 and HSS T3 columns were tested. e HSS T3 (2.1 mm × 100 mm, 1.8 μm) column had better separation and chromatographic peaks than BEH C18 and was chosen as the chromatographic column. is experiment also tried various mobile phases, including methanolwater, acetonitrile-water, methanol-0.1% formic acid, and acetonitrile-0.1% formic acid. Results showed that acetonitrile-0.1% formic acid had the best chromatographic peak shape.

Conclusion
In this study, a UPLC-MS/MS method for the simultaneous determination of four antiepileptic drugs was established and applied to pharmacokinetics. e results showed that this method had good selectivity, high sensitivity, and an excellent linear relationship. Altogether, the presented work provides a reliable tool for monitoring antiepileptic drugs in plasma, which could be implemented in individual administration plan formulation, drug abuse monitoring, and pharmacokinetic studies.   Data Availability e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.