6
5. Anticonvulsants
Epilepsy is a chronic neurological disorder which is characterised by recurrent epileptic seizures whose frequency and rhythm are difficult to predict. For the pharmacological therapy of epilepsy, a variety of antiepileptic drugs (AEDs) are available today, most of which exhibit a pronounced intra- and inter- individual variability in pharmacokinetics. In many patients, it is necessary to use multiple drugs, however, as interactions between different AEDs also affect the pharmacokinetics there is a clear need for TDM [21].
AEDs as a therapeutic class show diverse chemistries because of their different mode of action. In addition, some benzodiazepines are also used in the treatment of seizures. However, the therapeutic concentrations are in lower ranges compared to most AEDs. Structures of representative compounds are presented in Figure 7.
The lower limit of quantification (LLOQ) was set as 5 times lower than the low reference concentration. The upper limit of quantification (ULOQ) was set as 1.5 times higher than the high reference concentration. Targeted calibration ranges can be found in Table 4.
Quality control samples were prepared. Concentration levels were LLOQ (QCLOQ), 3 times the LLOQ (QC A), 50% of the concentration range (QC B) and 90% of the concentration range (QC C). Five individual replicates were prepared per level.
Automatic sample preparation was performed using CLAM-2000 module (Shimadzu, Kyoto) coupled to UHPLC-MS/MS system (Nexera X2 and LCMS-8050, Shimadzu, Kyoto).
For sample preparation, 30 µL of plasma were mixed with 270 µL of SIL-ISTD solution (10 µg/mL in methanol). After 30 seconds of vortex, the samples were filtered during 1 min. The resulting extract was then automatically transferred into the LC-MS/MS autosampler for analysis. Then, 0.5µL of extract were injected on the LC column (Shim-Pack GIS C18 5µm 50x2.1mm Shimadzu, Japan), maintained at 45°C. Mobile phase were (A) ammonium formate buffer 3mM pH3.6 and (B) methanol. A gradient from 10%B to 90%B in 2 minutes was run at 0.6 mL/min. The total run time was of 3.5 minutes. Two transitions per compound were acquired for quantification and confirmation of identification (except for Valproic Acid). Mass spectrometry parameters are described in Tables 5 and 6.
5.2. Linearity
Linearity of the method was assessed by calculating the relative deviation of calibration standards against the calculated linear regression model. In all cases, deviation was within ±15%, meeting the acceptance criteria. Typical calibration curves are shown in Figure 8.
Figure 7: Structures of the most common antiepileptic drugs and benzodiazepines included in this study.
To meet this need a TDM method was developed to measure simultaneously a large panel of anticonvulsant drugs with different chemistries and target concentrations.
5.1. Materials and Methods C6
in positive and negative ionisation, respectively. The Table 4 summarises the list of compounds assayed.
-Zonisamide and D5
Calibration standard levels were prepared by spiking a blank plasma pool (EDTA-K3, 6 donors, mixed gender, BioreclamationIVT, USA). Seven levels were prepared. For each compound, the calibration range was determined using the therapeutic reference range.
5.3 Recovery
Recovery of the method was evaluated by comparing peak areas measured in QC samples prepared in plasma (n=6) to the ones measured in QC samples made in neat solution (n=5). Total recovery, combining extraction and matrix effect, was measured. Results are presented in Table 7. For all compounds, recovery was consistent across the concentration range and the average was greater than 80%.
Certified standard solutions of each compound were purchased from Cerilliant (Sigma- Aldrich, USA). 13
-Phenobarbital were used as the internal standard 5.3. Accuracy and Precision
Accuracy and precision of the QC samples were calculated across 5 replicates per concentration level. Results are reported in Table 8. A typical chromatogram of target compounds is shown in Figure 9. All QC samples were within the acceptance criteria for accuracy and precision.
Figure 9: Representative anticonvulsant chromatograms at the QC A level (3 times the LLOQ). Figure 8: Representative calibration curves of anticonvulsants.
LAB ASIA - JANUARY/FEBRUARY 2018
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