LC–MS-MS Determination of Oxcarbazepine and an Active Metabolite in Human Plasma for Clinical Application

LC–MS-MS Determination of Oxcarbazepine and an Active Metabolite in Human Plasma for Clinical... Abstract A bioanalytical method for the simultaneous determination of oxcarbazepine (OXC) and its pharmacologically active metabolite, 10, 11-dihydro-10-hydroxycarbamazepine (HOXC), in human plasma was developed using a high-performance liquid chromatography with tandem mass spectrometry. After protein precipitation by acetonitrile, the analytes (OXC and HOXC) and a stable-labeled isotope of OXC as an internal standard (IS) were chromatographed on a Synergi Hydro-RP column (2.0 mm × 50 mm, 4 μm) with a gradient elution at a flow rate 0.5 mL/min. Detection was performed in electrospray ionization in the positive mode by monitoring the selected ion transitions at m/z 253.1 → 180.2, m/z 255.1 → 192.2 and m/z 257.2 → 184.2 for OXC, HOXC and the IS, respectively. The method was validated according to current bioanalytical method validation guidelines. The calibration standard curve ranged from 0.02 to 10 μg/mL for OXC and 0.1–50 μg/mL for HOXC using only 0.05 mL of plasma. No interferences were detected in blank plasma and hemolyzed plasma did not have any impacts on the assay. Accuracy and precision in the intra- and inter-batch reproducibility were within 15%. Neither cross-analytes inter-conversion nor matrix effects were observed. The method was successfully applied to determine plasma concentrations of OXC and HOXC to support a clinical study. Introduction Oxcarbazepine (OXC) has been used for patients with partial seizures as monotherapy and as adjunctive therapy by a proposed mechanism of action, blockade of voltage-gated sodium channels (1). OXC is metabolized to sulfate and glucuronide conjugates along with a pharmacological active metabolite, 10, 11-dihydro-10-hydroxycarbamazepine (HOXC) (2). Systemic exposures of HOXC are much higher than OXC, which make HOXC a clinically relevant metabolite (3, 4). Thus, in order to fully assess pharmacological effects in humans dosed with OXC, quantification of HOXC along with OXC is important. A number of bioanalytical methods have been published for the determination of OXC itself and simultaneous assay of OXC and HOXC in human plasma (5–12). However, most methods utilized liquid–liquid extraction or solid phase extraction, which are labor-intensive when a large number of samples are processed in a short timeframe. Thus, in the present study, protein precipitation is employed as sample extraction since it is simpler with higher sample throughput. In addition, in the previously reported methods, relatively large volume of plasma (e.g., 0.3 mL (6), 0.5 mL (7)) was used to achieve higher sensitivity, which could be an issue since only limited volume of blood sampling is allowed in children and infants who have an opportunity to receive OXC (13). Furthermore, as OXC can be concomitantly used with other anti-epilepsy drugs, a highly selective method is required, which is often a challenge in high-performance liquid chromatography (HPLC) with UV detection. High-performance liquid chromatography with tandem mass spectrometry (LC–MS-MS) is a powerful platform for developing more sensitive and selective bioanalytical methods. In the present study, we have developed a bioanalytical method for simultaneous determination of OXC and HOXC in human plasma by LC–MS-MS using only 0.05 mL of plasma and the method was successfully applied to assay pharmacokinetic plasma samples supporting a clinical study. Even with the low volume of plasma (0.05 mL) used, the established method achieved the lower limit of quantification (LLOQ) at 20 and 100 ng/mL for OXC and HOXC, respectively, sufficiently quantifying clinically relevant plasma concentrations. The LLOQ of OXC in the present method was equal to or lower than that in the previously reported methods; 580 ng/mL (7), 0.2 μmol/L (ca. 50 ng/mL) (11), 20 ng/mL (8), 78 ng/mL (10), while higher than 9.58 ng/mL using 0.3 mL of plasma (6). Other than the LLOQ, the present method has following advantages over previous methods: simple sample preparation and low volume of plasma used for the assay. This article demonstrates a simple and reproducible validated bioanalytical method for simultaneous determination of OXC and HOXC in human plasma and its successful application in clinical bioanalysis. Experimental Materials OXC, HOXC and a stable labeled isotope of OXC used as an internal standard (IS) (chemical structures in Figure 1) were obtained from Toronto Research Chemicals Inc (Toronto, Ontario, Canada) or TLC Pharmachem (Mississauga, Ontario, Canada). HPLC grade acetonitrile and methanol, and special grade formic acid were purchased from Fisher scientific (Fair Lawn, NJ, USA) or Sigma Aldrich (St. Louis, MO, USA). Drug-free blank plasma with K2EDTA as an anticoagulant was obtained from Bioreclamation Inc (Westbury, NY, USA). Figure 1. View largeDownload slide Chemical structures of oxcarbazepine (A), 10, 11-dihydro-10-hydroxycarbamazepine (B), and a stable labeled isotope of oxcarbazepine (C). Figure 1. View largeDownload slide Chemical structures of oxcarbazepine (A), 10, 11-dihydro-10-hydroxycarbamazepine (B), and a stable labeled isotope of oxcarbazepine (C). Chromatographic and mass spectrometric conditions Chromatographic separation was performed by a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) using gradient elution of mobile phase (A) water/formic acid (100/0.1, v/v) and (B) acetonitrile/methanol/formic acid (50/50/0.1, v/v/v). A linear increase in mobile phase (B) from 30 to 70% for 2.5 min at a flow rate of 0.5 mL/min was utilized to elute the analytes and the IS. The system was flushed immediately with 95% (B) for 1.4 min at a flow rate of 1.0 mL/min and then reversed to the initial composition of 30% (B) at a flow rate of 0.5 mL/min for 1.2 min for re-equilibration. The total run time per assay was 5.2 min. Chromatographic separation was achieved on a Synergi Hydro-RP column (2.0 mm × 50 mm, 4 μm) at ambient temperature. Electrospray ionization in the positive ion mode was employed for quantification with triple quadrupole mass spectrometer using API4000 (Sciex, CA, USA). Optimized mass spectrometer conditions are represented in Table I. The voltage, temperature, curtain gas flow and nebulizing gas flow were 5,000 V, 500°C, 25 psi and 50, respectively. Multiple reaction monitoring mode was used for detection by monitoring the transition pairs of m/z 253.1/180.2, m/z 255.1/194.2 and m/z 257.2/184.2 as precursor ion/product ion for OXC, HOXC and the IS, respectively. Table I. Optimized Mass Spectrometry Conditions for the Assay of Oxcarbazepine (OXC), 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) and the Internal Standard (IS) Analytes Q1 mass (m/z) Q3 mass (m/z) Dwell time (ms) Declustering potential (V) Collision energy (V) Entrance potential (V) Collision cell exit potential (V) OXC 253.1 180.2 200 50 43 10 10 HOXC 255.1 194.2 200 48 53 10 10 IS 257.2 184.2 200 50 43 10 10 Analytes Q1 mass (m/z) Q3 mass (m/z) Dwell time (ms) Declustering potential (V) Collision energy (V) Entrance potential (V) Collision cell exit potential (V) OXC 253.1 180.2 200 50 43 10 10 HOXC 255.1 194.2 200 48 53 10 10 IS 257.2 184.2 200 50 43 10 10 Table I. Optimized Mass Spectrometry Conditions for the Assay of Oxcarbazepine (OXC), 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) and the Internal Standard (IS) Analytes Q1 mass (m/z) Q3 mass (m/z) Dwell time (ms) Declustering potential (V) Collision energy (V) Entrance potential (V) Collision cell exit potential (V) OXC 253.1 180.2 200 50 43 10 10 HOXC 255.1 194.2 200 48 53 10 10 IS 257.2 184.2 200 50 43 10 10 Analytes Q1 mass (m/z) Q3 mass (m/z) Dwell time (ms) Declustering potential (V) Collision energy (V) Entrance potential (V) Collision cell exit potential (V) OXC 253.1 180.2 200 50 43 10 10 HOXC 255.1 194.2 200 48 53 10 10 IS 257.2 184.2 200 50 43 10 10 Preparation of calibration and quality control samples A stock solution of OXC and HOXC was separately prepared by dissolving in methanol to yield a concentration of 1 and 10 mg/mL, respectively, and then mixed with a volume ratio of 2:1 to make a mixed stock solution. The working standard solutions were prepared by diluting the mixed stock solution with methanol. A stock IS solution (1 mg/mL) was prepared then diluted 1,000-fold with methanol to make an IS working solution (1 μg/mL). The stock and working solutions were stored at −20°C. Calibration samples were prepared by fortifying working standard solution to 50 μL of drug-free blank human plasma at 20/100, 40/200, 100/500, 500/2,500, 1,000/5,000, 5,000/25,000, 8,000/40,000 and 10,000/50,000 ng/mL for OXC/HOXC. Quality control (QC, OXC/HOXC) samples at four concentration levels, LLOQ (20/100 ng/mL), QC at low concentration (LQC; 60/300 ng/mL), QC at middle concentration (MQC; 1,500/7,500 ng/mL), and QC at high concentration (HQC; 7,500/37,500 ng/mL), were prepared and stored at −20°C until use. Sample extraction procedures To the calibration and QC samples, 20 μL of the IS working solution (1 μg/mL) was spiked while 20 μL of methanol to blank samples. Acetonitrile (500 μL) was added for extraction then vortexed for 10 min. After centrifugation at 4,000 × g for 5 min, a 50-μL aliquot of the supernatant was transferred to each well in a 96-well plate, and then mixed with 500 μL of 0.1% formic acid. An aliquot of 10 μL was injected into the LC–MS-MS system. Method validation Linearity Peak area ratios of the analytes (OXC and HOXC) to the IS were plotted against corresponding nominal concentrations. The least square regression with 1/(concentration)2 as a weighting factor was used to have a calibration curve. Linear and quadratic regressions were used for OXC and HOXC, respectively. Accuracy as relative error (RE) was determined to ensure that RE was within ±15%. Precision as relative standard deviation (RSD) was calculated at each concentration in 19 separate assay runs. Selectivity The selectivity was assessed by analyzing drug-free blank plasma from six separate individuals to find any potential interfering peaks at the retention times of the analytes (OXC and HOXC) and the IS. The interfering peaks’ area should be within 20% of that of the analytes at the LLOQ level and 5% of that of the IS. Accuracy and precision Accuracy and precision in intra- and inter-batch reproducibility were evaluated using QC samples at four concentration levels (LLOQ, LQC, MQC and HQC). Six replicates per concentration were determined to calculate accuracy as RE and precision as RSD. RE and RSD values for LQC, MQC and HQC should be within ±15 and 15%, respectively (±20 and 20% for the LLOQ). Dilution integrity was examined by diluting QC samples by 10-fold to ensure that RE and RSD values were within ±15 and 15%, respectively. Hemolysis effects Potential impacts of hemolyzed plasma were evaluated using hemolyzed plasma prepared by fortifying the analytes at the low QC level (60/300 ng/mL for OXC/HOXC) in plasma/blood mixture (95/5, v/v). Hemolyzed QC samples were quantified using the calibration samples prepared from non-hemolyzed plasma, then RE and RSD values were calculated. RE and RSD values should be within ±15 and 15%, respectively. Extraction recovery and matrix effect Extraction recovery was determined at three different concentrations covering low, medium and high ranges of calibration curve by dividing peak areas of QC samples by those of blank extract to which analytes at the same concentration were fortified (reference samples). Matrix factors of OXC and HOXC were calculated in six individual lots of plasma by comparing peak areas in reference samples with those of neat solution at the LQC level. RSD value was calculated to ensure that it was within 15%. Cross-analytes inter-conversion Potential inter-conversions between OXC and HOXC (from OXC to HOXC and vice versa) were evaluated using HQC samples in which only one analyte (OXC or HOXC) was fortified. Three replicates were put on bench for 6 h at room temperature and then extracted. The formed counterpart analyte was assayed to ensure that peak areas were within 20% of those at the LLOQ level. Stability Stability in human plasma was assessed at two concentrations using LQC and HQC samples. The stability assessment included bench-top stability for 6 and 24 h at ambient temperature, freeze/thaw stability up to five cycles from −20 or −70°C to ambient temperature, and long-term frozen stability for 181 days at −20°C and 194 days at −70°C, as well as processed sample stability for 70 h at 4°C. Bench-top stability in whole blood was also assessed for 2 h at room temperature. Stability in standard solutions was investigated at −20°C (380/364 days for OXC/HOXC). All the stability assessments were performed in three replicates per concentration and RE values should be within ±15%. Clinical application A clinical study was performed in subjects with refractory partial onset seizures who received a subject-dependent stable dose of OXC for at least 1 month with or without perampanel. The study was approved by the ethics committee and performed with informed consent from subjects. Blood samples were obtained from subjects in weeks 0, 10, 14 and 19 in tubes with K2EDTA as an anticoagulant. Plasma samples were obtained by centrifugation (ca. 2000 × g, 15 min) of blood samples and then were stored at −20°C until they were assayed. Results Method validation RE values at each concentration of calibration curves in 19 separate assay runs were within ±3.8 and ±2.4% for OXC and HOXC, respectively (Table II), which was within the pre-defined acceptance criteria recommended in the bioanalytical method validation guidelines by the European Medicines Agency (14) and US Food and Drug Administration (15). RSD values of OXC and HOXC were within 3.1 and 3.5%, respectively, in 19 assay runs. Representative chromatograms of OXC, HOXC and the IS are shown in Figure 2A and B. No interfering peaks were observed in six different lots of blank plasma and in zero concentration samples fortified only with the IS. OXC, HOXC and the IS were eluted at ca. 2.2, 1.7 and 2.2 min, respectively. Table II. Linearity of Calibration Samples for the Simultaneous Assay of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma OXC (ng/mL) % RE % RSD HOXC (ng/mL) % RE % RSD 20 −1.5 1.8 100 −0.5 1.6 40 1.6 3.1 200 0.2 3.5 100 3.1 2.4 500 2.0 2.5 500 1.3 2.8 2,500 −0.6 2.3 1,000 2.3 1.7 5,000 −0.5 2.3 5,000 −1.0 1.5 25,000 −1.3 2.0 8,000 −3.8 2.6 40,000 −1.6 2.8 10,000 −1.9 2.9 50,000 2.4 2.5 OXC (ng/mL) % RE % RSD HOXC (ng/mL) % RE % RSD 20 −1.5 1.8 100 −0.5 1.6 40 1.6 3.1 200 0.2 3.5 100 3.1 2.4 500 2.0 2.5 500 1.3 2.8 2,500 −0.6 2.3 1,000 2.3 1.7 5,000 −0.5 2.3 5,000 −1.0 1.5 25,000 −1.3 2.0 8,000 −3.8 2.6 40,000 −1.6 2.8 10,000 −1.9 2.9 50,000 2.4 2.5 % RE and % RSD represent percentage of relative error and percentage of relative standard deviation, of 19 separate assay runs, respectively. Table II. Linearity of Calibration Samples for the Simultaneous Assay of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma OXC (ng/mL) % RE % RSD HOXC (ng/mL) % RE % RSD 20 −1.5 1.8 100 −0.5 1.6 40 1.6 3.1 200 0.2 3.5 100 3.1 2.4 500 2.0 2.5 500 1.3 2.8 2,500 −0.6 2.3 1,000 2.3 1.7 5,000 −0.5 2.3 5,000 −1.0 1.5 25,000 −1.3 2.0 8,000 −3.8 2.6 40,000 −1.6 2.8 10,000 −1.9 2.9 50,000 2.4 2.5 OXC (ng/mL) % RE % RSD HOXC (ng/mL) % RE % RSD 20 −1.5 1.8 100 −0.5 1.6 40 1.6 3.1 200 0.2 3.5 100 3.1 2.4 500 2.0 2.5 500 1.3 2.8 2,500 −0.6 2.3 1,000 2.3 1.7 5,000 −0.5 2.3 5,000 −1.0 1.5 25,000 −1.3 2.0 8,000 −3.8 2.6 40,000 −1.6 2.8 10,000 −1.9 2.9 50,000 2.4 2.5 % RE and % RSD represent percentage of relative error and percentage of relative standard deviation, of 19 separate assay runs, respectively. Figure 2. View largeDownload slide Representative LC–MS-MS chromatograms of the analytes (upper panel) oxcarbazepine (A and B) and 10, 11-dihydro-10-hydroxycarbamazepine (C and D) as well as the internal standard (lower panel). Chromatograms of blank plasma fortified only with the IS (A and C) and an LLOQ sample with the IS (B and D) are represented. Figure 2. View largeDownload slide Representative LC–MS-MS chromatograms of the analytes (upper panel) oxcarbazepine (A and B) and 10, 11-dihydro-10-hydroxycarbamazepine (C and D) as well as the internal standard (lower panel). Chromatograms of blank plasma fortified only with the IS (A and C) and an LLOQ sample with the IS (B and D) are represented. Possible cross-analytes inter-conversions were assessed using QC samples fortified only with OXC or HOXC at the HQC level. The inter-conversion was <1% of the LLOQ from OXC to HOXC while none from HOXC to OXC, suggesting no impacts by inter-conversion on the assays of OXC and HOXC. Results in the intra- and inter-batch assay reproducibility are represented in Table III. In the intra-batch assay, RE and RSD values in LQC, MQC and HQC samples were within ±5.2 and 2.7%, respectively, for OXC assay, and were within ±8.3 and 2.1% for HOXC, respectively. The RE and RSD values in inter-batch assay were within ±7.9 and 4.0%, respectively, for OXC and HOXC. At the LLOQ, RE and RSD values were within ±10.6 and 7.6%, respectively, for both analytes in intra- and inter-batch reproducibility. These data demonstrated that QC samples including the LLOQ were within the acceptance criteria recommended by the bioanalytical method validation guidelines. Data of the dilution integrity with 10-fold dilution showed that RE and RSD values for OXC and HOXC were within ±4.6 and 1.9%, respectively. Table III. Intra- and Inter-Batch Accuracy and Precision for the Simultaneous Assay of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma Quality control OXC HOXC % RE % RSD % RE % RSD Intra-batch  LLOQ −7.9 4.7 −10.6 5.3  LQC −5.2 2.7 −7.3 2.1  MQC −3.3 1.1 −5.4 1.7  HQC −3.3 0.8 −8.3 1.2 Inter-batch  LLOQ −4.6 6.4 −3.5 7.6  LQC −6.2 2.7 −7.7 4.0  MQC −5.0 2.2 −5.5 2.1  HQC −5.3 2.1 −7.9 1.8 Quality control OXC HOXC % RE % RSD % RE % RSD Intra-batch  LLOQ −7.9 4.7 −10.6 5.3  LQC −5.2 2.7 −7.3 2.1  MQC −3.3 1.1 −5.4 1.7  HQC −3.3 0.8 −8.3 1.2 Inter-batch  LLOQ −4.6 6.4 −3.5 7.6  LQC −6.2 2.7 −7.7 4.0  MQC −5.0 2.2 −5.5 2.1  HQC −5.3 2.1 −7.9 1.8 HQC, quality control at high concentration; LLOQ, lower limit of quantification; LQC, quality control at low concentration; MQC, quality control at middle concentration. RE and %RSD represent percentage of relative error and percentage of relative standard deviation, respectively. Table III. Intra- and Inter-Batch Accuracy and Precision for the Simultaneous Assay of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma Quality control OXC HOXC % RE % RSD % RE % RSD Intra-batch  LLOQ −7.9 4.7 −10.6 5.3  LQC −5.2 2.7 −7.3 2.1  MQC −3.3 1.1 −5.4 1.7  HQC −3.3 0.8 −8.3 1.2 Inter-batch  LLOQ −4.6 6.4 −3.5 7.6  LQC −6.2 2.7 −7.7 4.0  MQC −5.0 2.2 −5.5 2.1  HQC −5.3 2.1 −7.9 1.8 Quality control OXC HOXC % RE % RSD % RE % RSD Intra-batch  LLOQ −7.9 4.7 −10.6 5.3  LQC −5.2 2.7 −7.3 2.1  MQC −3.3 1.1 −5.4 1.7  HQC −3.3 0.8 −8.3 1.2 Inter-batch  LLOQ −4.6 6.4 −3.5 7.6  LQC −6.2 2.7 −7.7 4.0  MQC −5.0 2.2 −5.5 2.1  HQC −5.3 2.1 −7.9 1.8 HQC, quality control at high concentration; LLOQ, lower limit of quantification; LQC, quality control at low concentration; MQC, quality control at middle concentration. RE and %RSD represent percentage of relative error and percentage of relative standard deviation, respectively. Potential hemolysis effects were investigated by quantification of hemolyzed QC samples against calibrators prepared by non-hemolyzed plasma. RE and RSD values of OXC and HOXC in hemolyzed samples were within ±7.4 and 3.9%, respectively, indicating no hemolysis effects. Mean extraction recoveries of OXC and HOXC at three concentrations are shown in Table IV. The mean recovery at each concentration was almost complete (ca. 100%) and the overall recovery as the mean at the three concentrations was 102.3% for OXC and 102.1% for HOXC. Potential matrix effects of OXC/HOXC in six different lots of plasma were evaluated at the low level (60/300 ng/mL) and results are represented in Table IV. IS-normalized matrix factors were 99% for OXC and 106% for HOXC with %RSD of 3.0 and 1.9%, respectively, indicating no matrix effects. Table IV. Mean Extraction Recovery and Matrix Effect of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma Analyte Concentration (ng/mL) % Mean recovery IS-normalized matrix factor OXC  LQC 60 108.4 0.99 ± 0.03 (% RSD: 3.0)  MQC 1,500 97.4  HQC 7,500 101.0 HOXC  LQC 300 106.4 1.06 ± 0.02 (% RSD; 1.9)  MQC 7,500 98.8  HQC 37,500 101.1 Analyte Concentration (ng/mL) % Mean recovery IS-normalized matrix factor OXC  LQC 60 108.4 0.99 ± 0.03 (% RSD: 3.0)  MQC 1,500 97.4  HQC 7,500 101.0 HOXC  LQC 300 106.4 1.06 ± 0.02 (% RSD; 1.9)  MQC 7,500 98.8  HQC 37,500 101.1 HQC, quality control at high concentration; LQC, quality control at low concentration; MQC, quality control at middle concentration. Table IV. Mean Extraction Recovery and Matrix Effect of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma Analyte Concentration (ng/mL) % Mean recovery IS-normalized matrix factor OXC  LQC 60 108.4 0.99 ± 0.03 (% RSD: 3.0)  MQC 1,500 97.4  HQC 7,500 101.0 HOXC  LQC 300 106.4 1.06 ± 0.02 (% RSD; 1.9)  MQC 7,500 98.8  HQC 37,500 101.1 Analyte Concentration (ng/mL) % Mean recovery IS-normalized matrix factor OXC  LQC 60 108.4 0.99 ± 0.03 (% RSD: 3.0)  MQC 1,500 97.4  HQC 7,500 101.0 HOXC  LQC 300 106.4 1.06 ± 0.02 (% RSD; 1.9)  MQC 7,500 98.8  HQC 37,500 101.1 HQC, quality control at high concentration; LQC, quality control at low concentration; MQC, quality control at middle concentration. Table V summarizes stability data of OXC and HOXC at low and high concentrations. OXC and HOXC were stable in human plasma for at least 181 and 194 days at −20 and −70°C, respectively, and even after five freeze/thaw cycles from below −20 or −70°C to ambient temperature. Processed sample stability of OXC and HOXC was ensured up to 70 h at 4°C. Bench-top stability in plasma was ensured up to 6 h for OXC while up to 24 h for HOXC at ambient temperature. Bench-top stability in whole blood was confirmed for 2 h at ambient temperature for OXC and HOXC. The analytes in the stock solutions were stable for at least 364 days at −20°C. Table V. Stability of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma and Whole Blood Stability test Matrix Condition Quality control samples OXC HOXC % RE Bench-top Plasma After 6 h at RT LQC −9.5 −5.5 HQC −7.2 −6.7 Bench-top Whole blood After 2 h at RT LQC 0.3 −1.1 HQC −1.0 −1.8 Freeze/thaw Plasma After five cycles LQC −11.3 −1.5 HQC −9.1 −6.7 Long-term frozen Plasma After 181 days at −20°C LQC −13.1 2.6 HQC −7.0 −3.2 Processed Plasma After 70 h at 4°C LQC −6.8 −6.3 HQC −5.4 −8.7 Stability test Matrix Condition Quality control samples OXC HOXC % RE Bench-top Plasma After 6 h at RT LQC −9.5 −5.5 HQC −7.2 −6.7 Bench-top Whole blood After 2 h at RT LQC 0.3 −1.1 HQC −1.0 −1.8 Freeze/thaw Plasma After five cycles LQC −11.3 −1.5 HQC −9.1 −6.7 Long-term frozen Plasma After 181 days at −20°C LQC −13.1 2.6 HQC −7.0 −3.2 Processed Plasma After 70 h at 4°C LQC −6.8 −6.3 HQC −5.4 −8.7 HQC, quality control at high concentration; LQC, quality control at low concentration; RT, room temperature. Table V. Stability of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma and Whole Blood Stability test Matrix Condition Quality control samples OXC HOXC % RE Bench-top Plasma After 6 h at RT LQC −9.5 −5.5 HQC −7.2 −6.7 Bench-top Whole blood After 2 h at RT LQC 0.3 −1.1 HQC −1.0 −1.8 Freeze/thaw Plasma After five cycles LQC −11.3 −1.5 HQC −9.1 −6.7 Long-term frozen Plasma After 181 days at −20°C LQC −13.1 2.6 HQC −7.0 −3.2 Processed Plasma After 70 h at 4°C LQC −6.8 −6.3 HQC −5.4 −8.7 Stability test Matrix Condition Quality control samples OXC HOXC % RE Bench-top Plasma After 6 h at RT LQC −9.5 −5.5 HQC −7.2 −6.7 Bench-top Whole blood After 2 h at RT LQC 0.3 −1.1 HQC −1.0 −1.8 Freeze/thaw Plasma After five cycles LQC −11.3 −1.5 HQC −9.1 −6.7 Long-term frozen Plasma After 181 days at −20°C LQC −13.1 2.6 HQC −7.0 −3.2 Processed Plasma After 70 h at 4°C LQC −6.8 −6.3 HQC −5.4 −8.7 HQC, quality control at high concentration; LQC, quality control at low concentration; RT, room temperature. Clinical application Plasma concentrations of OXC and HOXC were determined in a clinical study, where the standard treatment by OXC was processed with or without perampanel for patients with refractory partial onset seizures. Typical plasma concentrations of OXC and HOXC in a subject who received 900 mg daily oral dose (450 mg b.i.d) are represented in Figure 3. Plasma concentration levels of HOXC were much higher than those of OXC and the concentration ratio of HOXC to OXC was similar to the previous papers (4, 5, 16). Figure 3. View largeDownload slide Plasma concentrations of oxcarbazepine (open circle; OXC) and 10, 11-dihydro-10-hydroxycarbamazepine (closed circle; HOXC) in a subject with refractory partial onset seizures who received oral dose of OXC (450 mg b.i.d.) Figure 3. View largeDownload slide Plasma concentrations of oxcarbazepine (open circle; OXC) and 10, 11-dihydro-10-hydroxycarbamazepine (closed circle; HOXC) in a subject with refractory partial onset seizures who received oral dose of OXC (450 mg b.i.d.) Discussion Method development An assay method for the simultaneous determination of OXC and HOXC in human plasma has been developed based on a previously developed bioanalytical method of OXC in human plasma. In the OXC assay, OXC was eluted at the retention time of 4.4 min on Cadenza CD-C18 (75 × 3.0 mm i.d., 3 μm) at a flow rate of 0.4 mL/min, where gradient elution was employed using mobile phases (A) 0.1% formic acid and (B) acetonitrile. The total run time was 6.0 min in the OXC assay, thus to shorten the run time despite the additional analyte, HOXC, HPLC conditions were modified and optimized in the present assay (see the optimized conditions in Experimental). A clear baseline peak separation was achieved for OXC and HOXC along with sharp peaks using the optimized conditions. A shortened run time may contribute in higher sample throughput which is important from bioanalytical perspective to support clinical trials, where a large number of samples are to be assayed in a short timeframe. This assay method utilized a simple protein precipitation for the extraction of the analytes, while previously reported methods used solid phase extraction (6, 10, 11), or liquid–liquid extraction by methyl tert-butyl ether (5) or diethyl ether (8), which would be more labor intensive than protein precipitation. Simpler extraction methods such as protein precipitation may sometimes cause matrix effects issues, however, in the present study, no matrix effects were noted using a stable isotope labeled IS. Although it is ideal to use two stable isotopes for OXC and HOXC, stable isotope of HOXC was not available at the moment, thus the stable isotope of OXC was used for the assay of HOXC as well. Data of the method validation parameters including linearity, reproducibility, extraction recovery and matrix effects in this study clearly demonstrate that the IS works well for HOXC assay along with OXC assay. The full-scan spectrum of the precursor ion of the analytes produced the most abundant protonated molecules at m/z 253.1 and 255.1 for OXC and HOXC, respectively. The product ion spectrum provided the highest signals at m/z 180.1 for OXC and 194.1 for HOXC (Figure 4). In the mass transition of HOXC, a less sensitive transition m/z 255.1 → 192.2 was selected given that plasma levels of HOXC were much higher than those of OXC in the simultaneous assay method supporting clinical trials. Figure 4. View largeDownload slide Representative product ion spectrum of oxcarbazepine (A) and 10, 11-dihydro-10-hydroxycarbamazepine (B) at precursor ions m/z 253.1 and 255.1, respectively. Figure 4. View largeDownload slide Representative product ion spectrum of oxcarbazepine (A) and 10, 11-dihydro-10-hydroxycarbamazepine (B) at precursor ions m/z 253.1 and 255.1, respectively. Method validation and clinical application In terms of response function of calibration curve, the bioanalytical guidelines by European Medicines Agency and US Food and Drug Administration suggest that a simple model that adequately describes the concentration–response relationship should be used, implying that the other regression than linear one can be used with justification. In the present study, although linear regression was used for the assay of OXC, quadratic regression was used for the assay of HOXC. In the HOXC assay, linear regression was not selected as the optimal regression since RE value of some samples was out of the criteria in some assay runs, while quadratic regression with a weighting factor of 1/concentration2 was able to produce the best fit throughout the assay runs. In addition, linearity evaluation revealed that back calculated data at higher calibration HOXC samples (e.g., 40,000 and 50,000 ng/mL) gave negative bias in every assay run when the linear regression was used. Given that random bias even at higher concentrations in the case of quadratic regression and potential signal saturation of HOXC at higher concentrations due to 5-time higher calibration sample concentrations of HOXC, it was rational to use quadratic regression for the HOXC assay in this 2-in-1 assay. The previous reports used quadratic regression (8) and linear regression (6, 9) for the OXC assay. In the assay of HOXC, both quadratic regression (8) and linear regression (6, 9) were used. As a validation study parameter, potential cross-analytes inter-conversion was assessed in this study and results demonstrated that there was no inter-conversion which may impact the assay of both OXC and HOXC. To the best of our knowledge, no reports have been available in which cross-analytes inter-conversions between OXC and HOXC were assessed. It is important to assess cross-analytes inter-conversions for accurate determination of more than one analyte especially when large differences in concentrations are expected as in the case of OXC and HOXC. A slight inter-conversion of one analyte with higher concentration levels to the other(s) may significantly impact assay of the other analyte with lower levels. Bench-top stability data of OXC in plasma was not ensured for 24 h at both low (% RE: −20.1) and high (% RE: −17.1) concentrations although stability was ensured up to 6 h at room temperature (Table V). It was reported that OXC in human plasma was stable up to 7 h at room temperature (8) and 24 h at 4°C (9). Although reasons or mechanism of instability of OXC in human plasma at room temperature remains to be investigated, it is recommended to store plasma samples at 4°C when sample processing times would take more than 6 h. Validation parameters of the established method assessed in this study supported that the method was robust and reproducible, thus the method was applied to the simultaneous determination of OXC and HOXC in human plasma to support a clinical trial. Plasma levels of HOXC were much higher than those of OXC in a subject, which was also the case in other subjects as well. The finding of higher HOXC levels observed in this study was also supported by previous reports; The AUC ratio of HOXC to OXC was 29.2–37.2 (6), 32.3 (16), 44.8 (2) and 48.8 (5). Conclusions The established LC–MS-MS method for the simultaneous determination of OXC and HOXC concentrations in human plasma has been validated and found to be simple and reproducible. The developed bioanalytical method has been successfully applied to pharmacokinetic sample assay in order to support a clinical trial. Acknowledgments The author acknowledges that Frontage Inc (Shanghai, China) conducted the method validation and assayed clinical pharmacokinetic samples. The author also thanks Jagadeesh Aluri (Eisai Inc) for language editorial assistance with this article. References 1 Kalis , M.M. , Huff , N.A. ; Oxcarbazepine, an antiepileptic agent ; Clinical Therapeutics , ( 2001 ); 23 : 680 – 700 . Google Scholar CrossRef Search ADS PubMed 2 Flesch , G. ; Overview of the clinical pharmacokinetics of oxcarbazepine ; Clinical Drug Investigation , ( 2004 ); 24 : 185 – 203 . 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Google Scholar CrossRef Search ADS 11 Rouan , M.C. , Decherf , M. , Le Clanche , V. , Lecaillon , J.B. , Godbillon , J. ; Automated microanalysis of oxcarbazepine and its monohydroxy and transdiol metabolites in plasma by liquid chromatography ; Journal of Chromatography B , ( 1994 ); 658 : 167 – 172 . Google Scholar CrossRef Search ADS 12 Mohamed , F.A. , Bakr , M.F. , Rageh , A.H. , Mostafa , A.M. ; The use of separation techniques in the analysis of some antiepileptic drugs: a critical review ; Journal of Liquid Chromatography & Related Technologies , ( 2016 ); 39 : 783 – 798 . Google Scholar CrossRef Search ADS 13 Bourgeois , B.F. , D’Souza , J. ; Long-term safety and tolerability of oxcarbazepine in children: a review of clinical experience ; Epilepsy Behavior , ( 2005 ); 7 : 375 – 382 . Google Scholar CrossRef Search ADS PubMed 14 European Medicines Agency . ( 2011 ) Guideline on Bioanalytical Method Validation, Committee for Medicinal Products for Human Use, 2011. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf. 15 US Food and Drug Administration . ( 2001 ) Guidance for Industry: Bioanalytical Method Validation. http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf. 16 Antunes , N.J. , Wichert-Ana , L. , Coelho , E.B. , Della Pasqua , O. , Alexandre , V. , Jr. , Takayanagui , O.M. , et al. . ; Influence of verapamil on the pharmacokinetics of oxcarbazepine and of the enantiomers of its 10-hydroxy metabolite in healthy volunteers ; European Journal of Clinical Pharmacology , ( 2016 ); 72 : 195 – 201 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

LC–MS-MS Determination of Oxcarbazepine and an Active Metabolite in Human Plasma for Clinical Application

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Abstract

Abstract A bioanalytical method for the simultaneous determination of oxcarbazepine (OXC) and its pharmacologically active metabolite, 10, 11-dihydro-10-hydroxycarbamazepine (HOXC), in human plasma was developed using a high-performance liquid chromatography with tandem mass spectrometry. After protein precipitation by acetonitrile, the analytes (OXC and HOXC) and a stable-labeled isotope of OXC as an internal standard (IS) were chromatographed on a Synergi Hydro-RP column (2.0 mm × 50 mm, 4 μm) with a gradient elution at a flow rate 0.5 mL/min. Detection was performed in electrospray ionization in the positive mode by monitoring the selected ion transitions at m/z 253.1 → 180.2, m/z 255.1 → 192.2 and m/z 257.2 → 184.2 for OXC, HOXC and the IS, respectively. The method was validated according to current bioanalytical method validation guidelines. The calibration standard curve ranged from 0.02 to 10 μg/mL for OXC and 0.1–50 μg/mL for HOXC using only 0.05 mL of plasma. No interferences were detected in blank plasma and hemolyzed plasma did not have any impacts on the assay. Accuracy and precision in the intra- and inter-batch reproducibility were within 15%. Neither cross-analytes inter-conversion nor matrix effects were observed. The method was successfully applied to determine plasma concentrations of OXC and HOXC to support a clinical study. Introduction Oxcarbazepine (OXC) has been used for patients with partial seizures as monotherapy and as adjunctive therapy by a proposed mechanism of action, blockade of voltage-gated sodium channels (1). OXC is metabolized to sulfate and glucuronide conjugates along with a pharmacological active metabolite, 10, 11-dihydro-10-hydroxycarbamazepine (HOXC) (2). Systemic exposures of HOXC are much higher than OXC, which make HOXC a clinically relevant metabolite (3, 4). Thus, in order to fully assess pharmacological effects in humans dosed with OXC, quantification of HOXC along with OXC is important. A number of bioanalytical methods have been published for the determination of OXC itself and simultaneous assay of OXC and HOXC in human plasma (5–12). However, most methods utilized liquid–liquid extraction or solid phase extraction, which are labor-intensive when a large number of samples are processed in a short timeframe. Thus, in the present study, protein precipitation is employed as sample extraction since it is simpler with higher sample throughput. In addition, in the previously reported methods, relatively large volume of plasma (e.g., 0.3 mL (6), 0.5 mL (7)) was used to achieve higher sensitivity, which could be an issue since only limited volume of blood sampling is allowed in children and infants who have an opportunity to receive OXC (13). Furthermore, as OXC can be concomitantly used with other anti-epilepsy drugs, a highly selective method is required, which is often a challenge in high-performance liquid chromatography (HPLC) with UV detection. High-performance liquid chromatography with tandem mass spectrometry (LC–MS-MS) is a powerful platform for developing more sensitive and selective bioanalytical methods. In the present study, we have developed a bioanalytical method for simultaneous determination of OXC and HOXC in human plasma by LC–MS-MS using only 0.05 mL of plasma and the method was successfully applied to assay pharmacokinetic plasma samples supporting a clinical study. Even with the low volume of plasma (0.05 mL) used, the established method achieved the lower limit of quantification (LLOQ) at 20 and 100 ng/mL for OXC and HOXC, respectively, sufficiently quantifying clinically relevant plasma concentrations. The LLOQ of OXC in the present method was equal to or lower than that in the previously reported methods; 580 ng/mL (7), 0.2 μmol/L (ca. 50 ng/mL) (11), 20 ng/mL (8), 78 ng/mL (10), while higher than 9.58 ng/mL using 0.3 mL of plasma (6). Other than the LLOQ, the present method has following advantages over previous methods: simple sample preparation and low volume of plasma used for the assay. This article demonstrates a simple and reproducible validated bioanalytical method for simultaneous determination of OXC and HOXC in human plasma and its successful application in clinical bioanalysis. Experimental Materials OXC, HOXC and a stable labeled isotope of OXC used as an internal standard (IS) (chemical structures in Figure 1) were obtained from Toronto Research Chemicals Inc (Toronto, Ontario, Canada) or TLC Pharmachem (Mississauga, Ontario, Canada). HPLC grade acetonitrile and methanol, and special grade formic acid were purchased from Fisher scientific (Fair Lawn, NJ, USA) or Sigma Aldrich (St. Louis, MO, USA). Drug-free blank plasma with K2EDTA as an anticoagulant was obtained from Bioreclamation Inc (Westbury, NY, USA). Figure 1. View largeDownload slide Chemical structures of oxcarbazepine (A), 10, 11-dihydro-10-hydroxycarbamazepine (B), and a stable labeled isotope of oxcarbazepine (C). Figure 1. View largeDownload slide Chemical structures of oxcarbazepine (A), 10, 11-dihydro-10-hydroxycarbamazepine (B), and a stable labeled isotope of oxcarbazepine (C). Chromatographic and mass spectrometric conditions Chromatographic separation was performed by a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) using gradient elution of mobile phase (A) water/formic acid (100/0.1, v/v) and (B) acetonitrile/methanol/formic acid (50/50/0.1, v/v/v). A linear increase in mobile phase (B) from 30 to 70% for 2.5 min at a flow rate of 0.5 mL/min was utilized to elute the analytes and the IS. The system was flushed immediately with 95% (B) for 1.4 min at a flow rate of 1.0 mL/min and then reversed to the initial composition of 30% (B) at a flow rate of 0.5 mL/min for 1.2 min for re-equilibration. The total run time per assay was 5.2 min. Chromatographic separation was achieved on a Synergi Hydro-RP column (2.0 mm × 50 mm, 4 μm) at ambient temperature. Electrospray ionization in the positive ion mode was employed for quantification with triple quadrupole mass spectrometer using API4000 (Sciex, CA, USA). Optimized mass spectrometer conditions are represented in Table I. The voltage, temperature, curtain gas flow and nebulizing gas flow were 5,000 V, 500°C, 25 psi and 50, respectively. Multiple reaction monitoring mode was used for detection by monitoring the transition pairs of m/z 253.1/180.2, m/z 255.1/194.2 and m/z 257.2/184.2 as precursor ion/product ion for OXC, HOXC and the IS, respectively. Table I. Optimized Mass Spectrometry Conditions for the Assay of Oxcarbazepine (OXC), 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) and the Internal Standard (IS) Analytes Q1 mass (m/z) Q3 mass (m/z) Dwell time (ms) Declustering potential (V) Collision energy (V) Entrance potential (V) Collision cell exit potential (V) OXC 253.1 180.2 200 50 43 10 10 HOXC 255.1 194.2 200 48 53 10 10 IS 257.2 184.2 200 50 43 10 10 Analytes Q1 mass (m/z) Q3 mass (m/z) Dwell time (ms) Declustering potential (V) Collision energy (V) Entrance potential (V) Collision cell exit potential (V) OXC 253.1 180.2 200 50 43 10 10 HOXC 255.1 194.2 200 48 53 10 10 IS 257.2 184.2 200 50 43 10 10 Table I. Optimized Mass Spectrometry Conditions for the Assay of Oxcarbazepine (OXC), 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) and the Internal Standard (IS) Analytes Q1 mass (m/z) Q3 mass (m/z) Dwell time (ms) Declustering potential (V) Collision energy (V) Entrance potential (V) Collision cell exit potential (V) OXC 253.1 180.2 200 50 43 10 10 HOXC 255.1 194.2 200 48 53 10 10 IS 257.2 184.2 200 50 43 10 10 Analytes Q1 mass (m/z) Q3 mass (m/z) Dwell time (ms) Declustering potential (V) Collision energy (V) Entrance potential (V) Collision cell exit potential (V) OXC 253.1 180.2 200 50 43 10 10 HOXC 255.1 194.2 200 48 53 10 10 IS 257.2 184.2 200 50 43 10 10 Preparation of calibration and quality control samples A stock solution of OXC and HOXC was separately prepared by dissolving in methanol to yield a concentration of 1 and 10 mg/mL, respectively, and then mixed with a volume ratio of 2:1 to make a mixed stock solution. The working standard solutions were prepared by diluting the mixed stock solution with methanol. A stock IS solution (1 mg/mL) was prepared then diluted 1,000-fold with methanol to make an IS working solution (1 μg/mL). The stock and working solutions were stored at −20°C. Calibration samples were prepared by fortifying working standard solution to 50 μL of drug-free blank human plasma at 20/100, 40/200, 100/500, 500/2,500, 1,000/5,000, 5,000/25,000, 8,000/40,000 and 10,000/50,000 ng/mL for OXC/HOXC. Quality control (QC, OXC/HOXC) samples at four concentration levels, LLOQ (20/100 ng/mL), QC at low concentration (LQC; 60/300 ng/mL), QC at middle concentration (MQC; 1,500/7,500 ng/mL), and QC at high concentration (HQC; 7,500/37,500 ng/mL), were prepared and stored at −20°C until use. Sample extraction procedures To the calibration and QC samples, 20 μL of the IS working solution (1 μg/mL) was spiked while 20 μL of methanol to blank samples. Acetonitrile (500 μL) was added for extraction then vortexed for 10 min. After centrifugation at 4,000 × g for 5 min, a 50-μL aliquot of the supernatant was transferred to each well in a 96-well plate, and then mixed with 500 μL of 0.1% formic acid. An aliquot of 10 μL was injected into the LC–MS-MS system. Method validation Linearity Peak area ratios of the analytes (OXC and HOXC) to the IS were plotted against corresponding nominal concentrations. The least square regression with 1/(concentration)2 as a weighting factor was used to have a calibration curve. Linear and quadratic regressions were used for OXC and HOXC, respectively. Accuracy as relative error (RE) was determined to ensure that RE was within ±15%. Precision as relative standard deviation (RSD) was calculated at each concentration in 19 separate assay runs. Selectivity The selectivity was assessed by analyzing drug-free blank plasma from six separate individuals to find any potential interfering peaks at the retention times of the analytes (OXC and HOXC) and the IS. The interfering peaks’ area should be within 20% of that of the analytes at the LLOQ level and 5% of that of the IS. Accuracy and precision Accuracy and precision in intra- and inter-batch reproducibility were evaluated using QC samples at four concentration levels (LLOQ, LQC, MQC and HQC). Six replicates per concentration were determined to calculate accuracy as RE and precision as RSD. RE and RSD values for LQC, MQC and HQC should be within ±15 and 15%, respectively (±20 and 20% for the LLOQ). Dilution integrity was examined by diluting QC samples by 10-fold to ensure that RE and RSD values were within ±15 and 15%, respectively. Hemolysis effects Potential impacts of hemolyzed plasma were evaluated using hemolyzed plasma prepared by fortifying the analytes at the low QC level (60/300 ng/mL for OXC/HOXC) in plasma/blood mixture (95/5, v/v). Hemolyzed QC samples were quantified using the calibration samples prepared from non-hemolyzed plasma, then RE and RSD values were calculated. RE and RSD values should be within ±15 and 15%, respectively. Extraction recovery and matrix effect Extraction recovery was determined at three different concentrations covering low, medium and high ranges of calibration curve by dividing peak areas of QC samples by those of blank extract to which analytes at the same concentration were fortified (reference samples). Matrix factors of OXC and HOXC were calculated in six individual lots of plasma by comparing peak areas in reference samples with those of neat solution at the LQC level. RSD value was calculated to ensure that it was within 15%. Cross-analytes inter-conversion Potential inter-conversions between OXC and HOXC (from OXC to HOXC and vice versa) were evaluated using HQC samples in which only one analyte (OXC or HOXC) was fortified. Three replicates were put on bench for 6 h at room temperature and then extracted. The formed counterpart analyte was assayed to ensure that peak areas were within 20% of those at the LLOQ level. Stability Stability in human plasma was assessed at two concentrations using LQC and HQC samples. The stability assessment included bench-top stability for 6 and 24 h at ambient temperature, freeze/thaw stability up to five cycles from −20 or −70°C to ambient temperature, and long-term frozen stability for 181 days at −20°C and 194 days at −70°C, as well as processed sample stability for 70 h at 4°C. Bench-top stability in whole blood was also assessed for 2 h at room temperature. Stability in standard solutions was investigated at −20°C (380/364 days for OXC/HOXC). All the stability assessments were performed in three replicates per concentration and RE values should be within ±15%. Clinical application A clinical study was performed in subjects with refractory partial onset seizures who received a subject-dependent stable dose of OXC for at least 1 month with or without perampanel. The study was approved by the ethics committee and performed with informed consent from subjects. Blood samples were obtained from subjects in weeks 0, 10, 14 and 19 in tubes with K2EDTA as an anticoagulant. Plasma samples were obtained by centrifugation (ca. 2000 × g, 15 min) of blood samples and then were stored at −20°C until they were assayed. Results Method validation RE values at each concentration of calibration curves in 19 separate assay runs were within ±3.8 and ±2.4% for OXC and HOXC, respectively (Table II), which was within the pre-defined acceptance criteria recommended in the bioanalytical method validation guidelines by the European Medicines Agency (14) and US Food and Drug Administration (15). RSD values of OXC and HOXC were within 3.1 and 3.5%, respectively, in 19 assay runs. Representative chromatograms of OXC, HOXC and the IS are shown in Figure 2A and B. No interfering peaks were observed in six different lots of blank plasma and in zero concentration samples fortified only with the IS. OXC, HOXC and the IS were eluted at ca. 2.2, 1.7 and 2.2 min, respectively. Table II. Linearity of Calibration Samples for the Simultaneous Assay of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma OXC (ng/mL) % RE % RSD HOXC (ng/mL) % RE % RSD 20 −1.5 1.8 100 −0.5 1.6 40 1.6 3.1 200 0.2 3.5 100 3.1 2.4 500 2.0 2.5 500 1.3 2.8 2,500 −0.6 2.3 1,000 2.3 1.7 5,000 −0.5 2.3 5,000 −1.0 1.5 25,000 −1.3 2.0 8,000 −3.8 2.6 40,000 −1.6 2.8 10,000 −1.9 2.9 50,000 2.4 2.5 OXC (ng/mL) % RE % RSD HOXC (ng/mL) % RE % RSD 20 −1.5 1.8 100 −0.5 1.6 40 1.6 3.1 200 0.2 3.5 100 3.1 2.4 500 2.0 2.5 500 1.3 2.8 2,500 −0.6 2.3 1,000 2.3 1.7 5,000 −0.5 2.3 5,000 −1.0 1.5 25,000 −1.3 2.0 8,000 −3.8 2.6 40,000 −1.6 2.8 10,000 −1.9 2.9 50,000 2.4 2.5 % RE and % RSD represent percentage of relative error and percentage of relative standard deviation, of 19 separate assay runs, respectively. Table II. Linearity of Calibration Samples for the Simultaneous Assay of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma OXC (ng/mL) % RE % RSD HOXC (ng/mL) % RE % RSD 20 −1.5 1.8 100 −0.5 1.6 40 1.6 3.1 200 0.2 3.5 100 3.1 2.4 500 2.0 2.5 500 1.3 2.8 2,500 −0.6 2.3 1,000 2.3 1.7 5,000 −0.5 2.3 5,000 −1.0 1.5 25,000 −1.3 2.0 8,000 −3.8 2.6 40,000 −1.6 2.8 10,000 −1.9 2.9 50,000 2.4 2.5 OXC (ng/mL) % RE % RSD HOXC (ng/mL) % RE % RSD 20 −1.5 1.8 100 −0.5 1.6 40 1.6 3.1 200 0.2 3.5 100 3.1 2.4 500 2.0 2.5 500 1.3 2.8 2,500 −0.6 2.3 1,000 2.3 1.7 5,000 −0.5 2.3 5,000 −1.0 1.5 25,000 −1.3 2.0 8,000 −3.8 2.6 40,000 −1.6 2.8 10,000 −1.9 2.9 50,000 2.4 2.5 % RE and % RSD represent percentage of relative error and percentage of relative standard deviation, of 19 separate assay runs, respectively. Figure 2. View largeDownload slide Representative LC–MS-MS chromatograms of the analytes (upper panel) oxcarbazepine (A and B) and 10, 11-dihydro-10-hydroxycarbamazepine (C and D) as well as the internal standard (lower panel). Chromatograms of blank plasma fortified only with the IS (A and C) and an LLOQ sample with the IS (B and D) are represented. Figure 2. View largeDownload slide Representative LC–MS-MS chromatograms of the analytes (upper panel) oxcarbazepine (A and B) and 10, 11-dihydro-10-hydroxycarbamazepine (C and D) as well as the internal standard (lower panel). Chromatograms of blank plasma fortified only with the IS (A and C) and an LLOQ sample with the IS (B and D) are represented. Possible cross-analytes inter-conversions were assessed using QC samples fortified only with OXC or HOXC at the HQC level. The inter-conversion was <1% of the LLOQ from OXC to HOXC while none from HOXC to OXC, suggesting no impacts by inter-conversion on the assays of OXC and HOXC. Results in the intra- and inter-batch assay reproducibility are represented in Table III. In the intra-batch assay, RE and RSD values in LQC, MQC and HQC samples were within ±5.2 and 2.7%, respectively, for OXC assay, and were within ±8.3 and 2.1% for HOXC, respectively. The RE and RSD values in inter-batch assay were within ±7.9 and 4.0%, respectively, for OXC and HOXC. At the LLOQ, RE and RSD values were within ±10.6 and 7.6%, respectively, for both analytes in intra- and inter-batch reproducibility. These data demonstrated that QC samples including the LLOQ were within the acceptance criteria recommended by the bioanalytical method validation guidelines. Data of the dilution integrity with 10-fold dilution showed that RE and RSD values for OXC and HOXC were within ±4.6 and 1.9%, respectively. Table III. Intra- and Inter-Batch Accuracy and Precision for the Simultaneous Assay of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma Quality control OXC HOXC % RE % RSD % RE % RSD Intra-batch  LLOQ −7.9 4.7 −10.6 5.3  LQC −5.2 2.7 −7.3 2.1  MQC −3.3 1.1 −5.4 1.7  HQC −3.3 0.8 −8.3 1.2 Inter-batch  LLOQ −4.6 6.4 −3.5 7.6  LQC −6.2 2.7 −7.7 4.0  MQC −5.0 2.2 −5.5 2.1  HQC −5.3 2.1 −7.9 1.8 Quality control OXC HOXC % RE % RSD % RE % RSD Intra-batch  LLOQ −7.9 4.7 −10.6 5.3  LQC −5.2 2.7 −7.3 2.1  MQC −3.3 1.1 −5.4 1.7  HQC −3.3 0.8 −8.3 1.2 Inter-batch  LLOQ −4.6 6.4 −3.5 7.6  LQC −6.2 2.7 −7.7 4.0  MQC −5.0 2.2 −5.5 2.1  HQC −5.3 2.1 −7.9 1.8 HQC, quality control at high concentration; LLOQ, lower limit of quantification; LQC, quality control at low concentration; MQC, quality control at middle concentration. RE and %RSD represent percentage of relative error and percentage of relative standard deviation, respectively. Table III. Intra- and Inter-Batch Accuracy and Precision for the Simultaneous Assay of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma Quality control OXC HOXC % RE % RSD % RE % RSD Intra-batch  LLOQ −7.9 4.7 −10.6 5.3  LQC −5.2 2.7 −7.3 2.1  MQC −3.3 1.1 −5.4 1.7  HQC −3.3 0.8 −8.3 1.2 Inter-batch  LLOQ −4.6 6.4 −3.5 7.6  LQC −6.2 2.7 −7.7 4.0  MQC −5.0 2.2 −5.5 2.1  HQC −5.3 2.1 −7.9 1.8 Quality control OXC HOXC % RE % RSD % RE % RSD Intra-batch  LLOQ −7.9 4.7 −10.6 5.3  LQC −5.2 2.7 −7.3 2.1  MQC −3.3 1.1 −5.4 1.7  HQC −3.3 0.8 −8.3 1.2 Inter-batch  LLOQ −4.6 6.4 −3.5 7.6  LQC −6.2 2.7 −7.7 4.0  MQC −5.0 2.2 −5.5 2.1  HQC −5.3 2.1 −7.9 1.8 HQC, quality control at high concentration; LLOQ, lower limit of quantification; LQC, quality control at low concentration; MQC, quality control at middle concentration. RE and %RSD represent percentage of relative error and percentage of relative standard deviation, respectively. Potential hemolysis effects were investigated by quantification of hemolyzed QC samples against calibrators prepared by non-hemolyzed plasma. RE and RSD values of OXC and HOXC in hemolyzed samples were within ±7.4 and 3.9%, respectively, indicating no hemolysis effects. Mean extraction recoveries of OXC and HOXC at three concentrations are shown in Table IV. The mean recovery at each concentration was almost complete (ca. 100%) and the overall recovery as the mean at the three concentrations was 102.3% for OXC and 102.1% for HOXC. Potential matrix effects of OXC/HOXC in six different lots of plasma were evaluated at the low level (60/300 ng/mL) and results are represented in Table IV. IS-normalized matrix factors were 99% for OXC and 106% for HOXC with %RSD of 3.0 and 1.9%, respectively, indicating no matrix effects. Table IV. Mean Extraction Recovery and Matrix Effect of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma Analyte Concentration (ng/mL) % Mean recovery IS-normalized matrix factor OXC  LQC 60 108.4 0.99 ± 0.03 (% RSD: 3.0)  MQC 1,500 97.4  HQC 7,500 101.0 HOXC  LQC 300 106.4 1.06 ± 0.02 (% RSD; 1.9)  MQC 7,500 98.8  HQC 37,500 101.1 Analyte Concentration (ng/mL) % Mean recovery IS-normalized matrix factor OXC  LQC 60 108.4 0.99 ± 0.03 (% RSD: 3.0)  MQC 1,500 97.4  HQC 7,500 101.0 HOXC  LQC 300 106.4 1.06 ± 0.02 (% RSD; 1.9)  MQC 7,500 98.8  HQC 37,500 101.1 HQC, quality control at high concentration; LQC, quality control at low concentration; MQC, quality control at middle concentration. Table IV. Mean Extraction Recovery and Matrix Effect of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma Analyte Concentration (ng/mL) % Mean recovery IS-normalized matrix factor OXC  LQC 60 108.4 0.99 ± 0.03 (% RSD: 3.0)  MQC 1,500 97.4  HQC 7,500 101.0 HOXC  LQC 300 106.4 1.06 ± 0.02 (% RSD; 1.9)  MQC 7,500 98.8  HQC 37,500 101.1 Analyte Concentration (ng/mL) % Mean recovery IS-normalized matrix factor OXC  LQC 60 108.4 0.99 ± 0.03 (% RSD: 3.0)  MQC 1,500 97.4  HQC 7,500 101.0 HOXC  LQC 300 106.4 1.06 ± 0.02 (% RSD; 1.9)  MQC 7,500 98.8  HQC 37,500 101.1 HQC, quality control at high concentration; LQC, quality control at low concentration; MQC, quality control at middle concentration. Table V summarizes stability data of OXC and HOXC at low and high concentrations. OXC and HOXC were stable in human plasma for at least 181 and 194 days at −20 and −70°C, respectively, and even after five freeze/thaw cycles from below −20 or −70°C to ambient temperature. Processed sample stability of OXC and HOXC was ensured up to 70 h at 4°C. Bench-top stability in plasma was ensured up to 6 h for OXC while up to 24 h for HOXC at ambient temperature. Bench-top stability in whole blood was confirmed for 2 h at ambient temperature for OXC and HOXC. The analytes in the stock solutions were stable for at least 364 days at −20°C. Table V. Stability of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma and Whole Blood Stability test Matrix Condition Quality control samples OXC HOXC % RE Bench-top Plasma After 6 h at RT LQC −9.5 −5.5 HQC −7.2 −6.7 Bench-top Whole blood After 2 h at RT LQC 0.3 −1.1 HQC −1.0 −1.8 Freeze/thaw Plasma After five cycles LQC −11.3 −1.5 HQC −9.1 −6.7 Long-term frozen Plasma After 181 days at −20°C LQC −13.1 2.6 HQC −7.0 −3.2 Processed Plasma After 70 h at 4°C LQC −6.8 −6.3 HQC −5.4 −8.7 Stability test Matrix Condition Quality control samples OXC HOXC % RE Bench-top Plasma After 6 h at RT LQC −9.5 −5.5 HQC −7.2 −6.7 Bench-top Whole blood After 2 h at RT LQC 0.3 −1.1 HQC −1.0 −1.8 Freeze/thaw Plasma After five cycles LQC −11.3 −1.5 HQC −9.1 −6.7 Long-term frozen Plasma After 181 days at −20°C LQC −13.1 2.6 HQC −7.0 −3.2 Processed Plasma After 70 h at 4°C LQC −6.8 −6.3 HQC −5.4 −8.7 HQC, quality control at high concentration; LQC, quality control at low concentration; RT, room temperature. Table V. Stability of Oxcarbazepine (OXC) and 10, 11-Dihydro-10-Hydroxycarbamazepine (HOXC) in Human Plasma and Whole Blood Stability test Matrix Condition Quality control samples OXC HOXC % RE Bench-top Plasma After 6 h at RT LQC −9.5 −5.5 HQC −7.2 −6.7 Bench-top Whole blood After 2 h at RT LQC 0.3 −1.1 HQC −1.0 −1.8 Freeze/thaw Plasma After five cycles LQC −11.3 −1.5 HQC −9.1 −6.7 Long-term frozen Plasma After 181 days at −20°C LQC −13.1 2.6 HQC −7.0 −3.2 Processed Plasma After 70 h at 4°C LQC −6.8 −6.3 HQC −5.4 −8.7 Stability test Matrix Condition Quality control samples OXC HOXC % RE Bench-top Plasma After 6 h at RT LQC −9.5 −5.5 HQC −7.2 −6.7 Bench-top Whole blood After 2 h at RT LQC 0.3 −1.1 HQC −1.0 −1.8 Freeze/thaw Plasma After five cycles LQC −11.3 −1.5 HQC −9.1 −6.7 Long-term frozen Plasma After 181 days at −20°C LQC −13.1 2.6 HQC −7.0 −3.2 Processed Plasma After 70 h at 4°C LQC −6.8 −6.3 HQC −5.4 −8.7 HQC, quality control at high concentration; LQC, quality control at low concentration; RT, room temperature. Clinical application Plasma concentrations of OXC and HOXC were determined in a clinical study, where the standard treatment by OXC was processed with or without perampanel for patients with refractory partial onset seizures. Typical plasma concentrations of OXC and HOXC in a subject who received 900 mg daily oral dose (450 mg b.i.d) are represented in Figure 3. Plasma concentration levels of HOXC were much higher than those of OXC and the concentration ratio of HOXC to OXC was similar to the previous papers (4, 5, 16). Figure 3. View largeDownload slide Plasma concentrations of oxcarbazepine (open circle; OXC) and 10, 11-dihydro-10-hydroxycarbamazepine (closed circle; HOXC) in a subject with refractory partial onset seizures who received oral dose of OXC (450 mg b.i.d.) Figure 3. View largeDownload slide Plasma concentrations of oxcarbazepine (open circle; OXC) and 10, 11-dihydro-10-hydroxycarbamazepine (closed circle; HOXC) in a subject with refractory partial onset seizures who received oral dose of OXC (450 mg b.i.d.) Discussion Method development An assay method for the simultaneous determination of OXC and HOXC in human plasma has been developed based on a previously developed bioanalytical method of OXC in human plasma. In the OXC assay, OXC was eluted at the retention time of 4.4 min on Cadenza CD-C18 (75 × 3.0 mm i.d., 3 μm) at a flow rate of 0.4 mL/min, where gradient elution was employed using mobile phases (A) 0.1% formic acid and (B) acetonitrile. The total run time was 6.0 min in the OXC assay, thus to shorten the run time despite the additional analyte, HOXC, HPLC conditions were modified and optimized in the present assay (see the optimized conditions in Experimental). A clear baseline peak separation was achieved for OXC and HOXC along with sharp peaks using the optimized conditions. A shortened run time may contribute in higher sample throughput which is important from bioanalytical perspective to support clinical trials, where a large number of samples are to be assayed in a short timeframe. This assay method utilized a simple protein precipitation for the extraction of the analytes, while previously reported methods used solid phase extraction (6, 10, 11), or liquid–liquid extraction by methyl tert-butyl ether (5) or diethyl ether (8), which would be more labor intensive than protein precipitation. Simpler extraction methods such as protein precipitation may sometimes cause matrix effects issues, however, in the present study, no matrix effects were noted using a stable isotope labeled IS. Although it is ideal to use two stable isotopes for OXC and HOXC, stable isotope of HOXC was not available at the moment, thus the stable isotope of OXC was used for the assay of HOXC as well. Data of the method validation parameters including linearity, reproducibility, extraction recovery and matrix effects in this study clearly demonstrate that the IS works well for HOXC assay along with OXC assay. The full-scan spectrum of the precursor ion of the analytes produced the most abundant protonated molecules at m/z 253.1 and 255.1 for OXC and HOXC, respectively. The product ion spectrum provided the highest signals at m/z 180.1 for OXC and 194.1 for HOXC (Figure 4). In the mass transition of HOXC, a less sensitive transition m/z 255.1 → 192.2 was selected given that plasma levels of HOXC were much higher than those of OXC in the simultaneous assay method supporting clinical trials. Figure 4. View largeDownload slide Representative product ion spectrum of oxcarbazepine (A) and 10, 11-dihydro-10-hydroxycarbamazepine (B) at precursor ions m/z 253.1 and 255.1, respectively. Figure 4. View largeDownload slide Representative product ion spectrum of oxcarbazepine (A) and 10, 11-dihydro-10-hydroxycarbamazepine (B) at precursor ions m/z 253.1 and 255.1, respectively. Method validation and clinical application In terms of response function of calibration curve, the bioanalytical guidelines by European Medicines Agency and US Food and Drug Administration suggest that a simple model that adequately describes the concentration–response relationship should be used, implying that the other regression than linear one can be used with justification. In the present study, although linear regression was used for the assay of OXC, quadratic regression was used for the assay of HOXC. In the HOXC assay, linear regression was not selected as the optimal regression since RE value of some samples was out of the criteria in some assay runs, while quadratic regression with a weighting factor of 1/concentration2 was able to produce the best fit throughout the assay runs. In addition, linearity evaluation revealed that back calculated data at higher calibration HOXC samples (e.g., 40,000 and 50,000 ng/mL) gave negative bias in every assay run when the linear regression was used. Given that random bias even at higher concentrations in the case of quadratic regression and potential signal saturation of HOXC at higher concentrations due to 5-time higher calibration sample concentrations of HOXC, it was rational to use quadratic regression for the HOXC assay in this 2-in-1 assay. The previous reports used quadratic regression (8) and linear regression (6, 9) for the OXC assay. In the assay of HOXC, both quadratic regression (8) and linear regression (6, 9) were used. As a validation study parameter, potential cross-analytes inter-conversion was assessed in this study and results demonstrated that there was no inter-conversion which may impact the assay of both OXC and HOXC. To the best of our knowledge, no reports have been available in which cross-analytes inter-conversions between OXC and HOXC were assessed. It is important to assess cross-analytes inter-conversions for accurate determination of more than one analyte especially when large differences in concentrations are expected as in the case of OXC and HOXC. A slight inter-conversion of one analyte with higher concentration levels to the other(s) may significantly impact assay of the other analyte with lower levels. Bench-top stability data of OXC in plasma was not ensured for 24 h at both low (% RE: −20.1) and high (% RE: −17.1) concentrations although stability was ensured up to 6 h at room temperature (Table V). It was reported that OXC in human plasma was stable up to 7 h at room temperature (8) and 24 h at 4°C (9). Although reasons or mechanism of instability of OXC in human plasma at room temperature remains to be investigated, it is recommended to store plasma samples at 4°C when sample processing times would take more than 6 h. Validation parameters of the established method assessed in this study supported that the method was robust and reproducible, thus the method was applied to the simultaneous determination of OXC and HOXC in human plasma to support a clinical trial. Plasma levels of HOXC were much higher than those of OXC in a subject, which was also the case in other subjects as well. The finding of higher HOXC levels observed in this study was also supported by previous reports; The AUC ratio of HOXC to OXC was 29.2–37.2 (6), 32.3 (16), 44.8 (2) and 48.8 (5). Conclusions The established LC–MS-MS method for the simultaneous determination of OXC and HOXC concentrations in human plasma has been validated and found to be simple and reproducible. The developed bioanalytical method has been successfully applied to pharmacokinetic sample assay in order to support a clinical trial. Acknowledgments The author acknowledges that Frontage Inc (Shanghai, China) conducted the method validation and assayed clinical pharmacokinetic samples. The author also thanks Jagadeesh Aluri (Eisai Inc) for language editorial assistance with this article. References 1 Kalis , M.M. , Huff , N.A. ; Oxcarbazepine, an antiepileptic agent ; Clinical Therapeutics , ( 2001 ); 23 : 680 – 700 . Google Scholar CrossRef Search ADS PubMed 2 Flesch , G. ; Overview of the clinical pharmacokinetics of oxcarbazepine ; Clinical Drug Investigation , ( 2004 ); 24 : 185 – 203 . 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( 2011 ) Guideline on Bioanalytical Method Validation, Committee for Medicinal Products for Human Use, 2011. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf. 15 US Food and Drug Administration . ( 2001 ) Guidance for Industry: Bioanalytical Method Validation. http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf. 16 Antunes , N.J. , Wichert-Ana , L. , Coelho , E.B. , Della Pasqua , O. , Alexandre , V. , Jr. , Takayanagui , O.M. , et al. . ; Influence of verapamil on the pharmacokinetics of oxcarbazepine and of the enantiomers of its 10-hydroxy metabolite in healthy volunteers ; European Journal of Clinical Pharmacology , ( 2016 ); 72 : 195 – 201 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. 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Published: Sep 1, 2018

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