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  • 1 Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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Abstract

Epilepsy is one of the most prevalent neurological conditions and antiepileptic drugs are the mainstay of epilepsy treatment. High variation in pharmacokinetic profiles of several antiepileptic drugs highlights the importance of therapeutic drug monitoring to estimate pharmacokinetic properties and consequently individualize drug posology. In this work, a simple, rapid and robust liquid chromatography-tandem mass spectrometry method was developed for simultaneous quantification of carbamazepine and its metabolite carbamazepine-10,11-epoxide, gabapentin, levetiracetam, lamotrigine, oxcarbazepine and its metabolite mono-hydroxy-derivative metabolite, phenytoin, topiramate, and valproic acid in human plasma for therapeutic drug monitoring. d 6-Levetiracetam, d 4-gabapentin and d 6-valproic acid were used as internal standards. After addition of internal standards along with two-step protein precipitation and dilution sample preparation, plasma samples were analyzed on a C18 column using a gradient elution in 5 min without interference. The calibration curves were linear over a 100-fold concentration range, with determination coefficients (r 2) greater than 0.99 for all analytes. The limit of quantification was 0.5 μg mL−1 (0.1 μg mL−1 for oxcarbazepine, 2 μg mL−1 for levetiracetam, and 10 μg mL−1 for valproic acid) with precision and accuracy ranging from 3% to 9% and from 94% to 112%, respectively. Intra- and inter-day precision and accuracy values were within 15% at low, medium and high quality control levels. No significant matrix effect was observed in the normal, hemolyzed, lipemic, and hyperbilirubin blood samples. This method was successfully used in the identification and quantitation of antiepileptic drugs in patients undergoing mono- or polytherapy for epilepsy.

Abstract

Epilepsy is one of the most prevalent neurological conditions and antiepileptic drugs are the mainstay of epilepsy treatment. High variation in pharmacokinetic profiles of several antiepileptic drugs highlights the importance of therapeutic drug monitoring to estimate pharmacokinetic properties and consequently individualize drug posology. In this work, a simple, rapid and robust liquid chromatography-tandem mass spectrometry method was developed for simultaneous quantification of carbamazepine and its metabolite carbamazepine-10,11-epoxide, gabapentin, levetiracetam, lamotrigine, oxcarbazepine and its metabolite mono-hydroxy-derivative metabolite, phenytoin, topiramate, and valproic acid in human plasma for therapeutic drug monitoring. d 6-Levetiracetam, d 4-gabapentin and d 6-valproic acid were used as internal standards. After addition of internal standards along with two-step protein precipitation and dilution sample preparation, plasma samples were analyzed on a C18 column using a gradient elution in 5 min without interference. The calibration curves were linear over a 100-fold concentration range, with determination coefficients (r 2) greater than 0.99 for all analytes. The limit of quantification was 0.5 μg mL−1 (0.1 μg mL−1 for oxcarbazepine, 2 μg mL−1 for levetiracetam, and 10 μg mL−1 for valproic acid) with precision and accuracy ranging from 3% to 9% and from 94% to 112%, respectively. Intra- and inter-day precision and accuracy values were within 15% at low, medium and high quality control levels. No significant matrix effect was observed in the normal, hemolyzed, lipemic, and hyperbilirubin blood samples. This method was successfully used in the identification and quantitation of antiepileptic drugs in patients undergoing mono- or polytherapy for epilepsy.

Introduction

Epilepsy is one of the most prevalent neurological conditions, affecting nearly 50 million people of all ages worldwide [1]. Antiepileptic drugs (AEDs) are the mainstay of the epilepsy treatment, making about 60% patients with epilepsy have seizure controlled effectively [2]. Pharmacokinetic profiles are different between AEDs, even the same dose of the same AED may lead to different plasma concentrations in different patients, which highlights the importance of therapeutic drug monitoring (TDM) to estimate pharmacokinetic properties and consequently individualize drug posology [3].

In psychiatry and neurology, patients who may benefit from TDM particularly are children and adolescents, pregnant women, elderly patients, individuals with intellectual disabilities, patients with substance abuse disorders, forensic psychiatric patients or patients with known or suspected pharmacokinetic abnormalities [3]. Non-response at therapeutic doses, uncertain drug adherence, suboptimal tolerability, or pharmacokinetic drug-drug interactions are indications for TDM.

In the latest consensus guideline for therapeutic drug monitoring in neuropsychopharmacology, TDM is strongly recommended for first‐generation AEDs, such as carbamazepine (CBZ) and its metabolite carbamazepine-10,11-epoxide (CBZ-epoxide), phenytoin (PHT), valproic acid (VPA), to identify an individual reference concentration and improve treatment safety and efficacy [3]. In addition, TDMs of second‐generation AEDs were defined as ‘recommended’ for lamotrigine (LTG), oxcarbazepine (OXC) and its metabolite mono-hydroxy-derivative (MHD), and as ‘useful’ for gabapentin (GBP) and topiramate (TPM). Although the recommendation level of TDM for levetiracetam (LEV) decreased from ‘recommended’ [4] to ‘potentially useful’ [3] owing to its predictable dose-serum concentration relationship and broad therapeutic window in general population, TDM for LEV may be beneficial for some populations with altered pharmacokinetics, such as critically ill patients, pregnant women, pediatrics or elderly [5].

AEDs quantification has been performed with several analytical methods, such as immunoassay and high‐performance liquid chromatography (HPLC). However, immunoassays are restricted to single drug detection and prone to cross-reaction between metabolites of the drugs [6, 7], and several antiepileptics, e.g. valproic acid and gabapentin, exhibit no UV absorption [8]. In recent years, liquid chromatography tandem mass spectrometry (LC-MS/MS) has become popular for its high specificity, sensitivity, reliability and simultaneous quantification. Several LC-MS/MS methods were developed for simultaneous determination of AEDs [9]. However, some of them required time‐consuming sample preparation [10], large sample volume [9] or extended run‐time [11, 12]. Some lacked internal standards [12], or had obvious matrix effect for certain AED [13]. Although several LC-MS/MS methods have improved analytical utility in regard to complex sample preparation, long run times and poor sensitivity [14, 15], the stability of AEDs in the whole TDM process was not fully evaluated in these method, such as the whole blood stability before sample preparation and solution stability.

To meet the requirements of physicians to adapt the treatment of AEDs using TDM, in this work, we developed and validated a simple, rapid and robust LC-MS/MS method for the simultaneous quantification of eight antiepileptic drugs and two metabolites in human plasma.

Experimental

Chemicals and materials

CBZ, GBP, LTG, OXC, TPM and VPA were purchased from National Institutes for Food and Drug Control (Beijing, China). CBZ-epoxide, d 6-LEV, d 4-GBP and d 6-VPA were purchased from Toronto Research Chemicals (North York, ON, Canada). LEV, MHD and PHT were purchased from ZZBIO Company (Shanghai, China). HPLC-grade acetonitrile and methanol were purchased from TEDIA (USA). Water was prepared using Elga Purelab Felx 3 water purification system (ELGA, High Wycombe, UK). All other chemicals and solvents were of the analytical grade available. Drug free plasma and blood were donated by Phase I Clinical Laboratory of Tongji Hospital.

LC-MS/MS

LC-20AD HPLC system (Shimadzu, Kyoto, Japan) was comprising a binary pump, a thermostatted autosampler and a thermostatted column compartment. An Ultimate XB-C18 column (5 μm, 50 × 2.1 mm, Welch, China) maintained at 40°C was used for separation. A gradient programme was used with the mobile phase, combining acetonitrile (solvent A) and 2 mM ammonium acetate in water (solvent B) at a flow rate of 0.35 mL min−1 and with a total run time of 5 min as follows: 20% A (0–1.0 min), 30%–50% A (1.01–3.5 min), 50%–90% A (3.51–3.8 min), 20% A (3.81–5 min).

The HPLC system was connected to a QTRAP 5500 mass spectrometer (AB Sciex, Singapore) equipped with an electrospray ionization (ESI) source. The Turbo-Ion-Spray interface was operated in both positive- and negative-ion modes with nitrogen as the nebulizing, turbo spray, and curtain gas, with the optimum values set at 40, 50, and 20 psi, respectively. The turbo-gas temperature was set at 400°C, and the ESI needle voltages in positive- and negative-ion modes were adjusted to 5500V and −4500V, respectively. Instrument control and data processing was carried out by the Analyst 1.6.2 software.

Calibrators, quality control and internal standards preparation

Stock solutions of CBZ, CBZ-epoxide, GBP, LEV, LTG, MHD, OXC, PHT, TPM and VPA were prepared by dissolving these compounds in methanol at 1, 1, 1, 5, 1, 1, 1, 1, 1 and 5 mg mL−1, respectively. The stock solutions were further diluted with methanol to obtain working standard solutions at several concentrations. The calibration curves were obtained using eight calibration standards, i.e., blank plasma samples prepared by the addition of the working solutions to drug-free blank plasma, giving final concentrations of 2, 4, 8, 20, 50, 100, 160 and 200 μg mL−1 for LEV, 10, 20, 40, 100, 250, 500, 800 and 1,000 μg mL−1 for VPA, 0.5, 1, 2, 5, 12.5, 25, 40 and 50 μg mL−1 for CBZ, CBZ-epoxide, GBP, LTG, MHD, PHT and TPM, 0.1, 0.2, 0.4, 1, 2.5, 5, 8 and 10 μg mL−1 for OXC. Calibration curves for AEDs in human plasma were derived from their peak area ratios relative to that of internal standard (IS) using linear regression with 1/x 2 as a weighting factor [9, 14, 15]. Quality control (QC) samples were prepared at four concentration levels (lower limit of quantification, low, medium and high) for final concentrations of 2, 6, 40 and 150 μg mL−1 for LEV, 10, 30, 200 and 750 μg mL−1 for VPA, 0.5, 1.5, 10 and 37.5 μg mL−1 for CBZ, CBZ-epoxide, GBP, LTG, MHD, PHT and TPM, 0.1, 0.3, 2 and 7.5 μg mL−1 for OXC. A mixed stock solution of d 6-LEV, d 4-GBP and d 6-VPA was diluted to 24 μg mL−1, 20 μg mL−1 and 40 μg mL−1 in methanol for routine use as a mixed IS. All prepared plasma samples and stock solutions were stored at −20°C.

Calibration curves were extracted in duplicate by adding the following volumes to 40 μL of blank plasma: 40 μL of the standards, 20 μL of the mixed IS solution and 700 μL of acetonitrile. The standards were vortexed for 30 s and centrifuged for 10 min at 12,000g. An aliquot of 40 μL of the supernatant was transferred to LC vial and diluted with 960 μL of water-acetonitrile (80/20: v/v). A 5 μL aliquot of the diluted supernatant was injected and analyzed by LC-MS/MS.

Patient sample preparation

To a 40 μL of patient plasma sample, 20 μL of the mixed IS solution and 740 μL of acetonitrile were added. The samples were vortexed for 30 s and centrifuged for 10 min at 12,000 g. A 40 μL of the supernatant was transferred to an LC vial and diluted with 960 μL of water-acetonitrile (80/20: v/v). A 5 μL of the diluted supernatant was injected and analyzed by LC-MS/MS. All prepared samples were kept in an autosampler at 4°C before injection.

Method validation

Method validation was carried out according to the latest United States Food and Drug Administration's bioanalytical method validation procedures [16].

Selectivity

Selectivity was evaluated by comparing chromatograms of six different batches of drug free plasma to ensure that no interfering peaks were present at the respective retention times of the analytes at the lower limit of quantification (LLOQ). The visible interferences were tested with blank plasma samples and plasma samples spiked with AEDs at LLOQ.

Carryover

Carryover was assessed by injecting a blank plasma sample after a calibration standard at the upper limit of quantification (ULOQ) for five times. Carryover in the blank sample following the ULOQ should not exceed 20% of LLOQ.

Linearity

Linearity was investigated over the designated concentration range for all analytes. Calibration curves were obtained using eight calibration standards for three different days and processed by weighed (1/x 2) least-squares linear regression analysis.

Accuracy and precision

The accuracy and precision (presented as the coefficient of variation; CV) of the assay were assessed by analyzing QC samples at four different concentrations. Accuracy (%) was determined from the percentage ratio of the measured to nominal concentration (mean of measured/nominal × 100). The CV and accuracy for the intra-day assays were evaluated based on the analysis of five samples. The CV and accuracy for the inter-day assays were assessed at the same concentration and repeated on three different days.

Recovery and matrix effects

Extraction recovery of all analytes, expressed as a percentage, was determined for three QC concentrations (low, medium and high) by comparing peak areas of six extracted spiked samples with six pre-extracted samples.

Besides normal blood sample, matrix effects in the hemolyzed, lipemic, and hyperbilirubin blood samples were also assessed. Matrix effect (MF) was evaluated for each analyte and IS by calculating the ratio of peak area in the presence of matrix (blank matrix spiked after extraction with analyte) to the peak area in the absence of matrix (diluted stock solution) at the same concentration. Six lots of matrix were spiked at three QC concentrations (low, medium and high). IS was added in all samples. The IS-normalized MF was calculated as the ratio of MF of the analyte to the MF of the IS. The CV of the IS-normalized MF should not be higher than 15%.

Stability

In our institution, the whole blood samples were collected at clinic and stored at 4°C immediately, which reached the pharmacological laboratory within a maximum of 24 h following collection. The blood samples were then centrifuged (2,000 g, 4°C for 10 min) and prepared for analysis, the rest of which would be immediately stored at −80°C. Based on the above clinical practice, the stability study was carried on using drug-free matrix (plasma or blood) spiked in triplicate with all AEDs at two different concentrations (low and high). The whole blood stability was determined at 4°C for 24 h compared with freshly spiked blood samples. The bench‐top stability was determined at room temperature for 4 h and compared with freshly spiked plasma samples. Long‐term stability was assessed after storage of QC samples at −80°C for one month. Freeze-thaw stability was determined after three freeze-thaw cycles (−80°C to room temperature). Autosampler stability was determined post treatment after storage at 4°C for 24 h. Solution stability at −20°C for 4 months was also assessed. Analytes were considered stable when the difference of concentration between the fresh sample and the stability testing sample did not exceed 15%.

Clinical application

The clinical application of the present method was evaluated by analyzing plasma taken from 22 patients undergoing mono- or poly-therapy for the treatment of epilepsy, which was approved by the Institutional Review Board of Tongji Hospital (NO. TJ-IRB20211015).

Results and discussion

Chromatography and mass spectrometry

Analytes investigated in this study generated prominent protonated molecular ion [M+H]+ in positive-ionmode or deprotonated molecular ion [M−H] in negative-ion mode. Based on signal intensity and fragmentation pattern, all AEDs were divided into two ionization groups: positive-ion mode for CBZ, CBZ-epoxide, GBP, LEV, LTG, MHD, OXC, and negative-ion mode for PHT, TPM and VPA. The multiple reaction monitoring (MRM) transition and optimized collision-induced dissociation conditions were shown in Table 1.

Table 1.

Multiple reaction monitoring parameters for the AEDs

CompoundIon modePrecursor ion (m/z)Product ion (m/z)DP (V)CE (V)
CBZpositive237.1194.211045
CBZ-epoxidepositive253.2180.26624
GBPpositive172.2154.28020
LEVpositive170.1125.96020
LTGpositive256.1211.19036
MHDpositive255.1194.27045
OXCpositive253.1208.18030
PHTnegative250.8101.8−60−30
TPMnegative338.277.8−100−40
VPAnegative143.1143.1−60−10
d6-LEVpositive177.3132.33220
d4-GBPpositive176.1158.15020
d6-VPAnegative149.0149−60−10

Originally, d 6-LEV was chosen as the IS for all the positive-ion-mode analytes and d 6-VPA for all the negative-ion-mode ones. However, low absolute recovery and obvious matrix inhibition were observed in GBP when d 6-LEV as its IS. Similar matrix inhibition in GBP was reported in another LC-MS/MS method when d 10-PHT was used as IS [13]. Thus, d 4-GBP was introduced as IS for GBP in this study.

Sample preparation and chromatographic conditions were optimized for simple, rapid, and practical quantitative analysis. Acetonitrile was chosen as the protein precipitation solution, which could result in better chromatographic peak shape for GBP than methanol. Simple protein precipitation was sufficient to detect all AEDs within the therapeutic reference range. The large dilution of plasma with acetonitrile minimized matrix effects. Although acid modifiers brought a better chromatographic peak shape for VPA, it also greatly extended the retention time of VPA and significantly lowered the MS detection responses of PHT and OXC. Finally, the combination of acetonitrile and water (2 mM ammonium acetate) resulted in higher ionization efficiency, better chromatographic separation and shorter running time than other combinations.

Among analytes, CBZ-epoxide and OXC had the same precursor ion at m/z 253 and showed a common fragment ion at m/z 182 in their product-ion spectra (Fig. 1A and 1B) [13]. Besides, in-source pyrolysis occurred in MHD [17], which was observed in the MRM channel of CBZ (Fig. 1C and 1D). Therefore, CBZ-epoxide and OXC, as well as CBZ and MHD had to be chromatographically separated to avoid interference. Several commercially available C18 columns were tested to optimize the chromatographic peak shape, peak width, and separation. Finally, Ultimate XB-C18 column (5 μm, 50 × 2.1 mm, Welch, China) was selected owing to its good peak shape, narrow peak width, optimal peak separation and low cost. Under the optimized chromatographic condition, CBZ-epoxide and OXC were successfully separated with retention times of 2.9 and 3.1 min. Meanwhile, CBZ and MHD were successfully separated with retention times of 3.6 and 2.4 min (Fig. 2).

Fig. 1.
Fig. 1.

Product-ion mass spectra of CBZ-epoxide (A), OXC (B), CBZ (C) and MHD (D)

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01024

Fig. 2.
Fig. 2.

Representative chromatograms of blank plasma, blank plasma spiked with LLOQ levels of antiepileptic drugs and the IS, and plasma samples obtained from patients undergoing treatment

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01024

As shown in Table 2, all analytes were separated and analyzed within a 5-min run time. To our best knowledge, this run time is faster than most of the reported LC-MS/MS methods for simultaneous determination of AEDs. Although the run time of a few LC-MS/MS methods are less than 5 min [15], the chromatographic performance of certain AEDs, such as VPA, was not as good as that in this method.

Table 2.

HPLC retention time, calibration range, and determination coefficient for AEDs in this study

CompoundISRetention time (min)Calibration range (ug/mL)r2Therapeutic reference range (μg mL−1)Laboratory alert level (μg mL−1)
CBZd6-LEV3.60.5–500.99344–1220
CBZ-epoxided6-LEV2.90.5–500.9942
GBPd4-GBP0.60.5–500.99722–2025
LEVd6-LEV0.72–2000.993710–4050
LTGd6-LEV2.00.5–500.99913–1520
MHDd6-LEV2.40.5–500.998510–3540
OXCd6-LEV3.10.1–100.9952nana
PHTd6-VPA3.60.5–500.996310–2025
TPMd6-VPA3.20.5–500.99652–1016
VPAd6-VPA1.710–1,0000.997850–100120
d6-LEV-0.7nananana
d4-GBP-0.6nananana
d6-VPA-1.7nananana

Method validation

Selectivity and carryover

As shown in Fig. 2, no interference was observed in drug free plasma samples at the retention times of the target drugs. No carry-over effect was observed in this method (data not shown). Typical chromatograms for blank plasma, plasma spiked with LLOQ of the AEDs, and plasma collected from patients being treated for epilepsy are presented in Fig. 2.

Linearity, accuracy and precision

As shown in Table 2, the calibration ranges for each AED were from 4 to 20 fold lower to 1.4-10 fold higher than the concentration of the reference ranges for TDM [3]. The calibration curves for the drugs were linear over the concentration ranges, with determination coefficients (r 2) higher than 0.99.

The intra- and inter-day variations of target drug determinations in human plasma over the entire concentration ranges are summarized in Table 3. As shown, the intra-day coefficients of variation were between 2% and 8%, and accuracies ranged from 92% to 112%. The inter-day coefficients of variation were between 2% and 9%, and the accuracy was between 92% and 109%.

Table 3.

Accuracy and precision of the LC-MS/MS method for AEDs (n = 5)

CompoundNominal Concentration (μg mL−1)Inter‐dayIntra‐day
Accuracy (%)Precision (%)Accuracy (%)Precision (%)
CBZ0.597.46.3101.23.5
1.5107.24.7109.82.0
10109.34.2111.03.4
37.591.64.294.01.8
CBZ-epoxide0.595.89.095.64.4
1.5106.47.1109.64.2
10106.83.4106.93.7
37.598.22.399.42.2
GBP0.5102.26.497.14.0
1.5101.84.5100.25.5
10101.23.699.32.4
37.597.93.4100.92.5
LEV294.15.599.63.1
6102.42.6102.71.9
40106.22.1104.51.7
15094.32.792.92.2
LTG0.5106.78.7112.32.9
1.5107.66.3105.24.4
10109.13.8106.42.6
37.5101.55.4108.42.2
OXC0.1102.18.8109.17.4
0.3101.64.8100.44.2
2102.83.4103.44.7
7.5103.73.5107.13.1
PHT0.594.88.198.47.5
1.5100.74.0102.43.1
10100.75.593.72.6
37.593.73.591.62.7
MHD0.595.88.8103.82.8
1.598.87.897.12.2
10100.85.795.81.7
37.5101.34.0101.23.0
TPM0.598.04.999.04.4
1.5102.03.7103.64.2
10100.93.696.82.3
37.596.92.097.41.8
VPA1098.84.699.55.2
30100.74.3105.14.6
200101.02.0101.71.5
75095.62.497.41.8

Recovery and matrix effects

As shown in Table 4, the relative extraction recovery of all AEDs was between 96% and 106%. The IS-normalized MF value of all AEDs ranged from 94% to 111% in normal blood sample, from 96% to 107% in hemolyzed blood sample, from 95% to 111% in lipemic blood sample, and from 96% to 112% in hyperbilirubin blood sample. No significant matrix effect was observed.

Table 4.

Extraction recovery and matrix effect for AEDs (n = 6)

CompoundNominal Concentration (μg mL−1)Recovery (%)IS-normalized MF value (%)
Normal sampleHemolyzed sampleLipemic sampleHyperbilirubin sample
CBZ1.5103.4±3.9111.3±3.7102.5±1.3105.1±2.2110.6±3.1
1098.4±4.9108.9±5.5102.0±2.6101.1±1.8103.9±3.7
37.5100.8±2.9107.8±2.2101.1±1.6101.9±2.3102.2±2.4
CBZ-epoxide1.5101.5±5.0108.3±3.0103.8±3.7106.3±2.5110.6±2.6
10100.9±4.2105.7±4.6104.3±2.4105.8±1.6105.4±2.8
37.5100.0±3.2107.8±2.4101.1±1.9102.7±2.1103.3±2.1
GBP1.598.7±2.899.8±2.1101.4±2.4102.6±1.4102.5±2.7
1096.2±4.7102.0±3.7100.4±2.298.1±2.899.2±2.5
37.598.8±2.697.7±2.7100.7±1.9100.2±2.2100.8±2.0
LEV6102.9±2.7101.0±1.197.5±2.0104.5±1.6102.3±2.0
40100.2±1.3101.5±2.0100.4±2.3100.9±1.9103.0±0.8
150100.2±2.6103.1±1.898.9±1.2101.7±2.2102.1±2.3
LTG1.599.4±4.4111.0±3.6101.4±3.3100.3±1.8109.5±2.1
1097.1±3.1109.8±2.9106.4±2.8102.9±2.6105.3±1.5
37.599.9±4.0105.4±2.2101.9±1.5102.0±2.8102.6±2.2
MHD1.599.9±7.3108.8±2.9102.8±4.6110.9±3.1111.2±1.4
10102.0±4.0109.4±3.9106.0±3.6110.6±3.4111.8±2.8
37.5103.3±4.2109.7±2.5101.9±3.1108.9±1.4110.3±1.6
OXC0.3101.2±4.3111.1±0.899.8±4.7107.7±3.5108.1±3.7
2101.4±4.2109.7±4.2107.1±2.2107.7±1.5110.6±1.0
7.5104.4±4.8107.0±2.1102.0±3.7105.1±2.6104.8±1.3
PHT1.5103.9±4.894.1±3.098.6±5.1100.1±5.596.0±3.6
10102.8±4.695.3±4.299.1±1.694.6±1.2100.8±2.9
37.5102.0±4.399.2±2.197.3±2.195.0±1.7100.13±3.1
TPM1.5103.0±4.8100.4±3.597.1±4.797.8±2.296.7±3.0
10106.4±1.596.1±1.4101.4±2.395.4±0.999.6±1.9
37.5104.2±3.3100.2±2.696.8±1.498.2±2.2101.1±2.1
VPA3098.9±2.998.8±1.895.76±3.898.8±2.5100.6±1.6
20095.8±1.197.0±2.199.6±0.597.2±1.797.8±1.2
75097.2±1.899.3±1.697.9±1.896.2±0.995.9±1.1

Stability

Based on the clinical practice in our institution, we evaluated the whole blood stability at 4°C for 24 h, the bench‐top stability at room temperature for 4h, the long‐term stability at −80°C for one month, the freeze-thaw stability after three cycles (−80°C to room temperature), the autosampler stability after storage at 4°C for 24 h, and the solution stability at −20°C for 4 months.

As shown in Table 5, no significant degradation of AEDs occurred in whole blood sample after storage at 4°C for 24 h. Besides, no significant degradation of each analyte was observed in human plasma after short-term storage at room temperature for 4h, long-term stability for one month at −80°C, three freeze-thaw cycles, post-treatment storage in autosampler for 24 h and solution stability for 4 month at −20°C, within ±15% deviation from the nominal concentrations.

Table 5.

Stability results of all AEDs under different conditions (n=3)

CompoundNominal Concentration (μg mL−1)Accuracy (%)
Blood at 4°C for 24 hPlasma at room temperature for 4 hPost-treatment in autosampler for 24 hThree freeze-thaw cyclesSolution stability at −20°C for 4 monthsPlasma after storage at −80°C for 1 month
CBZ1.591.4±3.095.9±1.793.8±1.9109.1±2.1101.0±2.695.2±3.6
37.595.6±3.4103.4±1.899.7±2.891.4±1.4111.4±1.7111.4±1.3
CBZ-epoxide1.593.3±4.099.3±4.198.0±10.4104.0±1.696.6±2.392.6±5.0
37.599.9±3.9101.7±1.5101.2±2.389.1±1.2107.2±2.098.8±1.6
GBP1.590.3±2.199.8±4.2100.1±3.5106.3±3.2107.2±4.187.7±2.3
37.588.9±2.192.3±0.6105.9±2.897.4±1.8104.0±2.097.4±1.6
LEV693.5±1.599.0±1.194.2±4.0103.9±2.9104.4±1.699.5±1.8
15096.5±4.196.5±1.197.6±0.893.0±1.7100.0±1.496.1±1.8
LTG1.590.9±1.8114.1±0.799.2±5.896.3±2.6101.2±2.4100.3±1.8
37.593.0±4.8106.1±2.099.4±3.086.6±1.2102.8±1.4110.6±3.0
MHD1.595.3±2.9108.5±2.497.4±3.8110.7±2.0103.8±2.686.2±1.0
37.590.9±2.6110.1±1.799.5±2.0107.2±4.1112.8±2.087.4±2.2
OXC1.590.8±2.489.4±2.590.8±1.895.0±1.391.3±6.193.2±5.3
37.591.8±4.2103.5±0.998.4±1.888.7±3.0113.0±1.9101.0±3.3
PHT0.3102.8±2.7101.7±4.296.4±5.8104.3±7.4102.8±6.399.4±5.1
7.5102.3±4.892.6±3.093.8±1.6102.8±2.4106.4±3.1103.0±3.8
TPM1.5106.8±4.698.8±4.093.9±5.7111.8±2.6107.0±4.397.7±3.6
37.5102.8±4.294.6±0.997.3±2.2105.7±2.4103.7±2.596.0±3.0
VPA3090.6±2.696.2±5.7104.1±7.9107.2±2.490.3±4.486.2±1.0
750101.0±4.488.8±0.899.4±2.395.8±0.992.4±2.197.4±1.6

It was reported that OXC was not stable at room temperature for up to 24 h in the whole blood, serum and plasma, which showed a loss of about 40% of the nominal concentrations [11]. However, in some other studies, OXC was stable in plasma at room temperature for 4 h and 6 h [13, 18], which was consistent with the results in our method.

Clinical application

The validated method was applied to drug monitoring of AEDs in plasma collected from 22 patients of epilepsy, and the estimated concentrations of the drugs are given in Table 6. Among 22 patients, two drugs were monitored in 13 of the 22 patients. VPA was detected in 18 patients with concentrations ranging from <10 to 94 μg mL−1, 10 below the therapeutic reference range. Besides, LEV was detected in 11 patients with concentrations ranging from <2 to 35 μg mL−1, 8 below the therapeutic reference range.

Table 6.

AEDs levels measured by the validated LC-MS/MS

PatientNumber of AEDs detectedConcentrations of AEDs (μg mL−1)
Patient 12LEV (7.1), VPA(12.0)
Patient 21VPA (94.3)
Patient 32LEV (7.4), VPA (33.8)
Patient 42LEV (6.6), VPA (13.9)
Patient 52LEV (7.8), VPA (10.2)
Patient 62LEV (6.3), VPA (33.7)
Patient 71VPA (28.2)
Patient 82MHD (24.1), OXC (0.2), VPA (72.1)
Patient 91VPA (86.6)
Patient 102LEV (<2), VPA (49.9)
Patient 112LEV (24.3), VPA (54.1)
Patient 122LEV (15.2), VPA (51.9)
Patient 132CBZ (6.0), CBZ-epoxide (1.4), VPA (51.6)
Patient 142LEV (7.8), VPA (42.4)
Patient 152LEV (8.5), VPA (<10)
Patient 161VPA (13.7)
Patient 172LEV (35.0), VPA (53.5)
Patient 181VPA (81.4)
Patient 191GBP (5.4)
Patient 201LTG (13.0)
Patient 211PHT (13.7)
Patient 221TPM (13.3)

Conclusions

A LC-MS/MS method for simultaneous quantification of eight AEDs (CBZ, GBP, LEV, LTG, OXC, PHT, TPM and VPA) and two active metabolite (CBZ-epoxide, MHD) in human plasma was developed with acceptable accuracy and precision. This method is rapid (5 min per sample) compared to most of previously reported simultaneous quantitation methods for monitoring AEDs. The quantitation can be performed using small volumes of human plasma (40 μL), allowing efficient use of limited blood samples. The method was fully validated and successfully applied to the identification and quantitation of plasma samples from patients receiving AEDs treatment, which would be useful for TDM of co-administered epileptic drugs in plasma as well as pharmacokinetic studies of these drugs.

Acknowledgements

This work was supported by the National Natural Science Foundation of China under Grant No. 21572073 and the National Major Scientific and Technological Special Project for Significant New Drugs Development under Grant No. 2017ZX09304022.

References

  • 1.

    Cendes, F. Lancet Neurol. 2021, 20, 333334.

  • 2.

    Wang, Y. ; Chen, Z. Pharmacol. Ther. 2019, 201, 7793.

  • 3.

    Hiemke, C. ; Bergemann, N. ; Clement, H. W. ; Conca, A. ; Deckert, J. ; Domschke, K. ; Eckermann, G. ; Egberts, K. ; Gerlach, M. ; Greiner, C. ; Grunder, G. ; Haen, E. ; Havemann-Reinecke, U. ; Hefner, G. ; Helmer, R. ; Janssen, G. ; Jaquenoud, E. ; Laux, G. ; Messer, T. ; Mossner, R. ; Muller, M. J. ; Paulzen, M. ; Pfuhlmann, B. ; Riederer, P. ; Saria, A. ; Schoppek, B. ; Schoretsanitis, G. ; Schwarz, M. ; Gracia, M. S. ; Stegmann, B. ; Steimer, W. ; Stingl, J. C. ; Uhr, M. ; Ulrich, S. ; Unterecker, S. ; Waschgler, R. ; Zernig, G. ; Zurek, G. ; Baumann, P. Pharmacopsychiatry 2018, 51, 962.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Hiemke, C. ; Baumann, P. ; Bergemann, N. ; Conca, A. ; Dietmaier, O. ; Egberts, K. ; Fric, M. ; Gerlach, M. ; Greiner, C. ; Grunder, G. ; Haen, E. ; Havemann-Reinecke, U. ; Jaquenoud, S. E. ; Kirchherr, H. ; Laux, G. ; Lutz, U. C. ; Messer, T. ; Muller, M. J. ; Pfuhlmann, B. ; Rambeck, B. ; Riederer, P. ; Schoppek, B. ; Stingl, J. ; Uhr, M. ; Ulrich, S. ; Waschgler, R. ; Zernig, G. Pharmacopsychiatry 2011, 44, 195235.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Jarvie, D. ; Mahmoud, S. H. J. Pharm. Pharm. Sci. 2018, 21, 149s176s.

  • 6.

    Dasgupta, A. ; McNeese, C. ; Wells, A. Am. J. Clin. Pathol. 2004, 121, 418425.

  • 7.

    McMillin, G. A. ; Juenke, J. M. ; Tso, G. ; Dasgupta, A. Am. J. Clin. Pathol. 2010, 133, 728736.

  • 8.

    Juenke, J. M. ; Brown, P. I. ; Johnson-Davis, K. L. ; McMillin, G. A. Ther. Drug Monit. 2011, 33, 209213.

  • 9.

    Yin, L. ; Wang, T. ; Shi, M. ; Zhang, Y. ; Zhao, X. ; Yang, Y. ; Gu, J. J. Sep. Sci. 2016, 39, 964972.

  • 10.

    Subramanian, M. ; Birnbaum, A. K. ; Remmel, R. P. Ther. Drug Monit. 2008, 30, 347356.

  • 11.

    Deeb, S. ; McKeown, D. A. ; Torrance, H. J. ; Wylie, F. M. ; Logan, B. K. ; Scott, K. S. J. Anal. Toxicol. 2014, 38, 485494.

  • 12.

    Shibata, M. ; Hashi, S. ; Nakanishi, H. ; Masuda, S. ; Katsura, T. ; Yano, I. Biomed. Chromatogr. 2012, 26, 15191528.

  • 13.

    Kim, K. B. ; Seo, K. A. ; Kim, S. E. ; Bae, S. K. ; Kim, D. H. ; Shin, J. G. J. Pharm. Biomed. Anal. 2011, 56, 771777.

  • 14.

    Liu, T. ; Kotha, R. R. ; Jones, J. W. ; Polli, J. E. ; Kane, M. A. J. Pharm. Biomed. Anal. 2019, 176, 112816.

  • 15.

    Qi, Y. ; Liu, G. Biomed. Chromatogr. 2021, 35, e5090.

  • 16.

    FDA. Bioanalytical Method Validation Guidance for Industry, 2018.

  • 17.

    Corso, G. ; D'Apolito, O. ; Paglia, G. Rapid Commun. Mass Spectrom. 2007, 21, 269272.

  • 18.

    Dupouey, J. ; Doudka, N. ; Belo, S. ; Blin, O. ; Guilhaumou, R. Biomed. Chromatogr. 2016, 30, 20532060.

  • 1.

    Cendes, F. Lancet Neurol. 2021, 20, 333334.

  • 2.

    Wang, Y. ; Chen, Z. Pharmacol. Ther. 2019, 201, 7793.

  • 3.

    Hiemke, C. ; Bergemann, N. ; Clement, H. W. ; Conca, A. ; Deckert, J. ; Domschke, K. ; Eckermann, G. ; Egberts, K. ; Gerlach, M. ; Greiner, C. ; Grunder, G. ; Haen, E. ; Havemann-Reinecke, U. ; Hefner, G. ; Helmer, R. ; Janssen, G. ; Jaquenoud, E. ; Laux, G. ; Messer, T. ; Mossner, R. ; Muller, M. J. ; Paulzen, M. ; Pfuhlmann, B. ; Riederer, P. ; Saria, A. ; Schoppek, B. ; Schoretsanitis, G. ; Schwarz, M. ; Gracia, M. S. ; Stegmann, B. ; Steimer, W. ; Stingl, J. C. ; Uhr, M. ; Ulrich, S. ; Unterecker, S. ; Waschgler, R. ; Zernig, G. ; Zurek, G. ; Baumann, P. Pharmacopsychiatry 2018, 51, 962.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Hiemke, C. ; Baumann, P. ; Bergemann, N. ; Conca, A. ; Dietmaier, O. ; Egberts, K. ; Fric, M. ; Gerlach, M. ; Greiner, C. ; Grunder, G. ; Haen, E. ; Havemann-Reinecke, U. ; Jaquenoud, S. E. ; Kirchherr, H. ; Laux, G. ; Lutz, U. C. ; Messer, T. ; Muller, M. J. ; Pfuhlmann, B. ; Rambeck, B. ; Riederer, P. ; Schoppek, B. ; Stingl, J. ; Uhr, M. ; Ulrich, S. ; Waschgler, R. ; Zernig, G. Pharmacopsychiatry 2011, 44, 195235.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Jarvie, D. ; Mahmoud, S. H. J. Pharm. Pharm. Sci. 2018, 21, 149s176s.

  • 6.

    Dasgupta, A. ; McNeese, C. ; Wells, A. Am. J. Clin. Pathol. 2004, 121, 418425.

  • 7.

    McMillin, G. A. ; Juenke, J. M. ; Tso, G. ; Dasgupta, A. Am. J. Clin. Pathol. 2010, 133, 728736.

  • 8.

    Juenke, J. M. ; Brown, P. I. ; Johnson-Davis, K. L. ; McMillin, G. A. Ther. Drug Monit. 2011, 33, 209213.

  • 9.

    Yin, L. ; Wang, T. ; Shi, M. ; Zhang, Y. ; Zhao, X. ; Yang, Y. ; Gu, J. J. Sep. Sci. 2016, 39, 964972.

  • 10.

    Subramanian, M. ; Birnbaum, A. K. ; Remmel, R. P. Ther. Drug Monit. 2008, 30, 347356.

  • 11.

    Deeb, S. ; McKeown, D. A. ; Torrance, H. J. ; Wylie, F. M. ; Logan, B. K. ; Scott, K. S. J. Anal. Toxicol. 2014, 38, 485494.

  • 12.

    Shibata, M. ; Hashi, S. ; Nakanishi, H. ; Masuda, S. ; Katsura, T. ; Yano, I. Biomed. Chromatogr. 2012, 26, 15191528.

  • 13.

    Kim, K. B. ; Seo, K. A. ; Kim, S. E. ; Bae, S. K. ; Kim, D. H. ; Shin, J. G. J. Pharm. Biomed. Anal. 2011, 56, 771777.

  • 14.

    Liu, T. ; Kotha, R. R. ; Jones, J. W. ; Polli, J. E. ; Kane, M. A. J. Pharm. Biomed. Anal. 2019, 176, 112816.

  • 15.

    Qi, Y. ; Liu, G. Biomed. Chromatogr. 2021, 35, e5090.

  • 16.

    FDA. Bioanalytical Method Validation Guidance for Industry, 2018.

  • 17.

    Corso, G. ; D'Apolito, O. ; Paglia, G. Rapid Commun. Mass Spectrom. 2007, 21, 269272.

  • 18.

    Dupouey, J. ; Doudka, N. ; Belo, S. ; Blin, O. ; Guilhaumou, R. Biomed. Chromatogr. 2016, 30, 20532060.

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Senior editors

Editor(s)-in-Chief: Kowalska, Teresa

Editor(s)-in-Chief: Sajewicz, Mieczyslaw

Editors(s)

  • Danica Agbaba (University of Belgrade, Belgrade, Serbia)
  • Ivana Stanimirova-Daszykowska (University of Silesia, Katowice, Poland)
  • Monika Waksmundzka-Hajnos (Medical University of Lublin, Lublin, Poland)

Editorial Board

  • R. Bhushan (The Indian Institute of Technology, Roorkee, India)
  • J. Bojarski (Jagiellonian University, Kraków, Poland)
  • B. Chankvetadze (State University of Tbilisi, Tbilisi, Georgia)
  • M. Daszykowski (University of Silesia, Katowice, Poland)
  • T.H. Dzido (Medical University of Lublin, Lublin, Poland)
  • A. Felinger (University of Pécs, Pécs, Hungary)
  • K. Glowniak (Medical University of Lublin, Lublin, Poland)
  • B. Glód (Siedlce University of Natural Sciences and Humanities, Siedlce, Poland)
  • A. Gumieniczek (Medical University of Lublin, Lublin, Poland)
  • U. Hubicka (Jagiellonian University, Kraków, Poland)
  • K. Kaczmarski (Rzeszow University of Technology, Rzeszów, Poland)
  • H. Kalász (Semmelweis University, Budapest, Hungary)
  • K. Karljiković Rajić (University of Belgrade, Belgrade, Serbia)
  • I. Klebovich (Semmelweis University, Budapest, Hungary)
  • A. Koch (Private Pharmacy, Hamburg, Germany)
  • Ł. Komsta (Medical University of Lublin, Lublin, Poland)
  • P. Kus (Univerity of Silesia, Katowice, Poland)
  • D. Mangelings (Free University of Brussels, Brussels, Belgium)
  • E. Mincsovics (Corvinus University of Budapest, Budapest, Hungary)
  • G. Morlock (Giessen University, Giessen, Germany)
  • A. Petruczynik (Medical University of Lublin, Lublin, Poland)
  • R. Skibiński (Medical University of Lublin, Lublin, Poland)
  • B. Spangenberg (Offenburg University of Applied Sciences, Germany)
  • T. Tuzimski (Medical University of Lublin, Lublin, Poland)
  • Y. Vander Heyden (Free University of Brussels, Brussels, Belgium)
  • A. Voelkel (Poznań University of Technology, Poznań, Poland)
  • B. Walczak (University of Silesia, Katowice, Poland)
  • W. Wasiak (Adam Mickiewicz University, Poznań, Poland)
  • I.G. Zenkevich (St. Petersburg State University, St. Petersburg, Russian Federation)

 

KOWALSKA, TERESA
E-mail: kowalska@us.edu.pl

SAJEWICZ, MIECZYSLAW
E-mail:msajewic@us.edu.pl

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