MS method for simultaneous determination of carbamazepine and its seven major metabolites in serum of epileptic patients

Accepted Manuscript UHPLC-MS/MS method for simultaneous determination of carbamazepine and its seven major metabolites in serum of epileptic patients

Wei Jiang, Tianyi Xia, Yunlei Yun, Mingming Li, Feng Zhang, Shouhong Gao, Wansheng Chen PII: DOI: Reference:

S1570-0232(17)31585-4 https://doi.org/10.1016/j.jchromb.2018.12.016 CHROMB 21467

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

11 September 2017 9 November 2018 13 December 2018

Please cite this article as: Wei Jiang, Tianyi Xia, Yunlei Yun, Mingming Li, Feng Zhang, Shouhong Gao, Wansheng Chen , UHPLC-MS/MS method for simultaneous determination of carbamazepine and its seven major metabolites in serum of epileptic patients. Chromb (2018), https://doi.org/10.1016/j.jchromb.2018.12.016

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ACCEPTED MANUSCRIPT UHPLC-MS/MS method for simultaneous determination of carbamazepine and its seven major metabolites in serum of epileptic patients Wei Jiang1, Tianyi Xia1, Yunlei Yun, Mingming Li, Feng Zhang, Shouhong Gao*,

authors contributed equally to this work.

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1These

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Wansheng Chen*

Affiliation:

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Department of Pharmacy, Changzheng Hospital, Second Military Medical University,

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Shanghai 200003, P. R. China

*Corresponding Author:

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Associate professor Shouhong Gao, Department of Pharmacy, Changzheng Hospital,

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Second Military Medical University, No. 415, Fengyang Road, Shanghai 200003, China

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Tel.: +86–21–60748783, fax: +86–21–60748767.

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E–mail: [email protected]

Professor Wansheng Chen, Department of Pharmacy, Changzheng Hospital, Second Military Medical University, No. 415, Fengyang Road, Shanghai 200003, China Tel.: +86-21-81886181, fax: +86-21-33100038. E-mail address: [email protected]

ACCEPTED MANUSCRIPT

Abstract: Carbamazepine (CBZ) was considered as the drug of choice in the treatment for various forms of epilepsy, yet the non-negligible adverse effects of CBZ suspend as the major concern for rational and efficient clinical medication. This study developed a method

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for the simultaneous determination of CBZ and its seven metabolites acridine (AI),

carbamazepine

(CBZ-DiOH),

2-

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dihydro-10,11-trans-dihy-droxy-

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iminostilbene (IM), acridone (AO), carbamazepine-10,11-epoxide (CBZ-EP), 10,11-

hydroxycarbamazepine (2-OH-CBZ) and 3-hydroxycarbamazepine (3-OH-CBZ) with

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phenacetin as the internal standard (IS) using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Plasma samples were

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purified with the one-step 96-well protein precipitation plate. Chromatographic separation was achieved on an Agilent Eclipse XDB-C18 (1.8μm, 2.1 mm×50 mm)

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chromatography column carrying the mobile phrase of acetonitrile and 0.1% formic

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acid in a gradient elution at a flow rate 0.3 mL/min. Agilent G6460 MS/MS in the positive MRM mode using electrospray ionization (ESI) was applied for quantification

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of target compounds. CBZ and its seven metabolites showed good linear correlation coefficient (r>0.990). Intra-day and inter-day precision (CV) were no more than 15%.

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This method was successfully accomplished within 5 min which was especially applicable for therapeutic drug monitoring (TDM) of CBZ and its metabolites in epileptic patients, and it provided insights for toxicological studies to achieve a rational and effective individualized administration in clinical practice.

Key words: Epilepsy, Carbamazepine, Metabolites, UHPLC-MS/MS; Serum

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1. Introduction Carbamazepine (5H-dibenzo[b, f]azepine-5-carbox-amide, CBZ), as one of the prevalently used anti-epileptic drugs (AEDs), has become frequently prescribed in monotherapy or polytherapy for the treatment of epileptic disorders since it was

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introduced onto the market during the Nineties [1-3]. It is mainly used for partial and

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generalized tonic-clonic epileptic seizures as well as trigeminal neuralgia and

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psychiatric disorders [4-8]. The daily dose of CBZ pre os for epilepsy patients is set at 4-12 µg/mL examined through fluorescence polarization immunoassay (FPIA) in

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clinical practice [9-11], which is considered to be safe to obtain satisfactory therapeutic responses [12].

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Although therapeutic drug monitoring (TDM) on epileptic patients is widely applied in clinical practice for appropriate CBZ medication, taking account of the varied

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pharmacokinetic and pharmacodynamics profiles (PK/PD) due to microsomal-enzyme

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inducing effects or drug-drug interactions of CBZ [13-16], adverse effects, however, come along with its therapeutic actions including diplopia, ataxia, somnolence etc. The

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non-negligible adverse effects suspend as the major concern for rational and in-depth clinical applications [17-19], and the underlying mechanism of various CBZ-toxic

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effects remained unsolved. Taking an overview of the PK/PD profiles, CBZ concentration decreased by hepatic metabolism through the cytochrome P450 system especially CYP 3A4 and CYP 2C8 subtypes after oral administration [20, 21]. Among the metabolites that have been verified, the most important secondary metabolite is the pharmacologically active compound carbamazepine-10,11-epoxide (CBZ-EP) and its subsequent metabolite 10,11-dihydro-10,11-trans-dihy-droxy-carbamazepine

(CBZ-DiOH).

This

major

ACCEPTED MANUSCRIPT pathway catalyzed by epoxide hydrolases accounts for more than half the total metabolism [22, 23]. The other two metabolic routes in liver are the formation of 2hydroxycarbamazepine (2-OH-CBZ) and 3-hydroxycarbamazepine (3-OH-CBZ), and the production of iminostilbene (IM) [24-28]. Besides liver as the major site of the drug metabolism, the leucocytes, along with its immune functions, have been involved in the

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oxidation of CBZ [29]. The myeloperoxidase takes a great part in the formation of a

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series of products of CBZ like acridine (AI) and acridone (AO) (structures of the

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metabolites see Fig.1). Seemingly CBZ-EP has attracted most attention for its anticonvulsant activity and toxic effects. However, evidence has been proposed that

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adverse effects still happen even without the presence of CBZ-EP [30]. To explore for the toxicological candidates of CBZ for rational clinical intervention, it was highly

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advisable to outline the metabolomics profiles of CBZ and its major metabolites. So, establishing an appropriate analytical method for efficient and accurate quantification

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was highlighted as a priority [31].

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Advanced analytical methods based on chromatographic seperation with satisfactory sensitivity and selectivity gained an advantage over routine immunological methods to

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reduce false positive results [32]. In recent years, liquid chromatography (LC) has attracted more attention than gas spectrometry (GC), since the former common method

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in determination of CBZ with tedious derivatization procedures [33, 34]. A number of LC methods with ultra-violet (UV) and mass spectrometry (MS) have been published for determination of CBZ in human liquids（plasma, urine, cerebrospinal fluid and milk serum) [35-38]. Most of them focus on the concentration of the two main metabolites CBZ-EP and CBZ-DiOH，just occasionally together with other minor ones, and these studies recruited only healthy volunteers or a few epileptic patients. Although Hélène

ACCEPTED MANUSCRIPT Breton has established a method to simultaneously quantify eight metabolites of CBZ [3], the tedious analyzing time for 50 min can’t meet the need for timely TDM results. To carry out toxicological studies for efficient clinical management of CBZ medication with better efficacy and less adverse effects, the present study aimed to establish an ultra-high-performance liquid chromatography-tandem mass spectrometry

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(UHPLC-MS/MS) method for the simultaneous determination of CBZ and its seven

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metabolites. The 96-well protein precipitation (PPT) plate was applied for sample

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preparation instead of solid-phase extraction (SPE) and liquid-liquid extraction (LLE) with spared time and high output [39, 40], and the well-validated method was furtherly

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applied on authentic plasma samples of 95 epileptic patients enrolled from Shanghai

2. Experimental 2.1 Chemicals and reagents

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Changzheng Hospital for preliminary applications.

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Standard references of CBZ and phenacetin (IS) (purity of both>99.0%) were

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provided by the National Institutes for Food and Drug Control (Beijing, China). CBZEP, IM and AI were offered by SIGMA-ALDRICH (St. Louis, MO, USA). CBZ-DiOH,

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2-OH-CBZ, 3-OH-CBZ and AO were supplied by Toronto Research Chemicals Inc (Toronto, Canada). Methanol and acetonitrile of analytical grade were purchased from

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Merck Company (Darmstadt, Germany), and formic acid was supplied by Tedia Company Inc. (Fairfield, USA). Ultrapure water was produced with a Milli-Q Reagent Water System (Millipore, MA, USA) and Ostro 96-well protein precipitation plates were obtained from Waters company (Waters, Zellik, Belgium).

2.2 Instrumentation An Agilent 1200 series high-performance liquid chromatography interfaced to an

ACCEPTED MANUSCRIPT Agilent 6460 triple-quadrupole mass spectrometer (Agilent Corporation, MA, USA) was applied for UHPLC-MS/MS analysis. Data acquisition and analyzing progress was achieved by means of Agilent 6460 Quantitative Analysis Version B.01.02 analyst data processing software (Agilent Corporation, MA, USA).

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2.3 Chromatographic and mass spectrometric conditions

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The target compounds were separated on an Agilent 1200 series ultra-highperformance liquid chromatograph (UHPLC). The mobile phase was acetonitrile (A)

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and 0.1% formic acid in water (B) following gradient elution: 15%A→30%A at 0-3.0

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min, 30%A→95%A at 3.0-5.0 min. Agilent Eclipse XDB-C18 column (1.8μm, 2.1mm×50 mm) was instrumented for liquid chromatographic separation with the

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column temperature maintained at 25 °C and the injection volume set at 2μl. Overall run time for each injection was 5 min.

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Quantification was achieved in the positive multiple reaction monitoring (MRM)

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mode. Parameters were set as follows: drying gas temperature, 325 °C; drying gas flow, 10 l/min; nebulizer pressure, 40 psi. Compound-dependent parameters in mass

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spectrometry analysis were set as listed in Table S1.

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2.4 Analytical procedures 2.4.1 Preparation of standard and quality control (QC) solutions The stock solution of CBZ, AI, IM, AO, CBZ-EP, CBZ-DiOH, 2-OH-CBZ, 3-OHCBZ and the IS were individually prepared by dissolving the accurately weighed standards into a final concentration of 1 mg/ml for each analysis. The stock solutions were further diluted with 10% methanol to produce working solutions at concentrations of 500, 1000, 2000, 5000, 10000, 20000, 50000 ng/ml for CBZ; 100, 200, 500, 1000,

ACCEPTED MANUSCRIPT 2000, 5000, 10000 ng/ml for AI; 1000, 2000, 5000, 10000, 20000, 50000, 100000 ng/ml for IM; 2, 5, 10, 20, 50, 100, 200 ng/ml for AO; 500, 1000, 2000, 5000, 10000, 20000, 50000 ng/ml for CBZ-EP; 1000, 2000, 5000, 10000, 20000, 50000, 100000 ng/ml for CBZ-DiOH; 100, 200, 500, 1000, 2000, 5000, 10000 ng/ml for 2-OH-CBZ ; 100, 200, 500, 1000, 2000, 5000, 10000 ng/ml for 3-OH-CBZ and 1000 ng/ml for IS respectively.

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Calibration standards were prepared by a 1:9 dilution of the corresponding working

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solution with drug-free human plasma to obtain a final concentration of 50-5000 ng/ml

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for CBZ, 10-1000 ng/ml for AI, 100-1000 ng/ml for IM, 0.2-20 ng/ml for AO, 50-5000 ng/ml for CBZ-EP, 100-10000 ng/ml for CBZ-DiOH and 10-1000 ng/ml for 2-OH-CBZ

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and 3-OH-CBZ. The volume of plasma added was of the same amount to get an equal proportion to the total volume for constant matrix effect.

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Quality control (QC) samples were prepared in the same way using fresh stock solutions to get the concentrations of 100, 500, 2000 ng/ml for CBZ; 20, 100, 500 ng/ml

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for AI; 200,1000,5000 ng/ml for IM; 0.5, 2, 10 ng/ml for AO; 100, 500, 2000 ng/ml for

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CBZ-EP; 2000, 1000, 5000 ng/ml for CBZ-DiOH; 20, 100, 500 ng/ml for 2-OH-CBZ and 3-OH-CBZ representing the low, medium, high concentrations. It was the results

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of the QC samples (coefficient of variation, CV 20%) that provided scale of the veracity of the acceptance or ignorance of each analytical run. Both the calibration

days.

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standards and the QC samples were freshly prepared in the three consecutive validation

2.4.2 Sample preparation Sample pre-treatment was achieved by protein precipitation. To achieve efficient protein and phospholipid removal, the protein precipitation procedure was achieved on Ostro 96-well protein precipitation plate. 100μl plasma was loaded together with 20μl IS (100 ng/ml) and 300μl acetonitrile. After thoroughly pipetting and mixing the

ACCEPTED MANUSCRIPT content for 3 times, the plate was placed in a positive pressure device (Agilent, USA) at 60 psi for 5 min for separation of the supernatant and the precipitate, after which the eluate was transferred to a 1.5 ml micro-centrifuge tube drying into concentrate by a gentle stream of nitrogen at 45 °C. The residue was re-constituted by 100μl initial mobile phase followed by vortex mixing for 1.0 min and centrifugation at 2000×g for

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10 min. A 10μl aliquot of supernatant was injected into UHPLC-MS/MS system for

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analysis.

2.5 Method validation

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Full validation of the method was performed according to the recommendations published by FDA.

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2.5.1 Specificity

The specificity of the method was investigated by comparing the chromatography of

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six different batches of drug-free human serum and the QC samples (n=5) to determine

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whether the endogenous substances would be co-eluted with the target compounds which might lead to interference. Satisfactory specificity was proved to be a requisite

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of a reliable and reproducible method. 2.5.2 Lower limit of quantification (LLOQ) and linearity

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LLOQ was defined as the lowest concentration point on standard curve at which the response should be more than 10 times the blank biological matrix interferences response value (S/N ≥10), and the precision expressed as relative error (RE) for accuracy should be ≤ ±20%. Calibrations curves were obtained by plotting the peak area ratios of analyte/internal standard versus the analyte concentrations in spiked serum with 1/x2 weighted leastsquares linear regression analysis. The criteria for acceptance were a correlation

ACCEPTED MANUSCRIPT coefficient (r) no less than 0.99, and the concentration of each analyte must be within ±15% deviation from the spiked value (±20% at the LLOQ). 2.5.3 Matrix effects and extraction recovery Matrix effects were caused by endogenous substances like phospholipids and cholesterol in biological samples. These endogenous substances competed with target

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compounds in the gasification process resulting in the decrease or enhancement of

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ionization efficiency, as well as the chromatographic response especially in samples

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after protein precipitation procedure. To evaluate matrix effects and extraction recovery, the matrix was obtained by processing the blank plasma according to the pre-treatment

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procedures. samples spiked after extraction were prepared in triplicates by spiking standard solution in matrix. The ratios of peak areas of samples spiked after extraction

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to those of the neat standards (diluted with water) at corresponding concentrations were defined as the absolute matrix effect.

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Extraction recoveries of CBZ and its seven metabolites were determined by

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comparing the peak area of the QC samples with that of the samples spiked after extraction. Extraction recovery of the IS was examined using the same method.

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2.5.4 Precision and accuracy

Intra-day precision (RE) was obtained by comparing the peak area ratio of five

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replicates of QC samples at three levels to the IS in one day, and inter-day precision was carried out through five replicates of three batches in a series of three days. Accuracy (RE) was determined as the percentage bias from the nominal value except at LLOQ, where it should not deviate by more than 20%, and precision was expressed as (CV) with acceptable ranges within ±15% except for the LLOQ, where it should not exceed 20% of the CV. 2.5.5 Stability

ACCEPTED MANUSCRIPT The stability of target compounds in plasma was assessed by five replicates of QC samples at three levels stored for 6 h in ambient atmosphere for bench-top stability, 3 months at -80 °C for long-term stability, 3 freeze-thaw cycles at -20 °C and in the autosampler (4 °C) for 24 h for processed sample stability. Concentrations of the target

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compounds after storage were compared with those of freshly prepared samples.

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2.6 Application of the analytical method

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95 epileptic patients administrating CBZ with a median age of 35.1 (range 18-65) and a mean body weight of 61.3 kg (range 52.6-71.9 kg) from Shanghai Changzheng

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Hospital were enrolled in the experiment. All the experimental procedures were reviewed and approved by the Ethical Committee of Changzheng Hospital, and were

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constructed in accordance with Declaration of Helsinki. Blood samples from epileptic patients were collected at 8 a.m. in steady state of plasma concentration after oral

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administration of CBZ (two weeks after first medication or one week after dose

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adjustment) without co-administration of other antiepileptic drugs (phenobarbital, lamotrigine, clonazepam, phenytoin, or olanzapine). Plasma samples were obtained by

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pipetting the blood sample into coagulation cubes before centrifugation at 3000×g for

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10 min. The upper plasma was aliquoted and frozen at -80 °C till analysis.

3. Results and discussion 3.1 LC-MS/MS optimization Choosing MS/MS as a detector precursor ions and product ions could be selectively detected (shown in Fig.2). Full scan and product ion spectra of the target compounds were fully scrutinized finding that positive mode showed higher sensitivities than negative mode. In the positive ESI mode, the richest response was chosen after the

ACCEPTED MANUSCRIPT optimization of the fragmentor energy (F) and collision energy (CE). For detection of CBZ and its metabolites with possibly the same product ions in serum, an Agilent Eclipse XDB-C18 column (1.8μm, 2.1 mm×50 mm) of higher efficiency was applied for better separation efficiency. A proper concentration of formic acid was added to the mobile phase which can enhance the abundance of the stable +

ions and improve chromatographic behaviors. After injection, the mobile

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[M+H]

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phase was not piped into spectrometer for analysis until 1.0 min to spare voltage life

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and reduce interference. Additionally, to avoid the “add-up” effects of the matrix caused mainly by lipids like phosphatidylcholine and lysophosphatidylcholine, proper elution

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was needed in the analyzing process.

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with high percentage of organic phase and timely flush port (Agilent 6460) progress

3.2 Selection of internal standard

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Internal standard could correct the errors generated during each sample preparation

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procedure, particularly important when using MS/MS as the detector for the instrument could generate errors due to instability. It was often required of similar structures and

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physicochemical properties for the target compounds and internal standard, yet herein, phenacetin was chosen because the matrix effect, recovery, ionization response and

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chromatographic behaviors proved to be similar those of target compounds. These characteristics contributed to its good performance as an internal standard to ensure the accuracy of detection, together with feasibility and reduced cost in clinical practice.

3.3 Sample preparation

Sample preparation was a crucial procedure requiring of both reliability and convenience. The SPE method was recognized as the most efficient one of eliminating

ACCEPTED MANUSCRIPT interferences like proteins (even lipids). LLE was another common method for seperation of lipophilic compounds using proper extraction solvents. However, compared with the time/organic solvent consuming SPE and LLE, the simple and rapid protein precipitation method was considerably applicable for TDM in clinical practice. The application of Ostro 96-well protein precipitation plate was preliminarily and

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efficiently applied with high output, and ACN was selected for efficient protein precipitation after comparison with several deproteinization agents. To be specific, by

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virtue of the efficient protein precipitation method, the need for batch analysis in the clinical settings could be satisfied with preparation time allocated for each sample less

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than 2 min and UHPLC-MS/MS analysis within 5 min, taking consideration of all the key steps for protein preparation including loading, mixing, elution, evaporation and

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re-constitution. Additionally, satisfactory and stable results were obtained for all the

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target compounds.

3.4 Method validation

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3.4.1 Specificity

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The peak position of the CBZ and its seven metabolites and the IS showed no interference from the endogenous substances, and good baseline separation was

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achieved for each component (shown in Fig.3). Under the established conditions the retention time turned out to be approximately 3.3 min for CBZ, 1.6 min for AI, 4.4 min

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for IM, 4.2 min for AO, 3.7 min for CBZ-EP, 2.5 min for CBZ-DiOH, 3.4 min for 2OH-CB, 3.7 min for 3-OH-CBZ and 3.4 min for the IS. 3.4.2 LLOQ and linearity of calibration curves The present method offered LLOQ results of 50 ng/ml for CBZ and CBZ-EP, 10 ng/ml for AI, 3-OH-CBZ and 2-OH-CBZ, 100 ng/ml for IM and CBZ-DiOH, 0.2 ng/ml for AO with an acceptable RE within ±20%. Calibration curves were obtained of 50-5000 ng/ml for CBZ, 10-1000 ng/ml for AI,

ACCEPTED MANUSCRIPT 100-10000 ng/ml for IM, 0.2-20 ng/ml for AO, 50-5000 ng/ml for CBZ-EP, 100-10000 ng/ml for DiOH-CBZ, 10-1000 ng/ml for 2-OH-CBZ and 3-OH-CBZ respectively with linearity results showed in Table 1. The concentration of each calibration standards was back-calculated from the calibration curve equation. The accuracies were within ±15%

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nominal values for eight target compounds.

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3.4.3 Extraction recovery and matrix effect

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The matrix effect of the target compounds and the IS was accepted with 88.52%105.5%, and the values were consistent at three QC levels. The above results indicated

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that the endogenous compounds practically had no influence on the determination of target compounds. The extraction recoveries were excellent for three levels of QC

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samples of 74.70%-93.48% and 85.92% for the IS which proved the method reliable. (shown in Table S2).

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3.4.4 Precision and accuracy

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Good performance with low deviation and consistent accuracy was observed at three QC levels in the validation. As was shown in Table S3, the intra- and inter-day precision

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was less than 20%, and the accuracy was within 20%, indicating that the a precise and accurate method for determination of target compounds in plasma.

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3.4.5 Stability

The stability results (Table S4) showed that the target compounds, at three concentrations studied, had acceptable stabilities after three freeze-thaw cycles, at room temperature (20 ºC) for 6 h and three months at -80 ºC in human plasma and 24 h in auto-sampler (4 ºC) after protein precipitation.

3.5 Application

ACCEPTED MANUSCRIPT The established method was successfully applied to perform the determination of concentrations of CBZ and its seven metabolites in 95 authentic plasma samples obtained from epileptic patients in monotherapy with CBZ. Mean concentration of target compounds were shown in Fig.4. Results indicated that CBZ-EP and CBZ-DiOH were main metabolites CBZ with a great individual variability in plasma concentrations.

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The minor pathway product 2-OH-CBZ and 3-OH-CBZ were identified in 27 and 74

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patients respectively and some values detected lay lower than LLOQ. AI and AO were

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found with trace levels of 16.20 ng/ml and 0.8 ng/ml on average. It was noteworthy that IM, the product of direct oxidation catalyzed by CYP450 system, took the largest

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proportion of all the target compounds.

It could be observed from the results that concentration of CBZ determined by

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UHPLC-MS/MS seemed to have no significant difference from FPIA results in clinical range. 2-OH-CBZ was of lower concentration than its isomer 3-OH-CBZ probably

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because it had exclusive pathway to produce iminoquinone (CBZ-IQ). AI and AO, two

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oxidized compounds resulting from activated leukocytes differed in concentration relating to activity of chlorine and myeloperoxidase. Apparently, the concentration of

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IM discovered in this study ranged higher than those of similar investigations. This might be attributed to CYP450 diversity and the metabolic stage of individualized

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patients. Since AI, AO, 3-OH-CBZ, 2-OH-CBZ and IM were all thought to be toxic candidates in the pathogenesis of CBZ-induced adverse effects.

4. Conclusion A simultaneous UHPLC-MS/MS method for the determination CBZ and its seven metabolites, AI, IM, AO, CBZ-EP, CBZ-DiOH, 2-OH-CBZ, 3-OH-CBZ were described in this study, which had the advantage of being rapid using the 96-well protein

ACCEPTED MANUSCRIPT precipitation plate with high output and taking the total analysis time of 5 min for the determination of CBZ and its seven metabolites altogether. Since mass spectrum had become a prevailing detection method for laboratory analysis especially in Western countries, the established method could be a promising alternative for FPIA. The results obtained by this study demonstrate its action on determination of CBZ and its

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metabolites. Through abortive validation of the established method, it proved feasible

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especially for the determination of toxic candidates of CBZ in the routine monitoring

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and pharmacokinetic studies.

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reactions: An update, Pharmacological Reports, 67 (2015) 426-434. [20] L. Shi, X.L. Dang, X.Y. Liu, H.M. Wei, M.M. Yang, Y. Zhang, Effect of Sophora

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flavescens on the pharmacokinetics of carbamazepine in rats, Archives of Pharmacal Research, 37 (2014) 1617.

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[21] A. Liu, C. Wang, M. Hehir, T. Zhou, J. Yang, In vivo induction of CYP in mice by carbamazepine is independent on PXR, Pharmacological Reports, 67 (2015) 299-304.

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[22] R. Hartley, W.I. Forsythe, B. Mclain, P.C. Ng, M.D. Lucock, Daily variations in

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steady-state plasma concentrations of carbamazepine and its metabolites in epileptic children, Clinical Pharmacokinetics, 20 (1991) 237-246. [23] R. Hartley, M.D. Lucock, P.C. Ng, W.I. Forsythe, B. Mclain, C.J. Bowmer, Factors

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influencing plasma level/dose ratios of carbamazepine and its major metabolites in epileptic children, Therapeutic Drug Monitoring, 12 (1990) 438.

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[24] J. Csetenyi, K.M. Baker, A. Frigerio, P.L. Morselli, Iminostilbene--a metabolite of carbamazepine isolated from rat urine, The Journal of Pharmacy and Pharmacology, 25 (1973) 340.

[25] S.M. Furst, J.P. Uetrecht, The effect of carbamazepine and its reactive metabolite, 9-acridine carboxaldehyde, on immune cell function in vitro, International Immunopharmacology, 17 (1995) 445-452. [26] S. Eto, N. Tanaka, H. Noda, A. Noda, 9-Hydroxymethyl-10-carbamoylacridan in human serum is one of the major metabolites of carbamazepine, Biological & Pharmaceutical Bulletin, 18 (1995) 926-928.

ACCEPTED MANUSCRIPT [27] N. Wad, C. Guenat, G. Krämer, Carbamazepine: detection of another metabolite in serum, 9 hydroxymethyl-10-carbamoyl acridan, Therapeutic Drug Monitoring, 19 (1997) 314. [28] J.M. Potter, A. Donnelly, Carbamazepine-10,11-epoxide in therapeutic drug monitoring, Therapeutic Drug Monitoring, 20 (1998) 652.

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[29] O. Mathieu, O. Dereure, D. Hillairebuys, Presence and ex vivo formation of acridone in blood of patients routinely treated with carbamazepine: exploration of the

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9-acridinecarboxaldehyde pathway, Xenobiotica, 41 (2011) 91-100.

metabolite syndromes, Lancet, 356 (2000) 1587.

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[30] S.R. Knowles, J. Uetrecht, N.H. Shear, Idiosyncratic drug reactions: the reactive

[31] R. Mandrioli, N. Ghedini, F. Albani, E. Kenndler, M.A. Raggi, Liquid

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chromatographic determination of oxcarbazepine and its metabolites in plasma of

253-263.

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epileptic patients after solid-phase extraction, Journal of Chromatography B, 783 (2003)

[32] J.T. Burke, J.P. Thénot, Determination of antiepileptic drugs, Journal of

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Chromatography, 340 (1985) 199.

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[33] M.E. Queiroz, S.M. Silva, D. Carvalho, F.M. Lanças, Determination of lamotrigine simultaneously with carbamazepine, carbamazepine epoxide, phenytoin, phenobarbital, and primidone in human plasma by SPME-GC-TSD, Journal of Chromatographic

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Science, 40 (2002) 219.

[34] B.R. Ramaswamy, G. Shanmugam, G. Velu, B. Rengarajan, D.G. Larsson, GC-

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MS analysis and ecotoxicological risk assessment of triclosan, carbamazepine and parabens in Indian rivers, Journal of Hazardous Materials, 186 (2011) 1586-1593. [35] V. Kimiskidis, M. Spanakis, I. Niopas, D. Kazis, C. Gabrieli, F.I. Kanaze, D. Divanoglou,

Development

and

validation

of

a

high

performance

liquid

chromatographic method for the determination of oxcarbazepine and its main metabolites in human plasma and cerebrospinal fluid and its application to pharmacokinetic study, Journal of Pharmaceutical & Biomedical Analysis, 43 (2007) 763-768. [36] S. Rani, A.K. Malik, B. Singh, Novel micro-extraction by packed sorbent

ACCEPTED MANUSCRIPT procedure for the liquid chromatographic analysis of antiepileptic drugs in human plasma and urine, Journal of Separation Science, 35 (2012) 359-366. [37] B.R. Lopes, J.C. Barreiro, P.T. Baraldi, Q.B. Cass, Quantification of carbamazepine and its active metabolite by direct injection of human milk serum using liquid chromatography tandem ion trap mass spectrometry, Journal of Chromatography

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B, 889-890 (2012) 17. [38] S. Deeb, D.A. Mckeown, H.J. Torrance, F.M. Wylie, B.K. Logan, K.S. Scott,

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Simultaneous analysis of 22 antiepileptic drugs in postmortem blood, serum and plasma using LC-MS-MS with a focus on their role in forensic cases, Journal of Analytical

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Toxicology, 38 (2014) 485-494.

[39] Y. Zhu, H. Chiang, M. Wulster-Radcliffe, R. Hilt, P. Wong, C.B. Kissinger, P.T.

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Kissinger, Liquid chromatography/tandem mass spectrometry for the determination of carbamazepine and its main metabolite in rat plasma utilizing an automated blood

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sampling system, Journal of Pharmaceutical & Biomedical Analysis, 38 (2005) 119. [40] Y. Kouno, C. Ishikura, M. Homma, K. Oka, Simple and accurate high-performance

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liquid chromatographic method for the measurement of three antiepileptics in

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therapeutic drug monitoring, Journal of Chromatography B, 622 (1993) 47-52.

ACCEPTED MANUSCRIPT Legends: Table 1 Calibration curves (n=5) and LLOQ in method validation

Fig.1 Chemical structures of the analytes and the internal standard Fig.2 MS/MS transitions and parameters for the detection of the target compounds and

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the internal standard

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Fig.3 Typical chromatograms (in MRM mode) of drug free blank serum (A), drug free

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blank serum spiked with CBZ-DiOH, 2-OH-CBZ, 3-OH-CBZ, CBZ, CBZ-EP, AO, IM, AI, IS at 100 ng/ml (B) and serum from authentic epileptic patient treated with CBZ (C)

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Fig.4 Mean serum concentration profiles of the target compounds in epileptic patients

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administrated with CBZ

ACCEPTED MANUSCRIPT Table 1 MS/MS transitions and parameters for the detection of the target compounds and the IS Molecular

Precursor ion

Product ion

Fragmentor

CE

compound

mass

(m/z)

(m/z)

(V)

(V)

CBZ

236.1

237.1

194.0

124

17

AI

179.0

180.0

151.0

176

42

IM

193.1

194.1

179.0

148

34

AO

195.0

196.0

167.1

CBZ-EP

236.0

237.0

181.1

CBZ-DiOH

270.0

271.0

2-OH-CBZ

252.0

253.0

3-OH-CBZ

252.0

253.0

Phenacetin

179.0

180.0

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Target

40

100

24

180.1

100

26

210.1

140

14

210.1

140

14

110.1

80

22

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146

Table 2 Results of calibration curves (n=5) Linear range Target compound

D

(ng/mL)

(r)

50.0-5000.0

y=7.889x+3.398

0.9994

10.0-1000.0

y=2.919x-0.1328

0.9948

100.0-10000.0

y=0.1509x+0.08330

0.9985

0.2-20.0

y=25.68x-0.003500

0.9941

50.0-5000.0

y=0.1919x+0.04500

0.9970

CBZ-DiOH

100.0-10000.0

y=0.6286+0.09480

0.9982

2-OH-CBZ

10.0-1000.0

y=0.8065x+0.1141

0.9949

3-OH-CBZ

10.0-1000.0

y=0.8846x+0.01860

0.9998

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CBZ

Correlation coefficient

Linear regression equation

AI

AO

AC

CBZ-EP

CE

IM

ACCEPTED MANUSCRIPT Table 3 The extraction recovery and the matrix effect of the target compounds and the IS in human serum (n=3) Extraction recovery

Matrix effect

Nominal concentration

compound

(ng/mL)

Mean±S.D.%

R.S.D%

Mean±S.D.%

R.S.D%

100

88.33±3.620

4.1

101.4±4.380

4.3

500

83.70±1.750

2.1

103.8±3.990

3.9

2000

84.70±2.380

2.8

0.5

81.90±2.450

3.0

2

77.03±7.310

10

74.70±2.380

20

83.30±5.260

100 500

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9.5

6.3

100.5±4.780

4.8

86.97±3.540

4.1

100.7±5.100

5.1

91.20±3.360

3.7

101.5±4.650

4.6

92.17±3.270

3.6

99.87±4.100

4.1

86.03±3.310

3.9

104.7±5.750

5.5

86.30±9.190

11

100.1±10.06

10

92.33±2.630

2.9

101.2±3.590

3.6

500

91.40±3.380

3.7

103.9±6.000

5.8

2000

90.53±3.760

4.2

97.57±7.360

7.6

200

88.10±3.580

4.1

88.50±1.930

2.2

1000

89.73±5.120

5.7

98.63±5.590

5.7

5000

87.13±3.930

4.5

100.1±6.430

6.4

20

84.33±5.000

5.9

105.4±6.230

5.9

100

89.70±1.670

1.9

101.8±3.100

3.1

500

86.20±7.450

8.6

101.0±7.060

7.0

20

93.40±2.630

2.8

92.37±4.380

4.7

100

88.70±4.520

5.1

91.20±3.030

3.3

500

87.90±3.860

4.4

95.20±10.10

11

1000

85.92±7.640

8.9

98.72±4.320

4.4

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99.87±9.470

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100

CE

AC 2-OH-CBZ

3-OH-CBZ

Phenacetin

D

5000

CBZ-DiOH

5.2

3.2

1000

CBZ-EP

98.57±5.110

5.8

200 AO

8.0

105.4±6.060

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IM

99.53±7.920

9.5

AI

MA

CBZ

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Target

ACCEPTED MANUSCRIPT

Table 4 Intra-day and inter-day precision and accuracy of the target compounds Intra-day (n=5)

Inter-day (n=5)

Added Measured Target compound

Accuracy (RE%a)

108.1±9.180

8.5

8.1

575.0±15.56

2.7

15

1.4

1904±46.57

2.4

-4.8

1.6

19.76±0.4600

2.3

-1.2

2.1

96.98±1.790

1.9

-3.0

-1.2

0.90

507.8±11.52

2.3

1.6

4.4

9.4

213.2±16.04

7.5

6.6

1148±26.48

C C

15

2.3

1162±25.58

2.2

16

4237±61.70

-15

1.5

4125±105.8

2.6

-18

0.4900±0.02000

-2.0

4.7

0.4500±0.04000

9.9

-10

2

2.140±0.04000

7.0

1.9

2.080±0.06000

2.7

4.0

10

9.590±0.1300

-4.1

1.4

9.490±0.1600

1.7

-5.1

Precision (R.S.D.)

Accuracy (RE%a)

I R

Precision (R.S.D.)

concentration

concentration

(ng/mL) (mean±S.D.,ng/mL)

CBZ

AI

IM

(mean±S.D.,ng/mL)

100

114.0±3.080

14

2.7

500

571.2±20.95

14

3.7

2000

1950±26.40

-2.5

20

20.14±0.3100

0.70

100

95.72±1.990

500

493.8±4.190

200

208.9±19.63

0.5

D E

T P E

1000 5000

AO

T P

Measured

concentration

A

-4.3

U N

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A M

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ACCEPTED MANUSCRIPT

CBZ-EP

CBZ-DiOH

2-OH-CBZ

3-OH-CBZ

a

100

102.6±5.210

2.6

5.1

100.7±6.680

6.6

0.70

500

583.4±12.02

17

2.1

576.9±11.89

2.1

15

2000

1832±25.07

-8.4

1.4

1802±45.98

2.6

-9.9

200

180.7±14.52

-9.7

8.0

186.1±15.94

8.6

-7.0

1000

1091±23.63

9.2

2.2

1076±20.08

1.9

7.6

5000

4724±174.6

-5.5

3.7

4550±170.1

3.7

-9.0

20

18.85±1.950

-5.8

10

19.91±2.270

11

-0.50

100

109.6±7.340

9.6

6.7

100.7±13.60

14

0.70

500

461.8±33.81

-7.6

7.3

450.6±32.01

7.1

-9.9

20

19.64±1.840

9.4

19.74±1.790

9.1

-1.3

100

115.5±5.440

16

4.7

101.6±12.24

12

1.5

500

435.8±27.29

-13

6.3

434.0±26.88

6.2

-13

T P E

D E -1.8

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U N

A M

C C

RE is expressed as [(mean measured concentration)/(added concentration)-1]×100%

A

24

I R

T P

ACCEPTED MANUSCRIPT

Table 5 Stability of the target compounds at different QC levels(n=5) Freeze-thaw stability

Short-term stability

Long-term stability

Nominal Target

Measured

Measured

concentration compound

Precision

concentration

concentration

(ng/mL)

CBZ

AI

IM

I R

(R.S.D.)

Precision concentration

(R.S.D.)

(ng/mL)

(ng/mL)

(R.S.D.) (ng/mL)

106.9

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2.4

105.0

2.5

562.4

1.3

571.2

2.6

0.90

1948

1.9

1876

1.2

1.2

19.75

2.7

19.71

2.1

2.2

97.15

1.8

97.05

1.8

509.0

0.70

502.9

3.0

508.9

1.6

7.0

215.6

2.7

219.8

3.9

221.1

4.3

4.0

1153

4.2

1163

1.6

1157

3.1

4256

3.9

4330

4.7

4225

2.6

4270

3.5

0.4400

4.3

0.4300

2.7

0.4500

1.5

0.4400

2.6

2

2.060

2.0

2.030

3.2

2.090

2.5

2.060

2.3

10

9.580

2.1

9.470

1.2

9.670

1.5

9.570

1.4

100

107.6

3.1

100.3

2.7

500

563.7

5.8

587.7

1.2

2000

1860

1.5

1819

20

20.04

3.0

19.35

100

97.04

2.1

500

514.8

20

227.9

100

1155

500 0.5 AO

Measured

Precision

concentration

(R.S.D.) (ng/mL)

T P

Measured

Precision

Post-preparative stability

C C

T P E

A

D E

1.7

96.95

U N

A M

25

ACCEPTED MANUSCRIPT

CBZ-EP

100

100.1

3.6

96.04

1.3

86.08

3.4

94.07

2.6

500

561.1

1.7

548.0

2.9

563.4

1.6

557.5

1.9

2000

1762

2.3

1737

0.70

1795

2.1

1765

1.5

200

194.1

6.1

201.0

7.4

181.6

5.9

192.2

6.3

1000

1062

1.7

1049

2.2

1039

2.1

1050

1.8

5000

4424

1.0

4336

0.80

4553

1.7

4438

1.0

20

19.98

8.2

21.06

1.7

5.2

19.74

4.8

100

89.95

8.9

100.1

98.57

2.0

96.21

5.8

500

434.8

9.5

453.8

N A

18.18

5.9

458.9

9.4

449.2

8.1

100

18.22

5.3

9.6

19.70

6.0

18.99

6.8

500

96.93

88.85

8.7

94.45

12

93.41

8.4

2000

448.2

440.8

6.2

441.4

8.3

443.5

7.3

CBZ-

I R

C S U

DiOH

2-OH-CBZ

3-OH-CBZ

T P E

D E

4.8 8.1

19.04

7.2

M

C C

A

26

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ACCEPTED MANUSCRIPT Highlights 

An UHPLC-MS/MS method for simultaneous determination of CBZ, AI, IM, AO, CBZ-EP, CBZ-DiOH, 2-OH-CBZ and 3-OH-CBZ



Concentration of CBZ and its metabolites varied in a wide range from person to person AI, AO, 3-OH-CBZ and 2-OH-CBZ were in trace amount and IM took the largest

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

proportion of all the target metabolites

TDM of the metabolites as well as CBZ was proposed of individual epileptic

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

patients

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Quantification of CBZ and its metabolites could be applied in clinical toxicology

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study

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

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Figure 1

Figure 2

Figure 3

Figure 4