Abstract
A simple, sensitive, selective, accurate and precise method was developed and fully validated for determination of oxcarbazepine (OXC) in presence of their preservatives and determination of oxcarbazepine (OXC) in human plasma. A reversed phase liquid chromatography (RP-HPLC) with UV detection techniques were applied for separation and quantification of studied drug OXC. Successful separation of the drug from methyl paraben (M.P.), propyl paraben (P.P.) and potassium sorbate (P.ST.) was achieved on a Kromasil C18 column (5 μm particle size, pore size 300 Å, l × I.D. 250 × 4.6 mm). The mobile phase that contain aqueous 0.05M potassium dihydrogen phosphate buffer (pH 7): acetonitrile, (50: 50, %v/v). The method was linear over concentration ranges 5.0–50 μg mL−1 for OXC. Bioanalytical validation of the developed method was carried out according to US-FDA guidelines and revealed a good linear relations over a range of (5.0–50), (0.5–10), (0.05–0.15), and (1.0–10) μg mL−1 for OXC, M.P, P.P, and P.ST, respectively, with a correlation coefficient (R2) of more than 0.999. Limit of detection (LOD) were 1.15, 0.03, 0.01 and 0.04 μg mL−1 for OXC, M.P, P.P, and P.ST, respectively, Intra and inter-day precisions, calculated as percentage relative standard deviation (% RSD), were lower than 2.0%. The developed method can be applied for routine drug analysis, therapeutic drug monitoring and bioequivalence studies through the analysis of plasma samples taken from blood bank.
1 Introduction
Oxcarbazepine (OXC) [1] is a carbamazepine keto analogue, Fig. 1. OXC is antiepileptic drug that has been approved for treatment of partial seizures and trigeminal neuralgia in the United States and the European Union as well as more than 50 countries worldwide. OXC is less toxic and has a more favorable pharmacokinetic profile than carbamazepine [2]. It has a good oral bioavailability and rapidly metabolized to form the pharmacologically active 10-monohydroxy derivative [2]. Pharmaceutical preparations, syrups and suspensions, contain an aqueous vehicle which may affect product stability or could be infectious to patient, thus requires safeguards from microbial contamination [3]. Addition of anti-microbial agents to formulation is required to inhibit the growth of microorganisms during drug manufacture and use [4]. Preservatives are effective to control mold and yeast growth including limited agents as p-hydroxybenzoic acid esters: methyl paraben (M.P.) C6H4 (OH) COOCH3, propyl paraben (P.P.) C6H4 (OH) COOC3H7 and potassium sorbate (P.ST.) C6H7KO2, Fig. 1 [1]. These preservatives characterized by their broad antimicrobial spectrum, good stability and non-volatility [5]. A synergetic effect obtained when both M.P. and P.P are used in combination [6].
OXC was analyzed and validated in many of literature articles using different mobile phases, column and detector. (Supplementary data: Table S1). In 2019, quantification of OXC in tablets was carried out with stability indicating HPLC method (HPLC with photodiode array detector) [7]. Determination of OXC was conducted by M.L. Qi et al. in new pharmaceutical preparation [8]. A simple method to monitor plasma concentrations of oxcarbazepine, carbamazepine, their main metabolites and lamotrigine in epileptic patients [9]. Oxcarbazepine was also determined using UHPLC technique [10].
A three structurally related antiepileptic drugs; carbamazepine, OXC, eslicarbazepine acetate and their main metabolites (i.e. carbamazepine-10,11-epoxide, 10,11-trans-dihydroxy-10,11-dihydro carbamazepine, and licarbazepine) was validate using HPLC-UV detection method [11]. A high-performance liquid chromatographic determination of OXC and its active metabolite in human serum and plasma was also reported in the literature [12–16]. In 2019, oxcarbazepine was clinically quantified in human plasma using a nondestructive tool called Surfaced-Enhanced Raman Spectroscopy (SERS) combined with chemometrics analysis [17].
Methyl parapen, propyl parapen and potassium sorbate were determined using High Performance Liquid Chromatography LC–UV, LC–MS/MS, HPLC–UV, RPC and gradient RP-HPLC methods [18–22]. RP-HPLC method was assigned by Muhammad F. et al. for determination of methyl paraben sodium (MPS) and propyl paraben sodium iron protein succinylate syrup [23].
Parabens were determined in biological samples i.e. blood and urine using FPSE-HPLC-PDA techniques [24]. Up to date no validated analytical method for the determination of OXC, methyl paraben, propyl paraben and potassium sorbate in oral suspension formulation was available in literature. Here, RP-HPLC method was used because of its unique characters, as it is environmentally and economically benign than other methods [25–30]. Up to date in 2021 Kwabena F. et al. discussed common chromatographic principles, requirements and/or conditions for HPLC as applied to assay of oxacarbazepine and 27 antiepileptic drugs in six biological matrices [31].
Herein, a proposed method for accurate, selective, and reliable HPLC-UV method quantification of OXC and its preservatives in plasma was developed. The development of a single assay for the determination of the studied antiepileptic drug saved time and was cost effective, representing an advantage of this research.
2 Materials and methods
2.1 Reagents and chemicals
Oxcarbazepine OXC, methyl paraben (M.P.), propyl paraben (P.P.) and potassium sorbate (P.ST.) were obtained from Simco pharma (Egypt). Oxaleptal 60 mg mL−1® oral suspension was purchased from the Egypt market. Plasma samples were taken from Egyptian blood bank, Shebin Elkome Hospital (Egypt). Methanol (HPLC grade optained from Scharlau), KH2PO4 and sodium hydroxide were obtained from Panreac (USA), 0.45 μm Whatman filter were purchased from Sigma-Aldrich for syringe filter. Ultrapure water (Milli-Q) (Millipore Corporation, Billerica, MA, USA) was used.
2.2 Instrumentation and chromatographic conditions
2.2.1 HPLC-UV analyses
The HPLC system (Waters, USA) was equipped with auto sampler, Binary HPLC Pumps, Dual lamp Absorbance Detector and In-Line Degasser ISA Card. Data acquisition was performed on Empower software. The detector was set to 250 nm. The HPLC separation and quantitation were achieved on Kromasil C18 column (5 μm particle size, pore size 300 Å, l × I.D. 250 × 4.6 mm). All determinations were performed at 30 ºC. The mobile phase buffer solution was prepared by transferring accurately weighed about 6.8 g of Potassium dihydrogen phosphate into 1,000 mL volumetric flask. Add 500 mL purified water mixed to dissolve. The volume was completed by the same solvent. The Mobile phase composed of buffer: Acetonitrile (50: 50, %v/v) and pH adjusted to 7 with 1M NaOH, which was run Isocratic. Flow rate was 1.5 mL min−1 and injection volume was 20 μl. (Supplementary data: Table S2).
2.3 Preparation of standard solutions
(Solution A): To 33 mg of OXC working standard in 50 mL volumetric flask, 25 mL acetonitrile was added, stirred till complete dissolution then mobile phase was used to complete to the final volume. (Solution B): Transfer methyl paraben (19.8 mg) of working standard into volumetric flask (100 mL), add mobile phase to complete to a required volume and the mixture shake well to dissolve. (Solution C): 5 mL from solution B was transferred to volumetric flask (50 mL) then completed by mobile phase to required volume. (Solution D): Propyl paraben (22 mg) working standard transferred into volumetric flask (100 mL), then final volume obtained by mobile phase and shake well to dissolve. (Solution E): 1 mL from solution D was taken into 100 mL volumetric flask then completed to volume by mobile phase. (Solution F): 22 mg of potassium sorbate working standard was transferred into volumetric flask (100 mL), and then completed to volume by mobile phase and shake well to dissolve. (Solution G): 5 mL from solution F was transferred into volumetric flask (50 mL) then completed to volume by mobile phase.
Standard solution: 5 mL from each of solutions A, C, E and G was transferred into volumetric flask (100 mL) then completed to volume by mobile phase. (Conc. of OXC, methyl paraben, propyl paraben and potassium sorbate is 33, 0.99, 0.11 and 1.1 μg mL−1).
2.4 Preparation of sample solutions
Oxaleptal oral suspension (11 mL) was accurately measured and transferred to a volumetric flask (200 mL), added water (10 mL), stirred for 5 min; then acetonitrile (50 mL) was added, stirred for 30 min then the volume was completed by mobile phase.
1 mL was transferred from the previous solution into volumetric flask (100 mL), the volume was completed by mobile phase, mixed and centrifuged. (Conc. of OXC, methyl paraben, propyl paraben and potassium sorbate is 33, 0.99, 0.11 and 1.1 μg mL−1).
2.4.1 Forced degradation conditions
Acid degradation: the mixture of OXZ., M.P., P.P. and P.ST: was prepared as previously described in the preparation of working standard solution but 5 mL of 5M HCl was added to the analytes, shaking for 5 min and leaving for 1 h at room temperature, the mixture was neutralized by 5 mL of 5M NaOH before volumetric dilution with 100 mL.
Alkaline degradation: Prepared by adding 5 mL of 5M NaOH to the mixture of OXZ., M.P., P.P. and P.ST. standard solutions, containing equal amounts of working standard solutions, and shaking for 5 min. The solution was then kept at room temperature for 1 h and neutralized with 5 mL of 5M HCl. The volume was diluted to 100 mL using the mobile phase.
Oxidative degradation: prepared by adding 5 mL of 30% H2O2 to the same concentration of a mixture of OXZ. M.P., P.P. and P.ST: standard solution used in acid and alkaline degradation. A 5-min mixture shake was followed by 1 h of dark storage at room temperature, then 100 mL of the mobile phase was added.
Heating Hydrolysis of OXZ., M.P., P.P. and P.ST: To 11 gm of oxaleptal oral suspension in 100 mL volumetric flask, 10 mL of purified water and 50 mL methanol were added. They were shaken well for 30 min at 60 C° for 3 h in a water bath. As the flask cooled to room temperature, the mobile phase was mixed with the acquired volume and filtered. Transfer 5 mL into volumetric flask (50 mL) and add mobile phase to achieve desired volume, and then 5 mL was transferred into 100 mL flask and then completed to volume with Mobile phase.
Day light Hydrolysis of OXZ., M.P., P.P. and P.ST Suspension:
Carried out as mentioned in heat hydrolysis & put in day light for 6 h instead of heating.
2.5 Method validation
2.5.1 Accuracy
Accuracy estimated by applying of the proposed study to standard solution of OXC, M.P., P.P. and P.ST to which known quantities of analyte have been spiked within the range of the calibration curve. Accuracy should be assessed using at least three concentrations (80, 100 and 120%) with average recovery percent's ranging from 98% to 102% of spiked drug in plasma.
2.5.2 Precision
Precision of the proposed method is the degree of agreement among results of individual test when it applied repeatedly and it is expressed as the relative standard deviation RSD of a series of measurements. It should be assessed using at least six quantitation's at 100% of the test concentration. Obtained Relative Standard Deviation (RSD %) are within the accepted range (NMT 2%) indicating that the method is Precise and repeatable.
2.5.3 Linearity, LOD and LOQ
The linearity represents the ability to get results that are directly proportional to the concentration of drug in samples within specific range. A minimum of five concentrations should be used. If appears to be a linear relation, calculate regression coefficient and y-intercept which should be higher than 0.99 and near to zero respectively.
Linearity of the method represents its ability to get a direct relation between the obtained results and the given concentration of the drug. At the optimized HPLC conditions, linearity was obtained by injection of standard solution series of OXC, M.P., P.P. and P.ST at five different levels; 60, 80, 100, 120 and 140% of the selected concentration range. Plotting the peak area against the selected concentration was used estimate the calibration curves of each sample. The Slope (b), intercept (a) and correlation R2 were estimated. LOD was determined from the formula; LOD = 3.3 σ/SD, where (σ) is SD of the intercept and (S) is the slope of regression equation. LOQ was calculated similarly from the equation LOQ = 10 σ/S.
2.5.4 Robustness
The robustness of a method is its ability to be not affected by small variations in its parameters as temperature, wavelength, and change in mobile phase. This reproducibility under the normal parameters compared to the precision to get a measure of the robustness of the analytical method.
2.6 Application to human plasma samples
Into 10 mL centrifuge tube, serial working solutions were prepared then an accurate volume of 200 μl OXC, M.P., P.P. and P.ST standard solution was quantitatively added to 500 μl of blank plasma samples taken from Egyptian blood bank (Ethical Approval Number ZA-AS/PH/22/C/2023), then 2.0 mL acetonitrile were added as a protein precipitation, and vortex mixed for two minutes followed by centrifugation at 5,000 rpm for 20 min. Further supernatant filtration was done using membrane filter of cellulose acetate (0.45-m), then 20 µl was applied to the HPLC-UV system.
3 Results and discussion
3.1 Method development
Different mobile phase compositions and flow rates were investigated to determine the best optimum mobile phase for separation of OXC, M.P, P.P, and P.ST. It was discovered that separated peaks were obtained. As a result, the mobile phase was tested with four components to achieve the best resolution. Different ratios of acetonitrile and 0.05M KH2PO4 buffer were investigated. As shown in Fig. 2, phosphate buffer (0.05 M, pH 7): acetonitrile (50:50; % v/v) was optimal for separation.
The pH range of 0.05 M phosphate buffer was tested from 5.0 to 9.0 to ensure good separation of OXC, M.P, P.P, and P.ST. According to Fig. 3, pH 7 was optimal for separation.
Moreover, the effect of flow rate was investigated in order to achieve sharp symmetric peaks of the cited drug in a reasonable amount of time. Flow rates ranging from 1.0 to 2.0 mL min−1 were found to provide good separation between the OXC, M.P, P.P, and P.ST with sharp symmetric peaks, so 1.5 mL min−1 was determined to be the appropriate flow rate. (Supplementary data: Fig. S1) illustrated the comparison of OXC and placebo.
Despite the proposed method is simple, accurate and rapid, but it still has limitations include multiple steps need to be optimized and the run time need to be shorter or the separation need to be quicker.
3.2 Method validation
The HPLC method was validated using ICH [32] rules and bio analytically using US-FDA guidelines [33].
3.2.1 Accuracy, precision
The proposed approach's accuracy was assessed using % targeting concentrations of 80, 100, and 120%, and the percent recovery was (33, 0.99, 0.11, and 1.1 μg mL−1) by three times injection of each concentration on the HPLC-UV system for OXC, M.P., P.P., and P.ST. (Supplementary data: Table 1). Table 1 represents the conclusions, which show that the proposed method is highly accurate. The HPLC-UV method's high sensitivity ensures its suitability for tracing OXC and its preservative in oral suspension.
Accuracy of OXC and its preservatives in oxaleptal sample solution
% of targeting concentration | OXC % recovery ± SD | M.P. % recovery ± SD | P.P. % recovery ± SD | P.ST. % recovery ± SD |
80% | 98.99 ± 0.067 | 99.88 ± 0.10 | 99.29 ± 1.196 | 99.03 ± 0.444 |
100% | 99.88 ± 0.037 | 101.01 ± 0.12 | 99.47 ± 0.097 | 99.28 ± 0.018 |
120% | 101.5 ± 0.061 | 100.94 ± 0.12 | 99.73 ± 0.23 | 101.46 ± 0.086 |
SD: Standard deviation
US-FDA criteria was used to bioanalytical validate the explored approach, with the accuracy and precision tested in human plasma. OXC; three concentrations, were assessed intraday (n = 6) and interday (n = 9) using low quality control samples (LQC), medium quality control samples (MQC), and high-quality control samples (HQC). Table 2 shows that human plasma has good repeatability, with a percent RSD less than 2.0 and a percent recovery ranging from 96.88 to 97.77%.
Accuracy and precision of the proposed method for determination of studied drug in human plasma
Conc. taken (µg mL−1) | Intra-day assay (n = 6) | Inter-day assay (n = 9) | ||
Accuracy (%Recovery) | Precision (%RSD) | Accuracy (%Recovery) | Precision (%RSD) | |
OXC | ||||
15 | 96.99 | 1.19 | 96.88 | 1.18 |
25 | 97.77 | 0.06 | 97.54 | 0.12 |
35 | 97.29 | 0.44 | 97.73 | 0.08 |
3.2.2 Selectivity
Analysis of Oxaleptal oral suspension (60 mg mL−1) was confirmed selectivity of the proposed method. No interference with the target analytes was observed in presence of the excipients. The representative chromatogram of OXC, M.P., P.P. and P.ST in Oxaleptal oral suspension showed no interfering peaks from excipient components (Fig. 4).
3.2.3 Linearity, LOD and LOQ
The linearity concentration ranges for OXC, M.P, P.P, and P.ST were found to be (5.0–50), (0.5–10), (0.05–0.15), and (1.0–10) μg mL−1, respectively. The linearity range and the sensitive parameters were summarized (Supplementary data: Table 3). The calculated results, summarized in Table 3, indicate sensitive HPLC method as other reported methods [12–16].
Calibration parameters of 60, 80, 100, 120 and 140% of target concentrations of the linearity concentration ranges of OXC, M.P, P.P and P.ST is 33, 0.99, 0.11 and 1.1 μg mL−1)
Analytes | Range (µg mL−1) | LOD (µg mL−1) | LOQ (µg mL−1) | Slope | Intercept ± SD | r2 |
OXC | 5.0–50 | 1.15 | 3.47 | 772 | 179 ± 55 | 0.9995 |
M.P. | 0.5–10 | 0.03 | 0.1 | 245 | 112 ± 50 | 0.9994 |
P.P. | 0.05–0.15 | 0.01 | 0.04 | 170 | 313 ± 75 | 0.9994 |
P.ST. | 1.0–10 | 0.04 | 0.11 | 457 | 254 ± 65 | 0.9993 |
To investigate the matrix effect and selectivity in human plasma, three quality control samples (LQC, MQC, and HQC) (15, 25, and 35 μg mL−1) for OXC were used. The recovery percent range was between 96.25 ± 0.88 and 97.19 ± 1.25. The results' lack of plasma matrix effect confirms the technique's outstanding selectivity, as shown in Table S3.
3.2.4 Robustness
To evaluate the robustness of the suggested HPLC-UV technique, the impacts of minor deviations from ideal chromatographic parameters, such as (mobile phase system ratio, pH value, mobile phase flow rate, and detection wavelength), were studied. The performance of the developed technique was demonstrated to be unaffected by this minor change in the experimental settings, guaranteeing the method's reliability.
Based on FDA [33] recommendations, incurred sample reanalysis (ISR) was investigated as a parameter for bio-analytical validations to confirm the accuracy and precision of incurred samples. ISR is calculated as (% difference between initial and incurred samples/Mean) x 100. Based on the HPLC analysis, the percentage variation between samples ranged from 3.20 to 4.11%.The findings concern the suitability and sensitivity of the HPLC method for estimating OXC (Table S4).
3.2.5 Applications of HPLC in human plasma
The recovery percentage for the tested procedures at five different concentration levels was discovered to be between 95.12 and 98.24 percent, as shown in Table 4. The percent RSD value for the examined OXC was 0.83–1.12. These findings are consistent with other methods reported and fall within the acceptable range of analytical method variability due to different matrix effects.
Analysis of OXC and their preservatives in spiked human plasma using the proposed HPLC-UV method
OXC, Conc (µg mL−1) | %Recovery* ± SD | M.P, Conc (µg mL−1) | %Recovery* ± SD | P.P, Conc (µg mL−1) | %Recovery* ± SD | P.ST, Conc (µg mL−1) | %Recovery* ± SD |
5.0 | 98.24 ± 1.12 | 0.5 | 98.99 ± 0.11 | 0.05 | 99.24 ± 1.16 | 1.0 | 99.32 ± 0.14 |
10 | 96.33 ± 1.50 | 1.5 | 100.05 ± 0.25 | 0.06 | 99.67 ± 0.09 | 2.5 | 101.63 ± 0.18 |
20 | 95.12 ± 0.83 | 3.5 | 101.14 ± 0.17 | 0.07 | 98.83 ± 0.43 | 4.5 | 102.16 ± 0.08 |
40 | 97.20 ± 0.98 | 6.6 | 99.89 ± 0.33 | 0.09 | 101.42 ± 0.17 | 6.5 | 99.48 ± 0.017 |
50 | 95.88 ± 1.07 | 10 | 99.64 ± 0.40 | 0.15 | 97.86 ± 1.20 | 10 | 98.66 ± 1.23 |
*Average of five replicates.
The purpose of this work was to create and validate an HPLC-UV technique for measuring OXC, M.P., P.P. and P.ST in plasma (Fig. 5). The design of a single assay for the determination of the examined antiepileptic drug and its preservatives saved time and cost, which was a value of this study. A validated analytical approach is required to produce data that allow for adequate patient monitoring during therapy. Furthermore, in a typical laboratory where a high number of samples must be tested on a routine basis, short analysis times, simple instrumentation, and simple chromatographic conditions are required. Thus, a speedy, simple, selective, accurate, exact, and sensitive HPLC-UV technique for determining OXC and its preservatives is presented here. The recommended HPLC method can be used to evaluate OXC for quality control.
3.2.6 Forced degradation studies
Samples of OXC, M.P., P.P. (and P.ST) standard solutions were subjected to forced degradation in NaOH, HCl, H2O2, heat, and light. The degradation samples were analyzed using the proposed method. Minor degradations of OXC, M.P., P.P. and P.ST were observed under acidic, oxidative, heat and light conditions at room temperature. At room temperature, OXC, M.P., P.P. and P.ST peaks showed approximately 20%, 11%, 11% and 14% degradation under the alkaline condition as shown in Fig. 6, respectively. All degradation products were chromatographically resolved from target analytes resolution between every two successive peaks was greater than 2.
4 Conclusions
A quick, accurate, and simple HPLC analytical method has been developed for the current study. For the routine analysis of OXC in oral suspension dosage form and spiked human plasma, this method was bio analytically validated. A reversed phase liquid chromatography (RP-HPLC) with UV detection techniques were applied for separation and quantification of studied drug OXC. Successful separation of the drug from methyl paraben (M.P.), propyl paraben (P.P.) and potassium sorbate (P.ST.) was achieved and bioanalytical validation of the developed method was carried out according to US-FDA guidelines and revealed a good linear relations over a range of (5.0–50), (0.5–10), (0.05–0.15), and (1.0–10) μg mL−1 for OXC, M.P, P.P, and P.ST, respectively. The developed method can be applied for routine drug analysis, therapeutic drug monitoring and bioequivalence studies through the analysis of plasma samples taken from blood bank. Further investigations are still in place to get more economically and environmentally benign methods for separation and quantification of studied drug OXC.
Ethics approval and consent to participate
The faculty of pharmacy, Al-Azhar University, Assuit Branch research ethical committee certifies that the research protocol presented by. Dr Ali Fouad, is compatible with the faculty scientific research ethic regulations, Dr Ali Fouad, was considered as preventative for all of the authors in the study.
Approval number ZA-AS/PH/22/C/2023.
Validity of approval 20/01/2023 to 19/01/2024.
Consent for publication
Not applicable.
Availability of data and materials
Not applicable.
Competing interests
The authors have declared no conflict of interest.
Funding
This article was self-funded.
Authors' contributions
Ali Fouad and Hany A. Batakoushy: research idea conceptualization; supervised the study; data analysis; manuscript writing, revision, and editing. Ahmed S. Abdelkhalek: Methodology, writing – original draft, data curation, validation, revision and editing. Hisham Elrefay: Methodology, writing – original draft and validation. Moustafa K. Soltan: Writing – review & editing.
Authors' information
Not applicable.
Acknowledgments
Not applicable.
Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1556/1326.2023.01157.
List of abbreviations
UHPLC | Ultra-High-Performance Liquid Chromatography |
LC-MS-MS | Liquid Chromatography with tandem mass spectrometry |
RPC | Remote procedure call |
FPSE | Fabric phase sorptive extraction |
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