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Concurrent detection of cabozantinib as an anticancer agent and its major metabolites in human serum using fluorescence-coupled micellar liquid chromatography
⁎Corresponding authors at: No. 85, Jinzhou South Road, Hanbin District, Ankang, Shanxi 72500, China (M. Yu). myu.ach@aol.com (Mei Yu), suliman.khan18@mails.ucas.ac.cn (Suliman Khan)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
A novel, highly sensitive, simple, and rapid strategy was designed and developed for simultaneous determination of cabozantinib (CBZ) as an anticancer agent and its main metabolites including monohydroxy sulfate (EXEL-1646), N-oxide (EXEL-5162(, amide cleavage product (EXEL-5366), and 6-desmethyl amide cleavage product sulfate) EXEL-1644). Measurements were done through a micellar liquid chromatography (MLC) method coupled with fluorescence detection. The high-performance liquid chromatography (HPLC) was performed using a Kinetex C18 100 Å column as well as acetonitrile, cetyltrimethylammonium bromide (CTAB; 0.2 mol.L−1), and tris buffer (pH 8.5) solutions as the mobile phase at a 40:50:10 (v/v) ratio. The method’s linearity (20 to 700 ng.mL−1), limit of detection (LOD; 2.11 to 3.69 ng.mL−1), limit of quantification (LOQ; 20 to 30 ng.mL−1), intra- and inter-day precisions (RSD < 4.00%), selectivity, recovery, and robustness were fully evaluated. According to the obtained results, the developed method can be used for simple and rapid (∼35 min) quantification of CBZ as an anticancer drug and its major metabolites in human serum samples with high sensitivity and low cost.
Keywords
Cabozantinib
Metabolites
Micellar chromatography
Fluorescence detection
Cetyltrimethylammonium bromid
1 Introduction
Cabozantinib (CBZ; XL184; N-{4-[(6,7-dimethoxyqui nolin-4-yl)-oxy]phenyl}-Ń-(4-fluorophenyl)-cyclopropane-1,1-dicarboxamide, (2S)-hydroxybutanedioate) is an efficient, novel tyrosine kinase inhibitor capable of suppressing the receptors and pathway targets for CBZ including MET (mesenchymal–epithelial transition), RET (Rearranged during Transfection), VEGFR-2 (vascular endothelial growth factor receptor 2), KIT (receptor tyrosine kinase), FLT-3 (Fms-like tyrosine kinase 3), TIE-2 (Angiopoietin-1 receptor), TRKB (Tropomyosin receptor kinase B), and AXL receptor tyrosine kinase (Yakes et al., 2011; You et al., 2011; Wang et al., 2015). CBZ is recognized as an antitumor agent which can significantly curb size, growth rate, metastasis, and angiogenesis of tumors (Wang et al., 2015; Fay et al., 2015). CBZ has been approved by the Food and Drug Administration (FDA) for treatment of thyroid cancer and renal cell carcinoma; also, it has shown a significant antitumor activity against breast cancer (Winer et al., 2012), hepatocellular carcinoma (Choueiri et al., 2015), non-small cell lung cancer (Drilon et al., 2013; Drilon et al., 2016), prostate cancer (Clyne, 2014; Smith et al., 2013), and pancreatic cancer (Wiecek and Karcher, 2016; Hage et al., 2013). Moreover, CBZ has been investigated for treatment of advanced and progressing cases of hepatocellular carcinoma (Abou-Alfa et al., 2018), and it has been researched through a phase 2 placebo-controlled randomized discontinuation study in patients suffering from hepatocellular carcinoma (Kelley et al., 2017). CBZ is mainly metabolized in liver by the enzyme cytochrome P450 3A4 (CYP3A4) (Schwartz et al., 2020), and its major metabolites are as follows: EXEL-1646 (2-[1-({4-[(6,7 dimethoxyquinolin-4yl)oxy]phenyl}carbamoyl) cyclopropane carboxamido 5-fluorophenyl hydrogen sulfate]), EXEL-5162 (4-(4-{1-[(4fluorophenyl) carbamoyl] cyclopropanecarboxamido}-6,7-dimethoxyquinoline 1-oxide), EXEL-5366 (1-{[({4-[(6,7 dimethoxyquinolin-4-yl)]oxy}phenyl) carbamoyl] cyclopropane carboxylic acid}), and EXEL-1644 (1-[(4-{[7-methoxy-6-(sulfooxy) quinolin-4-yl]phenyl}carbamoyl) cyclopropane carboxylic acid]) (Scheme 1). To the best of our knowledge, reported methods for determination of CBZ are as follows: LC-MS/MS (Su et al., 2015), UPLC-MS/MS (Wang et al., 2015), and micelle-enhanced spectrofluorimetry (Darwish et al., 2015). In addition, no method has been reported so far for simultaneous separation and determination of CBZ and its serum metabolites.
CBZ and its main metabolites (Lacy et al., 2015).
Su et al. designed and evaluated a fast, sensitive LC-MS/MS method to quantify CBZ in rat plasma using acetonitrile–water (45:55, v/v) as the eluting system at pH 5.5 and flow rate of 0.4 mL/min (Su et al., 2015). As they reported, the lower limit of quantification (LLOQ) was determined 0.5 ng.mL−1, and Intra- and inter-day accuracy and precision were found < 15%. In another study, Wang et al. developed and studied a simultaneous method to measure receptor tyrosine kinases (RTKs) inhibitors, i.e., lapatinib, CBZ, imatinib, dasatinib, sorafenib, crizotinib, erlotinib, and histone deacetylase (HDAC) inhibitor SAHA in rat plasma and applied acetonitrile for extraction of plasma samples. The employed mobile phase consisted of acetonitrile and water + 0.1% formic acid, and intra- and inter-day precisions were ascertained < 14% with an accuracy between 85.4 and 112.2% (Wang et al., 2015). Darwish et al. also reported a novel, sensitive method of micelle-enhanced spectrofluorimetry for CBZ determination in spiked human plasma. The proposed method was based on evaluation of fluorescence spectral behavior of CBZ in a micellar system of Cremophor RH 40 which could noticeably amplify the fluorescence intensity of CBZ. The obtained lower detection limit was 13.34 ng.mL−1, and the recovery value of CBZ for the spiked human plasma was determined 100.44 ± 3.91% (Darwish et al., 2015).
Micellar liquid chromatography (MLC) is categorized under reversed-phase high-performance liquid chromatography (RP-HPLC) that applies aqueous surfactants at above critical micelle concentration (CMC) as the mobile phase. This method can be used as an efficient strategy in order to determine free drug concentrations in biological samples. Some of the merits of MLC method are as follows: (i) ability to make solutions of insoluble and poorly soluble species owing to micelle phase formation, (ii) a good option for monitoring the concentration of a therapeutic agent in serum, as well as (iii) a low-cost and environment-friendly operation. In addition, this technique allows the direct injection of real samples via the solubilizing sample proteins (Carda-Broch et al., 2002; Ruiz-Angel et al., 2002; Ruiz-Angel et al., 2003). More importantly, no need to any extraction or removing of proteins as interfering agents is considered as the momentous benefit of MLC method in comparison with other analytical methods that depend on solubility of proteins in micelle aggregations in the mobile phase. Given the above-mentioned points, the extraction step before MLC can improve the method's efficiency to operate at lower limit of detections (LODs), a higher sensitivity, and a shorter separation time (Carda-Broch et al., 2007). Due to reducing steps of analysis and an accurate determination process, especially for biological samples, MLC has been introduced as an efficient, versatile analytical procedure (Esteve-Romero et al., 2016; Ruiz-Angel et al., 2009). According to studies, other advantages of MLC method include high accuracy in prediction of samples' retention behavior (Kawczak and Bączek, 2012) and simultaneous multiple drug detection in serum and urine samples (Gil-Agusti et al., 2003; Romero-Cano et al., 2015; Abd El-Hady and Albishri, 2014).
Spectrofluorimetry is a powerful, fast, and versatile (Albano et al., 2018; Albano et al., 2020) method in pharmaceutical analysis which offers sufficient selectivity, high sensitivity, and high accuracy (Farnoudian-Habibi et al., 2015; Farnoudian-Habibi and Jaymand, 2016; Wang et al., 2016). However, signal amplification may be needed for detection of weakly fluorescent samples in order to improve sensitivity, accuracy, and other analytical parameters. In the past few decades, some attractive signal amplification approaches have been developed using metallic nanoparticles (Jiang et al., 2014), surfactants (e.g., sodium dodecyl sulphate (SDS) and CTAB) (Ghosh et al., 2015), ionic liquids (ILs) (Wang et al., 2014), and host–guest interaction systems (e.g., beta-cyclodextrin (β-CD) and its derivatives) (Liu et al., 2015).
In this study, a novel chromatographic approach using acetonitrile, CTAB solution (the cationic surfactant; 0.2 mol.L−1), and tris buffer (pH 8.5) as the micellar mobile phase was developed for a fast, accurate, and sensitive quantification of CBZ and its main metabolites in serum samples (Scheme 2). It should be mentioned that the micellar phase acted as both separation agent and fluorescence intensity amplifier. This novel chromatographic method was validated according to the guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) (Clapham, 2017) in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), intra- and inter-day precisions, selectivity, recovery, and robustness.
The overall strategy for simultaneous determination of CBZ and its major serum metabolites (Fluorescence intensity *100).
2 Experimental
2.1 Reagents and chemicals
Cabozantinib, EXEL-1646, EXEL-5162, EXEL-5366, and EXEL-1644 (purity ≥ 99.99%) were purchased from the Exelixis Inc., (South San Francisco, CA, USA). Acetonitrile, CTAB, and diethyl ether were provided by Sigma-Aldrich (St. Louis, MO, USA). Millipore Milli-Q® UF Plus purification system (Millipore, Bedford, MA, USA) was used to produce ultrapure water with an 18 MΩ.cm−1 specific resistivity. The serum was prepared according to the HUPO BBB SOP guidelines (Rai et al., 2005).
2.2 Preparation of standard solutions
Analyte standard solutions (1 mg.mL−1) were prepared in methanol followed by sonication for 5 min in amber flasks to avoid photochemical degradation. Daily calibration curves were prepared by spiking different analyte concentrations of 20–700 ng.mL−1. Also, the quality control (QC) samples were similarly spiked with different analyte concentrations of 50–100 ng.mL−1.
2.3 Sample preparation
The whole human volunteers' blood samples were collected from healthy volunteers (HV) into plain glass vacutainers and were centrifuged at 10,000 g to separate serum and cellular components immediately in order to prevent clotting. The samples were stored in a freezer (−86 °C) prior to use. The serum (400 µL) was spiked with the standard solution for each analyte (20 µL; 200 ng.mL−1) followed by the addition of acetonitrile (100 µL) to precipitate proteins and other components of the serum causing interference. The resulting mixture was then centrifuged for 10 min at 8000 rpm. Afterwards, 400 µL of the supernatant was collected and added to a new tube containing water (1 mL) and diethyl ether (3 mL). The tube’s content was vortexed for 10 min followed by centrifugation for 5 min at 5000 rpm at room temperature. After, the organic phase was transferred to a 5 mL tube and exposed to a nitrogen stream to let it dry out. The obtained residue was reconstituted in the mobile phase (70 µL) from which, 20 µL of the sample was fed into the chromatography column.
2.4 Chromatography conditions
We used the Agilent 1100 series high-performance liquid chromatography (HPLC) system (Agilent Technologies, Palo Alto, CA, USA). The isocratic mobile phase contained acetonitrile, CTAB (0.2 mol.L−1), and tris buffer (pH 8.5) (40:50:10 v/v) at the flow rate of 1 mL.min−1. It should be pointed out that the first and second critical micelle concentrations of CTAB in water were about 8.9 × 10−4 and 2.1 × 10−2 mol.L−1, respectively (Parkinson et al., 1989). Auto-sampler was used for injection of 20-μL samples into the column. The mobile phase filtration was performed using filter membranes with a 0.45 μm pore size (Micron Separations, Westboro, MA, USA) prior to use. The column temperature was set at 35 ± 0.5 °C. The excitation and emission wavelength ranges at 245–295 and 342–483 nm, respectively, were used for sample detection by a fluorescence detector (G1321A).
3 Results and discussion
3.1 Mobile phase selection
CBZ and its afore-mentioned metabolites converted to their anionic forms at pH 8.5, and it was expected that the analytes would be rapidly extracted through MLC method. It is well known that CTAB is a stable cationic surfactant and can form stable micelles. Thus, its addition to the mobile phase resulted in obtaining a suitable separation with a retention time of ∼35 min. After evaluating the mobile phase candidates through configuration, a mixture of acetonitrile, CTAB (0.2 mol.L−1), and tris buffer (pH 8.5) at the ratio of 40:50:10 (v/v) was selected and isocratically pumped as the eluting system [LCGC North America, 2013, Volume 31, Issue 11 Page: 966]. The analytes (i.e., CBZ and its metabolites) were the only factors considered in mobile phase optimization because the only signal obtained from the blank serum sample was related to the protein bands at the retention times of 2 and 30 min. Mobile phase optimization led to a maximum resolution with no overlapping signals or interference and a short retention time of ∼ 35 min. Fig. 1 depicts chromatograms of the blank serum sample and the serum spiked with CBZ and its metabolites (100 ng.mL−1).
The chromatograms of aqueous solutions of CBZ and its metabolites, all at 100 ng.mL−1 as a model concentration (Fluorescence intensity *100).
3.2 Effect of pH on separation
As recognized, pH is an important factor in MLC method which controls the ionic form and the balance between ionic and nonionic forms of analytes (Carda-Broch et al., 2002; Ruiz-Angel et al., 2002). Also, selecting a proper pH value relies on the nature of the involved surfactant and analytes, so that if an appropriate pH is set given the contrast in ionic form of the surfactant and analytes, the separation space widens and thus favors the resolution. In all separation processes, the efficiency depends on the balance between column and mobile phase affinities for the analytes. In this study, all analytes were assumed to be in an anionic form at pH 8.5 given the dominance of negative OH molecules, so they could be attracted by the cationic mobile phase (i.e., CTAB). Nevertheless, the important point here was the balance between each analyte's affinity for the stationary and mobile phases. Therefore, both electrostatic attraction and affinity played pivotal roles in the separation process.
3.3 Effect of CTAB concentration on separation efficiency
In this study, it was found that micellar phases could simultaneously enhance the separation efficiency and the fluorescence intensity which resulted in low LOD and LOQ values as well as a suitable sensitivity. As the main element involved in separation, the effect of CTAB concentration in mobile phase on the separation efficiency and retention time was investigated. Based on the obtained results (Table 1), applying CTAB at the volume of 50% (0.2 mol.L−1) was found suitable for the efficient simultaneous separation of CBZ and its metabolites. Thus, CTAB concentration was recognized as a critical parameter in separation of CBZ and its major serum metabolites. TR: Retention time. SE%: Separation efficiency (%).
Analytes
25%
50%
TR
SE%
TR
SE%
XL1644 (M2a)
6.5
94.1
4.6
99.2
XL-184 (CBZ)
10.3
97.5
8.7
99.5
XL-1646 (M9)
14.2
95.6
13.9
98.7
XL-5162 (M19)
24.7
93.6
20
95.4
XL-5366 (M7)
27.7
94.9
25
96.0
3.4 Validation of the analytical chromatography method
HPLC method validation was performed according to ICH guidelines (Clapham, 2017) using a matrix by spiking the serum samples. Validated parameters included selectivity, sensitivity (i.e., LOD and LOQ), and linearity as well as accuracy and robustness. The repeatability of the HPLC method was assessed by the two following assessment procedures: within-assay precision (intra-day: five replicates for each concentration within one day) and between-assay precision (inter-day: five replicates for each concentration during five consecutive days.
3.4.1 Analytical method’s specificity
As mentioned, acetonitrile was used for precipitation of interferences regarding proteins and other components of serum, and extraction by diethyl ether was subsequently carried out prior to conducting HPLC separation. These process could remove several interferences from the matrix of serum influencing separation and determination of analytes. The chromatogram of the blank serum sample did not show any interference peaks in the range of 2 to 30 min (Data not shown), which was the desired time domain in the separation process. However, in range of the dead time (i.e., 0 to 2 min), several peaks were observed which did not cause any interferences to the process. In order to determine the analytical method’s specificity, serum samples (n = 10) were examined under the optimum conditions (Fig. 1). Once the method was proved to be interferences-free, a serum sample was spiked with 100 ng.mL−1 of CBZ and its metabolites. As depicted in Fig. 2, analytes were eluted with no overlapping signals.![The chromatogram of the spiked serum sample. All of spiked concentrations were similar to concentrations of the analytes in cancer patients' serum samples [XL-1644 (115 ngmL−1), XL-5366 (22 ngmL−1), XL-1646 (92 ngmL−1), XL-5162 (26 ngmL−1), and XL-184(100 ngmL−1)] (Fluorescence intensity *100).](/content/184/2021/14/7/img/10.1016_j.arabjc.2021.103206-fig4.png)
The chromatogram of the spiked serum sample. All of spiked concentrations were similar to concentrations of the analytes in cancer patients' serum samples [XL-1644 (115 ngmL−1), XL-5366 (22 ngmL−1), XL-1646 (92 ngmL−1), XL-5162 (26 ngmL−1), and XL-184(100 ngmL−1)] (Fluorescence intensity *100).
3.4.2 Analytical method’s sensitivity and linearity
To ascertain the relationship between the signal intensity and the analytes' concentrations, a blank serum sample was spiked with nine different concentrations of CZB and its four metabolites at the range of 20–700 ng.mL−1. Table 2 presents calibration parameters, i.e., slope, intercept, and determination coefficient values (r2), which were obtained by plotting the signal against the corresponding concentration (n = 5). Also, LOD and LOQ were calculated using standard deviation (SD) of the blank’s response (n = 5) and the calibration curve’s slope (Clapham, 2017).
Compound
Linear range(ng.mL−1)
Slop (ng.mL−1)
Intercept
r2
LOD (ng.mL−1)
LOQ (ng.mL−1)
XL-1644(M2a)
25–700
10.38 ± 4.7
28.13 ± 7.4
0.999
2.14
25
XL-184 (CBZ)
20–700
10.08 ± 6.1
134.18 ± 7.1
0.997
2.11
20
XL-1646 (M9)
25–700
10.57 ± 3.3
13.94 ± 8.6
0.999
2.44
25
XL-5162 (M19)
25–700
8.69 ± 4.1
100.99 ± 9.5
0.998
3.28
25
XL-5366 (M7)
30–700
8.85 ± 5.9
12.78 ± 10.9
0.998
3.69
30
3.4.3 Analytical method’s precision and accuracy
First, three concentrations of the analytes (50, 75, and 100 ng.mL−1) were used for spiking the blank serum samples. Then, to determine the accuracy, these standard solutions were added to serum samples, five times for each concentration (n = 5), and the accuracy was reported as the error (%) of the found concentrations of the analytes relative to their added concentrations. The recovery of analytes was calculated via dividing the value obtained from spiked samples by the added amount and then multiplying the resulting number by 100.
The inter- and intra-day precisions were evaluated applying the analysis of standard and spiked sample solutions. For this propose, three standard solutions and ten spiked sample solutions were used, and intra-day precision was calculated as the average of five individually prepared solutions for each analyte during two weeks. The obtained results showed high accuracy and high precision for all analytes, as listed in Table 3, and were in agreement with ICH guidelines (≤15%), suggesting that the proposed analytical method was applicable for routine analysis.
Compound
Founded (ng.mL−1)
Intra-day RSD% (n = 5)
Inter-day RSD% (n = 5)
XL-1644(M2a)
46.75
2.7
3.1
74.36
1.5
1.9
96.81
2.8
2.6
XL-184 (CBZ)
49.00
3.0
3.7
73.65
1.7
2.9
96.5
2.0
3.4
XL-1646 (M9)
48.85
1.6
3.6
73.20
2.0
3.4
98.30
3.5
4.0
XL-5162 (M19)
49.50
2.2
3.3
76.50
3.3
5.0
97.50
2.3
1.2
XL-5366 (M7)
48.00
2.3
3.0
69.97
2.9
3.2
95.60
1.0
1.7
3.4.4 Matrix effect factor and carryover
For assessing the carryover effect, serum samples were spiked with 100 ng.mL−1 of each analyte. As observed, analyte-free blank serum sample’s chromatogram did not show any signal in the area corresponding to the studied analytes. In order to avoid any interference, since ranges of concentrations had already been investigated, the remaining analyte residues in the needle were removed before injection.
3.4.5 Analytical method’s robustness
Robustness was evaluated by studying the vulnerability of the presented analytical method to variations of the analytical parameters which could affect separation and determination efficiencies of the method. To appraise robustness of the method, a standard solution of CZB and its serum metabolites (100 ng.mL−1; n = 5) was analyzed considering the values of three main variables including CTAB concentration, acetonitrile amount, and flow rate of the mobile phase. Table 4 summarizes variations observed in peak area and retention time of each analyte. As shown, low variations indicated that peak area and retention time remained constant during the analysis, which could prove high consistency of the proposed analytical method in determining and quantifying the analytes.
Parameter
Compound
Retention time (min) (RSD%)
Peak area (a.u) (RSD%)
The recommended condition
XL-1644(M2a)
4.9 ± 2. 2
620 ± 45 (3.1)
XL-184 (CBZ)
11.7 ± 1.3
734 ± 56 (2.8)
XL-1646 (M9)
20.1 ± 0.95
712 ± 74 (4.3)
XL-5162 (M19)
29.0 ± 0.64
670 ± 32 (3.8)
XL-5366 (M7)
33.7.0 ± 1.7
692 ± 48 (3.3)
Ratio of acetonitrile: CTAB: water 50:20:30
XL-1644(M2a)
5.2 ± 4.5
630 ± 45 (3.4)
XL-184 (CBZ)
11.2 ± 0.19
725 ± 56 (2.6)
XL-1646 (M9)
21.5 ± 2. 7
722 ± 74 (6.7)
XL-5162 (M19)
31.0 ± 0.16
685 ± 32 (2.8)
XL-5366 (M7)
35.1 ± 0.22
701 ± 48 (7.1)
Ratio of acetonitrile: CTAB: water 40:40:20
XL-1644(M2a)
5.9 ± 0.15
655 ± 45 (8.4)
XL-184 (CBZ)
13.3 ± 0.19
705 ± 56 (7.3)
XL-1646 (M9)
26.8 ± 0.14
736 ± 74 (2.7)
XL-5162 (M19)
29.8 ± 0.16
693 ± 36 (4.8)
XL-5366 (M7)
38.5 ± 0.32
688 ± 44 (1.7)
Flow rate 0.8 mLmin−1
XL-1644(M2a)
6.5 ± 0.15
706 ± 39 (3.9)
XL-184 (CBZ)
12.6 ± 0.11
711 ± 18 (6.3)
XL-1646 (M9)
24.7 ± 0.16
736 ± 70 (2.9)
XL-5162 (M19)
29.6 ± 0.24
693 ± 55 (7.8)
XL-5366 (M7)
36.7 ± 0.14
688 ± 40 (5.7)
3.5 Working solution’s stability
A blank serum sample was spiked with 100 ng.mL−1 of each analyte and kept in the dark at −20 °C. On consecutive days, the blank sample was thawed, used, and returned to the freezer for two weeks. No substantial decline in the peak area or alteration in the retention time was observed for each analyte. Therefore, all of analytes were found stable during this period. Thus, it was expected that under these conditions, the patients' extracted blood serum samples did not show any significant degradation problems.
3.6 Real sample analysis
The proposed analytical method was applied to serum samples spiked with the analytes at the concentrations close to those determined in cancer patients (Fig. 2). The considered concentrations were 115, 22, 92, 26, and 100 ng.mL−1 for XL-1644, XL-5366, XL-1646, XL-5162, and XL-184, respectively. These concentrations were respectively corresponding to 32%, 6%, 25.2%, 7%, and 27.2% of XL-1644, XL-5366, XL-1646, XL-5162, and XL-184 detected in patients' serum after one half-life of CBZ (Rai et al., 2005; Lacy et al., 2015). The gained results showed that the proposed method could cover all ranges of CBZ and its metabolites concentrations in serum. It is noteworthy that the maximum serum concentration of CBZ was in the range of 34.2–603 ng.mL−1 corresponding to the oral doses of 0.08–1.28 mg.kg−1 (Darwish et al., 2015). The obtained equations of calibration curve along with the corresponding r2 values for each analyte were as follows: XL-1644 (y = 10.377x + 28.128; r2 = 0.9994), XL-5366 (y = 8.8534x + 12.779; r2 = 0.9988), XL-1646 (y = 10.573x − 13.937; r2 = 0.9998), XL-5162 (y = 8.6903x + 100.99; r2 = 0.9981), and XL-184 (y = 10.082x + 134.18; r2 = 0.9972). In addition, some of the QC solutions were examined to ensure accurate detection and quantification of analytes in serum samples in bioequivalence and pharmacokinetics studies (Table 5).
Sample
XL1644 (M2a)
XL-184 (CBZ)
XL-1646 (M9)
XL-5162 (M19)
XL-5366 (M7)
QC1
50
50
50
50
50
T1
49.6 ± 0.05
49.6 ± 1.60
49.8 ± 1.58
49.8 ± 1.25
49.5 ± 0.03
T2
49.9 ± 0.06
49.7 ± 1.08
49.9 ± 2.51
49.7 ± 2.51
49.3 ± 0.07
T3
49.5 ± 1.06
49.4 ± 2.00
49.8 ± 3.16
49.5 ± 2.47
49.2 ± 0.08
T4
49.8 ± 2.01
49.8 ± 1.53
49.6 ± 2.12
49.3 ± 2.70
49.5 ± 0.01
T5
49.8 ± 0.09
49.5 ± 0.32
49.7 ± 0.64
49.5 ± 1.35
49.0 ± 0.06
QC2
75
75
75
75
75
T6
74.2 ± 1.04
74.8 ± 0.55
74.4 ± 1.77
74.9 ± 2.65
74.7 ± 2.33
T7
74.7 ± 2.03
74.8 ± 1.50
74.6 ± 2.11
74.7 ± 1.55
74.4 ± 1.95
T8
74.0 ± 1.05
74.5 ± 2.03
74.7 ± 2.26
74.1 ± 2.02
74.1 ± 3.04
T9
74.9 ± 0.07
74.2 ± 1.05
74.7 ± 1.50
74.4 ± 2.22
74.5 ± 2.65
T10
74.3 ± 0.17
74.6 ± 3.1
74.1 ± 2.36
74.5 ± 3.06
74.8 ± 1.24
QC3
100
100
100
100
100
T11
99.85 ± 3.02
99.8 ± 2.14
99.7 ± 3.02
99.7 ± 3.21
99.5 ± 0.25
T12
99.82 ± 0.80
99.9 ± 1.25
99.9 ± 0.98
99.8 ± 2.05
99.5 ± 1.34
T13
99.75 ± 0.28
99.2 ± 1.80
99.9 ± 1.53
99.6 ± 1.47
99.6 ± 2.05
T14
99.77 ± 0.85
99.6 ± 2.62
99.3 ± 2.01
99.9 ± 2.37
99.4 ± 1.27
T15
99.79 ± 0.47
99.7 ± 0.94
99.8 ± 2.51
99.4 ± 1.03
99.2 ± 2.56
3.7 Efficiency of the method
The efficiency data of the developed method in comparison with some other methods are summarized in Table 6. As seen, the developed method could be considered as a powerful, simple, and low-cost approach for determination of CBZ.
Method
LOD
LOQ
Linearity
Intra-day accuracy and precision (%)
Inter- day accuracy and precision (%)
Ref
LC-MS/MS
–
500 ng.mL−1
6–1500 ng.mL−1
3.5
3.87
(Aghai et al., 2021)
LC-MS/MS
0.32 ng.mL−1
0.97 ng.mL−1
1.0–100 ng.mL−1
3.37
2.61
(Abdelhameed et al., 2017)
Micelle-enhanced spectrofluorimetric
13.34 ng.mL−1
20 ng.mL−1
25–800 ng.mL−1
97.22
100.61
(Darwish et al., 2015)
LC-ESI-MS/MS
5 pg.(10 μL)−1
50 pg.mL−1
5.0–5000.0 pg.mL−1
1.95 to 2.37
2.93 to 9.3
(Inturi and Avula, 2018)
LC-MS/MS
–
7 µg.mL−1
75 to 5000 ng.mL−1
13.5
14
(Jolibois et al., 2019)
UPLC–MS/MS
–
99.9 µg.mL−1
100–5000 µg.mL−1
6.1
4.0
(Krens et al., 2020)
LC-MS/MS
–
0.50 ng.mL−1
0.500–5000 ng.mL−1
10.9
8.5
(Ren Lj et al., 2018;32(7):e4227.)
LC-MS/MS
–
0.5 ng.mL−1
0.5–1000 ng.mL−1
10.8
2.3
(Su et al., 2015)
UPLC–MS/MS
–
1.5 ng.mL−1
5–5000 ng.mL−1
4.3
2.3
(Wang et al., 2015)
MLC-FL
2.11 ng.mL−1
7.04 ng.mL−1
20–700 ng.mL−1
3.0
3.7
This study
4 Conclusion
A novel, highly sensitive, simple, and rapid approach was successfully developed and validated for the quantification of CBZ and its main metabolits (i.e., EXEL-1646, EXEL-5162, EXEL-5366, and EXEL-1644) in spiked human serum samples using the MLC method. A solution of CTAB (0.2 mol.L−1) was used to enhance both separation efficiency and fluorescence intensity. In comparison with previously reported methods (i.e., LC-MS/MS (Su et al., 2015) and micelle-enhanced spectrofluorimetry (Darwish et al., 2015) the proposed method for determination of serum concentrations of CBZ and its metabolites showed better LOD and LOQ values. Hence, it was concluded that this novel analytical method could be used for efficient analysis of CBZ and its four main metabolites in human serum samples, especially in hospital laboratories, mainly due to its noticeable sensitivity, simplicity, speedy operation (∼35 min), reproducibility, and cost effectiveness.
Consent for publication
All authors agreed and declared the consent for publication.
Funding
None.
Acknowledgements
The authors gratefully acknowledge the China Postdoctoral Science Foundation research grant NO. 2020M672291.
Declaration of Competing Interest
None.
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