Abstract
ASP3026 is a recently formulated and highly selective inhibitor designed to target the ALK kinase. ASP3026 efficiently inhibited ALK kinase activity and demonstrated superior selectivity at a panel of Tyr-kinases compared to crizotinib. The target of this investigation was to establish a highly accurate, fast, green, and highly sensitive Ultra-high performance liquid chromatography- Tandem mass spectrometry (UHPLC-MS/MS) technique for assessing the concentration of ASP3026 in human liver microsomes (HLMs). In vitro incubation, the metabolic stability of ASP3026 in HLMs was evaluated using this known approach. The validation steps for the UHPLC-MS/MS analytical technique in the HLMs were performed along with the bio-analytical method validation guidelines settled by the US-FDA. To increase the ecological sustainability of the current UHPLC-MS/MS system, a lower flow rate of 0.3 mL min−1, a shorter elution duration of 1 min, and a reduced consumption of ACN have been implemented. A screening of the chemical structure of ASP3026 for hazardous alerts and metabolic lability was performed by the StarDrop package, that includes the DEREK and P450 modules. The analytical separation of ASP3026 and fenebrutinib (FNB) on the reversed phase Eclipse Plus C18 column was performed using an isocratic mobile phase approach. The calibration curve produced by the ASP3026 showed a linear association over the level range of 1–3,000 ng mL−1. A study was conducted to evaluate the precision and accuracy of UHPLC-MS/MS technology in evaluating both intra-day and inter-day variations. The accuracy exhibited a range of −1.56%–7.33% across various days, and a range of −0.78%–10.66% within the same day. The ASP3026 underwent in vitro half-life and intrinsic clearance measurements, yielding values of 14.32 min and 56.62 mL min−1 kg−1, correspondingly. According to in silico software research, using minor modifications to the piperazine component or substituting the group in drug design has the potential to improve the metabolic safety and stability of novel derivatives in comparison to ASP3026.
1 Introduction
Lung cancer is the most often identified type of cancer and the major cause of cancer-related deaths universal [1]. As evidenced by long-term survival and the low rates of response, the effectiveness of conventional anticancer drugs in treating metastatic or advanced non-small cell lung cancer (NSCLC) remains insufficient [2]. Novel investigations into the molecular mechanisms of cancer development have recognized numerous cancer-promoting factors, including receptor tyrosine kinases (TK). These drivers have been employed in the advancement of targeted therapies for a number of forms of cancer, as well as NSCLC [3–6]. These medications have greatly increased the rates of treatment response in individuals with cancer and have substantially elevated their chances of survival. Furthermore, they signify a profound shift in the approach to cancer therapy.
The EML4-ALK gene is a fusion product resulting from the condensation of segments from the EML4 and ALK genes. Initially, it was identified in a certain subset of NSCLC cells [7, 8]. ALKoma, a term used to describe tumors with mutations in the ALK gene that significantly contribute to their growth, has been recognized in other types of cancer other than NSCLC [9]. In anaplastic large cell lymphoma, the oncogenicity of ALK is attributed to the formation of fusions with other proteins, including nucleophosmin (sometimes referred to as nucleolar phosphoprotein B23 or numatrin).
Crizotinib, a first-generation ALK TK inhibitor (TKI), has extensively revealed efficacy in the treatment of NSCLC with ALKr [10–12]. Thus, as per the National Comprehensive Cancer Network guideline [13], it is presently the suggested first therapy for advanced ALKr-positive NSCLC. Nevertheless, a subgroup of patients may progress resistance to crizotinib in 8–12 months of receiving therapy. The primary mechanisms by which resistance arises include the augmentation of the oncogenic fusion gene, acquisition of mutations in the ALK TK domain, activation of alternative signaling pathways, and manifestation in the CNS [14–16]. The more recent ALK TKIs, ceritinib [17] and alectinib [18], have shown effectiveness in treating patients with ALK-positive NSCLC who have not received crizotinib therapy, as well as in patients who have established resistance to crizotinib.
ASP3026 (Fig. 1) is an orally available, novel, targeted, and ATP-competitive second-generation ALK TKI. The pharmaceutical compound ASP 3026, now under development by Astellas Pharma in Japan, demonstrates anticancer properties against various solid tumours, including NSCLC [19]. The initial report of ASP3026 was published in 2011 [19, 20]. Furthermore, additional preclinical and clinical findings have been shown [19, 21]. ALK, TK non-receptor 1, ROS proto-oncogene 1, TK non-receptor 2, receptor TK, discoidin domain receptor TK 1, Src family TKs, fyn-related Src family TK, and receptor TK family are among the kinases on which ASP3026 demonstrates inhibitory effects. These findings contrast with the crizotinib kinase inhibition profile [19]. The most common symptoms reported were decreased appetite (34.5%), mild or severe fatigue (31.0% of the sample), and nausea (27.6%) [21].
The chemical structure of ASP3026 and fenebrutinib (Internal standard)
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01282
After conducting an extensive examination of the existing scholarly literature, it was found that there are no documented studies that have explicitly examined the quantification of ASP3026 in HLMs for the target of assessing its metabolic stability. Hence, it is imperative to establish an approach employing UHPLC-MS/MS that is characterized by its exceptional sensitivity and rapidity. Implementing this approach is crucial for precisely determining the quantity of ASP3026 in different matrices. The target of the current investigation was to develop a fast (completed within 1 min), very sensitive (capable of detecting low concentrations, 1 ng mL−1, in the HLMs matrix), and specific analytical technique for determining the metabolic stability of ASP3026 in the HLMs matrix. Further, the outcomes were confirmed by in vitro incubation tests and an in silico software screening using StarDrop P450 package (version 6.6) developed by Optibrium Ltd. (Cambridge, MA, USA).
In the present work, the UHPLC-MS/MS approach was employed to evaluate the intrinsic clearance (Clint) and the in vitro half-life (t1/2) of ASP3026 [22]. The above-mentioned numbers could then be used to estimate the rate of metabolism in living organisms using three models: venous equilibrium, parallel tube, and dispersion. The in vitro t1/2 and Clint values of ASP3026 were calculated using the well-stirred model in an in vitro laboratory setting [23, 24]. This definite model is mostly employed in investigations on drug metabolism due to its inherent simplicity. The current studies have shown that the ASP3026 exhibits a quite fast metabolic rate, resulting in a modest period of biological activity and a favorable bioavailability in living organisms. The Derek program was utilized to analyze structural features in the chemical structure of ASP3026, with the target of verifying the proposed hypothesis about its metabolic stability in a computer simulation, which corresponded to the results obtained from in vitro metabolic stability studies [25–29].
2 Materials and methods
2.1 Materials
All solvents utilized in the UHPLC-MS/MS method were of HPLC grade. The HPLC-grade H2O was attained in-house via a Milli-Q filteration apparatus produced by Millipore company (Billerica, MA, USA). The produced ultrapure water exhibits a resistivity of 18.2 MΩ cm at 25 °C, total organic carbon (TOC) levels of ≤2 ppb, absence of particles larger than 0.22 µm, and pyrogens (endotoxins) below 0.001 EU/mL. The investigation utilized reference powders, ASP3026 and fenebrutinib (FNB), which were of analytical grade. The analyte ASP3026 (Cat. No.: HY-13326) and the fenebrutinib (GDC-0853), internal standard (IS), were obtained from MedChem (Princeton, NJ, USA) with absolute purities of 99.73% and 99.15% respectively. The HLMs (20 mg mL−1) that were purchased from Sigma-Aldrich company, were stored in a refrigerator kept at −78 °C until they were set for use. The chemicals ammonium formate (NH4COOH), acetonitrile (ACN), formic acid (HCOOH), and HLMs were acquired from Sigma-Aldrich company, a well-established commercial entity (St. Louis, MO, USA).
2.2 UPLC-MS/MS instrumental parameters
The successful separation of the ASP3026 and FNB peaks by chromatography was achieved using the Acquity UPLC instrument, namely the UPH model with SN: H10UPH. Quantification of mass ions of ASP3026 and FNB was performed using the Acquity triple quadrupole (TQD) MS instrument, which is particularly designated by the model code TQD and SN: QBB1203, following the purification and extraction from the incubated HLMs matrix. As shown in Table 1, the UHPLC-MS/MS configurations were fine-optimized to achieve excellent resolution and optimum sensitivity of the ASP3026 and FNB chromatographic peaks. So as to attain good and sensitive separation of the analytical peaks of ASP3026 and FNB, the HPLC system was accurately adjusted. The alterations included changes in the components of the mobile phase, the pH, and the polarity of the stationary phase, as shown in Table 1. The molecules ASP3026 and FNB were separated using a ZORBAX C18 column (Eclipse Plus; 2.1 × 50 mm, 1.8 µm) obtained from Agilent Technologies Corporation (Santa Clara, CA, USA) under controlled conditions using Zorbax C18 (Eclipse Plus; 2.1 × 5 mm, 1.8 μm) as a guard column. An aqueous solution containing 15% (0.1% HCOOH in H2O) with a pH of 3.2 constituted mobile phase A. The second phase of the mobile component comprised 85% ACN and was controlled at a flow rate of 0.3 mL min−1. When testing a solution containing NH4COOH (10 mM) with a pH more than 3.2, it was observed that there was a phenomena of peak tailing and a prolonged running time of the ASP3026 analysis. Furthermore, when the concentration of ACN surpasses 85%, it becomes impractical to separate the analytical peaks of ASP3026 and FNB. Conversely, a lower ratio led to a prolonged elution times.
UHPLC–MS/MS system configurations
| UHPLC | MS/MS | ||
| Mobile phase (Isocratic) | 0.1% HCOOH in H2O (15%; pH: 3.2) | ESI | Positive mode |
| 85% ACN | Cone gas (100 L/H) | ||
| Injection volume: 5.0 μL | RF lens voltage (0.1 V) | ||
| 0.3 mL min−1. | Capillary voltage (4 KV) | ||
| Eclipse plus-C18 column | T: 23.0 ± 2.0 °C | Extractor voltage (3.0 V) | |
| Length: 50.0 mm | Nitrogen (350 °C; 100 L h−1) | ||
| PS: 1.8 μm | Mode | MRM | |
| i.d.: 2.1 mm | Collision cell | Argon gas (0.14 mL min−1) | |
An optimization of the mass spectrometric settings of the TQD MS analyzer was conducted to improve the sensitivity in detecting chromatographic peaks of ASP3026 and FNB. The ESI source was used in the positive ionization method to augment ionization by using the nucleophilic properties of nitrogen atoms in the targets (ASP3026 and FNB), that can capture protons and produce positively charged ions. Evaporation of the charged droplets of the isocratic mobile phase was performed in the ESI source by the aid of nitrogen gas provided by a nitrogen generator from Peak Scientific company (Scotland). The UHPLC-MS/MS instrument was operated using the MassLynx software suite, namely Version 4.1 with the SCN 805 designation. Two main components of the MassLynx software suite are QuanLynx and IntelliStart®. Analysis of the acquired results was conducted using the QuanLynx module. The MS calibration of ASP3026 (C29H40N8O3S) and FNB (C37H44N8O4) was performed using the IntelliStart® program. The calibration was conducted by promptly introducing the solutions at 10 μg mL−1 of ASP3026 and FNB into the isocratic mobile phase stream, employing a combination function methodology. A Multiple Reaction Monitoring (MRM) experiment was conducted using the detection capabilities of the TQD mass analyzer to quantify ASP3026 and FNB. The objective of this study was to rise the specificity and responsiveness of the current UHPLC-MS/MS methodology. In the collision cell of the TQD of the MS/MS analyzer, the analyte ions ASP3026 and FNB underwent fragmentation when reacting with the collision gas (99.999% argon gas). As a consequence of this fragmentation, several daughter ions were formed. The mass transition duration for ASP3026 and FNB, from precursor ions to daughter ions, was measured to be 0.025 s. Table 2 exhibits the MRM characteristics and mass transition profiles for ASP3026 and FNB (IS).
MRM-adjusted features for the assessment of ASP3026 and FNB
| Time (min) | Retention time (min) | Targets | MRM configurations | |
| Mass spectra segments | 0.0–0.6 | ASP3026 (0.47) | Analyte ASP3026 | 581.33→ 481.22 |
| CEa: 28 and CVb: 46 | ||||
| 581.33→ 421.20 | ||||
| CE: 36 and CV: 46 | ||||
| 0.6–1.0 | FNB (0.73) | : Fenebrutinib (FNB, IS) | 665.3 → 236.2 | |
| CV: 58 and CE: 58 | ||||
| 665.3 → 647.4 | ||||
| CV: 58 and CE: 26 |
a Collision energy, b cone voltage.
2.3 Metabolic lability assessment of ASP3026 using P40 software
as kw is the water formation rate constant and Ktotal is the sum of the rates for all potential sites of metabolism.
This study examines the CSL in both the P450 column dataset and the metabolic landscape. This evaluation measures the efficiency of the procedure used to generate the metabolite in the CYP3A4 metabolic cycle. A lower CSL score reveals a higher probability of enhanced stability.
2.4 In silico toxicity prediction of ASP3026 using DEREK toxicity software
The DEREK model was used to assess the proposed alerts of toxicity of ASP3026 The algorithm was used to characterize structural alerts related to ASP3026, with the target of proposing structural interventions that might alleviate the detected toxicity alert [32].
2.5 ASP3026 and FNB working dilutions
Dimethyl sulfoxide (DMSO) is the solvent used to dissolve both ASP3026 and FNB. In order to dissolve ASP3026 at 20 mg mL−1 (34.44 mM), ultrasonic treatment is required. In contrast, FNB is soluble at 23 mg mL−1 (34.60 mM). Thus, stock solutions of ASP3026 and FNB were prepared in DMSO at 1 mg mL−1 each. ASP3026 working solutions were generated at concentrations of 1 μg mL−1, 10 μg mL−1, and 100 μg mL−1, while the FNB stock solution was specifically prepared at 10 μg mL−1. To prepare the solutions, the initial stock solutions of ASP3026 and FNB, each at 1 mg mL−1, were diluted with the optimal mobile phase. A total of four quality controls (QCs) and seven calibration standards (CSs) for ASP3026 were developed utilizing the WKs.
2.6 Preparation of ASP3026 CSs and QCs
Before validating the present UHPLC-MS/MS approach, HLMs were made inactive by 2% DMSO, an organic solvent. The deactivation procedure was accompanied for 5 min at a temperature of 50 °C. The purpose of the deactivation stage was to mitigate the probable impact of HLMs on the levels of the analytes, ASP3026 and FNB, by blocking their metabolic pathways [33–35]. A specialized matrix was developed specifically for high-throughput screening evaluations to determine the metabolic stability of ASP3026. This approach involved combining 30 µL of HLMs (inactive) with a metabolic buffer consisting of a 0.1 M sodium phosphate solution at pH 7.4, together with MgCl2 (3.3 mM) and 1 mM NADPH (enzyme cofactor). The dilution step was conducted to replicate the circumstances that happen in the in vitro incubation of ASP3026 and HLMs. To decrease the potential influences of matrix dilution in the in vitro incubation investigation, the concentration matrix of HLMs was systematically upheld more than 90% for the whole experiment. QCs were used as demonstrative samples for indeterminate values. The level of these QCs were determined using the regression equation attained from the synchronized injection of ASP3026 CSs substances. To prepare each ASP3026 CS and QC sample, 1 mL of the sample was combined with 100 µL of FNB WK solution. The FNB WK solution, with a 10,000 ng mL−1, used as the IS.
2.7 Extraction of ASP3026 and FNB
Effective separation of ASP3026 and FNB from the HLMs matrix was achieved by the protein precipitating method. The present approach utilized ACN, an organic solvent, to induce protein precipitation and halt metabolic processes within the HLMs matrix. Accordingly, a 2 mL volume of ACN was added to the ASP3026 QCs and CSs. The materials were exposed to incessant agitation for 5 min to remove ASP3026 and FNB from the sediment, namely the proteins. Centrifugation was conducted for 12 min at 4 °C using a thermostatic centrifuge operating at 14,000 rpm. The centrifugation process was crucial for the partitioning of proteins and the purification of the supernatants. Each sample was subjected to filtration using a 13 mm syringe filter (0.22 µm, Nylon) to verify their suitability and reliability for mass spectrometric analysis. Following purification, the extracts were transferred into individual vials within the UHPLC-MS/MS system. In order to settle that the HLMs matrix does not influence the retention time of ASP3026 and FNB, negative and positive controls, both, were acquired using the standard approach. The experimental positive control comprised a sample containing FNB within the HLMs. An ASP3026 calibration curve was constructed by plotting the theoretical values of ASP3026 on the x-axis against the ratio of the peak areas of ASP3026 to FNB on the y-axis. The linearity range of ASP3026 CSs was determined by assessing several analytical validation properties and application of the linear regression equation (y = ax + b; r2) utilizing the UHPLC-MS/MS approach.
2.8 Validation of the established UHPLC-MS/MS methodology
The precision, extraction recovery, linearity, accuracy, matrix effect, sensitivity, stability, and specificity of the present UHPLC-MS/MS method were assessed to validate its performance. This validation method followed the phases of analytical technique validation stated in the FDA guidelines [36, 37].
2.8.1 Specificity
To evaluate the specificity of the new UHPLC-MS/MS method, six groups of blank HLMs samples were injected using the extraction methodology. The pure extracts were subjected to UHPLC-MS/MS analysis to determine if any intervention was observed with the analytical peaks of ASP3026 or FNB at the same retention time as the surrounding matrix. The collected data was then compared to spiked HLMs matrix which contained the particular analytes ASP3026 and FNB. To alleviate the carryover influence of the targets ASP3026 and FNB in the TQD analyzer, the MRM mode was employed. Validation was attained by analyzing the negative control HLMs, that did not contain ASP3026 and FNB, and then observing the resulting consequences.
2.8.2 Linearity and sensitivity
To assess the linearity of the UHPLC-MS/MS approach, the coefficient of variation (r2) was computed by the least squares methodology (y = ax + b).
2.8.3 Precision and accuracy
2.8.4 Matrix effect and extraction recovery
An investigation was made to assess the value of the ASP3026 extraction technique from the HLMs matrix and the influence of the HLMs matrix on the ionization capabilities of ASP3026. This was attained by introducing four QCs into the UHPLC-MS/MS system. The value of protein precipitation as the chosen extraction methodology for ASP3026 and FNB was determined by treating six sets of four QCs in the HLMs and comparing them with 4 other QCs generated in the mobile phase (A). The extraction recoveries of ASP3026 and FNB were estimated using the ratio of B to A and applying a multiplication factor of 100.
2.8.5 Stability
The objective of this study is to evaluate the stability of ASP3026 in the HLMs by measuring its concentration at two specific QC tiers (HQC and LQC). The inquiry aimed to validate the dependability and robustness of the UHPLC-MS/MS method at different storage circumstances. Five repetitions of the assessment were performed under several laboratory situations, comprising short and long-term storage, storage in the auto-sampler, and subjecting the samples to 3 cycles of thawing and freezing. The freeze-thaw stability of the HQC and LQC was assessed through 3 freeze-thaw cycles. Throughout each cycle, the samples were subjected to cryogenic freezing at a temperature of −80 °C. The samples were subsequently thawed at room temperature before proceeding with further dispensation. The HQC and LQC samples go through analysis and processing following an 8-hour storage period at room temperature on the tabletop, being conducted as part of short-term stability studies. Prior to the mass spectrometric measurement, it was crucial to freeze the spiked HLMs matrix at −80 °C for a duration of 3 months in order to evaluate its long-term stability. The stability of the HQC and LQC samples in the auto-sampler was assessed by subjecting them to a 24-hour storage period at 10 °C prior to use in the UHPLC-MS/MS system.
2.9 In vitro assessment of the ASP3026 metabolic stability
The in vitro t1/2 of ASP3026 was determined by analyzing the residual abundance of ASP3026 following in vitro metabolism, as well as Clint. In order to accomplish this, an active HLMs matrix including MgCl2 and NADPH (an enzyme cofactor) was established. The metabolic incubation reaction was conducted in vitro in four consecutive steps. Initially, 1 µL of ASP3026 was mixed with metabolic HLMs matrix and left to incubate. The procedure was conducted at 37 °C for 10 min using a water bath that was fine-tuned to sustain the required temperature. First, a concentration of one mM of NADPH was loaded into each sample. Then, the samples were reintroduced into a temperature-regulated moving water bath, configured at 37 °C. For the third stage, a 100 µL solution of FNB (1,000 ng mL−1) was added immediately before the ACN, that served as a solvent to restore equilibrium to the reaction. In order to maintain a constant level of the IS and minimize the effect of metabolic processes on its level, this approach was adopted. During the fourth stage, known as halting the metabolic process, 2 mL of ACN was added into each sample at prescribed time intermissions: 0, 2.5, 5, 7.5, 15, 20, 30, 40, 50, and 60 min. To commence the extraction procedure of ASP3026 and FNB, excess proteins were isolated by precipitation after halting the enzyme reaction, as outlined in Section 2.7. An identical procedure was used to conduct a negative control incubation of ASP3026 with HLMs in the absence of NADPH. The aim of this investigation was to evaluate the impact of incubation conditions or matrix components on the concentration of ASP3026 in in vitro experiments.
3 Results and discussions
3.1 In silico determination of ASP3026 metabolic lability
Based on the metabolic profile of ASP3026, the metabolic stability of the active parts in the chemical structure of ASP3026 concerning CYP3A4 enzymes was estimated, as shown in the pie chart [41–43]. Figure 2 illustrates the metabolic lability of ASP3026, which was determined using the CSL value of 0.9978. The metabolic lability of ASP3026 was evaluated by exposing it to the active HLMs matrix and utilizing the well-established UHPLC-MS/MS approach for analysis. The major enzyme instability of ASP3026 is commonly credited to the N-methyl piperazine ring, chiefly at positions C41 (92%), C37 and C39 (5%), and C36 and C40 (1%). Figure 2 illustrates the robust association between the CSL score (0.9978) and the metabolic lability. These results align with the conclusions drawn from the in vitro metabolic experiments, that will be presented in more detail in the following sections.
The CSL score of 0.9978 reveals that ASP3026 has a higher degree of lability susceptibility to metabolic enzymes
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01282
3.2 DEREK screening for ASP3026 toxicity alerts
To evaluate the toxicity of ASP3026, in silico analysis was conducted using the DEREK software (Fig. 3). ASP3026 has structural features indicative of skin sensitization (potential) caused by ortho or para amino or hydroxyl aniline. Furthermore, it demonstrates the potential inhibition of the HERG channel induced by the HERG pharmacophore I. Existing studies have established that the piperazine ring is accountable for both the toxic effects (Fig. 3A) and the instability of metabolic processes (Fig. 3B). Modifying or substituting the piperazine ring in drug design can enhance the safety properties and metabolic stability of new derivatives in comparison to ASP3026 (Fig. 3).
(A) The DEREK program has proposed structural alerts of ASP3026, which are highlighted in red. (B) The P450 lability at C41 (92%) suggests that the high metabolic instability of ASP3026
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01282
3.3 UHPLC-MS/MS approach
The work investigated distinct stationary phases that exhibited both nonpolar and polar characteristics. In this study, the stationary phases employed were a polar imidazole column (1.8 µm, 2.1 × 50 mm) supplied by Sepax Technologies (Newark, DE, USA), and a HILIC column (DIOL) manufactured by Fortis™ Technologies Ltd. in Neston, UK. However, the normal phase columns that used in this work, met challenges in efficiently isolating and retaining ASP3026 and FNB. The results obtained from the reversed C18 stationary phase column were the most beneficial. Insufficient separation of ASP3026 and FNB was seen in the C8 reversed column used in the UHPLC-MS/MS approach, resulting in analytical peak tailing, prolonged retention times, and the merging of base chromatographic peaks. The ZORBAX Eclipse Plus C18 reversed column, produced by Agilent Technologies with dimensions of 95 Å, 2.1 × 50 mm, and 1.8 µm, demonstrated exceptional presentation in terms of both analytical peak shape and elution time. Determination of ASP3026 and FNB was attained using the UHPLC-MS/MS technique with an analytical apparatus that maintained a consistent mobile phase composition at 0.3 mL min−1 for 1 min. The calibration curve established for ASP3026 exhibited a linear relation in the span of 1–3,000 ng mL−1. Utilizing the MRM detection mode, the UHPLC-MS/MS analytical method enhanced sensitivity in identifying and estimating ASP3026 and FNB. The aim of this approach was to reduce any probable disturbance by matrix constituents present in HLMs, as seen in Fig. 4.
MRM mass spectrum of ASP3026 [M+H]+ (A) and FNB [M+H]+ (B). The proposed dissociation behaviours are presented
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01282
For three specific reasons, the concentration of ASP3026 was determined using analytical UHPLC-MS/MS technology with the FNB serving as the IS. Purification of ASP3026 and FNB via protein precipitation is a very effective method, leading to a substantial increase in the yield. The ASP3026 yield was statistically calculated to be 101.91% with a CV of 2.71%, whilst the FNB yield was found to be 100.73% with an CV of 4.44%. The chromatographic peaks for the ASP3026 target (0.47 min) and FNB target (0.73 min) were efficaciously attained in less than 1 min. The results demonstrate that the UHPLC-MS/MS method used in the current investigation experiment is a very efficient analytical instrument that allows for rapid separation. By reducing the duration of the process to a mere 1 min and using a lower ratio of ACN, this technology greatly promotes ecological sustainability. Furthermore, it is crucial to recognize that ASP3026 and FNB were not simultaneously assigned to the same person. The present UHPLC-MS/MS technology is suitable for monitoring therapeutic drug concentrations and investigating the pharmacokinetics of ASP3026 circulating in the body. MRM chromatograms of HLMs controls for ASP3026 exhibited a minor degree of carry-over, as shown in both the negative (Fig. 5A) and positive (Fig. 5B) samples.
(A) The negative control sample revealed no chromatographic peaks at ASP3026 and FNB retention times. (B) The MRM chromatogram of the ASP3026 HQC (2,400 ng mL−1) at 0.47 min and FNB (1,000 ng mL−1) at 0.73 min
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01282
3.4 Validation parameters of the UHPLC-MS/MS method
3.4.1 Specificity
Successful differentiation of the analytical peaks of ASP3026 and FNB, as shown in Fig. 5, confirmed the validity of the UHPLC-MS/MS approach. The analytical peaks of ASP3026 and FNB did not reveal any interaction with the matrix constituents of the HLMs. An insignificant residual effect of ASP3026 was observed in the negative control samples.
3.4.2 Linearity and sensitivity
The range of linearity of the UHPLC-MS/MS method was determined as 1–3,000 ng mL−1. In order to accomplish this objective, we included seven ASP3026 CSs into the HLMs matrix and then analyzed them as unmeasured variables. A linear regression equation is given by y = 0.3619x + 0.628, with a r2 of 0.9973. The calibration curve for ASP3026 was made by employing statistical weighting, specifically by utilizing the 1/x ratio, to appropriately address the substantial inconsistency seen in the CSs. The CVs of the 6 replications of CSs were calculated to be lower than 7.67%, as shown in Table 3. Figure 6 illustrates the previously determined LOD and LOQ at 0.33 ng mL−1 and 1.1 ng mL−1, correspondingly.
A brief summary of the results obtained from back-calculation for six repeats of ASP3026 (CSs)
| ASP3026 (ng mL−1) | Average | SD | Accuracy (% E) | Precision (CV) | Recovery (%) |
| 1.00 | 1.08 | 0.05 | 7.67 | 4.19 | 107.67 |
| 15.00 | 15.36 | 0.41 | 2.42 | 2.64 | 102.42 |
| 50.00 | 51.00 | 1.70 | 1.99 | 3.34 | 101.99 |
| 200.00 | 202.94 | 3.16 | 1.47 | 1.56 | 101.47 |
| 500.00 | 502.52 | 10.62 | 0.50 | 2.11 | 100.50 |
| 1500.00 | 1491.83 | 14.72 | −0.54 | 0.99 | 99.46 |
| 3000.00 | 2995.46 | 8.72 | −0.15 | 0.29 | 99.85 |
| % Recovery | 101.91 ± 2.76 |
(A) The ASP3026LOQ (1 ng mL−1). (B) The FNB (IS) peak (1,000 ng mL−1)
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01282
3.4.3 Precision and accuracy
The precision and accuracy of the UHPLC-MS/MS method were assessed by conducting 12 independent sets, each consisting of 4 quality controls, in a 24-h period. Furthermore, 6 sets of QCs, each consisting of 4 samples, were carried out uninterrupted for 3 consecutive days. The observed outcomes met the validation standards set by the FDA [38]. As shown in Table 4, the UHPLC-MS/MS technique exhibited accuracy and precision values ranging from −0.78% to 10.66% and −1.56% to 7.33%, respectively.
Precision and accuracy of the settled UHPLC-MS/MS approach
| ASP3026 (ng mL−1) | Intra-Day | Inter-Day | ||||||
| QCs | 1 | 3 | 900 | 2400 | 1 | 3 | 900 | 2400 |
| Mean | 1.08 | 2.98 | 905.33 | 2408.11 | 1.07 | 2.95 | 891.46 | 2392.10 |
| SD | 0.05 | 0.13 | 8.71 | 10.66 | 0.04 | 0.09 | 1.75 | 7.09 |
| Precision (CV) | 4.19 | 4.33 | 0.96 | 0.44 | 3.27 | 3.07 | 0.20 | 0.30 |
| % Accuracy | 7.67 | −0.78 | 0.59 | 0.34 | 7.33 | −1.56 | −0.95 | −0.33 |
| Recovery (%) | 107.67 | 99.22 | 100.59 | 100.34 | 107.33 | 98.44 | 99.05 | 99.67 |
3.4.4 The HLMs ME and extraction recovery of ASP3026
The efficacy of the protein precipitation extraction approach in the UHPLC-MS/MS methodology was evaluated by analyzing 6 repeats (4 QCs) in the HLMs and comparing them with other repeats of QCs detected in the mobile phase. The data revealed a substantial extraction recovery rate for ASP3026, with a mean of 101.95% and a SD of 3.85% (CV less than 3.78%). The mean extraction recovery rate of FNB was 102.35%, with a SD of 7.27% (CV below 7.1%). An analysis of two sets of loaded samples of HLMs matrix showed no significant effect on ion generation from either ASP3026 or FNB species. Group 1 was created by the combination of ASP3026 at 3 ng mL−1 with FNB at 1,000 ng mL−1. Group 2 was created by exchanging the metabolic HLMs matrix in place of the mobile phase. The HLMs, consisting of ASP3026 and FNB, had ME values of 101.23 ± 2.85% and 103.63 ± 2.75%, correspondingly. The NME of the IS was 0.97, falling within the acceptable threshold specified by FDA regulations. The findings of the investigation show that the HLMs matrix has a minimal impact on the degree of FNB or ASP3026 ionization.
3.4.5 ASP3026 exhibits exceptional stability in both HLMs matrix and the stock solution
ASP3026 exhibited maximum stability when preserved in DMSO at −80 °C for 28 days. This stability was noted in both the original solution and when subjected to HLMs for the entire incubation period. The statistical results showed that the CV for all ASP3026 samples was lower than 3.70% in diverse storage circumstances, as listed in Table 5. The level of ASP3026 exhibited remarkable stability across various storage circumstances, encompassing long-term storage, three freeze-thaw cycles, auto-sampler, and short-term storage. The obtained data indicate that ASP3026 shown remarkable stability.
The stability validation features of ASP3026
| Features | Mean | SD | Precision (CV) | Accuracy (% E) | ||||
| Freeze–thaw Stability | 3.07 | 2385.87 | 0.01 | 8.12 | 0.38 | 0.34 | 2.44 | −0.59 |
| Auto-sampler Stability | 3.11 | 2420.83 | 0.05 | 2.75 | 1.47 | 0.11 | 3.67 | 0.87 |
| Long-term Stability | 3.07 | 2385.87 | 0.11 | 8.12 | 3.70 | 0.34 | 2.33 | −0.59 |
| Short-term Stability | 3.08 | 2409.09 | 0.04 | 12.88 | 1.31 | 0.53 | 2.78 | 0.38 |
3.5 Assessing the UHPLC-MS/MS greenness using the AGREE software
A sustainability evaluation of the ecological impact of the present UHPLC-MS/MS approach, referred to as “greenness,” was conducted by the in silico software AGREE. This program incorporates all twelve of the criteria set by the GAC organization [44]. The software employs a spectrum of mathematical values (0.0–1.0) to evaluate several characteristics of the GAC system, therefore initiating numerical scales that measure the degree of the current approach greenness. The data are graphically exhibited by a circular geometry that encompasses a diverse range of colors, ranging from red to dark green, symbolizing 12 different features. The ecological aspect of the UHPLC-MS/MS system being deployed is illustrated in Fig. 7. The ratings corresponding to each of the 12 attributes are presented in Table 6. The value of 0.76 was obtained by evaluating many characteristics related to the present research approach. The reading acts as a specific standard for evaluating the degree of ecological sustainability achieved by the UHPLC-MS/MS system. An evaluation value below 1.0 shows a higher level of ecological sustainability in the analytical methodology. The newly enhanced UHPLC-MS/MS method demonstrates a significant level of environmental accountability, as designated by eco-scale measurements in the range from 0.75 to 1.00.
The AGREE software results are displayed in a circular diagram represent 12 separate characteristics
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01282
A report for the UHPLC-MS/MS system showing its impact on the environment. The study uses individual scores based on the ethics listed in the GAC commendations. The marks are exhibited in a circular format, showcasing a varied array of colors. The color spectrum ranges from 0.0, indicating no green, to 1.0, reflecting the highest level of green intensity. Each of the colors specified above is related to twelve separate features, that are detailed in the adjacent table
 |
3.6 In vitro metabolic stability investigation of ASP3026 with HLMs matrix
No noteworthy decrease in the level of ASP3026 was measured in the control sample (negative control). The metabolic stability of ASP3026 was assessed in laboratory experiments using HLMs at 1 μM mL−1 under controlled conditions. To provide a clear linear correlation among the rate of ASP3026 metabolism and the time of in vitro incubation, this level was intentionally chosen to be less than the Michaelis-Menten constant. An HLMs protein concentration of 1 mg mL−1 was utilized to reduce non-specific protein binding. The initial metabolic stability curve for ASP3026 is depicted in Fig. 8A. The x-axis measures the duration of time points for stopping of metabolic process, in the range of 0–60 min. The vertical axis in the graph corresponds to the equivalent residual ratio of ASP3026. A specific linear segment was selected from the previous curve for the time points in the range of 0–30 min. The target of the previous steps was to establish a curve that shows the correlation between the LN of the residual ratio of ASP3026 and the intermittent pauses in the incubation time within the interval of 0–30 min. This trend is clearly seen in Fig. 8B. Based on the investigation, the estimated slope of the ASP3026 metabolic rate was 0.0484. These results were derived using the linear equation y = −0.0484x + 4.589, which exhibited a strong correlation with a r2 value of 0.9996 (Table 7). The in vitro t1/2 can be calculated by splitting the logarithm of 2 by the slope. The t1/2 of ASP3026 in an in vitro HLMs incubation was determined to be 14.32 min. The Clint value of ASP3026 was calculated to be 56.62 mL min−1 kg−1. ASP3026 is a pharmaceutical compound with a significant metabolic rate, as evidenced by the scaling method devised by McNaney et al. [39]. The in vivo pharmacokinetics of ASP3026 can be predicted using computer software algorithms such as simulation and Cloe PK softwares [45].
(A) The graph shows the ASP3026 % remaining over the time range (0–60 min) in a matrix of HLMs. (B) The graph shows the ln of the ASP3026 % remaining over the time range (0–30 min)
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01282
In vitro metabolic incubation of ASP3026 with HLMs (Metabolic stability of ASP3026)
| Time intervals | Meana | Xb | LN X | Regression line characteristics |
| 0.00 | 562.38 | 100.00 | 4.61 | y = −0.0484x + 4.589 |
| 2.50 | 490.11 | 87.15 | 4.47 | |
| 5.00 | 434.38 | 77.24 | 4.35 | R2 = 0. 9996 |
| 7.50 | 377.81 | 67.18 | 4.21 | |
| 15.00 | 267.97 | 47.65 | 3.86 | Slope: −0.0484 |
| 20.00 | 209.09 | 37.18 | 3.62 | |
| 30.00 | 130.42 | 23.19 | 3.14 | t1/2: 14.32 min and |
| 40.00 | 120.80 | 21.48 | 3.07 | Clint: 56.62 mL min−1 kg−1 |
| 50.00 | 105.67 | 18.79 | 2.93 | |
| 60.00 | 99.26 | 17.65 | 2.87 |
a Average of 3 readings; b X: Average of the % remaining of ASP3026.
4 Conclusions
The objective of this work was to create and verify a UHPLC-MS/MS method for quantifying the concentrations of ASP3026 in the HLMs incubation matrix. Furthermore, the present approach was utilized to assess the metabolic stability of ASP3026. The application of the UHPLC-MS/MS approach has shown advantageous characteristics like specificity, ecological reliability, increased sensitivity, and good recovery of ASP3026 and FNB from the HLMs matrix. The selected extraction technique was protein precipitation. To increase the ecological sustainability of the current UHPLC-MS/MS system, a lower flow rate of 0.3 mL min−1, a shorter elution duration of 1 min, and a reduced consumption of ACN have been implemented. The results generated by the metabolic P450 program were confirmed by the data obtained from the in vitro laboratory incubation investigation of HLMs. Analysis of metabolic stability revealed that ASP3026 has a t1/2 of 14.32 min and a comparatively high Clint of 56.62 mL min−1 kg−1. These outcomes proposed that ASP3026 can be classified as a medication with a fairly high extraction ratio. Thus, it is postulated that giving of ASP3026 to patients will not result in the manifestation of dosage accumulation in the body. Depending on the data acquired from DEREK software analysis and in silico P450 metabolic studies, it is recommended to execute slight changes to the piperazine and piperidine rings or replace these groups in the drug development phase (Fig. 9). This has the probability to improve the safety properties and metabolic stability of new compounds if compared to ASP3026. Possible future investigations could adopt the current approach, which involves using in vitro metabolic incubations and in silico programs. Effective implementation of these techniques is important for improving the development of novel pharmaceuticals, namely in terms of strengthening metabolic stability. The effectiveness of using various computational metabolic methods to optimize resource allocation and minimize exert was shown by laboratory experiments and computer simulations of ASP3026.
The metabolic lability percentage of ASP3026 (shown in blue) was created using P450 software. On the other hand, DEREK toxicity predictions for ASP3026 (shown in red) reveal that the piperazine and piperidine moieties is accountable for the ASP3026's metabolic instability
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01282
Ethics approval
Utilizing HLMs obtained from Sigma company excludes the requirement for ethical approval.
Authors' contributions
The current criteria were developed by M.W.A., A.S.A., and A.A.K. A.S.A. and M.W.A. conducted the in vitro metabolic incubations of ASP3026 with HLMs and participated in the initial writing of the paper. The contributions of A.S.A. and A.A.K. were crucial in the development of software applications and the formulation of the established approach. The experimental methodologies were performed internally, without the use of any external paper production facilities. The manuscript's final version has been subjected to a comprehensive assessment and has obtained unanimous approval from all contributing authors.
Funding
The Researcher Supporting Project Number (RSPD2025R750) of King Saud University in Riyadh, Saudi Arabia served as the funding source for this work.
Competing interests
There are no conflicting interests to disclose.
Data availability
We confirm that the data supporting the findings of this study are available within the main article.
Acknowledgments
The authors express their gratitude to King Saud University, Riyadh, Saudi Arabia, under Researcher Supporting Project Number (RSPD2025R750), for giving financial support for this research effort.
References
- 1.↑
Siegel, R.; Ward, E.; Brawley, O.; Jemal, A. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J. Clin. 2011, 61(4), 212–236. https://doi.org/10.3322/caac.20121. From NLM.
- 2.↑
Janku, F.; Stewart, D. J.; Kurzrock, R. Targeted therapy in non-small-cell lung cancer--is it becoming a reality? Nat. Rev. Clin. Oncol. 2010, 7(7), 401–414. https://doi.org/10.1038/nrclinonc.2010.64. From NLM.
- 3.↑
Moen, M. D.; McKeage, K.; Plosker, G. L.; Siddiqui, M. A. Imatinib: a review of its use in chronic myeloid leukaemia. Drugs 2007, 67(2), 299–320. https://doi.org/10.2165/00003495-200767020-00010. From NLM.
- 4.
Druker, B. J.; Sawyers, C. L.; Kantarjian, H.; Resta, D. J.; Reese, S. F.; Ford, J. M.; Capdeville, R.; Talpaz, M. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N. Engl. J. Med. 2001, 344(14), 1038–1042. https://doi.org/10.1056/nejm200104053441402. From NLM.
- 5.
Baselga, J. Clinical trials of single-agent trastuzumab (Herceptin). Semin. Oncol. 2000, 27, 20–26.
- 6.
Paez, J. G.; Jänne, P. A.; Lee, J. C.; Tracy, S.; Greulich, H.; Gabriel, S.; Herman, P.; Kaye, F. J.; Lindeman, N.; Boggon, T. J.; Naoki, K.; Sasaki, H.; Fujii, Y.; Eck, M. J.; Sellers, W. R.; Johnson, B. E.; Meyerson, M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004, 304(5676), 1497–1500. https://doi.org/10.1126/science.1099314. From NLM.
- 7.↑
Soda, M.; Choi, Y. L.; Enomoto, M.; Takada, S.; Yamashita, Y.; Ishikawa, S.; Fujiwara, S.; Watanabe, H.; Kurashina, K.; Hatanaka, H.; Bando, M.; Ohno, S.; Ishikawa, Y.; Aburatani, H.; Niki, T.; Sohara, Y.; Sugiyama, Y.; Mano, H. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007, 448(7153), 561–566. https://doi.org/10.1038/nature05945. From NLM.
- 8.↑
Rikova, K.; Guo, A.; Zeng, Q.; Possemato, A.; Yu, J.; Haack, H.; Nardone, J.; Lee, K.; Reeves, C.; Li, Y.; Hu, Y.; Tan, Z.; Stokes, M.; Sullivan, L.; Mitchell, J.; Wetzel, R.; Macneill, J.; Ren, J. M.; Yuan, J.; Bakalarski, C. E.; Villen, J.; Kornhauser, J. M.; Smith, B.; Li, D.; Zhou, X.; Gygi, S. P.; Gu, T. L. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 2007, 131(6), 1190–1203. https://doi.org/10.1016/j.cell.2007.11.025. From NLM.
- 9.↑
Mano, H. ALKoma: a cancer subtype with a shared target. Cancer Discov. 2012, 2(6), 495–502. https://doi.org/10.1158/2159-8290.Cd-12-0009. From NLM.
- 10.↑
Camidge, D. R.; Bang, Y.-J.; Kwak, E. L.; Iafrate, A. J.; Varella-Garcia, M.; Fox, S. B.; Riely, G. J.; Solomon, B.; Ou, S.-H. I.; Kim, D.-W. Activity and safety of crizotinib in patients with ALK-positive non-small-cell lung cancer: updated results from a phase 1 study. Lancet Oncol. 2012, 13(10), 1011–1019.
- 11.
Shaw, A. T.; Kim, D.-W.; Nakagawa, K.; Seto, T.; Crinó, L.; Ahn, M.-J.; De Pas, T.; Besse, B.; Solomon, B. J.; Blackhall, F. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. New Engl. J. Med. 2013, 368(25), 2385–2394.
- 12.
Solomon, B. J.; Mok, T.; Kim, D.-W.; Wu, Y.-L.; Nakagawa, K.; Mekhail, T.; Felip, E.; Cappuzzo, F.; Paolini, J.; Usari, T. First-line crizotinib versus chemotherapy in ALK-positive lung cancer. New Engl. J. Med. 2014, 371(23), 2167–2177.
- 13.↑
Ettinger, D. S.; Wood, D. E.; Akerley, W.; Bazhenova, L. A.; Borghaei, H.; Camidge, D. R.; Cheney, R. T.; Chirieac, L. R.; D'Amico, T. A.; Dilling, T. J. NCCN guidelines insights: non–small cell lung cancer, version 4.2016. J. Natl. Compr. Cancer Netw. 2016, 14(3), 255–264.
- 14.↑
Katayama, R.; Shaw, A. T.; Khan, T. M.; Mino-Kenudson, M.; Solomon, B. J.; Halmos, B.; Jessop, N. A.; Wain, J. C.; Yeo, A. T.; Benes, C. Mechanisms of acquired crizotinib resistance in ALK-rearranged lung cancers. Sci. Transl. Med. 2012, 4(120), 120ra117–120ra117.
- 15.
Doebele, R. C.; Pilling, A. B.; Aisner, D. L.; Kutateladze, T. G.; Le, A. T.; Weickhardt, A. J.; Kondo, K. L.; Linderman, D. J.; Heasley, L. E.; Franklin, W. A. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non–small cell lung cancer. Clin. Cancer Res. 2012, 18(5), 1472–1482.
- 16.
Awad, M. M.; Shaw, A. T. ALK inhibitors in non–small cell lung cancer: crizotinib and beyond. Clin. Adv. Hematol. & Oncol. H&O 2014, 12(7), 429.
- 17.↑
Shaw, A. T.; Kim, D.-W.; Mehra, R.; Tan, D. S.; Felip, E.; Chow, L. Q.; Camidge, D. R.; Vansteenkiste, J.; Sharma, S.; De Pas, T. Ceritinib in ALK-rearranged non–small-cell lung cancer. New Engl. J. Med. 2014, 370(13), 1189–1197.
- 18.↑
Gadgeel, S. M.; Gandhi, L.; Riely, G. J.; Chiappori, A. A.; West, H. L.; Azada, M. C.; Morcos, P. N.; Lee, R.-M.; Garcia, L.; Yu, L. Safety and activity of alectinib against systemic disease and brain metastases in patients with crizotinib-resistant ALK-rearranged non-small-cell lung cancer (AF-002JG): results from the dose-finding portion of a phase 1/2 study. Lancet Oncol. 2014, 15(10), 1119–1128.
- 19.↑
Mori, M.; Ueno, Y.; Konagai, S.; Fushiki, H.; Shimada, I.; Kondoh, Y.; Saito, R.; Mori, K.; Shindou, N.; Soga, T. The selective anaplastic lymphoma receptor tyrosine kinase inhibitor ASP3026 induces tumor regression and prolongs survival in non–small cell lung cancer model mice. Mol. Cancer Ther. 2014, 13(2), 329–340.
- 20.↑
Mori, M.; Kuromitsu, S.; Ueno, Y.; Tanaka, R.; Shimada, I.; Kondoh, Y.; Konagai, S.; Sakagami, H.; Fushiki, H.; Saito, R. ASP3026, a selective ALK inhibitor, induces tumor regression in a crizotinib-refractory model and prolongs survival in an intrapleurally xenograft model. Cancer Res. 2012, 72(8_Supplement), 866–866.
- 21.↑
Ono, A.; Murakami, H.; Seto, T.; Shimizu, T.; Watanabe, S.; Takeshita, S.; Takeda, K.; Toyoshima, J.; Nagase, I.; Bahceci, E.; Morishita, M.; Morita, S.; Fukuoka, M.; Nakagawa, K. Safety and antitumor activity of repeated ASP3026 administration in Japanese patients with solid tumors: a phase I study. Drugs R. D 2021, 21(1), 65–78. https://doi.org/10.1007/s40268-020-00331-2. From NLM.
- 22.↑
Marothu Vamsi, K.; Kantamaneni, P.; Gorrepati, M. In vitro metabolic stability of drugs and applications of LC-MS in metabolite profiling. In Drug Metabolism; Katherine, D., Ed. IntechOpen, 2021, Ch. 5.
- 23.↑
Houston, J. B. Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem. Pharmacol. 1994, 47(9), 1469–1479. https://doi.org/10.1016/0006-2952(94)90520-7. From NLM.
- 24.↑
Obach, R. S.; Baxter, J. G.; Liston, T. E.; Silber, B. M.; Jones, B. C.; MacIntyre, F.; Rance, D. J.; Wastall, P. The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data. J. Pharmacol. Exp. Ther. 1997, 283(1), 46–58, From NLM.
- 25.↑
Attwa, M. W.; Abdelhameed, A. S.; Alsaif, N. A.; Kadi, A. A.; AlRabiah, H. A validated LC-MS/MS analytical method for the quantification of pemigatinib: metabolic stability evaluation in human liver microsomes. RSC Adv. 2022, 12(31), 20387–20394. https://doi.org/10.1039/d2ra02885a, Scopus.
- 26.
Attwa, M. W.; Kadi, A. A.; Abdelhameed, A. S. Characterization of reactive intermediates formation in dacomitinib metabolism and bioactivation pathways elucidation by LC-MS/MS: in vitro phase I metabolic investigation. Rsc Adv. 2018, 8(68), 38733–38744. https://doi.org/10.1039/c8ra06709k. From NLM.
- 27.
Kadi, A. A.; Darwish, H. W.; Attwa, M. W.; Amer, S. M. Detection and characterization of ponatinib reactive metabolites by liquid chromatography tandem mass spectrometry and elucidation of bioactivation pathways. RSC Adv. 2016, 6(76), 72575–72585, 10.1039/C6RA09985H. https://doi.org/10.1039/C6RA09985H.
- 28.
Alsubi, T. A.; Attwa, M. W.; Bakheit, A. H.; Darwish, H. W.; Abuelizz, H. A.; Kadi, A. A. In silico and in vitro metabolism of ribociclib: a mass spectrometric approach to bioactivation pathway elucidation and metabolite profiling. RSC Adv. 2020, 10(38), 22668–22683. https://doi.org/10.1039/d0ra01624a. Scopus.
- 29.
Attwa, M. W.; Kadi, A. A.; Abdelhameed, A. S. Phase i metabolic profiling and unexpected reactive metabolites in human liver microsome incubations of X-376 using LC-MS/MS: bioactivation pathway elucidation and: in silico toxicity studies of its metabolites. RSC Adv. 2020, 10(9), 5412–5427. https://doi.org/10.1039/c9ra09115g, Scopus.
- 30.↑
Alrabiah, H.; Kadi, A. A.; Attwa, M. W.; Abdelhameed, A. S. A simple liquid chromatography-tandem mass spectrometry method to accurately determine the novel third-generation EGFR-TKI naquotinib with its applicability to metabolic stability assessment. Rsc Adv. 2019, 9(9), 4862–4869. https://doi.org/10.1039/c8ra09812c.
- 31.↑
Attwa, M. W.; Mostafa, G. A. E.; AlRabiah, H.; Kadi, A. A. An LC–MS/MS analytical method for quantifying Tepotinib in human liver microsomes: application to in vitro and in silico metabolic stability estimation. Separations 2023, 10(6), 330.
- 32.↑
Al-Shakliah, N. S.; Kadi, A. A.; Aljohar, H. I.; AlRabiah, H.; Attwa, M. W. Profiling of in vivo, in vitro and reactive zorifertinib metabolites using liquid chromatography ion trap mass spectrometry. RSC Adv. 2022, 12(32), 20991–21003, 10.1039/D2RA02848D. https://doi.org/10.1039/D2RA02848D.
- 33.↑
Busby, W. F., Jr.; Ackermann, J. M.; Crespi, C. L. Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450. Drug Metab. Dispos 1999, 27(2), 246–249, From NLM.
- 34.
Störmer, E.; Roots, I.; Brockmöller, J. Benzydamine N-oxidation as an index reaction reflecting FMO activity in human liver microsomes and impact of FMO3 polymorphisms on enzyme activity. Br. J. Clin. Pharmacol. 2000, 50(6), 553–561. https://doi.org/10.1046/j.1365-2125.2000.00296.x. From NLM.
- 35.
Fouin-Fortunet, H.; Tinel, M.; Descatoire, V.; Letteron, P.; Larrey, D.; Geneve, J.; Pessayre, D. Inactivation of cytochrome P-450 by the drug methoxsalen. J. Pharmacol. Exp. Ther. 1986, 236(1), 237–247, From NLM.
- 36.↑
Meesters, R.; Voswinkel, S. Bioanalytical method development and validation: from the USFDA 2001 to the USFDA 2018 guidance for industry. J. Appl. Bioanal. 2018, 4(3), 67–73. https://doi.org/10.17145/jab.18.010.
- 37.↑
Smith, G. European Medicines Agency guideline on bioanalytical method validation: what more is there to say? Bioanalysis 2012, 4(8), 865–868. https://doi.org/10.4155/bio.12.44.
- 38.↑
González, O.; Alonso, R. M. Chapter 6 - validation of bioanalytical chromatographic methods for the quantification of drugs in biological fluids. In Handbook of Analytical Separations, Hempel, G. Ed.; Vol. 7. Elsevier Science B.V., 2020; pp 115–134.
- 39.↑
McNaney, C. A.; Drexler, D. M.; Hnatyshyn, S. Y.; Zvyaga, T. A.; Knipe, J. O.; Belcastro, J. V.; Sanders, M. An automated liquid chromatography-mass spectrometry process to determine metabolic stability half-life and intrinsic clearance of drug candidates by substrate depletion. Assay Drug Dev. Technol. 2008, 6(1), 121–129. https://doi.org/10.1089/adt.2007.103. From NLM.
- 40.↑
Słoczyńska, K.; Gunia-Krzyżak, A.; Koczurkiewicz, P.; Wójcik-Pszczoła, K.; Żelaszczyk, D.; Popiół, J.; Pękala, E. Metabolic stability and its role in the discovery of new chemical entities. Acta Pharm. 2019, 69(3), 345–361. https://doi.org/10.2478/acph-2019-0024. From NLM.
- 42.
Hunt, P. A.; Segall, M. D.; Tyzack, J. D. WhichP450: a multi-class categorical model to predict the major metabolising CYP450 isoform for a compound. J. Comput. Aided Mol. Des. 2018, 32(4), 537–546. https://doi.org/10.1007/s10822-018-0107-0. From NLM.
- 43.
G Shin, Y.; Le, H.; Khojasteh, C.; ECA Hop, C. Comparison of metabolic soft spot predictions of CYP3A4, CYP2C9 and CYP2D6 substrates using MetaSite and StarDrop. Comb. Chem. High Throughput Screen. 2011, 14(9), 811–823.
- 44.↑
Pena-Pereira, F.; Wojnowski, W.; Tobiszewski, M. AGREE—analytical GREEnness metric approach and software. Anal. Chem. 2020, 92(14), 10076–10082. https://doi.org/10.1021/acs.analchem.0c01887.
- 45.↑
Leahy, D. E. Integrating invitro ADMET data through generic physiologically based pharmacokinetic models. Expert Opin. Drug Metab. Toxicol. 2006, 2(4), 619–628.
