Abstract
The first targeted Rho-associated protein kinase 2 inhibitor was Belumosudil (Rezurock). After at least two systemic treatments failed for chronic graft-versus-host disease in adults and children aged 12 and older, belumosudil (BLS) was approved on July 16, 2021. This study established a validated Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) system to estimate BLS in HLMs and assess metabolic stability. Agilent C18 column was used to separate BLS and encorafenib (internal standard) in HLMs using an isocratic mobile phase. The electrospray ionization (ESI) source produced BLS parent ions. Multiple reaction monitoring identified and measured BLS daughter ions. The methodology was validated following FDA standards that assessed linearity, selectivity, accuracy, sensitivity, precision, extraction recovery, matrix effects, and stability. The accuracy and precision ranged from −1.42 to 12.50% between days and from −0.89 to 10.5% within a day. The created BLS calibration curve displayed a linearity in the range from 1 ng mL−1 to 3,000 ng mL−1. The LLOQ of 0.64 ng mL−1 showed the method's sensitivity. The AGEE program displayed that the approach was eco-friendly. In vitro half-life and intrinsic clearance of BLS were 17.55 min and 46.21 mL min−1 kg−1, demonstrating a high extraction ratio drug. These features can improve the metabolic stability of new derivatives compared to BLS, making them important in medication design.
1 Introduction
Chronic graft versus host disease (cGVHD) is a disorder that affects multiple systems in individuals who have received allogeneic hematopoietic stem cell transplants (HSCT). It is characterized by a combination of inflammation and fibrosis [1]. Both the adaptive and innate immune systems were implicated in the pathogenesis based on experimental models. The development of the disease is believed to progress from an initial acute inflammatory response to tissue damage shortly after transplantation, which then leads to chronic inflammation and disruption of both B and T cells. This is followed by abnormal tissue healing and the formation of fibrotic tissue [2]. Untreated cGVHD can lead to life-threatening multisystem tissue damage as a result of persistent inflammation and fibrosis. The initial treatment often consists of administering steroids, either alone or in combination with a calcineurin inhibitor, which is determined based on the severity of the condition. Approximately 50% of patients diagnosed with cGVHD necessitate the utilization of three or more treatment regimens, comprising several off-label immunosuppressant medications [3].
Rho-associated coiled-coil inclosing kinase 2 (ROCK2) is a serine/threonine kinase that controls various cellular processes, such as the arrangement of the cytoskeleton, cell movement, and gene expression [4]. ROCK2 can be activated by the cleavage of the carboxyl terminus by apoptotic proteins, or by the collaboration of RhoA GTPase G protein and Rho signaling [5]. The expression of proinflammatory cytokines, specifically IL-17 and IL-21, as well as the profibrotic gene targets regulated by myocardin-related transcription factor (MRTF), is regulated by the ROCK2 signaling pathway. Dysregulation of these features has been linked to the development of cGVHD [6, 7].
Belumosudil (BLS; Fig. 1), also known as Rezurock, SLx-2119, or KD025, is a type of small molecule inhibitor specifically designed to target and inhibit the activity of ROCK2. BLS shown inhibitory effects on the secretion of Interleukin-21 (IL-21) and IL-17 in ex-vivo or in vitro experiments. This inhibition was observed in human peripheral blood mononuclear cells stimulated with CD3-CD28. BLS also decreased transcriptional activity and STAT3 phosphorylation, as well as MRTF-mediated gene expression [8, 9]. BLS demonstrated inhibitory effects on the mammalian target of rapamycin (mTOR) pathway and, to a lesser extent, on the ROCK1 isoform in vitro. Experiments conducted on live cGVHD animal models showed that administering BLS led to a significant improvement in clinical and histological abnormality scores associated to cGVHD in the lung and skin [10]. On July 16, 2021, the Food and Drug Administration decided approval for the use of belumosudil, a kinase inhibitor, in the treatment of cGVHD in patients aged 12 and above. This approval is specifically for patients who have not responded to at least two previous lines of systemic therapy [11].
Chemical structure of Belumosudil and the internal standard (Encorafenib; IS)
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01339
The primary objective of the present study is to assess the in vitro metabolic stability of BLS, using an Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) approach. The development of a swift, environmentally sustainable, and highly responsive UPLC-MS/MS approach for estimating BLS in several matrices is of considerable importance. An accurate examination of the specific medication (BLS in this study) is crucial for monitoring its therapeutic drug concentrations (TDM). Additionally, it is vital to acquire data regarding the relationship among BLS activity and its concentration to endorse the safe and reliable usage of this substance by patients. At now, there is absence of reported valuation about the metabolic stability of BLS in human liver microsomes (HLMs) employing UPLC-MS/MS technology. Assessing the metabolic stability of BLS in HLMs is crucial for understanding the kinetics of its metabolic processes and elimination [12–14].
The stability of a pharmacological molecule to metabolic enzymes denotes its vulnerability to metabolic procedures, evaluated by its in vitro half-life (t1/2) and intrinsic clearance (Clint) [15–17]. The term “t1/2” represents the interval necessary for 50% of the initial drug level to be metabolized. Clint denotes to the liver's ability to process a drug within the circulatory system. This research targeted to establish a highly sensitive, selective, fast, and green UPLC-MS/MS methodology for estimating the metabolic stability of BLS in HLMs. Recently, there has been a heightened emphasis on green analytical chemistry (GAC), that seeks to alleviate hazardous chemicals, lower energy usage, and diminish waste formation using numerous analytical methodologies [18, 19]. Numerous measurement attitudes have been employed to assess the level of greenness based on numerous analytical outcomes, with the target of achieving these goals. This collection comprises National Environmental Methods Index (NEMI), Analytical Eco-Scale (AES), Green Analytical Procedures Index (GAPI), the Analytical Green-ness Metric Approach (AGREE), and Red-Green-Blue (RGB) [18]. The NEMI, GAPI, AES, and RGB methods demonstrated a requirement on specific GAC standards, as illustrated. The research employed the “AGREE” procedure to appraise the sustainability of the environment by examining 12 GAC and assigning scores to each principle.
The UPLC-MS/MS methodology employed mobile phase of constant composition (isocratic) and achieved a total operational time of 1 min, yielding a very effective analytical system. The use of a low flow rate at 0.4 mL min−1 and a reduction of ACN to 50% markedly improved the greenness features of the present system. Moreover, the method utilized in this research exhibited a direct connection over the level range of 1–3,000 ng mL−1. The current strategy was developed to reduce expenses and decrease time. This study employed the UPLC-MS/MS technique to evaluate the Clint and in vitro t1/2 of BLS, adhering to the methodologies described in prior publications [20, 21]. These procedures can be employed to estimate the metabolic rate in living beings through using three diverse models: venous equilibrium, dispersion, and parallel tube. The t1/2 and Clint of BLS were estimated employing an in vitro procedure grounded in the well-stirred model [22, 23]. This model is repeatedly utilized in drug metabolism research due to its effortlessness and ease of implementation.
2 Methodology
2.1 Materials
The solvents employed in the UPLC-MS/MS methodology in this work were of UPLC quality. The solid chemicals employed in the current research, BLS and encorafenib (ENB), were acquired in analytical grade. This study specifically examines two analytes: BLS (SLx-2119 or KD025) and ENB (LGX818). The selected analytes were obtained from MedChem Express (Princeton, NJ, United States). The purity of BLS was 99.77%, whilst ENB showed a 99.63% purity. The chemicals utilized in the current research, specifically ammonium formate (NH4COOH), acetonitrile (ACN), HLMs (M0317; Microsomes from Liver, Pooled), and formic acid (HCOOH), were sourced from Sigma Aldrich company (St. Louis, Missouri, USA). The HLMs were conveyed on dry ice to confirm the conservation of their reliability. The HLMs at 20 mg mL−1 were maintained at −78 °C upon delivery until deemed ready for utilization.
2.2 Instruments
The Milli-Q system, created by Millipore company (Billerica, MA, USA), was utilized for water filtering to produce UPLC-grade water. The inquiry employed the Acquity UPLC (H10UPH) and the Acquity Triple Quadrupole (TQD) MS (QBB1203) and, analytical systems manufactured by Waters Corporation. The system is known as a UPLC-MS/MS instrument. The equipment was utilized to perform a thorough examination and discern the analytical peaks of the BLS and ENB subsequent to their extraction (using protein precipitation) from the incubation HLMs matrix. The UPLC-MS/MS approach employed a MassLynx operating program (Version 4.1, SCN 805). The vacuum in the TQD detection analyzer was primarily established employing a vacuum pump by Sogevac Corporation (Murrysville, PA, USA). The obtained results were analyzed and assessed employing the QuanLynx program. The optimization of MS features for the analytes BLS and ENB was directed utilizing the IntelliStart® software, an intelligent application integrated inside the MassLynx 4.1 operating software (version 4.1, SCN 805). The evaporation of droplets in the ESI source of the mobile phase was enhanced by employing nitrogen gas. The nitrogen gas was generated utilizing a nitrogen generator acquired from Peak Scientific Company, situated in Scotland, UK. The ions of interest, namely BLS and ENB, fragmented into their product ions in the second quadrupole (collision cell). The fragmentation route was enhanced by utilizing the argon gas at 99.999% purity [24, 25].
2.3 UPLC-MS/MS system characteristics
The UPLC-MS/MS features were refined to accomplish maximum detectability and resolution for the BLS and ENB peaks, as itemized in Table 1. A comprehensive analysis of many parameters, including pH level, mobile phase, and stationary phase features, was performed to increase the analytical specifications of the devised method. The goal of this tuning test was to augment the detectability and resolution of the BLS and ENB peaks, as detailed in Table 1. The isocratic mobile phase comprised two lines: line A, an aqueous solution of 0.1% formic acid in H2O (pH of 3.2), comprising 50%; and line B, consisted of acetonitrile (ACN), which also accounted for 50%. The flow rate was measured at 0.4 mL min−1. Upon reaching a pH value of roughly 3.2, the application of 10 mM NH4COOH in H2O resulted in analytical peak tailing in the BLS analysis and a lengthier elution time. When the concentration of ACN exceeds 50%, overlapping peaks are distinctly seen in the analytical chromatograms of BLS and ENB. A reduced percentage of ACN leads to an extended elution time. The ESI ionization source was utilized in positive ionization mode to enhance ionization, as the BLS and ENB analytes contained basic nitrogen atoms capable of capturing protons, leading to the creation of positive charge of the target analytes and their daughter ions [26].
UPLC-MS/MS tuned characteristics
| UPLC | TQD MS | ||
| Eclipse plus-C18 reversed column | Length: 50.0 mm | ESI | Positive ESI source |
| 2.1 mm ID | The extractor voltage: 3.0 (V) | ||
| 3.5 μm PS | Cone gas: 100 L/H flow rate | ||
| T: 22.0 ± 2.0 °C | The RF lens voltage: 0.1 (V) | ||
| Isocratic mobile phase system | 50% ACN | 100 L h−1 Nitrogen gas at 350 °C | |
| 0.1% HCOOH in Water (50%; pH: 3.2) | Capillary voltage: 4 KV | ||
| 5.0 μL (Injection volume) | Mode | MRM | |
| 0.4 mL min−1 (Flow rate) | Collision cell | 0.14 mL min−1 (Argon gas) | |
The tuning of mass spectrometry (MS) settings for BLS (C26H24N6O2) and ENB (C22H27ClFN7O4S) was effectively conducted utilizing the IntelliStart® application. This was attained by directly infusing BLS and ENB (10 μg mL−1) into the mobile phase. The MRM method was employed as the mass analyzer approach to evaluate BLS and ENB. The application of this method enhanced the detectability and specificity of the established UPLC-MS/MS technique [27]. The fragmentation of BLS and ENB ions into their corresponding product ions in the collision cell was attained employing argon gas at high-purity. The time for the alteration of mass ions from parent to fragments in BLS and ENB was recorded at 0.025 s. Table 2 presents a broad description of the many components connected with MRM and mass transitions in BLS and ENB.
MRM specific characteristics (parent to two daughter ions) for the determination of BLS and ENB
| Time segments | Elution time | Mass reaction transitions (m/z) | |
| MRM segments | 0.0–1.25 min | BLS (0.88 min) | 453→ 353 (CE*: 34 and CV**: 54) |
| 453→ 325 (CE: 40 and CV: 54) | |||
| 1.25–2.0 min | ENB (IS; 1.39 min) | 540→116 (CE: 56 and CV: 32) | |
| 540→359 (CE: 56 and CV: 36) |
*Collision energy.
**Cone voltage.
2.4 BLS and ENB working solutions
The highest solubility of BLS and ENB was recorded in DMSO at doses of 100 mg mL−1 (220.99 mM; needing ultrasonication) and 50 mg mL−1 (92.59 mM; attained with ultrasonication step), correspondingly. Thus, the BLS and ENB stock solutions, each with a concentration of 1 mg mL−1, were dissolved in DMSO. The mobile phase was adjusted to make working solutions (WKs) of BLS at concentrations of 100, 10, and 1 μg mL−1, and ENB at 10 μg mL−1, employing a multi-step dilution approach. The stock solutions of BLS and ENB were primarily prepared at 1 mg mL−1, followed by succeeding dilutions.
2.5 Preparation of BLS levels
Prior to commencing the validation for the UPLC-MS/MS method, the HLMs matrix was rendered inactive by including a 2% DMSO solution (2 mL in 100 mL H2O) and incubating at 50 °C for 5 min. The implementation of this defensive amount was to decrease the possible metabolic impacts of HLMs [28–30] on the analytes being examined, namely BLS and ENB. A certain matrix was developed for HLMs to evaluate the BLS metabolic stability. The investigational methodology compromised the addition of 30 µL of HLMs (inactive form), with a 1 mg protein mL−1, to 1 mL of the HLMs matrix. The metabolic buffer solution for the HLMs matrix was created by mixing a 0.1 M sodium phosphate with a pH of 7.4. The buffer solution comprised 1 mM of NADPH and 3.3 mM of MgCl2. The aim of employing these steps was to replicate the conditions of in vitro metabolic incubation for metabolic investigations conducted in vitro. The BLS calibration standards (CSs) were generated using a multistep dilution of BLS (WK3 and WK2) utilizing the inactive HLMs matrix. Consequently, a total of seven CSs were produced over the range of 1–3,000 ng mL−1. Furthermore, four quality controls (QCs) were established: 1 ng mL−1 (lower limit of quantification, LLOQ), 3 ng mL−1 (lower QC, LQC), 2,400 ng mL−1 (high QC, HQC), and 900 ng mL−1 (middle QC, MQC). In the dilution phase, rigorous protocols were adhered to sustain the HLMs matrix over 90% concentration. The goal of this activity was to alleviate the possible impacts of matrix dilution, therefore replicating the settings of in vitro metabolic incubation of BLS with HLMs. The QCs were employed as unknowns, computed employing the regression equation derived from the simultaneous BLS calibration curve. An IS comprising a 100 µL aliquot of ENB WK solution at 10,000 ng mL−1 was included into 1 mL of all BLS QCs and CSs.
2.6 The recovery of BLS and ENB from the HLMs matrix
The protein precipitation system was utilized to separate BLS and ENB from the HLMs incubation matrix. The organic solvent (ACN) was efficiently employed in this method to inhibit and induce the formation of solid protein particles within the HLMs matrix. Consequently, 2 mL of acetonitrile was incorporated into the BLS quality controls and calibration standards. The mixture was subsequently shaked for 5 min to enhance the extraction productivity of the BLS and ENB from the precipitated proteins. Subsequently, the mixture was centrifuged at 14,000 rpm (4 °C) for 12 min. The centrifugation method was applied to isolate proteins and provide a clean supernatant. A purification approach was conducted on all incubates to assess the stability and suitability of the samples designated for loading into the UPLC-MS/MS apparatus. The procedure utilized a 0.22 µm syringe filter. The purified extracts were introduced into vials for loading into a UPLC-MS/MS system. Two varieties of control samples were performed for the experiment. The positive control sample, designated as the second control sample, entailed of HLMs augmented with ENB. The negative control sample, the initial control sample, consisted of HLMs. The previously designated approaches were replicated to verify that the HLMs contents did not impact the separation of BLS and ENB. The technique of building a calibration curve for a BLS entailed plotting the given BLS data on the x-axis, whereas the y-axis depicted the peak area ratio of BLS to ENB. The linearity range of the produced BLS CSs was assessed by investigating the linear regression equation (y = ax + b; r2) and the validation features of the developed UPLC-MS/MS technique.
2.7 Validation features of the UPLC-MS/MS approach
The UPLC-MS/MS method employed in this research was authenticated in accordance with the FDA's criteria for bioanalytical procedures. The validation methodology incorporated the assessment of numerous attributes comprising features, accuracy, precision, linearity, sensitivity, specificity, stability, matrix effect, and extraction recovery [31–34].
2.7.1 Specificity
The UPLC-MS/MS method specificity was estimated by loading six sets of blank HLMs matrix samples after precipitating proteins, the chosen extraction approach. The purified extracts of samples were injected into a UPLC-MS/MS instrument and analyzed to characterize any interfering peaks that may occur from the HLMs matrix components at the same retention time as the chromatographic peaks (BLS or ENB). A relative analysis was subsequently done to determine the attained data concerning the spiked HLM samples containing BLS and ENB. The MRM analyzer mode was utilized to eradicate the carry over influences of the BLS and ENB in the TQD mass analyzer. The validation was attained through the analysis of the negative control samples outcomes that exhibited deficiencies in BLS and ENB.
2.7.2 Sensitivity and linearity
The linearity assessment in the current UPLC-MS/MS attitude employed statistical measurements, definitely the least squares method (y = ax + b) and the correlation coefficient (r2).
2.7.3 Precision and accuracy
2.7.4 Matrix effect and extraction recovery
The calculation of BLS extraction recovery and the analysis of HLMs' impact on the degree of BLS parent ionization were achieved alongside the implementation of four QCs. The validation of proteins precipitation as the optimal extraction approach for BLS and ENB was performed by loading six sets of 4 QCs in the HLMs matrix (B) and afterwards relating them with 4 QCs obtained in the selected mobile phase (A). The estimation of percent extraction recoveries for BLS and ENB comprised calculating the % by multiplying the quantity of B by A and then multiplying by 100.
2.7.5 Stability
The target of this research was to estimate the stability of BLS in HLM matrices and stock preparations under several laboratory situations, comprising pre-analysis cycles, short-term and long-term storage, and storage in an autosampler.
2.8 In vitro determination of the BLS metabolic stability
The valuation of in vitro t1/2 and Clint (clearance) of BLS involved quantifying the residual amount of BLS subsequent to its introduction to an in vitro metabolic incubation. The incubation process included a functional HLMs matrix, augmented with NADPH as a coenzyme and MgCl2. The in vitro metabolic incubation steps were executed employing a four-step approach. Throughout the preliminary step, a 1 µL aliquot of BLS underwent pre-incubation with a HLMs matrix. The above-stated approach was conducted using a water bath at 37 °C via thermostatic control for 10 min. At the commencement of the research, each sample was administered a solution comprising 1 mM NADPH. Thereafter, all samples were placed in a thermostatically controlled water bath with shaking functionality, maintained at 37 °C. In the third phase of the experiment, 100 µL of ENB (10,000 ng mL−1) was administered prior to the addition of ACN, that helped halting the metabolic reaction. The aim of this methodology was to attain a consistent concentration of the internal standard and to alleviate any possible impression of metabolic enzymatic reactions on the internal standard's level. In the fourth step, termed the quenching phase, a 2 mL aliquot of ACN was administered at specified intervals (0, 2.5, 7.5, 15, 20, 30, 40, 50, and 60 min) to halt the metabolic enzymes and precipitate excess unwanted proteins. The first stage of the extraction procedure for the BLS and ENB is designated as the first step, as detailed in Section 2.6. A negative control experiment was performed to examine the effect of excluding NADPH via the metabolic incubation of BLS with HLMs, adhering to the formerly documented methodology. The objective of the test was to evaluate the potential impact of incubation settings and HLM matrix effects on the level of BLS in the practical in vitro metabolic incubation studies.
3 Results and discussion
3.1 UPLC-MS/MS method
Various stationary phases of diverse natures were assessed, comprising HILIC columns. Neither the BLS nor the ENB were kept or separated. The use of a reversed C18 column has been found to produce useful outcomes as the preferred stationary phase. Utilizing the UPLC-MS/MS methodology to resolute BLS and ENB resulted in analyte retention inside the chromatographic system when a C8 column was employed. Nonetheless, the analyzed targets, particularly BLS and ENB, demonstrated inadequate resolution of the analyte peaks, extended peak tailing, and an elongated duration of operation. The usage of an Agilent C18 column (Eclipse plus), categorized by a 2.1 mm ID, a 150 mm length, and a 3.5 μm PS, yielded favorable results regarding elution time and the shape of analyte peaks. The existing UPLC-MS/MS methodology involves the separation of the current analytes, BLS and ENB, utilizing a mobile phase with a fixed configuration. The separation process was done at 0.4 mL min−1 for two minutes. The calibration curve for the BLS, developed using the specified methodology, established a linear relation throughout the entire level range of 1–3,000 ng mL−1. Table 3 exhibits a comprehensive collecting of different tests performed to augment and recognize the optimal features for the resolution, extraction, and assessment of BLS and ENB analytical peaks. The main aim of these research was to accomplish optimal features, comprising a clearly defined chromatographic peak form and a short elution time.
Optimized UPLC-MS/MS features
| Analytes | Mobile Phase | Recovery | Stationary System | |||
| Methanol | ACN | Solid Phase Extraction | Protein Precipitation Employing ACN | C18 reversed column | C8 reversed column | |
| BLS | 1.28 min | 0.85 min | 65.27% Low recovery | 102.7 ± 4.13% High recovery | 0.85 min | 0.75 min |
| Tailed | Good shape | Not accurate | RSD < 4.03% Precise | Perfect shape | Tailed peaks | |
| ENB | 1.45 min | 1.33 min | Good (89.89%) | High (101.61 ± 3.23% | 1.33 min | 0.95 min |
| Overlapped | Good shape | Not accurate | RSD < 3.18% Precise | Optimum shape | Optimum shape | |
To rise the detectability and sensitivity of the UPLC-MS/MS method, the MRM mass analysis mode was utilized to accurately characterize and estimate the quantities of BLS and ENB. The target of this experiment was to test any potential intervention caused by the matrix ingredients included in the HLMs (Fig. 2).
MRM mass spectra of BLS [M+H]+ displaying two mass reaction transitions (parent to two fragment ions) (A) and ENB [M+H]+ (B), PI mass spectrum of BLS and ENB are exhibited. The proposed fragmentation behaviours are elucidated
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01339
The UPLC-MS/MS process utilized ENB as an IS to determine BLS. This methodology depended on three crucial components. Precipitating proteins using organic solvent (ACN) as an extraction approach effectively extracts both BLS and ENB analytes, achieving 102.7 ± 4.13% with a relative standard deviation (RSD) below 4.03% for BLS, and 101.61 ± 3.23% (RSD less than 3.18%) for ENB. The chromatographic peaks of BLS (0.85 min) and ENB (1.33 min) were successfully eluted within two minutes. The aforementioned result demonstrates the efficacy of the developed UPLC-MS/MS technology as a competent and effective analytical implement. The employed technique not only successfully diminishes the total duration of the operation but also augments the consumption of ACN, therefore aligning with the green chemistry score. Also, it is crucial to acknowledge that the patient in a specific disease did not consume both drugs (BLS and ENB) together. Consequently, the existing UPLC-MS/MS methodology is appropriate for pharmacokinetic investigations of BLS. No discernible carry-over impact was observed in the MRM mass chromatograms of both positive (Fig. 3B) and negative controls (Fig. 3A) for BLS in the HLMs. Figure 3C illustrates the mass chromatograms derived from the MRM analysis of BLS CSs and ENB. The linearity span of the analytical method is from 1 ng mL−1 to 3,000 ng mL−1 for BLS CSs and 1,000 ng mL−1 for ENB.
The negative-control HLMs exhibited no interfering peaks during the elution periods of BLS and ENB (A). The MRM mass chromatogram of the positive control (Blank HLMs mixed with ENB at a concentration of 1,000 ng mL−1) (B). The overlaid MRM chromatograms of the seven BLS CSs and the three QCs (C). The chromatograms display the maxima of BLS CSs (0–3,000 ng mL−1) at 0.85 min and ENB (1,000 ng mL−1) at 1.33 min
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01339
3.2 Validation of the UPLC-MS/MS approach
3.2.1 Specificity
The UPLC-MS/MS approach's efficacy was evidenced by its efficacious separation of the BLS and ENB peaks, as illustrated in Fig. 3. Additionally, the analysis approved that the BLS and ENB peaks were not considerably affected by the ingredients of the HLMs matrix. The MRM chromatograms from the controls in the experiment exhibited no apparent carry-over influence from the preceding sample analysis accompanied with BLS.
3.2.2 Linearity and sensitivity validation features
Statistical examination was employed to illustrate the linearity of the established UPLC-MS/MS method, encompassing a concentration levels at 1 ng mL−1 to 3,000 ng mL−1 accomplished by incorporating BLS CSs into the HLMs matrix, which was then employed to compute the data as unidentified variables. The variables had a linear relationship, designated by a high r2 (0.9977) and a regression equation of y = 2.6314x + 1.4701. To address the issue of utilizing the extensive range of BLS level scores of the CSs, a weighting factor of (1/x) was employed in the formulation of the BLS calibration curve. Table 4 indicates that the RSD of the six replications, consisting of seven CSs, was below 4.02%. The LOQ) and LOD were computed to be 0.64 ng mL−1 and 0.21 ng mL−1, correspondingly.
Back-calculation data of 6 BLS levels (CSs)
| BLS (ng mL−1) | Mean | SD | % RSD | %E | Recovery (%) |
| 1.00 | 1.11 | 0.03 | 2.81 | 10.50 | 110.50 |
| 15.00 | 15.54 | 0.34 | 2.21 | 3.62 | 103.62 |
| 50.00 | 52.15 | 2.10 | 4.02 | 4.31 | 104.31 |
| 300.00 | 303.28 | 4.51 | 1.49 | 1.09 | 101.09 |
| 500.00 | 490.78 | 3.88 | 0.79 | −1.84 | 98.16 |
| 1500.00 | 1535.75 | 9.42 | 0.61 | 2.38 | 102.38 |
| 3000.00 | 2964.57 | 15.52 | 0.52 | −1.18 | 98.82 |
| % Recovery | 102.7 ± 4.13 | ||||
3.2.3 Accuracy and precision validation features
The valuation of the UPLC-MS/MS method's precision and accuracy entailed performing 12 series, each consisting of 4 QC samples, within a single day. Subsequently, six further sets, each encompassing 4 QCs, were done during the succeeding three days. The obtained data were believed to be in the predetermined adequate range, as outlined in the validation parameters provided by the FDA [37]. The applied UPLC-MS/MS procedure verified varying precision and accuracy ratings, both intra-day and inter-day. The outcomes are exhibited in Table 5, offering a thorough summary. The inter-day accuracy and precision covered a range from −1.42 to 12.50%, while the intra-day accuracy and precision ranged from −0.89 to 10.5%.
Precision and accuracy of the UPLC-MS/MS system
| BLS level (ng mL−1) | Intra-Day (12 BLS QCs in one day) | Inter-Day (6 BLS QCs in 3 following days) | ||||||
| BLS QCs | 1.00 | 3.00 | 900.00 | 2400.00 | 1.00 | 3.00 | 900.00 | 2400.00 |
| Average | 1.11 | 3.10 | 913.10 | 2378.60 | 1.13 | 3.16 | 922.15 | 2365.89 |
| SD | 0.03 | 0.06 | 8.18 | 7.41 | 0.01 | 0.06 | 7.34 | 9.84 |
| Precision (%RSD) | 2.81 | 1.77 | 0.90 | 0.31 | 0.51 | 1.80 | 0.80 | 0.42 |
| % Accuracy | 10.50 | 3.42 | 1.46 | −0.89 | 12.50 | 5.42 | 2.46 | −1.42 |
| Recovery (%) | 110.50 | 103.42 | 101.46 | 99.11 | 112.50 | 105.42 | 102.46 | 98.58 |
3.2.4 The HLMs metabolic matrix does not show any discernible impact on the extraction and recovery of BLS in the present UPLC-MS/MS system
The accomplishment of the chosen protein precipitation as an extraction system for BLS and ENB in the UPLC-MS/MS analytical methodology was computed by six replications, comprising 4 QCs, within the HLMs matrix. Subsequently, the acquired data was compared to the QCs created using the mobile phase. The investigation's outcomes designated a considerable recovery rate for BLS extraction (102.7 ± 4.13% with an RSD under 4.03%) and ENB (101.61 ± 3.23% with an RSD below 3.18%). The influence of the HLMs incubation matrix on ion generation, BLS or ENB, was analyzed by investigating two groups of HLMs samples. The results revealed that the influence was insignificant. Sample group 1 was formed by amalgamating LQC of BLS (3 ng mL−1) and ENB (1,000 ng mL−1), where sample set 2 was constituted by substituting the HLMs matrix with the mobile phase. The incubation matrix of the HLMs, comprising BLS and ENB, demonstrated a ME of 104.18 ± 5.05% for BLS and 99.74 ± 3.85% for ENB, respectively. The NME of the IS was assessed at 1.04, falling in the permitted parameters established by FDA regulatory standards. The research's outcomes indicate that there is no statistically significant relationship amid the HLMs matrix and the ionization level of ENB and BLS parent ions.
3.2.5 BLS was Stable in the DMSO and HLMs matrix
The stability valuation of the BLS in HLMs matrix and DMSO solvent indicated that best stability was attained by preserving the BLS in DMSO at a specific temperature of −80 °C for 28 days. Table 6 demonstrates that the RSD% for all BLS samples steadily remained below 3.13% throughout all storage circumstances. No important decrease in BLS level was gotten after introduction of the auto-sampler, short-term storage, three freeze-thaw cycles, and long-term storage. The findings presented in this study substantiate the considerable consistency exhibited by the BLS.
BLS stability analysis
| Stability parameters | 3.0 | 2400.0 | 3.0 | 2400.0 | 3.0 | 2400.0 | 3.0 | 2400.0 |
| Mean | SD | RSD (%) | Accuracy (%) | |||||
| Auto-Sampler Stability at 15 °C for 24 h | 3.07 | 2367.26 | 0.06 | 19.77 | 1.97 | 0.84 | 2.17 | −1.36 |
| Long-Term Stability at −80 °C for 28 days | 3.14 | 2365.60 | 0.05 | 21.12 | 1.45 | 0.89 | 4.67 | −1.43 |
| 3 cycles of Freeze–Thaw Stability at −80 °C and room T | 2.92 | 2375.66 | 0.07 | 6.74 | 2.36 | 0.28 | −2.58 | −1.01 |
| Short-Term Stability for 4 h at room T | 3.06 | 2363.77 | 0.10 | 26.15 | 3.13 | 1.11 | 1.92 | −1.51 |
3.3 Estimation of the UPLC-MS/MS analytical method greenness
The determination of the total greenness degree of the present UPLC-MS/MS method was accomplished employing the AGREE software as an in silico tool. The application incorporates all 12 measures stated by the Global Analytical Chemistry (GAC) community [18]. The in silico program uses a weighting method that assigns values in the range of 0.0–1.0 to specific fundamentals of the GAC approach. The above-mentioned approach produces analytical scores that are particularly efficient in evaluating greenness levels. The statistics are presented in a circular arrangement featuring a broad colors spectrum, from red to dark green, representing 12 separate characteristics. Figure 4 proves the degree to which the UPLC-MS/MS system exhibits greenness. The recorded scores for each of the 12 features were then compiled and listed in Table 7. The standing approach was assessed against numerous criteria, generating a data of 0.76. The data were employed as a quantitative evaluation of the degree of greenness responsibility achieved with the application of UPLC-MS/MS system. A number approaching 1.0 revealed an enhanced degree of greenness in the analytical methodology. The UPLC-MS/MS system, a recently established approach, exhibits a high degree of greenness, as shown by eco-scale data between 0.75 and 1.00.
The AGREE software was utilized to illustrate the greenness score profile of the current UPLC-MS/MS system, shown as a circular diagram comprising 12 distinct elements
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01339
The greenness report data sheet for the UPLC-MS/MS methodology in accordance with GAC requirements
| Criteria | Score | Weight |
| 1. To eliminate the need for sample pre-treatment, it is recommended to utilize direct analytical methods. | 0.3 | 2 |
| 2. The objectives of this study are to achieve a diminished quantity and smaller amount of samples. | 0.75 | 3 |
| 3. Preferably, it is advisable to operate measurements in situ whenever it is practical. | 0.66 | 1 |
| 4. Research indicates that the incorporation of analytical techniques and activities yields advantageous outcomes in energy conservation and the minimization of reagent consumption. | 1.0 | 2 |
| 5. It is advisable to choose automated and streamlined processes. | 0.75 | 2 |
| 6. It is prudent to advocate for the implementation of derivatization approaches. | 1.0 | 2 |
| 7. The necessity of minimizing the generation of significant analytical waste and implementing effective disposal methods is paramount. | 1.0 | 2 |
| 8. In analytical chemistry, there is a predilection for multi-analyte or multi-feature techniques rather than those that concentrate solely on only one analyte. | 1.0 | 2 |
| 9. Efforts should be undertaken to reduce energy use. | 0.0 | 2 |
| 10. It is prudent to organize the consumption of reagents derived from sources that are sustainable. | 1.0 | 2 |
| 11. The imperative of eliminating or substituting harmful reagents is of utmost importance. | 0.8 | 2 |
| 12. There is a necessity to enhance the safety regulations for employees. | 0.8 | 1 |
3.4 In vitro incubations of BLS with HLMs matrix
The sample designated as the negative control did not establish a statistically significant reduction in the quantity of BLS. An in vitro experiment was performed to evaluate the metabolic stability of BLS using a level of 1 μM mL−1 and an active human liver microsomes matrix. The selection to employ this certain level was determined by the target of maintaining the concentration below the Michaelis-Menten constant. This delineates a definitive correlation between the metabolic rate of BLS and the duration of incubation of the in vitro HLMs. This study utilized a level of 1 mg mL−1 of HLMs protein to lessen non-specific protein binding. The primary BLS metabolic stability curve was generated on the x-axis by charting certain time intervals from 0 to 60 min, during which the metabolic enzymes were inhibited. In Fig. 5A, the y-axis characterizes the residual proportion of BLS.
(A) BLS metabolic stability curve over the time points (0–60 min); (B) linear part from the time points (0–20 min) was employed to construct the LN calibration curve exhibiting the linearity equation
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01339
The linear part of the aforementioned graph was confined to the designated time points of 0–20 min. The aim of this study was to produce a curve illustrating the relation among various incubation time breaks (spanning 0–20 min) and the LN of the remaining ratio of BLS, as exhibited in Fig. 5B. The research outcomes revealed that the metabolic rate (slope) of the BLS was established at 0.0395 (SD: 0.002463), as proved by the equation y = −0.0395x + 4.615, with a r2 value of 0.9863 (Table 8). The Y-intercept is 4.615 (SD: 0.02684). The in vitro t1/2 can be computed employing the equation (LN2/slope). The recent study determined that the substance's half-life in an in vitro experiment was 17.55 min. The computed clearance of BLS was 46.21 mL min−1 kg−1 [35], categorizing BLS as a medication with high clearance, suggesting that it can be injected without worry for dose buildup in the human body. Employing software methodologies like as Cloe PK and modeling software can provide respected understandings into the in vivo pharmacokinetics of BLS, therefore elucidating significant physiological features [38].
Metabolic stability data of Belumosudil (BLS) after in vitro incubation with HLMs
| Time intervals (min.) | Mean* (ng mL−1) | X** | LN X | Linearity features |
| 0.00 | 438.95 | 100.00 | 4.61 | Linear regression line equation: y = −0.0395x + 4.615 R2 = 0.9863 Slope: −0.0395 t1/2: 17.55 min Clint: 46.21 mL min−1 kg−1 |
| 2.50 | 405.74 | 92.43 | 4.53 | |
| 5.00 | 360.87 | 82.21 | 4.41 | |
| 7.50 | 324.71 | 73.97 | 4.30 | |
| 15.00 | 261.34 | 59.54 | 4.09 | |
| 20.00 | 192.75 | 43.91 | 3.78 | |
| 30.00 | 154.23 | 35.14 | 3.56 | |
| 40.00 | 135.36 | 30.84 | 3.43 | |
| 50.00 | 128.64 | 29.31 | 3.38 | |
| 60.00 | 125.73 | 28.64 | 3.35 |
*Mean of 3 replicates.
**X: Average of the % residual BLS in 3 repeats.
4 Conclusions
The present study attentive on the establishment and evaluation of a UPLC-MS/MS method for determining BLS levels in HLMs. Additionally, the formerly indicated approach was employed for evaluation of the metabolic stability of BLS. The UPLC-MS/MS methodology shown exceptional characteristics due to its improved sensitivity, greenness, effective recovery, and selectivity of BLS and ENB from the HLMs matrix. The current data were obtained through the use of ACN as the chosen extraction system for protein precipitation. The UPLC-MS/MS approach was specifically designed to align with environmental sustainability, utilizing precise methods to reduce its ecological impact. The employed methods included a conservative flow rate of 0.4 mL min−1, a reduced ACN level at 50%, and a reduced analytical period of 2 min. An estimation of the degree of method greenness utilizing the AGREE program suggests that the UPLC-MS/MS methodology has environmentally responsive parameters and could be more suitable for the routine analysis of BLS, without inflicting damaging impacts on the surrounding environment. The outcomes on metabolic stability, featuring a half-life of 17.55 min and a high Clint of 46.21 mL min−1 kg−1, recommend that the BLS compound possesses characteristics like to those of a medicine with a high extraction ratio characteristic.
Ethics approval
The integration of HLMs obtained from Sigma business eliminates the necessity for ethical approval.
Authors' contributions
M.A., A.A., and A.K. conducted the in vitro analyses and were responsible for composing the initial version of the publication. A.A. and A.K. collaboratively developed and structured the present UPLC-MS/MS methodology, focusing specifically on software applications. The data and results in this study were exclusively obtained from internal sources, without reliance on other sources or unethical techniques such as paper mills. After an extensive review process, the final version of the work has garnered universal approval from all contributing authors.
Conflict of interest
No conflict of interest to report exist among the authors of this paper.
Funding
The Researcher Supporting Project Number (RSPD2025R760) of King Saud University in Riyadh, Saudi Arabia served as the funding source for this work.
Data availability statement
The data supporting the results of this investigation are accessible from the corresponding author upon an adequate request.
Acknowledgments
The authors express gratitude for the financial support provided by the Researcher Supporting Project Number (RSPD2025R760) from King Saud University in Riyadh, Saudi Arabia for this research endeavor.
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