Differential role of potential stressors, underlying degradation mechanism, characterization of degradants using LC-MS/MS, and establishment of a stability-indicating analytical method for duvelisib.

Duvelisib (DUV) was first approved globally in 2018. An extensive literature search revealed that the differential role of a potential degradation medium in altering the shelf-life of DUV due to its exposure during storage has not been identified till date. Moreover, its degradation impurities and degradation mechanism are not known. In addition, no analytical method has been reported for the quantification of DUV in the presence of its degradation impurities. Therefore, the aim of this study was to identify the impact of different potential degradation media on the stability of DUV, establish the degradation mechanism, and identify its major degradation impurities. The aim was also to establish a stability-indicating analytical method for the quantification of DUV in the presence of its degradation impurities. This study is the first to report the structure of degradation impurities and the step-by-step degradation mechanism of DUV. This information will be useful for the scientific community and manufacturers in optimizing the formulation parameters and/or storage conditions. The validated method can be employed for analysis of stability study and routine quality control samples of newer DUV formulations in pharmaceutical industries. The identified impurities may serve as impurity standards for specifying their limits in the drug after required qualification studies.

Delineation of prototypical degradation mechanism, characterization of unknown degradation impurities by liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry and stability-indicating analytical method of selumetinib.

Selumetinib (SELU) was recently approved by the US Food and Drug Administration (US FDA) in 2020. However, the degradation impurities of SELU have not been characterized or identified to date. The mechanism for impurity formation and the degradation behavior have not been previously studied. This study aims to elucidate the prototypical degradation mechanism of SELU. Furthermore, the degradation impurities have been identified using LC-quadrupole-time-of-flight tandem mass spectrometry and are reported in this article for the first time. In addition, a stability-indicating analytical method (SIAM) has been developed for this drug. Forced degradation studies revealed the degradation of SELU under various stress conditions, including hydrolytic stress (acid and base), oxidative stress, and photolytic stress (ultraviolet and visible). Three degradation impurities were identified. This article presents the first validated SIAM, capable of accurately quantifying SELU in the presence of its degradation impurities. Furthermore, we have proposed the degradation pathway for SELU and its degradation impurities, a first in the field. The developed SIAM can find applications in process development and quality assurance of SELU in both research laboratories and pharmaceutical industries. Moreover, the identified degradation impurities may serve as impurity standards for quality control testing in pharmaceutical industries.

Ultra-high-performance liquid chromatography-quadrupole time of flight tandem mass spectrometry based in vitro metabolite profiling of DK-GV-04P, a novel anticancer molecule under drug discovery.

DK-GV-04P, chemically identified as 3-cinnamyl-2-(4-methoxyphenyl) quinazolin-4(3H)-one, is an investigational molecule synthesized at the Chemical Biology Laboratory of the National Institute of Pharmaceutical Education and Research-Ahmedabad. The compound has shown potential anticancer activity against squamous CAL27 cell lines. Metabolite identification and characterization are critical in drug discovery, providing key insights into a compound's pharmacokinetics, pharmacodynamics safety, and metabolic fate. The primary aim of the study was to identify and characterize the in vitro metabolites of DK-GV-04P. In silico identification of the site of metabolism was also carried out using xenosite online software. The molecule was incubated with human liver microsomes and human S9 liver fraction to generate in vitro metabolites, which were further identified and characterized using ultra-high-performance liquid chromatography-quadrupole time of flight tandem mass spectrometry. A total of nine metabolites (four phase I and five phase II) were identified and characterized through tandem mass spectrometry. The major biotransformation pathways involved in metabolism of DK-GV-04P were hydroxylation, O-demethylation and glucuronidation. In addition to this, a detailed biotransformation pathway of DK-GV-04P has been established in this study.

Comprehensive characterization and preclinical assessment of an imidazopyridine-based anticancer lead molecule.

Imidazopyridine scaffold holds significant pharmacological importance in the treatment of cancer. An in-house synthesized imidazopyridine-based molecule was found to have promising anticancer activity against breast cancer, lung cancer, and colon cancer. The molecule is an inhibitor of pyruvate kinase M2, the enzyme that elevates tumor growth, metastasis and chemoresistance by directly controlling tumor cell metabolism. Screening of the physicochemical properties of any lead molecules is essential to avoid failure in late-stage drug development. In this research, the physicochemical properties of the molecule including log P, log D, pKa, and plasma protein binding were assessed to check its drug-likeness. Plasma and metabolic stability of the molecule were also evaluated. Moreover, pharmacokinetic profiles of the lead molecule in Sprague-Dawley rats and in vitro metabolite identification studies were also performed. Finally, an in silico software, Pro-Tox-II, was used to predict toxicity of the molecule and its metabolites. Log P, Log D (pH 7.4), pKa, and plasma protein binding of the molecule were found to be 2.03%, 2.42%, 10.4%, and 98%, respectively. The molecule was stable in plasma and metabolic conditions. A total of nine new metabolites were identified and characterized. Cmax and t½ of this molecule were found to be 4016 ± 313.95 ng/mL and 9.57 ± 3.05 h, respectively. Based on the previously reported study and this finding, the molecule can be considered as a promising anticancer lead with potential drug-likeness properties. Further preclinical and clinical drug discovery studies may be initiated in continuation of this study in search of a potential anticancer lead.

LC-QQQ-MS based intracellular quantification of bictegravir in peripheral blood mononuclear cells and plasma.

Most antiretrovirals (ARVs) have intracellular therapeutic target sites and therefore, their plasma concentration may be misleading when relating to their efficacy or toxicity. A bioanalytical method for quantification of the ARV drug bictegravir (BTG) in its target site peripheral blood mononuclear cells (PBMCs) is not available till date. This is the first time to establish a sufficiently sensitive mass spectrometry-based bioanalytical method to quantify BTG in both rat PBMCs and plasma. The developed method was validated over the range of 1 ng/ml to 100 ng/ml and 0.005 ng-10ng/sample for plasma and PBMCs, respectively. For PBMCs, average accuracy and precision at four quality control levels were found to be 93.30%-110.00% and 6.52%-8.25%, respectively. Plasma and intracellular pharmacokinetics of BTG was evaluated by the developed method in rats and a lack of accumulation of BTG in the PBMCs was observed. Pearson correlation coefficient data analysis indicated a moderated correlation between plasma and PBMC concentration of BTG. Therefore, it will be beneficial to include a quantification plan for BTG in its actual therapeutic target site during all its future research and development work. This reported method can be useful for site-specific monitoring of BTG in research laboratories and pharmaceutical industries.

Mechanism of capmatinib degradation in stress conditions including degradation product characterization using ultra-high-performance liquid chromatography-quadrupole-time of flight mass spectrometry and stability-indicating analytical method development.

RATIONALE: Capmatinib (CMT) has been recently approved for the treatment of non-small cell lung cancer by the United States Food and Drug Administration (USFDA). Till date, the degradation mechanism of CMT in different stress conditions is not known. Moreover, degradation products (DPs) of the drug are yet to be identified. Characterization study on degradation products of CMT has not been reported before. Furthermore, no previously reported literature is available on the stability-indicating method of CMT. METHODS: Owing to the lack of such scientific reports, we developed a sensitive, stability-indicating method for CMT which can resolve it from all its degradation products. The method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH Q2 [R1]) guideline. We studied and established the degradation mechanism of CMT in different stress conditions. One degradation product (DP2) was isolated and characterized using 1 H NMR. RESULTS: The degradation products (DP1, DP2 and DP3) of the drug have been identified and characterized for the first time by using high-resolution mass spectrometry and 1 H NMR spectroscopy. CMT was found to become degraded under acidic, basic and photolytic stress conditions in the solution phase to yield three major DPs. The drug was found to be stable in neutral hydrolysis, oxidation and thermal stress conditions. CONCLUSIONS: DP1 was formed under acidic and basic hydrolytic conditions, whereas DP2 and DP3 were formed under photolytic conditions. Characterization of all the DPs has been carried out to establish their structures and understand the molecular mechanism behind the degradation of the drug. Few studies reported quantitative analysis of CMT and its metabolites in biological fluids. However, this is the first study to identify the unknown DPs of CMT and the mechanism of its degradation. Moreover, this article reports a stability-indicating analytical method for CMT which has not yet been reported in any literature.

Assessment of metabolic stability and pharmacokinetics by LC-MS/MS and establishment of the safe dose of IMID-2, a novel anticancer molecule under drug discovery.

Pyruvate kinase (PK) M2 activators ramp up glycolysis in cancer cells, leading to a reversal of the Warburg effect in cancer cells. A promising PKM2 activator molecule, IMID-2, developed by the National Institute of Pharmaceutical Education and Research-Ahmedabad showed promising anticancer activity against MCF-7 and COLO-205 cell lines, which represent breast and colon cancer. Its physicochemical properties, like solubility, ionization constant, partition coefficient and distribution constant, have already been established. Its metabolic pathway is also well established through in vitro and in vivo metabolite profiling and reported previously. In this study, we have evaluated the metabolic stability of IMID-2 using LC-MS/MS and investigated the safety aspect of the molecule through an acute oral toxicity study. In vivo studies in rats confirmed that the molecule is safe even at a dose level of 175 mg/kg. Furthermore, a pharmacokinetic study of IMID-2 was also carried out using LC-MS/MS to understand its absorption, distribution, metabolism, and excretion profile. The molecule was found to have promising bioavailability through the oral route. This research work is thus another step in the drug testing of this promising anticancer molecule. The molecule can be considered to be a potential anticancer lead based on the earlier report substantiated by current findings.

Implication of metabolomics and transporter modulation based strategies to minimize multidrug resistance and enhance site-specific bioavailability: a needful consideration toward modern anticancer drug discovery.

Induction of drug-metabolizing enzymes and efflux transporters (DMET) through activation of pregnane x receptor (PXR) is the primary factor involved in almost all bioavailability and drug resistance-related problems of anticancer drugs. PXR is a transcriptional regulator of many metabolizing enzymes and efflux transporters proteins like p-glycoprotein (p-gp), multidrug resistant protein 1 and 2 (MRP 1 and 2), and breast cancer resistant protein (BCRP), etc. Several anticancer drugs are potent activators of PXR receptors and can modulate the gene expression of DMET proteins. Involvement of anticancer drugs in transcriptional regulation of DMET can prompt increased metabolism and efflux of their own or other co-administered drugs, which leads to poor site-specific bioavailability and increased drug resistance. In this review, we have discussed several novel strategies to evade drug-induced PXR activation and p-gp efflux including assessment of PXR ligand and p-gp substrate at early stages of drug discovery. Additionally, we have critically discussed the chemical structure and drug delivery-based approaches to avoid PXR binding and inhibit the p-gp activity of the drugs at their target sites.

High resolution mass spectrometry-driven metabolite profiling of baricitinib to report its unknown metabolites and step-by-step reaction mechanism of metabolism.

RATIONALE: Metabolite profiling is an integral part of the drug development process for selecting candidates with high therapeutic efficacy and low risk. Baricitinib (BARI) was approved in 2018 by the US Food and Drug Administration to treat rheumatoid arthritis. According to the available literature, no systematic study has been reported on the metabolite profiling of BARI. The biotransformation pathway of the drug has also not been established until recently. This study aims to identify BARI metabolites generated in in vitro matrices. METHODS: The in vitro metabolism study was carried out using rat liver microsome, human liver microsomes, and human S9 fraction. Ultra high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (U-HPLC-Q/TOF) and ultra-high-performance liquid chromatography/linear ion trap-Orbitrap mass spectrometry (U-HPLC/LTQ-Orbitrap-MS/MS) were used to identify and characterize the metabolites of BARI. The in silico toxicity of BARI and its metabolite was studied using ProTox-II toxicity predictor software. RESULTS: A total of five new metabolites have been identified amongst which two (M1 and M2) were detected on both U-HPLC/LTQ-Orbitrap-MS/MS and U-HPLC-Q/TOF and two additional metabolites (M4 and M5) were detected on U-HPLC/LTQ-Orbitrap-MS/MS. Moreover, one metabolite (M3) was only detected on LC-QTOF. CONCLUSIONS: The major metabolic changes were found to be N-dealkylation, demethylation, hydroxylation, and hydrolysis. Metabolites M3 and M4 were found to have the potential for carcinogenicity. The novelty of the study can be justified by the unavailability of any previous research on in vitro metabolite profiling of BARI. Furthermore, this is the first time the biotransformation pathway of BARI and the toxicity potential of its metabolites have been reported.

UHPLC-Q-TOF-MS/MS-based metabolite profiling of duvelisib and establishment of its metabolism mechanisms.

Duvelisib is a dual inhibitor of phosphoinositide 3 kinase that received global approval by the US Food and Drug Administration in 2018 to treat follicular lymphoma after at least two prior systemic therapies. An extensive literature search revealed that, to date, metabolites of duvelisib have not been characterized and information on them is not available in any of the literature. Moreover, the metabolism pathway is yet to be established. This study aimed to investigate and characterize the metabolites of duvelisib generated in microsomes and S9 fractions. In this study, five duvelisib metabolites were identified using UHPLC-Q-TOF-MS/MS analysis technique. The structural characterization of the metabolites was performed by comparing the fragmentation pattern of duvelisib and its metabolites through an accurate mass measurement technique. Three metabolites were generated through phase I hydroxylation and dechlorination reactions. The other two metabolites were generated through a phase II glucuronidation reaction. The metabolism mechanism established through this study can be useful to improve the safety profile of drugs of similar categories in the future after establishment of the toxicity profile of the identified metabolites.

Identification and characterization of the rat in-vivo and in-vitro metabolites of tazemetostat using LC-QTOF-MS.

In drug discovery, metabolite profiling unveils biotransformation pathways and potential toxicant formation, guiding selection of candidates with optimal pharmacokinetics and safety profiles. Tazemetostat (TAZ) is employed in treating locally advanced or metastatic epithelioid sarcoma. Identification of drug metabolites are of significant importance in improving safety, efficacy and reduced toxicity of drugs. The current study aimed to investigate the comprehensive metabolic fate of TAZ using different in vivo (rat) and in vitro (RLM, HLM, HS9) models. For in vivo studies, drug was orally administered to Sprague-Dawley rats with subsequent analysis of plasma, feces and urine samples. A total of 21 new metabolites were detected across various matrices and were separated on Phenomenex kinetex C18 (2.5 µm; 150 × 4.6 mm) column using acetonitrile and 0.1% formic acid in water as mobile phase. LC-QTOF-MS/MS and NMR techniques were employed to identify and characterize the metabolites from extracted samples. The major metabolic routes found in biotransformation of TAZ were hydroxylation, N-dealkylation, N-oxidation, hydrogenation, hydrolysis and N-acetylation. In silico toxicity revealed potential immunotoxicity for TAZ and few of its metabolites. This research article is the first time to discuss the complete metabolite profiling including identification and characterization of TAZ metabolites as well as its biotransformation mechanism.

Induction effect of antiretroviral bictegravir on the expression of Abcb1, Abcg2 and Abcc1 genes associated with P-gp, BCRP and MRP1 transporters present in rat peripheral blood mononuclear cells.

BACKGROUND: Antiretrovirals have the potential to cause drug interactions leading to inefficacy or toxicity via induction of efflux transporters through nuclear receptors, altering drug concentrations at their target sites. RESEARCH DESIGN AND METHODS: This study used molecular dynamic simulations and qRT-PCR to investigate bictegravir's interactions with nuclear receptors PXR and CAR, and its effects on efflux transporters (P-gp, BCRP, MRP1) in rat PBMCs. PBMC/plasma drug concentrations were measured using LC-MS/MS to assess the functional impact of transporter expression. RESULTS: Bictegravir significantly increased the expression of ABC transporters, with Car identified as a key mediator. This suggests that bictegravir's influence on nuclear receptors could affect drug transport and efficacy at the cellular level. CONCLUSIONS: Bictegravir activates nuclear receptors enhancing efflux transporter expression. Understanding these interactions is crucial for preventing drug-drug interactions and reducing toxicity in clinical use. Combining CAR antagonists with bictegravir may prevent drug resistance and toxicity. However, these findings are based on preclinical data and necessitate further clinical trials to confirm their applicability in clinical settings.

Development of an effective cleaning technique and ancillary analytical method for estimation of residues of selected kinase inhibitors from stainless steel and glass surfaces by swab sampling.

Importance of cleaning validation in the pharmaceutical industry cannot be overstated. It is essential for preventing cross-contamination, ensuring product quality & safety, and upholding regulatory standards. The present study involved development of an effective cleaning method for five selected kinase inhibitors binimetinib (BMT), selumetinib (SMT), brigatinib (BGT), capmatinib (CPT), and baricitinib (BRT). For checking the effectiveness of the developed cleaning technique, a sensitive and specific RP-HPLC based analytical method employing a diode array detector has been established to quantitate drug residue on glass and stainless steel surfaces. A reproducible swab sampling protocol utilizing TX714A Alpha swabs wetted with an extracting solvent has been developed to collect representative samples from both surfaces. Chromatographic separation of selected kinase inhibitors was achieved in gradient mode using an Agilent Zorbax eclipsed C18 column with acetonitrile and 10 mM ammonium formate as the mobile phase. The analytes were chromatographically separated in a 12 min run time. The mean swab recovery for each drug from glass and stainless steel surfaces exceeded 90%. Cleaning with IPA (70%) and acetone (70%) effectively removed residues for all five drugs. A solution comprising 10 mM SDS with 20% IPA demonstrated good efficacy in cleaning residues of BGT, BRT, and CPT, but exhibited lower efficacy for SMT and BMT.

ProteoExcelTP: Development of a simple excel-based tool for surrogate peptide selection in mass spectrometry based targeted proteomics.

Selection of surrogate peptides plays a major role to achieve reproducible and accurate quantification of desired proteins in targeted proteomics. Currently, available peptide selection tools suffer from the limitation of not covering entire proteins including all the species and inflexibility in applying the exclusion criteria. Here, we have developed an excel-based ProteoExcelTP tool which can automatically select the most appropriate surrogate peptides with high flexibility in terms of addition, deletion or changing the exclusion criteria. The developed ProteoExcelTP tool has also been validated by comparison of obtained peptides from the tool with those selected in previously reported works. This is the first time to develop an excel based tool for quick and accurate selection of surrogate peptides for entire protein family of all the species. The tool is having the unique advantage of a highly user-friendly nature. It can be customized according to the specific need of the researchers. ProteoExcelTP tool will significantly enhance the throughput of the quantitative proteomic analysis. The tool can immensely help the scientists working in the field of proteomics by significantly minimizing their effort in accurate selection of surrogate peptides for quantification of endogenous proteins.

Innovation in Strategies for Sensitivity Improvement of Chromatography and Mass Spectrometry Based Analytical Techniques.

Chromatography and mass spectrometry based techniques are the most commonly employed procedures to quantitate the analytes in pharmaceutical research. However, sensitivity of analytical methods significantly varies due to the difference in physicochemical characteristics of analytes. Sensitivity of methods greatly affects the quality of analytical results. Establishment of a sufficiently sensitive method ensures the suitability of a technique for its intended purpose. Although various types of advancement in chromatographic science are witnessed, issues related to sensitivity remain a major challenge for the analyte with low detection limit. Highly sensitive analytical methods are specifically essential to quantitate the analytes in the samples from dissolution study of sustained release formulations, cross-contamination study, impurity analysis, metabolite profiling, bioanalysis of highly potent and low bioavailable drugs. In recent years, huge involvement of researchers toward sensitivity enhancement of quantitative methods is evidenced. Wide verities of approaches are being reported in the field. Derivatization technique, introduction of ion-pairing reagents, sample pretreatment, and utilization of innovative methods such as 2-dimensional liquid chromatography, nano liquid chromatography, 2-dimensional gas chromatography, supercritical fluid chromatography, use of microcolumn are some approaches that are being employed. Online sample preparation techniques can significantly improve the sensitivity of a method by reducing sample loss and degradation. This review summarizes and critically discussed the approaches to improve the sensitivity of chromatographic and mass spectrometry based analytical methods. This article can guide the researchers to select suitable approaches for achieving the desired detection limit of analytical and bioanalytical methods based on their specific requirements.

Progressive tools and critical strategies for development of best fit PBPK model aiming better in vitro-in vivo correlation.

Nowadays, conducting discriminative dissolution experiments employing physiologically based pharmacokinetic modeling (PBPK) or physiologically based biopharmaceutical modeling (PBBM) is gaining significant importance in quantitatively predicting oral absorption of drugs. Mechanistic understanding of each process involved in drug absorption and its impact on the performance greatly facilitates designing a formulation with high confidence. Unfortunately, the biggest challenge scientists are facing in current days is the lack of standardized protocol for integrating dissolution experiment data during PBPK modeling. However, in vitro-in vivo drug release interrelation can be improved with the consideration and development of appropriate biorelevant dissolution media that closely mimic physiological conditions. Multiple reported dissolution models have described nature and functionality of different regions of the gastrointestinal tract (GI) to more accurately design discriminative dissolution media. Dissolution experiment data can be integrated either mechanistically or without a mechanism depending primarily on the formulation type, biopharmaceutics classification system (BCS) class and particle size of the drug substance. All such parameters are required to be considered for selecting the appropriate functions during PBPK modeling to produce a best fit model. The primary focus of this review is to critically discuss various progressive dissolution models and tools, existing challenges and approaches for establishing best fit PBPK model aiming better in vitro-in vivo correlation (IVIVC). Strategies for proper selection of dissolution models as an input function in PBPK/PBBM modeling have also been critically discussed. Logical and scientific pathway for selection of different type of functions and integration events in the commercially available in silico software has been described through case studies.

Maresin-1 prevents blood-spinal cord barrier disruption associated with TRPV4 elevation in the experimental model of spinal cord injury.

Recently, we reported the TRPV4 ion channel activation and its association with secondary damage after spinal cord injury (SCI). TRPV4 activation is linked with blood-spinal cord barrier (BSCB) disruption, endothelial damage, and inflammation after SCI. Specialized pro-resolving mediators (SPM) are endogenous lipid mediators released for inflammation resolution. Studies suggest that SPM could act as an endogenous antagonist of ion channels directly or indirectly at the plasma membrane. Herein, we studied the effect of maresin-1, a docosahexaenoic acid (DHA)-derived SPM, in SCI-induced TRPV4 expression and subsequent associated damage. First, employing a particular agonist (4αPDD) in endothelial and neuronal cell lines, we examined the potential of maresin-1 to block TRPV4 activation. Then we quantify the DHA levels in plasma and epicenter of the spinal cord in sham and at 1, 3, 7, 14, 21, and 28-days post-injury (DPI) using LC-MS. Then, we exogenously administered maresin-1 using two dosing regimens i.e., single-dose (1 µg) and multiple-dose (1 µg/day for seven days), to confirm its role in the TRPV4 inhibition and its linked damage. After SCI, DHA levels decrease in the spinal cord epicenter area as well as in the plasma. Treatment with maresin-1 attenuates TRPV4 expression, inflammatory cytokines, and chemokines and impedes neutrophil infiltration. Furthermore, treatment with maresin-1 prevents BSCB disruption, alleviates glial scar formation, and improves functional recovery. Thus, our results suggest that maresin-1 could modulate TRPV4 expression and could be a safe and promising approach to target inflammation and BSCB damage after SCI.

Advancement in Analytical Strategies for Quantification of Biomarkers with a Special Emphasis on Surrogate Approaches.

Accurate quantification of biomarkers has always been a challenge for many bioanalytical scientists due to their endogenous nature and low concentration in biological matrices. Different analytical approaches have been developed for quantifying biomarkers including enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and chromatographic techniques assisted with mass spectrometry. Liquid chromatography-tandem mass spectrometry-based quantification of biomarkers has gained more attention over other traditional techniques due to its higher sensitivity and selectivity. However, the primary challenge lies with this technique includes the unavailability of a blank matrix for method development. To overcome this challenge, different analytical approaches are being developed including surrogate analyte and surrogate matrix approach. Such approaches include quantification of biomarkers in a surrogate matrix or quantification of an isotopically labeled surrogate analyte in an authentic matrix. To demonstrate the authenticity of the surrogate approach, it is mandatory to establish quantitative parallelism through validation employing respective surrogate analytes and surrogate matrices. In this review, different bioanalytical approaches for biomarker quantification and recent advancements in the field aiming for improvement in the specificity of the techniques have been discussed. Liquid chromatography-tandem mass spectrometry-based surrogate approaches for biomarker quantification and significance of parallelism establishment in both surrogate matrix and surrogate analyte-based approaches have been critically discussed. In addition, different methods for demonstrating parallelism in the surrogate method have been explained.

LC-MS/MS bioanalytical method for quantification of binimetinib and venetoclax, and their pharmacokinetic interaction.

Aim: Because of several prospective benefits, binimetinib (BMT)-venetoclax (VTC) combination can be a better therapeutic strategy to treat cancer. Results: An LC-MS/MS method for simultaneous quantification of BMT and VTC in rat plasma has been developed and validated. Specificity, accuracy, precision and stability results met the acceptance criteria for validation. Accuracy and precisions at all quality control levels were <15%. The study revealed that co-administration of BMT and VTC has no significant effect on their pharmacokinetics. Conclusion: The developed method can provide accurate results for quantification of BMT and VTC over the range of 5-500 ng/ml. The reported pharmacokinetic interaction study results will be useful for future consideration of the combined treatment of BMT and VTC in anticancer chemotherapy regimens.

Surrogate peptide selection and internal standardization for accurate quantification of endogenous proteins.

Relative quantification techniques have dominated the field of proteomics. However, biomarker discovery, mathematical model development and studies on transporter-mediated drug disposition still need absolute quantification of proteins. The quality of data of trace-level protein quantification is solely dependent on the specific selection of surrogate peptides. Selection of surrogate peptides has a major impact on the accuracy of the method. In this article, the advanced approaches for selection of surrogate peptides, which can provide absolute quantification of the proteins are discussed. In addition, internal standardization, which accounts for variations in the quantitation process to achieve absolute protein quantification is discussed.