Comprehensive degradation profiling and influence of different oxidizing reagents on tinoridine hydrochloride: Structural characterization of its degradation products using HPLC and HRMS.

RATIONALE: Stress testing on tinoridine hydrochloride was carried out using a multidimensional approach. This included different conditions: hydrolytic (acidic, alkaline, and neutral conditions), different oxidative reagents, thermal, photolytic conditions, HPLC method development, and structural elucidation using high-resolution mass spectrometry (HRMS). It provides the basis for quality control of tinoridine hydrochloride and its derivatives during storage conditions. METHODS: The tinoridine hydrochloride was subjected to a variety of stress conditions. A gradient reversed-phase HPLC method was developed on a X-Bridge C18 column (250 × 4.6 mm, 5 µm) to separate all the degradation products (DPs). HRMS studies have been performed to elucidate the structure of DPs. RESULTS: HPLC-PDA study revealed that significant degradation products were formed in hydrolytic, AIBN (radical initiator at 40°C), thermal, and solid-state photolight stress conditions, but the drug was stable under oxidative conditions (H2 O2, Fenton's reagent at room temperature and ferric chloride at 40°C). The structure of degradation products was elucidated, and mechanism of their formation was explained. CONCLUSION: Stress study was successfully carried out as per ICH Q1A (R2) guideline on tinoridine hydrochloride. A total of six new degradation products were characterized, DP 2 and DP 6 formed by the effect of co-solvent. This study provides the scientifically sound basis for quality monitoring and storage conditions of tinoridine hydrochloride.

iRGD conjugated nimbolide liposomes protect against endotoxin induced acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a deadly respiratory illness associated with refractory hypoxemia and pulmonary edema. The recent pandemic outbreak of COVID-19 is associated with severe pneumonia and inflammatory cytokine storm in the lungs. The anti-inflammatory phytomedicine nimbolide (NIM) may not be feasible for clinical translation due to poor pharmacokinetic properties and lack of suitable delivery systems. To overcome these barriers, we have developed nimbolide liposomes conjugated with iRGD peptide (iRGD-NIMLip) for targeting lung inflammation. It was observed that iRGD-NIMLip treatment significantly inhibited oxidative stress and cytokine storm compared to nimbolide free-drug (f-NIM), nimbolide liposomes (NIMLip), and exhibited superior activity compared to dexamethasone (DEX). iRGD-NIMLip abrogated the LPS induced p65 NF-κB, Akt, MAPK, Integrin ß3 and ß5, STAT3, and DNMT1 expression. Collectively, our results demonstrate that iRGD-NIMLip could be a promising novel drug delivery system to target severe pathological consequences observed in ARDS and COVID-19 associated cytokine storm.

Identification and characterization of novel metabolites of nintedanib by ultra-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry with in silico toxicological assessment.

RATIONALE: Nintedanib, an oral, triple angiokinase inhibitor, is used alongside docetaxel in the management of locally recurrent non-small-cell lung cancer and idiopathic pulmonary fibrosis. The present study deals with the identification and characterization of in vitro and in vivo stable and reactive (if any) metabolites of nintedanib and sheds light on some novel metabolites of the drug which have not been reported previously. METHODS: The study involved an oral administration of the drug to male Wistar rats, followed by collection of the biological matrices (urine, plasma and feces) at specific intervals for determination of in vivo metabolites. In addition, in vitro studies were performed on human and rat liver microsomes in the presence of appropriate co-factors. The samples were subjected to protein precipitation and nitrogen evaporation prior to ultra-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry analysis. The toxicities of all the metabolites were assessed in silico, employing ADMET Predictor™. RESULTS: A total of 18 metabolites of nintedanib were identified in all the matrices, of which nine were found to be novel and unreported previously. The unreported metabolites were elucidated as oxidative, demethylated and glucuronide conjugates of nintedanib. Interestingly, acetonitrile adducts of a few metabolites (low concentration) were also observed. No reactive metabolites were observed in this study. CONCLUSIONS: Characterization of hitherto unknown in vitro and in vivo metabolites of nintedanib adds to the existing knowledge on the metabolism of the drug. Identification on the basis of the solvated adducts can be a useful approach for characterization of minor metabolites, which remain undetected owing to sensitivity issues.

In vivo metabolic investigation of cetilistat in normal versus pseudo-germ-free rats using UPLC-QTOFMS/MS and in silico toxicological evaluation of its metabolites.

Cetilistat (CET) is a pancreatic lipase inhibitor approved for management of obesity after the serious adverse effects exhibited by its analogue orlistat. Exhaustive literature review reveals lack of comprehensive reports on its biotransformation. With a view to study the same, the present study reports the identification and characterization of metabolites of CET in rats using UPLC-MS/MS. As the small intestine is the site of action for CET, it is important that the role of microbial flora in the metabolism of CET be explored. To achieve this, the metabolic profile of CET was compared between normal and pseudo-germ-free rats. The study involved the administration of a drug suspension to male Sprague-Dawley pseudo-germ-free and normal untreated rats followed by collection of urine, feces, and blood at specific intervals. Sample preparation was performed using liquid-liquid extraction and concentration of samples followed by analysis using LC-MS/MS. Finally, an in silico study was performed on the drug and metabolites to predict their toxicological properties using ADMET PredictorTM software. Four metabolites of CET were observed in in vivo matrices. As expected, significant changes were observed both qualitatively and quantitatively, implying that formation of metabolites was both CYP enzymes and gut microflora mediated.

A comprehensive study on rearrangement reactions in collision-induced dissociation mass spectrometric fragmentation of protonated diphenyl and phenyl pyridyl ethers.

RATIONALE: Recently, we have reported a forced degradation study of a pharmaceutical drug regorafenib which contains a phenyl pyridyl ether derivative as building block. We observed interesting rearrangements in two of its degradation products in tandem mass spectrometry (MS/MS) experiments. As diphenyl ether derivatives are also molecular building blocks of biological importance and used as herbicides and flame retardants, we decided to investigate specifically the fragmentation behavior of these compounds along with phenyl pyridyl derivatives in detail using high-resolution electrospray ionization (ESI) MS/MS. METHODS: To understand the fragmentation reactions of protonated substituted diphenyl ethers and phenyl pyridyl ethers, ESI-MS/MS experiments were performed using a quadrupole time-of-flight (QTOF) mass spectrometer. RESULTS: In contrast to radical cations of diphenyl ether derivatives which do not eliminate CO, the [M + H]+ ions of substituted diphenyl ethers undergo rearrangement reactions after loss of neutral molecules (H2 O, HCl, etc.) to form a bicyclic structure containing a keto group and do eliminate CO. Similar rearrangement followed by fragmentation was observed for protonated phenyl pyridyl ethers and the degradation products formed from regorafenib and sorafenib. CONCLUSIONS: The protonated ions of substituted diphenyl ethers and phenyl pyridyl ethers on collision-induced dissociation have exhibited interesting rearrangement reactions, despite the nature of the substituent on both the aryl moieties. The proposed fragmentation patterns of these compounds give an insight into the understanding of gas-phase reactions in mass spectrometric studies of diphenyl ether and phenyl pyridyl ether derivatives.

Characterization of forced degradation products of canagliflozine by liquid chromatography/quadrupole time-of-flight tandem mass spectrometry and in silico toxicity predictions.

RATIONALE: Forced degradation studies are useful for better understanding of the stability of active pharmaceutical ingredients and drugs and to generate information about drug degradation pathways and formation of degradation products (DPs). Identification of DPs plays a vital role in establishing the safety and therapeutic benefit of a drug. METHODS: Canagliflozin (CAN) was subjected to different stress conditions as per International Conference on Harmonization guidelines (Q1A R2). All the DPs and the drug were well separated on an Aquity CSH C18 (100 × 2.1 mm, 1.7 µm) column using acetonitrile-methanol (70:30, v/v) and formic acid in gradient mode. The same UPLC method was employed for LC/HRMS for the characterization of DPs. In addition, in silico toxicity was predicted for all the DPs by using TOPKAT and DEREK software tools. RESULTS: CAN was found to degrade under oxidative stress condition and formed DP1 and DP2. This is a typical case of degradation where co-solvents acetonitrile-water (50:50, v/v) and methanol-water (50:50, v/v) react with CAN under acid hydrolytic conditions leading to the formation of pseudo-DPs, DP3 and DP4, respectively. Among these, DP2 and DP3 showed ocular irritancy whereas DP1 showed skin sensitization. CONCLUSIONS: The drug was labile under oxidative stress condition. CAN reacted with co-solvent under acid hydrolytic conditions and gave pseudo-DPs. All the DPs were separated using UPLC and characterized by LC/QTOF/MS/MS. Toxicity of DPs was evaluated using TOPKAT and DEREK software tools.

Identification and characterization of vilazodone metabolites in rats and microsomes by ultrahigh-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry.

RATIONALE: Vilazodone is a selective serotonin reuptake inhibitor (SSRI) used for the treatment of major depressive disorder (MDD). An extensive literature search found few reports on the in vivo and in vitro metabolism of vilazodone. Therefore, we report a comprehensive in vivo and in vitro metabolic identification and structural characterization of vilazodone using ultrahigh-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF/MS/MS) and in silico toxicity study of the metabolites. METHODS: To identify in vivo metabolites of vilazodone, blood, urine and faeces samples were collected at different time intervals starting from 0 h to 48 h after oral administration of vilazodone to Sprague-Dawley rats. The in vitro metabolism study was conducted with human liver microsomes (HLM) and rat liver microsomes (RLM). The samples were prepared using an optimized sample preparation approach involving protein precipitation followed by solid-phase extraction. The metabolites have been identified and characterized by using LC/ESI-MS/MS. RESULTS: A total of 12 metabolites (M1-M12) were identified in in vivo and in vitro matrices and characterized by LC/ESI-MS/MS. The majority of the metabolites were observed in urine, while a few metabolites were present in faeces and plasma. Two metabolites were observed in the in vitro study. A semi-quantitative study based on percentage counts shows that metabolites M11, M6 and M8 were observed in higher amounts in urine, faeces and plasma, respectively. CONCLUSIONS: The structures of all the 12 metabolites were elucidated by using LC/ESI-MS/MS. The study suggests that vilazodone was metabolized via hydroxylation, dihydroxylation, glucuronidation, oxidative deamination, dealkylation, dehydrogenation and dioxidation. All the metabolites were screened for toxicity using an in silico tool.

Chiral LC method development: Stereo-selective separation, characterization, and determination of cabotegravir and related RS, RR, and SS isomeric impurities on coated cellulose-based chiral stationary phase by HILIC-LC and LC-MS.

The first, sensitive, and rapid chiral method was developed for enatioseperations and determination of cabotegravir and its enantiomeric impurities by using HPLC and LC-MS. Cabotegravir is an antiretroviral medication used for the treatment of HIV/AIDS approved by the food and drugs administration (FDA) in the year 2021. The cabotegravir chiral separation was achieved on the coated cellulose-tris (4-chloro-3-methyl phenyl carbamate) (CHIRALCEL OX-3R) column in HILIC mode and the total run time is less than 15 min. The effects of mobile phase composition, elution mode, and percentage of organic modifier as well as the effect of mobile phase-additives and column temperature were investigated on selectivity, resolution, and peak symmetry. The mobile phase consisted of acetonitrile and water with 0.1% (v/v) addition of formic acid additive with the flow rate of 1 mLmin-1. UV detection was carried out at 220 nm. The calibration curves of cabotegravir and its enantiomers were linear over the concentration range of 0.04-1.125 µgmL-1. The limits of detection and quantification for cabotegravir and its enantiomer (RS isomer) were ≤ 0.02 and ≤ 0.06, and the RR and SS-isomers limits were ≤ 0.02 and 0.03 µgmL-1 respectively. It was demonstrated that the proposed method is selective, precise, and robust. Finally, the validated method was applied for the determination and identification of cabotegravir and its chiral enantiomers in the bulk drugs by using HPLC and LC-MS techniques.

Evaluation of Chiral Liquid Chromatographic Method for Separation and Quantification of Isomers of Brivaracetam.

A selective, sensitive and robust chiral analytical method was developed for the quantification of Brivaracetam (BRV) and its three isomers. Systematic chiral chromatographic elution process was executed in different modes on chiral columns of polysaccharide based to attain the finest condition. The analytical method was developed by utilizing immobilized polysaccharide chiral column (CHIRALPAK IG-U) with reversed phase under isocratic condition containing acetonitrile and 10 mM ammonium bicarbonate in the proportion of 40:60 (v/v). The mobile phase flow rate and column temperature were monitored at 0.3 mLmin-1 and 25°C with a resolution of more than 2.0. The eluted components from the column were processed at 212 nm UV detection. The limit of detection and limit of quantification values of BRV, 2R, 4S-Isomer, 2R, 4R-Isomer and 2S, 4S-Isomers were found to be 0.0066/0.02, 0.0035/0.0107, 0.0036/0.0109 and 0.005/0.0152 µgml-1 respectively. Precision, linearity, accuracy and robustness were conducted according to ICH guidelines and the findings were within the acceptable limits. The proposed analytical method was found to be precise, accurate and specific for the quantification of enantiomer and its diastereomers for drug product and drug substance of BRV.

Characterization of stress degradation products of nintedanib by UPLC, UHPLC-Q-TOF/MS/MS and NMR: Evidence of a degradation product with a structure alert for mutagenicity.

Nintedanib is an anti-cancer drug used for the treatment of idiopathic pulmonary fibrosis and non-small cell lung cancer. The purpose of this study was to explore its degradation chemistry under various stress conditions recommended in ICH guidelines Q1A R(2). The drug was subjected to hydrolytic, photolytic, thermal and oxidative (H2O2, AIBN, FeCl3 and FeSO4) stress conditions. The degradation products formed in stressed solutions were successfully separated on an ACQUITY UPLC CSH C18 (2.1 × 100 mm, 1.7 µm) column, using a gradient UPLC-PDA method, developed with acetonitrile:methanol (90:10) and 0.1 % formic acid (pH 3.0) as the mobile phase. The drug proved to be labile to acidic, neutral and alkaline hydrolytic, and H2O2/AIBN oxidative conditions. It was stable to photolytic and thermal stress conditions, and even in oxidative reaction solutions containing FeCl3 or FeSO4. Additionally, the drug exhibited instability when its powder with added sodium bicarbonate was stored at 40 °C/75 % RH for 3 months. In total, nine degradation products (DPs 1-9) were formed. To characterize them, a comprehensive mass fragmentation pathway of the drug was first established using UHPLC-Q-TOF/MS/MS data. Similarly, the mass studies were then carried out on the stressed samples using the developed UPLC method. All the degradation products were primarily characterized through comparison of their mass fragmentation profiles with that of the drug. To confirm the structure in one case (DP 3), additional nuclear magnetic resonance (NMR) studies were carried out on the isolated product. Subsequently, mechanisms for their formation were laid down. A significant finding was the formation of a degradation product upon acid hydrolysis having a free aromatic amine moiety, which is considered as a structural alert for mutagenicity. Furthermore, the physicochemical and ADMET properties of the drug and its degradation products were predicted using ADMET predictor™ software.

Identification and structural characterization of hydrolytic degradation products of alvimopan by LC/QTOF/MS/MS and NMR studies.

Alvimopan (ALV), a peripherally acting mu-opioid receptor (PAM-OR) antagonist used for the treatment of postoperative ileus, was examined for its degradation behaviour under different stress conditions. A total of five degradation products (DP1-DP5) were formed and identified using high-performance liquid chromatography (HPLC) method. Primarily, complete fragmentation pathways of the protonated drug and its degradation products (DP1-DP5) were elucidated by using liquid chromatography-mass spectrometry (LC/MS) and high-resolution mass spectrometry (HRMS) studies. Subsequently, three major degradation products (DP1-DP3) formed under acid hydrolytic stress conditions were isolated by preparative-high performance liquid chromatography (prep-HPLC) and subjected to nuclear magnetic resonance (NMR) experiments (1D and 2D NMR) for further confirmation of their structures. All the spectral data from LC/QTOF/MS/MS and NMR studies were used for the identification and structural characterization of degradation products (DP1-DP5).

Isolation and structural characterization of degradation products of afatinib dimaleate by LC-Q-TOF/MS/MS and NMR: cytotoxicity evaluation of afatinib and isolated degradation products.

Afatinib is an irreversible tyrosine kinase receptor inhibitor which was approved lately by USFDA for the treatment of metastatic non-small cell lung cancer (NSCLC). AFT was subjected to stress degradation studies under hydrolytic (acid, base and neutral), oxidative, thermal and photolytic conditions to investigate the inherent stability of the drug. The present study describes the simple and rapid HPLC method development for the selective separation of the AFT and its degradation products. The drug and degradation products were separated on Agilent Eclipse plus C18 (150 × 4.6 mm, 5 µ) column with ammonium acetate buffer (10 mM, pH 6.7) in gradient elution mode. The drug was found to be unstable in all the conditions studied. The developed chromatographic method was extended to tandem mass spectrometry (QTOF-MS) for the characterization of the degradation products. A total of 11 unknown degradation products were characterized using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS/MS). Two major degradation products (DP2 and DP3) were isolated using preparative HPLC and their structures were confirmed by conducting 1H and 13C NMR experiments. The isolated DPs were evaluated for their anticancer potential using non small cell lung cancer cell line A549. The IC50 values for AFT, DP2 and DP3 were found to be 15.02 ± 1.49, 25.00 ± 1.26 and 32.56 ± 0.11 respectively. The in silico toxicity studies were performed employing ProTox-II software for the assessment of toxicity potential of drug and its degradation products. Finally, the developed chromatographic method was validated as per the International Conference on Harmonization guideline Q2 (R1).

In vitroand in vivo investigation of metabolic fate of riociguat by HPLC-Q-TOF/MS/MS and in silico evaluation of the metabolites by ADMET predictor™.

Riociguat, a guanyl cyclase inhibitor, is one of its kind drug regimen approved for management of pulmonary arterial hypertension and chronic thromboembolism pulmonary hypertension. Extensive literature review indicates lack of comprehensive reports on its metabolic fate. The present study reports the in vivo and in vitro identification and characterization of metabolites of riociguat, using high-performance liquid chromatography-quadruple time-of-flight tandem mass spectrometry. In vitro studies were conducted by incubating the drug in human and rat liver microsomes in presence of respective cofactors. In vivo studies were undertaken by oral administration of suspension of drug to male Sprague-Dawley rats followed by collection of urine, feces and blood at specific intervals. A total of 18 metabolites were observed in in vivo and in vitro matrices which includes hydroxyl, N-oxide, desmethyl, defluorinated hydroxyl, glucuronides and N-acetyl cysteine conjugates. Presence of N-acetyl cysteine conjugates strongly points towards the formation of a reactive metabolite intermediate trapped through N-acetyl cysteine and can be considered a matter of concern as the reactive metabolites have been known to manifest toxicities. Their presence was mimicked in in vitro samples as well. The toxicological properties of drug and metabolites were evaluated by using ADMET Predictor ™ software.

LC-ESI-MS determination and pharmacokinetics of adrafinil in rats.

A highly sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for investigating the pharmacokinetics of adrafinil in rats was developed. Rat serum pretreated by solid-phase extraction (SPE) was analyzed by LC-MS/MS with an electrospray ionization (ESI) interface. The mobile phase consisted of acetonitrile:water:acetic acid (35:65:0.1, v/v/v) in an isocratic elution mode pumped at 1.0 ml/min. The analytical column (250 mm x 4.6 mm i.d.) was packed with Kromasil C(18) material (5.0 microm). The standard curve was linear from 16.5 to 5000 ng/ml. The assay was specific, accurate (R.S.D.<2.6%), precise and reproducible (within- and between-day precisions R.S.D. <7.0% and <9.0%, respectively). Adrafinil in rat serum was stable over three freeze-thaw cycles at ambient temperature for 6 h. The method had a lower limit of quantitation of 16.5 ng/ml, which offered high sensitivity for the determination of adrafinil in serum. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats.

Continuous counter current extraction, isolation and determination of solanesol in Nicotiana tobacum L. by non-aqueous reversed phase high performance liquid chromatography.

A method of continuous counter current extraction in a large-scale production of solanesol from tobacco leaves was developed. The crude extract containing 15-20% solanesol was subjected to a series of steps, viz., saponification, solvent recrystallization and column chromatography. The pure material was characterized by FT-IR, ESI-MS, 1H NMR and 13C NMR spectrometry. The analysis was carried out by a simple and rapid non-aqueous reversed-phase high performance liquid chromatographic method on a Hypersil BDS C18 column (250 mm x 4.6mm, particle size 5 microm) with isopropyl alcohol-methanol (60:40, v/v) as mobile phase and detection at 215 nm. The product purity was between 95 and 98% (w/w) as determined by HPLC.

LC/QTOF/MS/MS characterization, molecular docking and in silico toxicity prediction studies on degradation products of anagliptin.

Pharmaceutical drugs are potential molecules with specific biological activity. However, long-term use of these chemical molecules can affect the human physiological system because of their increased levels in the human body. Therefore, identification and structure elucidation of impurities or degradation products should be taken into consideration in order to assure drug safety. The present study assessed the degradation behaviour of dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin under different stress conditions as per ICH guidelines Q1A (R2) followed by elucidation of the structure of degradation products. All the stress samples were analysed by using UPLC/PDA. The superior separation of drug from its degradation products was attained with time programmed gradient elution on BEH C18 (100 mm × 2.1 mm, 1.7 µm) column using 10 mM ammonium formate (aqueous) and acetonitrile (organic) as the mobile phase components. All the degradation products of anagliptin were characterized using LC/QTOF/MS/MS. In addition, the activity and toxicity of degradation products were determined through molecular docking and in silico toxicity prediction studies, respectively. The developed UPLC/PDA method was validated as per ICH guidelines in terms of specificity, accuracy, precision, linearity and robustness.

First report on the pharmacokinetic profile of nimbolide, a novel anticancer agent in oral and intravenous administrated rats by LC/MS method.

Nimbolide is a novel, natural compound with promising potential as a drug candidate for anticancer activity. It is isolated from the Indian traditional medicinal plant Azadirachta indica popularly known as neem. The present study was undertaken to explore the oral bioavailability and pharmacokinetic characteristics of nimbolide in rats using the LC/QTOF/MS method. A simple protein precipitation method using acetonitrile was employed for extracting nimbolide from rat plasma. The chromatographic separation of nimbolide and the internal standard (regorafenib) was attained on an Aquity BEH C18 column (100 × 2.1 mm, 2.7 µm), using ACN and 0.1% of formic acid in water as mobile phase components in a gradient elution mode at a flow rate of 0.45 mL/min over a short run time of 4 min. A mass detection was carried out using target ions of [M + H]+ at m/z 467.2074 for nimbolide and m/z 483.0847 for the internal standard. The LC/MS method was validated and all the parameters were found well within the specified limits. The calibration curve was constructed in the range of 1-1000 ng/mL. The method shows good accuracy (91.66-97.12%) and precision (intra 2.21-6.92% CV and inter-day 2.56-4.62% CV). This developed LC/MS method was effectively applied to the pharmacokinetic study of nimbolide upon oral and intravenous administration in rats. In concordance with its physicochemical properties and high lipophilicity, we found that it shows poor oral absorption at different doses (10, 30 and 50 mg/kg). As expected, higher plasma levels were observed upon intravenous (10 mg/kg) administration. This method can be extended for evaluation of drug interaction and drug metabolism in rats as well as in humans. Moreover, our rapid and sensitive method may cater the need to accelerate the preclinical formulation development and lead optimization for future drug development of this potent anticancer agent. Further, our oral bioavailability studies demonstrated that nimbolide possesses poor oral absorption, which could be the probable reason for the delay in therapeutic translation of this promising agent for clinical use.

In vitro and in vivo metabolic investigation of the Palbociclib by UHPLC-Q-TOF/MS/MS and in silico toxicity studies of its metabolites.

Palbociclib (PAB) is a CDK4/6 inhibitor and U. S Food and Drug Administration (FDA) granted regular approval for the treatment of hormone receptor (HR) positive, metastatic breast cancer in combination with an aromatase inhibitor in postmenopausal women. Metabolite identification is a crucial aspect during drug discovery and development as the drug metabolites may be pharmacologically active or possess toxicological activity. As there are no reports on the metabolism studies of the PAB, the present study focused on investigation of the in vitro and in vivo metabolic fate of the drug. The in vitro metabolism studies were carried out by using microsomes (HLM and RLM) and S9 fractions (Human and rat). The in vivo metabolism of the drug was studied by administration of the PAB orally to the Sprague-Dawley rats followed by analysis of urine, faeces and plasma samples. The sample preparation includes simple protein precipitation (PP) followed by solid phase extraction (SPE). The extracted samples were analyzed by ultrahigh-performance liquid chromatography-quadruple time-of-flight tandem mass spectrometry (UHPLC/Q-TOF/MS/MS). A total of 14 metabolites were detected in in vivo matrices. The PAB was metabolized via hydroxylation, oxidation, sulphation, N-dealkylation, acetylation and carbonylation pathways. A few of the metabolites were also detected in in vitro samples. Metabolite identification and characterization were performed by using UHPLC/Q-TOF/MS/MS in combination with HRMS data. To identify the toxicity potential of these metabolites, in silico toxicity assessment was carried out using TOPKAT and DEREK softwares.

An overview of recent applications of inductively coupled plasma-mass spectrometry (ICP-MS) in determination of inorganic impurities in drugs and pharmaceuticals.

The recent applications of inductively coupled plasma-mass spectrometry (ICP-MS) in determination of trace level inorganic impurities in drugs and pharmaceuticals have been reviewed. ICP-MS coupled with LC, GC and CE was used for speciation of heavy metals in pharmaceutical products. The review covers the period from 1995 to 2005 during which the technique was applied not only for determination of metallic impurities but also the assay of various trace elements in pharmaceuticals.

Selective separation and characterisation of stress degradation products and process impurities of prucalopride succinate by LC-QTOF-MS/MS.

The present study reports the degradation behaviour of a new prokinetic agent, Prucalopride succinate, under various stress conditions as per International Conference on Harmonization guidelines (ICH, Q1A (R2)). The investigation involved monitoring decomposition of the drug under hydrolytic (acidic, basic and neutral), oxidative, photolytic and thermal stress conditions followed by characterization of the degradation products (DPs) and process related impurities (IMPs). A rapid, precise, accurate and robust reverse phase high performance liquid chromatography (RP-HPLC) method has been developed involving mobile phase of 20mM ammonium bicarbonate buffer and acetonitrile: methanol (80:20v/v) on a Waters Xbridge-C8 (150mm×4.6mm i.d., 3.5µm) column using gradient elution. The drug was found to be degraded in hydrolytic (acidic) and oxidative conditions, whereas it was stable under basic and neutral hydrolytic, photolytic and thermal stress conditions. The method was extended to LC-ESI-QTOF-MS/MS for the structural characterization of DPs and process related IMPs. Structural characterization was carried out based on the generated molecular formula of DPs and its fragment ions. It has been observed that two major DPs were formed under each acid hydrolysis and oxidative stress conditions. The most probable mechanisms involved in the formation of DPs were also proposed. Finally, the method was validated in the term of specificity, linearity, accuracy, precision, and robustness as per ICH guidelines, Q2 (R1).