Design of experiment-oriented development and validation of novel bioanalytical reverse phase high performance liquid chromatography method of palbociclib in rat plasma and tissues, and its application in pharmacokinetic studies.

The current research involved the development and validation of an easy, cost-effective, and sensitive bioanalytical reverse-phase high-performance liquid chromatography method for the assessment of palbociclib (PAL) in rat plasma and kidney, liver, spleen and heart. A response surface methodology-based Box-Behnken design was used to optimize critical chromatographic conditions such as buffer pH, organic phase concentration and flow rate to attain good sensitivity, tailing factor and retention time. The conditions were: pH of buffer, 4.5; organic phase concentration, 40%; Shimpac column with 1.0 ml/min flow rate. The responses were: tailing factor, 1.29 ± 0.03, sensitivity, 366,593 ± 8,592; and retention time, 4.5 ± 0.05 min. The samples were extracted by a protein precipitation method, and absolute recoveries were in the range of 88.99-95.08%. The linearity of the developed method was validated over the range 100-2,000 ng/ml (r2 ≥ 0.994) in all tested matrices. The developed bioanalytical method showed greater accuracy (0.98 and 4.01%) and precision (<4.88%). The method was optimized for the sensitive analysis of the PAL in rat plasma, and the kidney, liver, spleen and heart were effectively applied to pharmacokinetic studies.

Chemogenetics Reveal an Anterior Cingulate-Thalamic Pathway for Attending to Task-Relevant Information.

In a changing environment, organisms need to decide when to select items that resemble previously rewarded stimuli and when it is best to switch to other stimulus types. Here, we used chemogenetic techniques to provide causal evidence that activity in the rodent anterior cingulate cortex and its efferents to the anterior thalamic nuclei modulate the ability to attend to reliable predictors of important outcomes. Rats completed an attentional set-shifting paradigm that first measures the ability to master serial discriminations involving a constant stimulus dimension that reliably predicts reinforcement (intradimensional-shift), followed by the ability to shift attention to a previously irrelevant class of stimuli when reinforcement contingencies change (extradimensional-shift). Chemogenetic disruption of the anterior cingulate cortex (Experiment 1) as well as selective disruption of anterior cingulate efferents to the anterior thalamic nuclei (Experiment 2) impaired intradimensional learning but facilitated 2 sets of extradimensional-shifts. This pattern of results signals the loss of a corticothalamic system for cognitive control that preferentially processes stimuli resembling those previously associated with reward. Previous studies highlight a separate medial prefrontal system that promotes the converse pattern, that is, switching to hitherto inconsistent predictors of reward when contingencies change. Competition between these 2 systems regulates cognitive flexibility and choice.

Box-Behnken design aided optimization and validation of developed reverse phase HPLC analytical method for simultaneous quantification of dolutegravir sodium and lamivudine co-loaded in nano-liposomes.

A stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous estimation of dolutegravir sodium and lamivudine encapsulated in the nanoliposomal formulation was developed. The chromatographic parameters namely, organic phase ratio, flow rate, and sample injection volume were selected as independent factors and were optimized by multivariate Box-Behnken design. Responses analyzed were retention time, peak area, and resolution. The optimized chromatographic method with Hypersil BDS C8 CN column as stationary phase and methanol and acetonitrile mixture and acidified Milli-Q water (pH 2.8, adjusted with 0.02% v/v orthophosphoric acid) as the mobile phase in an isocratic elution mode was validated according to parameters of International Conference on Harmonization Q1(R2) guidelines. The validated reversed-phase high-performance liquid chromatography method exhibited specificity for both dolutegravir sodium and lamivudine in the presence of degradation products as well as the liposomal matrix. This method was effectively utilized to determine the amount of drug entrapped and drug loading efficiency of dolutegravir sodium and lamivudine in a nano-liposomal formulation.

In vitro metabolism of olaparib in liver microsomes by liquid chromatography/electrospray ionization high-resolution mass spectrometry.

RATIONALES: Olaparib is a Poly (ADP-ribose) Polymerase (PARP) inhibitor which has been developed as an anti-cancer agent. The purpose of this study was to characterize the metabolites of olaparib from liver microsomes and to reveal the interspecies differences between animals and humans. METHODS: Olaparib (20 µM) was incubated with different species of liver microsomes at 37°C for 1 h in the presence of NADPH. The incubation samples were analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) operated in positive ion mode. The metabolites were characterized by accurate masses, MS2 spectra and retention times. RESULTS: A total of 12 metabolites were detected and the structures of the metabolites were characterized based on their accurate masses, fragment ions and retention times. Four metabolites, i.e., M1, M10, M11 and M12, were unambiguously identified by using reference standards. The metabolic pathways of olaparib included hydroxylation, bis-hydroxylation, hydrolysis, dealkylation, dehydrogenation, and alcohol oxidation. CONCLUSIONS: Compared with animal species, no human-specific metabolite was found in HLM. Dog also had a closer metabolic profile to humans. This study will be helpful for a better understanding of the species difference in pharmacokinetics/pharmacodynamics.

Detection and quantification of synthetic cathinones and selected piperazines in hair by LC-MS/MS.

New psychoactive substances (NPS)-such as synthetic cathinones and piperazines-are defined as substances designed to replicate the effects of traditional illegal drugs, including cocaine, ecstasy and amphetamines. These substances are known to potentially be much more potent than their analogs. In the past, there were many poisonings and deaths associated with NPS. Because of this, NPS identification and quantification have become more important in forensic toxicology. The present work aimed to develop, validate and apply a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of detecting 35 synthetic cathinones and piperazines in hair samples. All target analytes were resolved in a 12 min run time and identified based on the quantifier ion, at least one product ion and the retention time. Depending on the analyte, the calibration curves were linear over a maximal range of 0.01-3 ng/mg. The limits of detection and quantification were within the ranges of 0.006-0.052 ng/mg and 0.008-0.095 ng/mg, respectively. The precision, bias and matrix effect were all within acceptable GTFCh thresholds and the method was free from interferences. The validated method was successfully used to identify synthetic cathinones and piperazines in authentic hair samples (n = 40) from forensic cases, demonstrating its suitability for the screening and quantification of a wide number of new stimulants in hair specimens.

Mass Spectrometry-Sensitive Probes Coupled with Direct Analysis in Real Time for Simultaneous Sensing of Chemical and Biological Properties of Botanical Drugs.

The development of botanical materials as therapeutic agents involves the meticulous assessment of safety, efficacy, and quality. Compared with small-molecule drugs, quality control of botanical drugs confronts with more significant challenges due to their inherent complexity. Current quality control methods for botanical drugs, either prevailing chemical tests or emerging biological assays, are not able to meet recent demands of multiplexing, sensitivity, and speed. Here, we propose an on-demand strategy based on a direct analysis in real time-mass spectrometry (DART-MS) platform, which is capable of simultaneously analyzing multiple constituents and bioactivities of botanical drugs. Notably, the bioactivities are assessed by a multiple-enzyme assay that adopts cleavable mass spectrometry probes as enzymatic substrates: these probes labeled with a piperazine tag make possible sensitive, multiplexed, and quantitative enzyme activity measurements. The concept is successfully demonstrated via a case study of Danshen (Salvia miltiorrhiza) Injection where simultaneous detection of 34 constituents and inhibitory activities on two target enzymes can be achieved in just minutes. This proof-of-concept application also gives evidence that combining MS-sensitive probes with DART-MS can provide an environmentally friendly, highly sensitive analytical approach for botanical quality control.

Single Chain Cationic Polymer Dot as a Fluorescent Probe for Cell Imaging and Selective Determination of Hepatocellular Carcinoma Cells.

This letter describes formation of single chain cationic polymer dots (Pdots) made of poly[1,4-dimethyl-1-(3-((2,4,5-trimethylthiophen-3-yl)oxy)propyl)piperazin-1-ium bromide] conjugated polyelectrolyte (CPE). The single chain Pdot formation relies on a simple process which is a rapid nanophase separation between CPE solution of ethylene glycol and water. Pdots show narrow monodisperse size distribution with a 3.6 nm in diameter exhibiting high brightness and excellent colloidal and optical stability. It has been demonstrated that photoluminescent Pdots provide selective nuclear translocation to hepatocellular carcinoma cells as compared to healthy liver cells. The Pdot labeling effectively discriminates cancer cells in the coculture media. Pdots hold great promise as a luminescent probe to diagnose cancer cells in histology and may guide surgeons during operations to precisely separate out cancerous tissue due to augmented fluorescence brightness.

Investigation of deep eutectic solvents as additives to ß-CD for enantiomeric separations of Zopiclone, Salbutamol, and Amlodipine by CE.

The enantioseparation of chiral drugs via CE was first investigated using ß-CD as chiral additive and deep eutectic solvents (DESs) as auxiliary additive. The results showed that improved separation of tested chiral drugs was obtained in the presence of DESs and ß-CD compared to the single ß-CD separation system. With the optimized condition, resolutions of DESs applied ß-CD separation system for rac-Zopiclone, rac-Salbutamol, and rac-Amlodipine increased 3-4.2 times as single ß-CD separation system. The resolutions reached 4.74, 6.37, and 9.67, respectively. The results demonstrate that DESs are viable additives to CD system in CE for the separation of the chiral drugs.

Development and validation of a bioanalytical method for the quantification of the CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib in human and mouse matrices using liquid chromatography-tandem mass spectrometry.

A novel method was developed and validated for the quantification of the three approved CDK4/6 inhibitors (abemaciclib, palbociclib, and ribociclib) in both human and mouse plasma and mouse tissue homogenates (liver, kidney, spleen, brain, and small intestine) using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). For all matrices, pretreatment was performed using 50 µL of sample by protein precipitation with acetonitrile, followed by dilution of the supernatant. Chromatographic separation of the analytes was done on a C18 column using gradient elution. A full validation was performed for human plasma, while a partial validation was executed for mouse plasma and mouse tissue homogenates. The method was linear in the calibration range from 2 to 200 ng/mL, with a correlation coefficient (r) ≥0.996 for each analyte. For both human and mouse plasma, the accuracy and precision were within ±15% and ≤15%, respectively, for all concentrations, except for the lower limit of quantification, where they were within ±20% and ≤20%, respectively. A fit-for-purpose strategy was followed for tissue homogenates, and the accuracy and precision were within ±20% and ≤20%, respectively, for all concentrations. Stability of all analytes in all matrices at different processing and storage conditions was tested; ribociclib and palbociclib were unstable in most tissue homogenates and conditions were modified to increase the stability. The method was successfully applied for the analysis of mouse samples from preclinical studies. A new ribociclib metabolite was detected in mouse plasma samples with the same m/z transition as the parent drug.

Ultra-performance liquid chromatography tandem mass spectrometry for the rapid, simultaneous analysis of ziprasidone and its impurities.

The separation and characterization of the unknown degradation product of second-generation antipsychotic drug ziprasidone are essential for defining the genotoxic potential of the compound. The aim of this study was to develop a simple UHPLC method coupled with tandem mass spectrometry (MS/MS) for chemical characterization of an unknown degradant, and the separation and quantification of ziprasidone and its five main impurities (I-V) in the raw material and pharmaceuticals. Chromatographic conditions were optimized by experimental design. The MS/MS fragmentation conditions were optimized individually for each compound in order to obtain both specific fragments and high signal intensity. A rapid and sensitive UHPLC-MS/MS method was developed. All seven analytes were eluted within the 7 min run time. The best separation was obtained on the Acquity UPLC BEH C18 (50 × 2.1 mm × 1.7 µm) column in gradient mode with ammonium-formate buffer (10 mm; pH 4.7) and acetonitrile as mobile phase, with the flow rate of 0.3 mL min-1 and at the column temperature of 30°C. The new UHPLC-MS/MS method was fully validated and all validation parameters were confirmed. The fragmentation pathways and chemical characterization of an unknown degradant were proposed and it was confirmed that there are no structural alerts concerning genotoxicity.

A quantitative LC-MS/MS method for determination of a small molecule agonist of EphA2 in mouse plasma and brain tissue.

Compound 27 {1, 12-bis[4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl]dodecane-1,12-dione} is a novel small molecule agonist of EphA2 receptor tyrosine kinase. It showed much improved activity for the activation of EphA2 receptor compared with the parental compound doxazosin. To support further pharmacological and toxicological studies of the compound, a method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC-MS/MS) has been developed for the quantification of this compound. Liquid-liquid extraction was used to extract the compound from mouse plasma and brain tissue homogenate. Reverse-phase chromatography with gradient elution was performed to separate compound 27 from the endogenous molecules in the matrix, followed by MS detection using positive ion multiple reaction monitoring mode. Multiple reaction monitoring transitions m/z 387.3 → 290.1 and m/z 384.1 → 247.1 were selected for monitoring compound 27 and internal standard prazosin, respectively. The linear calibration range was 2-200 ng/mL with the intra- and inter-day precision and accuracy within the acceptable range. This method was successfully applied to the quantitative analysis of compound 27 in mouse plasma and brain tissue with different drug administration routes.

Multi-Steps Fragmentation-Ion Trap Mass Spectrometry Coupled to Liquid Chromatography Diode Array System for Investigation of Olaparib Related Substances.

A high-performance liquid chromatography-diode array-mass spectrometric (LC-DAD-MS) method was developed and validated to investigate the related substances of olaparib (OLA) in bulk form. OLA was exposed to acid⁻base hydrolysis, boiling, oxidation with hydrogen peroxide, and UV light followed by LC-DAD-MS analysis. OLA and OLA-related substances were simultaneously and quantitatively monitored by DAD at 278 nm and triple quadrupole mass spectrometry (QQQ-MS). The investigated compounds were auto-scanned by an ion trap MS which applied positive and negative modes separately. The fragmentation pathway was confirmed by applying multi-steps fragmentation to identify the resulted cleaved ions and their parent ion. OLA was found to be sensitive to the alkaline hydrolysis and less sensitive to UV light. Two major hydrolytic degradation products, including the protonated molar ions m/z 299 and m/z 367, were identified. Three potential impurities were also characterized. The LC-MS limit of detection (LOD) and limit of quantification (LOQ) were 0.01 and 0.05 ng/µL, respectively. The quantitative results obtained by LC-DAD was comparable with that of LC-QQQ-MS. The proposed method shows good intra-day and inter-day precision with relative standard deviation (RSD) <2%.

A Fluorescent Probe with Improved Water Solubility Permits the Analysis of Protein S-Depalmitoylation Activity in Live Cells.

S-Palmitoylation is an abundant lipid post-translational modification that is dynamically installed on and removed from target proteins to regulate their activity and cellular localization. A dearth of tools for studying the activities and regulation of protein S-depalmitoylases, thioesterase "erasers" of protein cysteine S-palmitoylation, has contributed to an incomplete understanding of the role of dynamic S-palmitoylation in regulating proteome lipidation. Recently, we developed "depalmitoylation probes" (DPPs), small molecule probes that become fluorescent upon S-depalmitoylase enzymatic activity. To be suitable for application in live cells, the first-generation DPPs relied on a shorter lipid substrate (C8 vs naturally occurring C16), which enhanced solubility and cell permeability. However, the use of an unnatural lipid substrate on the probes potentially limits the utility of the approach. Herein, we present a new member of the DPP family, DPP-5, which features an anionic carboxylate functional group that increases the probe water solubility. The enhanced water solubility of DPP-5 permits the use of a natural, palmitoylated substrate (C16), rather than a surrogate lipid. We show that DPP-5 is capable of monitoring endogenous S-depalmitoylases in live mammalian cells and that it can reveal changes in S-depalmitoylation levels due to lipid stress. DPP-5 should prove to be a useful new tool for probing the regulation of proteome lipidation through dynamic S-depalmitoylation.

Determination of vortioxetine and its degradation product in bulk and tablets, by LC-DAD and MS/MS methods.

Vortioxetine hydrobromide (VOR), is a novel antidepressant used for the treatment of major depressive disorder. It has a chemical structure susceptible to degradation, therefore it is important to have suitable analytical methods to determine VOR in presence of its main degradation products (DP), because if the compound degrades, this could result in diminution of the therapeutic activity and safety. A simple HPLC method with photodiode array detection was developed and validated for determination of VOR in bulk and tablets, in the presence of its major DP. The drug was subjected to oxidative, hydrolytic, and photolytic stress conditions, showing significant degradation under oxidation with the formation of one DP, which was identified by ESI-MS/MS. A C18 column was used, with mobile phase consisting of acetonitrile and water with acetic acid and triethylamine in isocratic elution mode, with detection at 228 nm and 1.0 mL/min flow rate. The assay was linear in the 25-125 µg/mL concentration range. For precision, the RSD was <1.8%, the recovery was 100.0-101.6%, and the method demonstrated adequate selectivity. The method was successfully applied to quantify VOR in tablets. The results showed that the method is useful for routine analysis and for quality control purposes.

Residue and distribution of triforine in different cultivars and fruit periods of watermelon under field conditions.

The dissipation of triforine in the immature and mature fruit periods was investigated under field conditions. Residue levels of triforine in watermelon were determined by gas chromatography with an electron capture detector (GC-ECD). The decline curves of triforine residues in the watermelon corresponded with first-order kinetics. The half-lives of triforine in Dark Belle and Shiny Boy were 2.10-2.57 days and 2.31-2.67 days respectively. Meanwhile, the half-lives of triforine in the immature and mature fruit periods were 1.69-2.04 days and 2.89-3.85 days, respectively. In the terminal residue experiment, the terminal residues of triforine in the watermelon flesh and peel were below 0.01 mg/kg to 0.05 mg/kg and 0.03 mg/kg to 0.36 mg/kg, respectively. The dissipation rates of triforine varied in different cultivars of watermelon, and even in the same cultivar, the half-lives of triforine significantly varied in the different fruit periods. Although triforine is a fungicide within the suction, the terminal residues in the peel and flesh were very significant.

Optimization and validation of RP-HPLC method for simultaneous estimation of palbociclib and letrozole.

A simple, rapid, and robust RP-HPLC method have been developed and validated to measure palbociclib (PB) and letrozole (LT) at single wavelength (254 nm). A isocratic elution of samples performed on Intersil C8 (4.6 mm × 250 mm particle size 5 µm) column with mobile phase consisting 0.02 M sodium dihydrogen phosphate buffer (pH 5.5): acetonitrile: methanol (80:10:10 v/v/v) delivered at flow rate 1.0 mL min-1. A good linear response was achieved over the range of 5-50 µg mL-1. The LODs for PB and LT were found to be 0.098 and 0.0821 µg mL-1, while the LOQs for PB and LT were 0.381-0.315 µg mL-1, respectively. The method was quantitatively evaluated in terms of system suitability test, linearity, precision, accuracy (recovery) and robustness as per standard guidelines. The method is simple, convenient and suitable for the analysis of PB and LT in bulk drug.

Synthesis of some new 2-[4-(2-furoyl)-1-piperazinyl]-N-aryl/aralkyl acetamides as potent antibacterial agents.

In this work, a new series of 2-[4-(2-furoyl)-1-piperazinyl]-N-aryl/aralkyl acetamides has been synthesized and evaluated for their antibacterial potential. The synthesis was initiated by the reaction of different aryl/aralkyl amines (1a-u) with 2-bromoacetylbromide (2) to obtain N-aryl/aralkyl-2-bromoacetamides (3a-u). Equimolar quantities of different N-aryl/aralkyl-2-bromoacetamides (3a-u) and 2-furoyl-1-piperazine (4) was allowed to react in acetonitrile and in the presence of K2CO3, to form 2-[4-(2-furoyl)-1-piperazinyl]-N-aryl/aralkyl acetamides (5a-u). The structural elucidation was done by EI-MS, IR and 1H-NMR techniques of all the synthesized compounds. All of the synthesized molecules were active against various Gram positive and Gram negative bacterial strains. Among them 5o and 5c showed very excellent MIC values. The cytotoxicity of the molecules was also checked to find their utility as possible therapeutic agents, where 5c (0.51%) and 5g (1.32%) are found to be least toxic in the series.

Fungal artificial chromosomes for mining of the fungal secondary metabolome.

BACKGROUND: With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30-80 kb in size, breakthrough techniques are needed to characterize this SM wealth. RESULTS: Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery. CONCLUSION: The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery.

General, Label-Free Method for Determining K(d) and Ligand Concentration Simultaneously.

Some of the most commonly used affinity reagents (e.g., antibodies) are often developed and used in conditions where their input concentrations ([L]0) and affinities (K(d)) are not known. Here, we have developed a general approach to determine both [L]0 and K(d) values simultaneously for affinity reagents (small molecules, proteins, and antibodies). To do this, we perform quantitative equilibrium exclusion immunoassays with two different concentrations of target and fit the data simultaneously to determine K(d) and [L]0. The results give accurate and reproducible measures of both values compared to established methods. By performing detailed error analysis, we demonstrate that our fitting gives unique solutions and indicates where K(d) and [L]0 measures are reliable. Furthermore, we found that a divalent model of antibody binding gives accurate K(d) and [L]0 values in both the forward (antibody immobilized) and the reverse (target immobilized) assays-addressing the long-term problem of obtaining quantitative data from reverse assays.

An overview of recent developments in the analytical detection of new psychoactive substances (NPSs).

New psychoactive substances (NPSs), sometimes referred to as "legal highs" in more colloquial environments/the media, are a class of compounds that have been recently made available for abuse (not necessarily recently discovered) which provide similar effects to the traditional well studied illegal drugs but are not always controlled under existing local, regional or international drug legislation. Following an unprecedented increase in the number of NPSs in the last 5 years (with 101 substances discovered for the first time in 2014 alone) its, occasionally fatal, consequences have been extensively reported in the media. Such NPSs are typically marketed as 'not for human consumption' and are instead labelled and sold as plant food, bath salts as well as a whole host of other equally nondescript aliases in order to bypass legislative controls. NPSs are a new multi-disciplinary research field with the main emphasis in terms of forensic identification due to their adverse health effects, which can range from minimal to life threatening and even fatalities. In this mini-review we overview this recent emerging research area of NPSs and the analytical approaches reported to provide detection strategies as well as detailing recent reports towards providing point-of-care/in-the-field NPS ("legal high") sensors.