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.

Electrochemical degradation of antibiotic enoxacin using a novel PbO2 electrode with a graphene nanoplatelets inter-layer: Characteristics, efficiency and mechanism.

A novel PbO2 electrode was fabricated by adding graphene nanoplatelets (GNP) inter-layer into ß-PbO2 active layer (called GNP-PbO2) and utilized to degradation of antibiotic enoxacin (ENO). The GNP-PbO2 electrode had a much rougher surface than the typical PbO2 electrode, with smaller crystal size and lower charge-transfer resistance at the electrode/electrolyte interface. Notably, the GNP inter-layer increased the oxygen evolution potential of PbO2 electrode (2.05 V vs. SCE), which was very beneficial to inhibit oxygen evolution and promote ·OH production. The relatively best operating parameters for ENO removal and energy efficiency were current density of 20 mA cm-2, initial pH of 7, initial ENO concentration of 100 mg L-1 and electrode distance of 4 cm. Furthermore, indirect radical oxidation was found to be the main way during electrolysis process. Based on the observed analysis of intermediate products, the main reaction pathways of ENO included hydroxylation, defluorination and piperazine ring-opening. Finally, combinating with the electro-oxidation capability, stability and safety evaluation, we can conclude that GNP-PbO2 is a promising anode for treatment of various organic pollutants in wastewater.

Detection of unsuspected cathinone and piperazine-type drugs in urine samples positive for methamphetamine and amphetamine collected in emergency departments. / Detección no sospechada de catinonas y piperacinas en pacientes consumidores de metanfetamina y anfetamina atendidos en servicios de urgencias hospitalarios.

OBJECTIVES: To detect the presence of unsuspected and/or undeclared cathinone and piperazine-type designer drugs in methamphetamine (METH) and amphetamine users treated in emergency departments, and to compare clinical and toxicologic profiles. MATERIAL AND METHODS: Retrospective observational study of emergency department patients treated for confirmed acute intoxication by recreational drugs (METH and amphetamines) between March 2019 and December 2020. We ordered high-performance liquid chromatography with tandem mass spectrometry to detect cathinones (methylone, fluoromethcathinone, mexedrone, fluoromethamphetamine, mephedrone, methylenedioxypyrovalerone) and synthetic piperazines (meta-chlorophenylpiperazine and trifluoromethylphenylpiperazine). Demographic, clinical, and toxicologic variables were analyzed with SPSS software (version 23). RESULTS: Thirty-nine patients were included: 24 (61.5%) had used METH and 15 (38.5%) an amphetamine. Synthetic cathinones were detected in samples from 11 patients (28.2%), 10 (90.9%) in the METH group and 1 (9.1%) in the amphetamine group (P = .028). The METH users had taken mephedrone (8 patients) or methylone (2 patients); the amphetamine user had taken mephedrone. None of the patients had declared use of a cathinone; nor was use suspected. The mean (SD) number of substances involved was higher among users of cathinones (3.5 [1.13] vs 2.5 [1.40] in those who took no cathinones; P = .036). Among the cathinone users, 90.9% were men, 90.9% had used METH, and 45.5% had practiced chemsex. HIV positivity was significantly associated with cathinone use (in 45.5% vs 10.7% of those not using cathinones; P = .028). All 5 of the patients who had taken cathinones and also practiced chemsex were HIV positive. Significantly more patients who had taken cathinones presented with anxiety (72.7% vs 21.43%; P = .007). No differences in clinical management were found. CONCLUSION: Detection of METH in intoxicated patients should raise suspicion of probable use of a synthetic cathinone. Patients in whom new psychoactive substances are detected should be kept under observation, and clinical protocols should include referring them to addiction treatment centers.

OBJETIVO: Determinar la incidencia de catinonas y piperazinas, no sospechadas y/o declaradas en consumidores de metanfetamina (MANF) y anfetamina (ANF) atendidos en servicios de urgencias hospitalarios (SUH) y comparar los perfiles clínicos y toxicológicos. METODO: Estudio retrospectivo de pacientes con intoxicación aguda por drogas recreativas con MANF y ANF confirmadas analíticamente atendidos en 3 SUH entre marzo de 2019 y diciembre de 2020. Se detectaron por HPLC-MS/MS las catinonas [metilona, fluorometcatinona, mecedrona, fluorometanfetamina, mefedrona, metilendioxipirovalerona (MDPV)] y las piperazinas sintéticas [meta-clorofenilpiperazina (mCPP), trifluorometilfenilpiperazina (TFMPP)]. RESULTADOS: Se incluyeron 39 pacientes: 24 (61,5%) en el grupo MANF y 15 (38,5%) en el ANF. En 11 (28,2%), se detectaron catinonas sintéticas (grupo CAT), 10 en el grupo MANF (8 mefedrona, 2 metilona) y 1 en el grupo ANF (1 mefedrona) (90,9% vs 9,1%; p = 0,028). Ninguno de los pacientes declaró consumo de catinonas. El número de drogas implicadas en la intoxicación fue superior en el grupo CAT (3,5 [1,13] vs 2,5 [1,40]; p = 0,036). El perfil clínico del grupo CAT fue: varón (90,9%), consumidor de MANF (90,9%) y usuario de chemsex (45,5%). El diagnóstico de VIH se asoció significativamente al grupo CAT (45,5% vs 10,7%; p = 0,028). Los pacientes del grupo CAT presentaron mayor ansiedad (72,7% vs 21,4%; p = 0,007). No se hallaron diferencias en su manejo clínico. CONCLUSIONES: La detección de MANF debería considerarse un dato de sospecha de consumo de catinonas sintéticas, y en esos casos debería contemplarse la detección de nuevas sustancias psicoactivas de abuso.

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.

Quantification of KRAS inhibitor sotorasib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

Sotorasib is a KRAS inhibitor with promising anticancer activity in phase I clinical studies. This compound is currently under further clinical evaluation as monotherapy and combination therapy against solid tumors. In this study, a liquid chromatography-tandem mass spectrometric method to quantify sotorasib in mouse plasma and eight tissue-related matrices (brain, liver, spleen, kidney, small intestine, small intestine content, lung, and testis homogenates) was developed and validated. Protein precipitation using acetonitrile was utilized in 96-well format to extract sotorasib and erlotinib (internal standard) from mouse plasma and tissue homogenates. Separation of the analytes was performed on an Acquity UPLC® BEH C18 column by gradient elution of methanol and 0.1% formic acid in water at a flow rate of 0.6 ml/min. Sotorasib was detected by a triple quadrupole mass spectrometer with positive electrospray ionization in selected reaction monitoring mode. A linear calibration range of 2-2,000 ng/ml of sotorasib was achieved during the validation. Accuracy values were in the range of 90.7-111.4%, and precision values (intra- and interday) were between 1.7% and 9.2% for all tested levels in all investigated matrices. The method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of sotorasib in female wild-type mice.

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.

LC-MS/MS Estimation of the Anti-Cancer Agent Tandutinib Levels in Human Liver Microsomes: Metabolic Stability Evaluation Assay.

PURPOSE: Tandutinib (MLN518 or CT 53518) (TND) is a novel, oral, small-molecule inhibitor of type III receptor tyrosine kinases utilized for the treatment of acute myeloid leukemia (AML). MATERIALS AND METHODS: In silico prediction of hepatic drug metabolism for TND was determined using the StarDrop® WhichP450™ module to confirm its metabolic liability. Second, an efficient and accurate LC-MS/MS method was established for TND quantification to evaluate metabolic stability. TND and entrectinib (ENC) (internal standard; IS) were resolved using an isocratic elution system with a reversed stationary phase (C8 column). RESULTS: The established LC-MS/MS method exhibited linearity (5-500 ng/mL) with r2 ≥0.9999 in the human liver microsomes matrix. The method sensitivity was indicated by the limit of quantification (3.8 ng/mL), and reproducibility was revealed by inter- and intraday precision and accuracy (below 10.5%). TND metabolic stability estimation was calculated using intrinsic clearance (22.03 µL/min/mg) and in vitro half-life (29.0 min) values. CONCLUSION: TND exhibited a moderate extraction ratio indicative of good bioavailability. According to the literature, the approach developed in the present study is the first established LC-MS/MS method for assessing TND metabolic stability.

Concentrations of aripiprazole and dehydroaripiprazole in hair segments from deceased individuals with mental disorders.

Segmental hair analysis provides information regarding previous long-term drug exposure, which is useful in the evaluation of cause of death for individuals with mental disorders. The aim was to analyze postmortem concentrations of the antipsychotic drug aripiprazole and its active metabolite dehydroaripiprazole in hair segments from individuals with known aripiprazole intake. Hair samples were collected during autopsy. Each sample was segmented into one to six 1cm segments, depending on the length of the hair shaft. Pulverized hair was extracted and analyzed using a previously published ultra-high-performance liquid chromatography-tandem mass spectrometric method. The 10th-90th percentile of aripiprazole concentrations in all hair segments (n=78) from 17 individuals were 0.024ng/mg-11ng/mg with a median of 2.3ng/mg, and the 10th-90th percentile concentrations of dehydroaripiprazole were 0.020ng/mg-11ng/mg, with a median of 2.6ng/mg, in all hair segments (n=71). The metabolite-to-parent drug ratios ranged from 0.21 to 1.5, with a median of 0.72. The administered doses were calculated for each individual based on aripiprazole prescription data and pharmacy pickups, giving dose estimates of 1mg-32mg daily. A positive significant correlation was observed between concentrations in hair and blood, whereas no trends were observed between the concentrations in hair and the estimated doses. Besides aripiprazole, other antipsychotic drugs were found in several hair segments, indicating a high degree of polypharmacy among all subjects. The present study establishes concentrations of aripiprazole and dehydroaripiprazole in hair segments from 17 deceased individuals with long-term aripiprazole use. In addition, hair analysis demonstrates the possibility of evaluating polypharmacy.

Mass balance, metabolic disposition, and pharmacokinetics of [14C]ensartinib, a novel potent anaplastic lymphoma kinase (ALK) inhibitor, in healthy subjects following oral administration.

PURPOSE: Ensartinib is a novel, potent and highly selective inhibitor of anaplastic lymphoma kinase (ALK) that has promising clinical activity and low toxicity in patients with ALK-positive non-small cell lung cancer. This study was conducted to investigate the pharmacokinetics, metabolism and excretion of ensartinib following a single 200 mg/100 µCi oral dose of radiolabeled ensartinib to healthy subjects. METHODS: Six healthy male subjects were enrolled and administrated an oral suspension in a fasted state. Blood, urine and feces were collected. Radioactivity concentrations were measured by liquid scintillation counting and plasma concentrations of ensartinib by liquid chromatography-tandem mass spectrometry. Both techniques were applied for metabolite profiling and characterization. RESULTS: The mean total recovery was 101.21% of the radiolabeled dose with 91.00% and 10.21% excreted in feces and urine, respectively. Unchanged ensartinib was the predominant drug-related component in urine and feces, representing 4.39% and 38.12% of the administered dose, respectively. Unchanged ensartinib and its metabolite M465 were the major circulating components, accounting for the same 27.45% of the plasma total radioactivity (AUC0-24h pool), while other circulating metabolites were minor, accounting for less than 10%. Mean Cmax, AUC0-∞, T1/2 and Tmax values for ensartinib in plasma were 185 ng/mL, 3827 h ng/mL, 18.3 h and 3.25 h, respectively. The total radioactivity in plasma was cleared with terminal half-life of 27.2 h. Treatment with ensartinib was well tolerated, and no serious adverse events were reported. CONCLUSION: It was well tolerated in the six healthy male subjects following a single oral administration of 200 mg/100 µCi dose of ensartinib. Besides unchanged ensartinib, metabolite of M465 was the predominant circulating drug-related component. The drug was primarily eliminated in feces. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT03804541.

Development and characterisation of a monoclonal antibody to detect the mycotoxin roquefortine C.

Roquefortine, also known as roquefortine C (ROQC) is a fungal secondary metabolite (mycotoxin) that is produced by some of the same Penicillia as the tremorgen penitrem-A (PEN-A). The two mycotoxins have been linked to sporadic cases of toxicosis in dogs, cattle, and humans, leading some to consider ROQC as a biomarker of PEN-A. Reported here are the development of a monoclonal antibody (mAb) and associated competitive enzyme-linked immunosorbent assay (ELISA) for the screening of ROQC in extracts of nuts (nut "milks"), and dog serum. The ELISA was sensitive for ROQC, with a level of 0.117 ng ml-1 inhibiting colour development by 50% (IC50), a limit of detection of 0.026 ng ml-1, and a dynamic range (IC20 to IC80) of 0.038 to 0.289 ng ml-1 in buffer. The assay was tolerant to significant levels of methanol. Recoveries from 4 types of nut milks spiked over the range of 0.25 to 2 ng ml-1 were in the range of 83.5% to 116%. A small survey of commercial nut "milks" and "creamers" indicated 4 of 35 samples contained ROQC at levels so low that they are unlikely to be significant to human health (<0.6 ng ml-1). The assay was also applied to canine serum. Recoveries from serum spiked over the range of 0.2 to 5 ng ml-1 ranged from 98.1% to 123%. The results suggest the ELISA can be applied to the screening of food products, such as nut extracts, as well as for the screening of serum from dogs suspected to be suffering from mycotoxin-induced tremors.

The phenomenon of para-Fluorophenylpiperazine (pFPP) in combination with the synthetic cannabinoid AMB-FUBINACA in seized plant material in New Zealand.

New psychoactive substances have emerged as a vast and diverse group of illicit drugs over the past decade, with synthetic cannabinoids comprising the largest of the categories. Commonly, a single synthetic cannabinoid is applied to plant material, creating a product that is designed to be smoked by the user. The clandestine preparation process can result in an unevenly distributed product, with varying concentration within and between plant materials. This investigation describes the novel co-detection of the synthetic cannabinoid AMB-FUBINACA, with the piperazine para-fluorophenylpiperazine (pFPP), in a number of plant material samples analysed in New Zealand in 2017. Of 157 samples of plant material containing AMB-FUBINACA, pFPP was detected in 55 of them. A range of pFPP concentrations was observed between the plant material samples, as well as intra-batch variation. The presence of both drugs may be designed to enhance, prolong or balance the psychoactive effects caused from smoking the plant material. However the intended purpose has not been verified. This is the first reported combination of a synthetic cannabinoid and a piperazine in plant material.

Development of a specific fragmentation pattern-based quadrupole-Orbitrap™ mass spectrometry method to screen drugs in illicit products.

Over the past decade, illicit drugs have been founded in marketed products, which pose a risk to public health. In particular, newly designed analogues synthesized by chemical modification of parent compounds to avoid detection by authorities are frequently detected worldwide. Although many analytical methods for determination of drugs have been reported, analytical methods using high-resolution mass spectrometry, which has the advantage of rapid screening and accurate identification of new substances, are necessary to control illicit drugs in marketed products. In this study, a rapid analytical method using an Orbitrap™ mass spectrometer for identification of illicit drugs in marketed products was developed. The 32 drugs were classified as benzodiazepine-, synthetic cannabinoid-, amphetamine- and benzylpiperazine-type drugs according to their chemical structures, and from their fragmentation patterns in tandem mass spectrometry spectra of an established method. The method validation gave a limit of detection of 0.06-5.30 ng/mL and a limit of quantification of 0.18-16.50 ng/mL, high linearity (R2 > 0.994) and mean recoveries of spiked matrix-blank samples ranging from 83.7% to 117.1%. Approximately 71% of 21 samples collected over 3 years were found to individually contain one of four types of benzodiazepines or two different synthetic cannabinoids. In one case, levels as high as 827.2 mg/g were measured suggesting adulteration at high levels, which suggests that potential illicit products containing drugs should be regularly screened to protect public health.

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.

Distribution of zopiclone and main metabolites in hair following a single dose.

In forensic investigations, such as drug-facilitated crimes, reference values are useful for interpretation of hair results. The aim of this study was to establish levels of zopiclone and two main metabolites, N-desmethylzopiclone and zopiclone N-oxide, in hair after the administration of a single dose of zopiclone, as very limited data are published. A controlled study was performed, where 16 volunteers consumed either 5 or 10mg zopiclone. Hair was sampled prior to consumption and 14, 30, 60, and 120 days after intake. The deposition of drug in hair segments of all sampling time points was followed in small hair segments of 5-mm, using a validated ultra-high performance liquid chromatography-tandem mass spectrometry method. In all participants, hair segments corresponding to the time of intake were positive for zopiclone, but also with lower concentrations in the neighbouring segments. The highest zopiclone concentrations were detected in samples collected 30 or 60 days after intake. For all sampling time points maximum values for the 5-mg dose ranged from 5.0-370pg/mg for zopiclone and 5.4 to 300pg/mg for N-desmethylzopiclone, where the maximum values for the 10-mg dose ranged from 17 to 590pg/mg for zopiclone and 25-410pg/mg for N-desmethylzopiclone for all sampling time points. No significant difference in concentrations was found between the two dosing groups for either zopiclone or N-desmethylzopiclone. Almost half of the participants showed lower levels 14 days after intake than in the later sampling time points. The metabolite to parent drug ratio of N-desmethylzopiclone to zopiclone varied from 0.6 to 3.4 (median=1.2) for the maximum levels of all sampling time points. N-desmethylzopiclone are suggested to serve as an additional marker to confirm the intake of zopiclone. Traces of zopiclone N-oxide were detected in hair from only eight participants. This study showed, that it was possible to follow zopiclone and N-desmethylzopiclone in hair for 4 months even though the drugs was divided into several segments in the latest collected hair samples, and no obvious wash-out effect between the sampling time points by e.g. personal hygiene could be discerned because the cumulated amount at each sampling time point was similar. We conclude that the analysis of short segments e.g. segments of 5-mm can help determine the time of a single intake of zopiclone and that obtaining a sample 1-2 months after a drug exposure provide the best conditions to detect and interpret the results.

An Activatable Cancer-Targeted Hydrogen Peroxide Probe for Photoacoustic and Fluorescence Imaging.

Reactive oxygen species play an important role in cancer, however, their promiscuous reactivity, low abundance, and short-lived nature limit our ability to study them in real time in living subjects with conventional noninvasive imaging methods. Photoacoustic imaging is an emerging modality for in vivo visualization of molecular processes with deep tissue penetration and high spatiotemporal resolution. Here, we describe the design and synthesis of a targeted, activatable probe for photoacoustic imaging, which is responsive to one of the major and abundant reactive oxygen species, hydrogen peroxide (H2O2). This bifunctional probe, which is also detectable with fluorescence imaging, is composed of a heptamethine carbocyanine dye scaffold for signal generation, a 2-deoxyglucose cancer localization moiety, and a boronic ester functionality that specifically detects and reacts to H2O2. The optical properties of the probe were characterized using absorption, fluorescence, and photoacoustic measurements; upon addition of pathophysiologic H2O2 concentrations, a clear increase in fluorescence and red-shift of the absorption and photoacoustic spectra were observed. Studies performed in vitro showed no significant toxicity and specific uptake of the probe into the cytosol in breast cancer cell lines. Importantly, intravenous injection of the probe led to targeted uptake and accumulation in solid tumors, which enabled noninvasive photoacoustic and fluorescence imaging of H2O2. In conclusion, the reported probe shows promise for the in vivo visualization of hydrogen peroxide. SIGNIFICANCE: This study presents the first activatable and cancer-targeted hydrogen peroxide probe for photoacoustic molecular imaging, paving the way for visualization of hydrogen peroxide at high spatiotemporal resolution in living subjects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5407/F1.large.jpg.

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.

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.

An available strategy based on accurate mass by ultra high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry technology to characterization of metabolic profile of palbociclib in rat urine, feces and bile.

Palbociclib (named PD 0332991) is a novel highly selective cyclin-dependent kinase 4 and 6 (CDK 4/6) inhibitor, which has been approved by the Food and Drug Administration (FDA) for the treatment of hormone-receptor-positive advanced breast cancer. This present study developed a comprehensive strategy to investigate the metabolic profile of palbociclib in rat urine, feces and bile samples based on an ultra high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR MS). A total of 29 metabolites, including 18 phase I metabolites and 11 phase II metabolites, were detected and identified. The metabolic pathways included hydroxylation, oxidation, dehydrogenation, N-dealkylation, carbonylation, oxidative deamination, acetylation, glucuronidation, sulphate conjugation as well as the crossover of multiple metabolic pathways in vivo, and 16 of these metabolites were proposed for the first time. This study showed an insight into the metabolism of palbociclib in vivo, which may provide relevant chemical information for subsequent studies in the future.

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.