Zinc-Chelating Small Molecules Preferentially Accumulate and Function within Pancreatic ß Cells.

Diabetes is a hyperglycemic condition characterized by pancreatic ß-cell dysfunction and depletion. Whereas methods for monitoring ß-cell function in vivo exist, methods to deliver therapeutics to ß cells are lacking. We leveraged the rare ability of ß cells to concentrate zinc to preferentially trap zinc-binding molecules within ß cells, resulting in ß-cell-targeted compound delivery. We determined that zinc-rich ß cells and islets preferentially accumulated TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline) in a zinc-dependent manner compared with exocrine pancreas. Next, we asked whether appending a zinc-chelating moiety onto a ß-cell replication-inducing compound was sufficient to confer preferential ß-cell accumulation and activity. Indeed, the hybrid compound preferentially accumulated within rodent and human islets in a zinc-dependent manner and increased the selectivity of replication-promoting activity toward ß cells. These data resolve the fundamental question of whether intracellular accumulation of zinc-chelating compounds is influenced by zinc content. Furthermore, application of this principle yielded a proof-of-concept method for ß-cell-targeted drug delivery and bioactivity.

Metabolite identification of the antimalarial naphthoquine using liquid chromatography-tandem high-resolution mass spectrometry in combination with multiple data-mining tools.

Naphthoquine (NQ) is one of important partner drugs of artemisinin-based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2-4 h) and has a long elimination half-life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high-resolution LTQ-Orbitrap mass spectrometry (HRMSn ) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid-phase extraction. For data processing, multiple data-mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1-M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N-oxidation. This study demonstrates that LC-HRMSn in combination with multiple data-mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs.

An eco-friendly direct spectrofluorimetric method for the determination of irreversible tyrosine kinase inhibitors, neratinib and pelitinib: application to stability studies.

A new rapid and simple stability-indicating spectrofluorimetric method has been developed for the determination of two irreversible tyrosine kinase inhibitors (TKIs), neratinib (NER) and pelitinib (PEL). The method is based upon measurement of the native fluorescence intensity of both drugs at λex 270 nm in aqueous borate buffer solutions (pH 10.5). The fluorescence intensity recorded at 545 nm (NER) and 465 nm (PEL) were rectilinear over the concentration range of 0.1-10 µg/mL for both drugs with a high correlation coefficient (r > 0.999). The proposed method provided low limits of detection and of quantitation of 0.07, 0.11 µg/mL (NER) and 0.02, 0.05 µg/mL (PEL), respectively. The method was successfully applied for the determination of NER and PEL in bulk powder. The proposed methods were fully validated as per the International Conference on Harmonisation (ICH) guidelines. The application of the method was extended to stability studies of both NER and PEL under different forced-degradation conditions (acidic-induced, base-induced, oxidative, wet heat, and photolytic degradation). Moreover, the kinetics of the base-induced and oxidative degradation of both drugs was investigated and the pseudo-first-order rate constants and half-lives were estimated at different temperatures. Also, an Arrhenius plot was applied to predict the stability behaviour of the two drugs at room temperature. Copyright © 2016 John Wiley & Sons, Ltd.

New insights into the importance of prolactin in dairy ruminants.

In most mammals, prolactin (PRL) is essential for maintaining lactation, and the suppression of PRL inhibits lactation. However, the involvement of PRL in the control of ruminant lactation is less clear, because inconsistent effects on milk yield have been observed with the short-term suppression of PRL by bromocriptine. Therefore, several experiments have been conducted to assess the galactopoietic role of PRL. In an initial experiment, cows in early lactation received daily injections of the dopamine agonist quinagolide for 9 wk. Quinagolide reduced milking-induced PRL release and caused a faster decline in milk production. Quinagolide also reduced mammary epithelial cell activity, survival, and proliferation. In goats, cabergoline, another dopamine agonist, caused a 28% decrease in milk yield the day after injection. In another experiment, cows were injected for 5d with quinagolide, with quinagolide plus bovine PRL injected at milking time, or with vehicles only. Again, quinagolide reduced milk, protein, and lactose yields. Although PRL injections were not sufficient to restore milk yield, they tended to increase milk protein and lactose yields and increased the viability of mammary epithelial cells purified from milk. Recently, our team stimulated PRL secretion with daily injections of the dopamine antagonist domperidone for 5 wk. Milk production increased gradually and was greater in domperidone-treated cows during the last 4 wk of the treatment period. In most experiments where PRL secretion was manipulated, feed intake paralleled the changes of PRL concentration, supporting the idea that PRL increases feed intake to provide the nutrients necessary to support lactation in dairy ruminants. In late-lactation cows, quinagolide and cabergoline decreased milk production within the first day of treatment and induced more rapid changes in several markers of mammary gland involution after drying-off. In addition, quinagolide improved the resistance to intramammary infection, suggesting that PRL inhibition could be an alternative strategy for facilitating drying-off. Prolactin appears to directly affect mammary gland functions, but mammary gland responsiveness to PRL appears to be modulated by local and systemic factors. Therefore, the modulation of the number and isoforms of the PRL receptors as well as the expression of intracellular modulators of cell signaling in the mammary gland require further investigation. In conclusion, these data, combined with those from other studies, provide a good body of evidence that PRL is galactopoietic in dairy ruminants.

Attractive design: an elution solvent optimization platform for magnetic-bead-based fractionation using digital microfluidics and design of experiments.

There is great interest in the development of integrated tools allowing for miniaturized sample processing, including solid phase extraction (SPE). We introduce a new format for microfluidic SPE relying on C18-functionalized magnetic beads that can be manipulated in droplets in a digital microfluidic platform. This format provides the opportunity to tune the amount (and potentially the type) of stationary phase on-the-fly, and allows the removal of beads after the extraction (to enable other operations in same device-space), maintaining device reconfigurability. Using the new method, we employed a design of experiments (DOE) operation to enable automated on-chip optimization of elution solvent composition for reversed phase SPE of a model system. Further, conditions were selected to enable on-chip fractionation of multiple analytes. Finally, the method was demonstrated to be useful for online cleanup of extracts from dried blood spot (DBS) samples. We anticipate this combination of features will prove useful for separating a wide range of analytes, from small molecules to peptides, from complex matrices.

Liquid chromatography and mass spectrometry for the analysis of N-ß-methylamino-L-alanine with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

Numerous studies in the past decade have identified N-ß-methylamino-L-alanine (BMAA) as a putative environmental neurotoxin. Produced by cyanobacteria and accumulated at different levels of the trophic system, BMAA has been detected in the brain tissue of human patients that died from progressive neurodegenerative disease. Research into the mechanism of neurotoxicity has been hampered by conflicting results and disagreement in the literature over analytical methods used for quantification and detection. While several research approaches have been tested, the use of the derivatizing reagent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate is presented here as an effective and selective means for the analysis of BMAA and two co-occurring biological isomers, DAB and AEG, by liquid chromatography and tandem mass spectrometry.

Infrared imaging spectroscopy and chemometric tools for in situ analysis of an imiquimod pharmaceutical preparation presented as cream.

In the present work the homogeneity of a pharmaceutical formulation presented as a cream was studied using infrared imaging spectroscopy and chemometric methodologies such as principal component analysis (PCA) and multivariate curve resolution with alternating least squares (MCR-ALS). A cream formulation, presented as an emulsion, was prepared using imiquimod as the active pharmaceutical ingredient (API) and the excipients: water, vaseline, an emulsifier and a carboxylic acid in order to dissolve the API. After exposure at 45°C during 3 months to perform accelerated stability test, the presence of some crystals was observed, indicating homogeneity problems in the formulation. PCA exploratory analysis showed that the crystal composition was different from the composition of the emulsion, since the score maps presented crystal structures in the emulsion. MCR-ALS estimated the spectra of the crystals and the emulsion. The crystals presented amine and C-H bands, suggesting that the precipitate was a salt formed by carboxylic acid and imiquimod. These results indicate the potential of infrared imaging spectroscopy in conjunction with chemometric methodologies as an analytical tool to ensure the quality of cream formulations in the pharmaceutical industry.

Aberraciones cromosomales en bulbos de cebolla Allium cepa inducidas pormoléculas híbridas 4-aminoquinolínicas / Chromosomal aberrations in Allium cepa onion bulbs induced by 4-aminoquinoline hybrid molecules

Se estudiaron las propiedades toxicológicas de los trescompuestos híbridos quinolin-tiazolidinona (FR-72 y FR-121), y acridinepoxiisoindolindiona(FR-154) aplicándolos en el test de las raíces debulbos de cebolla Allium cepa. Las moléculas FR-72, FR-121 y FR-154 fueron sintetizadas de novo según protocolos experimentalesdescritos. Los bulbos de Allium cepa (2n = 16) limpios y sanos,previamente sumergidos en el agua destilada, se secaron con papeltoalla y se colocaron directamente en los tubos de ensayo llenos conla sustancia a evaluar. Los experimentos se realizaron a temperaturaambiente 20 ± 2°C y se mantuvieron en oscuridad. El periodo deexposición de los bulbos fue de 120 horas y las raíces que se utilizaronpara el montaje de genotoxicidad eran en promedio de 2 a 2.5 cmde longitud. La evaluación del comportamiento de las tres moléculasquinolínicas sobre la inhibición del crecimiento promedio de las raícesde bulbos de cebolla Allium cepa se realizó con los tres parámetrosde bioactividad (CE50, IM, ACs) en diferentes concentraciones. Lassustancias evaluadas pueden considerarse aneugénicas, ya que actúana nivel de estructuras celulares y moleculares e impiden la fijación delas fibras del huso acromático, por ende ocasionan el desplazamientode cromosomas en anafase o pérdidas de cromosomas, llegandoincluso a ser inductoras de la apoptosis al sobrepasar la capacidadhomeostática de la célula. El análisis preliminar indicó que la moléculaFR-121 a concentración 10-6 M y la molécula FR-154 a concentración10-3 M resultaron ser potentes agentes fitotóxicos provocando diversasaberraciones claustogénicas y aneugénicas...

The toxicological properties of three hybridcompounds, quinoline-thiazolidinone (FR-72 and FR-121)and acridin-epoxyisoindolindione (FR-154) were studiedby applying named compounds to the test of the rootsof Allium cepa onion bulbs. Molecules FR-72, FR-121 andFR-154 were synthesized de novo according to describedsynthetic protocols. Clean and healthy bulbs of Alliumcepa (2n = 16), previously immersed in distilled water,were dried with paper towels and placed directly into testtubes filled with the test substance. The experiments werecarried out at room temperature 20 ± 2°C and were keptin darkness. The period of exposure of bulbs was 120hours; the roots used for the genotoxicity evaluation wereon average of 2 to 2.5 cm in length. The evaluation of theeffect of the three quinolinic molecules on the growthof onion roots of Allium cepa bulbs was achieved usingdifferent concentrations of the three growth parameters(EC50, IM, ACs) The evaluated substances performedaneugenic actions, operating at cellular and molecularstructure level and preventing the fixing of mitoticspindle fibers, causing the movement of chromosomesin the anaphase or loss of chromosomes, even inducingapoptosis by exceeding the homeostatic capacity of thecell. The preliminary analysis indicated that molecule FR-121 at 10-6 M concentration and molecule FR-154 at 10-3M concentration, proved to be potent phytotoxic agentscausing various claustogenic and aneugenic aberrations...

Foram estudadas as propriedades toxicológicas dos trêscompostos híbridos, quinolina-tiazolidinona (FR-72 e FR-121)e hidroacridin-epoxiisoindolindiona (FR-154) aplicando-os noteste das raízes de bulbos de cebola Allium cepa. As moléculas FR-72, FR-121 e FR-154 foram sintetizadas de novo de acordo com osprotocolos experimentais já descritos. Os bulbos de Allium cepa (2n= 16) limpos e sádios, previamente imersos em água destilada, foramsecos com papel toalha e colocados diretamente em tubos de ensaio,cheios com a substância de teste. Os experimentos foram realizadosà temperatura ambiente 20 ± 2°C e mantiveram-se na escuridão. Operíodo de exposição dos bulbos foi de 120 horas e as raízes usadaspara a montagem de genotoxicidade tinham, em média, de 2 a 2,5 cmde comprimento. A avaliação do comportamento das três moléculasquinolínicas sobre a inibição do crescimento médio das raízes debulbos de cebola Allium cepa foi realizada com os três parâmetrosde bioatividade (EC50, IM, ACs) em diferentes concentrações. Assubstâncias avaliadas podem se considerar aneugénicas, já queatuam ao nível de estruturas celulares e moleculares, e impedem oestabelecimento de fibras do fuso acromático, provocando assim odeslocamento dos cromossomos na anáfase ou perda de cromossomos,chegando, inclusive, a serem indutoras da apoptose ao ultrapassar acapacidade homeostática da célula. A análise preliminar indicou que amolécula FR-121 em concentração de 10-6 M e a molécula de FR-154em concentração de 10-3 M resultaram ser potentes agentes fitotóxicosprovocando várias aberrações claustogénicas e aneugénicas...

Aminoquinolines as fluorescent labels for hydrophilic interaction liquid chromatography of oligosaccharides.

In this study, we investigated the potential of four different aminoquinoline (AQ) compounds as fluorescent labels for glycan analysis using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection (FLD). We confirmed the optimal excitation and emission wavelengths of 3-AQ and 6-AQ conjugated to glycan standards using three-dimensional fluorescent spectral scanning. The optimal excitation and emission wavelengths for 6-AQ were confirmed at λ(ex)=355 nm and λ(em)=440 nm. We concluded that the optimal wavelengths for 3-AQ were λ(ex)=355 nm and λ(em)=420 nm, which differed considerably from the wavelengths applied in previous reports. HILIC-FLD chromatograms using experimentally determined wavelengths were similar to 2-aminobenzamide controls, but the peak capacity and resolution differed significantly when published 3-AQ λ(ex/em) values were applied. Furthermore, we found that 5-AQ and 8-AQ labeled maltohexaose did not display any fluorescent properties when used as a carbohydrate tag for HPLC analysis. Finally, we applied experimentally determined wavelengths to 3-AQ labeled N-glycans released from human IgG to illustrate changes in retention time as well as to demonstrate that AQ labeling is applicable to complex sample analysis via exoglycosidase sequencing.

Sensor specific imaging of proteomic Zn2+ with zinquin and TSQ after cellular exposure to N-ethylmaleimide.

The impact of the thiol binding reagent N-ethylmaleimide (NEM) on proteomic Zn(2+) availability was investigated in rat glioma cells. Zinquin (ZQ) or TSQ, two related fluorescent sensors, were used to observe reactive Zn(2+). Control cells contained proteomic Zn(2+) but no detectable low molecular weight (LMW) Zn(2+). With either sensor, basal cellular fluorescence emission centered near 470 nm, indicative of sensor-Zn-proteins. ZQ sequestered 13% of proteomic Zn(2+) as Zn(ZQ)(2); TSQ reacted only with the Zn-proteome. NEM (100 µM) abolished LMW thiols, including glutathione (GSH) and lowered proteomic sulfhydryl content about 30%. In ZQ-treated cells, NEM exposure enhanced fluorescent intensity and the formation of Zn(ZQ)(2) (λ(MAX), 492 nm). Cells incubated with TSQ and NEM also displayed increased fluorescence without a spectral shift in wavelength maximum, consistent with increased formation of TSQ-Zn-protein adducts but not Zn(TSQ)(2). In neither experiment was Zn(2+) lost from cells. NEM altered Zn(2+) accessibility to sensors in membrane-nuclear and cytosolic fractions, but Zn(ZQ)(2) was only generated in the cytosol. Similar results were obtained when cell supernatant replaced cells. In contrast, when isolated proteome was reacted with ZQ and 100 µM NEM in the absence of GSH, 70% of the proteomic thiols underwent reaction. As a consequence, most of the ZQ-Zn-protein adducts were converted to Zn(ZQ)(2). Substituting TSQ for ZQ, only increased TSQ-Zn-proteins were observed. Evidently, the results of imaging cells with Zn(2+) sensors are dependent upon the specific chemical properties of the sensors and can only be understood after detailed chemical analysis.

Enantiomeric separation of antimalarial drugs by capillary electrophoresis using neutral and negatively charged cyclodextrins.

Capillary electrophoresis (CE) methods for chiral resolution of five antimalarial drugs (primaquine, tafenoquine, mefloquine, chloroquine and quinacrine) were developed by using a wide selection of neutral and anionic cyclodextrin (CD) derivatives. The use of sulfobutyl-ß-CD and carboxymethyl-ß-CD (CMBCD) resulted in good resolution of quinacrine and tafenoquine, respectively. New results are presented for resolutions of chloroquine and mefloquine. Application of carboxyalkyl- and sulfobutyl-CD derivatives provided improved resolution for primaquine. The impurity in primaquine sample detected by CE was identified as quinocide by MS and NMR. CMBCD provided not only the best separation of primaquine from quinocide but also the simultaneous complete resolution of both compounds.

Electrospray ionization mass spectrometry as a valuable tool in the characterization of novel primaquine peptidomimetic derivatives.

Novel primaquine-derived antimalarials have been extensively characterized by electrospray ionization-ion trap mass spectrometry (ESI-MS). Experiments by in-source collision-induced dissociation (CID) in the nozzle- skimmer region (NSR) or by tandem-MS are shown to be most valuable tools for the physicochemical characterization of these 8-aminoquinolinic drugs that also bear the biologically relevant imidazolidin-4-one scaffold. It was possible to find parallelism between compound stability in the NSR and its reactivity towards hydrolysis at physiological pH and T. Moreover, tandem-MS fragmentation patterns were characteristic for each family, providing a means for structural distinction of isomers and allowing to find interesting correlations between the relative abundance of particular fragments and relevant structure-activity determinants, such as Charton steric parameter, v. In conclusion, this work provides solid grounds to establish ESI-MS as a key tool for the physicochemical characterization of biopharmaceuticals bearing the 8-aminoquinoline and/or the imidazolidin-4-one moieties.

Ab initio, density functional theory and structural studies of 4-amino-2-methylquinoline.

The Fourier transform infrared (FTIR) and FT-Raman spectra of 4-amino-2-methylquinoline (AMQ) have been recorded in the range 4000-400 and 4000-100 cm(-1), respectively. The experimental vibrational frequency was compared with the wavenumbers obtained theoretically by ab initio HF and DFT-B3LYP gradient calculations employing the standard 6-31 G** and high level 6-311 ++G** basis sets for optimised geometry of the compound. The complete vibrational assignment and analysis of the fundamental modes of the compounds were carried out using the experimental FTIR and FT-Raman data, and quantum mechanical studies. The geometry and normal modes of vibration obtained from the HF and DFT methods are in good agreement with the experimental data. The potential energy distribution of the fundamental modes was calculated with ab initio force fields utilising Wilson's FG matrix method. The NH-pi interactions and the influence of amino and methyl groups on the skeletal modes are investigated.

Identification of 3-acetyl-2-aminoquinolin-4-one as a novel, nonpeptidic scaffold for specific calpain inhibitory activity.

A series of 3-acetyl-2-aminoquinolin-4-one derivatives selected from the Korean Chemical Bank were screened for calpain inhibitory activity by using a high-throughput fluorimetric calpain assay. We identified a potent and selective mu-calpain inhibitor, compound 17, whose specificity and efficacy for mu-calpain inhibition was better than MDL28170. Docking studies revealed that the efficacy of its inhibitory effect on calpain depended on the size and charge properties of the substitutions on the phenylamino ring.

Analysis of quinocide in unprocessed primaquine diphosphate and primaquine diphosphate tablets using gas chromatography-mass spectrometry with supersonic molecular beams.

Malaria is one of the most widespread and deadly diseases on the planet. Every year, about 500 million new cases are diagnosed, and the annual death toll is about 3 million. Primaquine has strong antiparasitic effects against gametocytes and can therefore prevent the spread of the parasite from treated patients to mosquitoes. It is also used in radical cures and prevents relapse. Consequently, primaquine is an often-used drug. In this study the separation of unprocessed primaquine from the contaminant quinocide based on gas chromatography-mass spectrometry with supersonic molecular beam (SMB) is presented and 7.5 mg primaquine diphosphate tablets were analyzed. We present a novel method for fast determination of quinocide which is an isomer of primaquine as the main contaminant in unprocessed primaquine and in its medical form as tablets by gas chromatography-mass spectrometry with SMB (also named supersonic GC-MS). Supersonic GC-MS provides enhanced molecular ion without any ion source related peak tailing plus extended range of compounds amenable for GC-MS analysis. In addition, major isomer mass spectral effects were revealed in the mass spectra of primaquine and quinocide which facilitated the unambiguous identification of quinocide in primaquine tablets. Fast GC-MS analysis is demonstrated with less then 2 min elution time of the drug and its main contaminants.

Determination of quinocide as impurity in primaquine tablets by capillary zone electrophoresis.

A capillary zone electrophoretic method has been developed and validated for the determination of the impurity quinocide (QC) in the antimalarial drug primaquine (PQ). Different buffer additives such as native cyclodextrins and crown ethers were evaluated. Promising results were obtained when either beta-cyclodextrin (beta-CD) or 18-crown-6 ether (18C6) were used. Their separation conditions such as type of buffer and its pH, buffer additive concentration, applied voltage capillary temperature and injection time were optimized. The use of 18C6 offers slight advantages over beta-CD such as faster elution times and improved resolution. Nevertheless, migration times of less than 5 min and resolution factors (R(s)) in the range of 2-4 were obtained when both additives were used. The method was validated with respect to selectivity, linearity, limits of detection and quantitation, analytical precision (intra- and inter-day variability) and repeatability. Concentrations of 2.12 and 2.71% (w/w) of QC were found in pharmaceutical preparations of PQ from two different manufacturers. A possible mechanism for the successful separation of the isomers is also discussed.

Optimization of separation and detection of 6-aminoquinolyl derivatives of amino acids by using reversed-phase liquid chromatography with on line UV, fluorescence and electrochemical detection.

The combined use of UV-absorbance, fluorescence and electrochemical detection was proposed for the analysis of a set of thirteen amino acids by reversed-phase liquid chromatography (RP-HPLC) using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as a precolumn derivatization reagent. The utility of using three detectors in series was demonstrated. The separation of all derivatized amino acids was optimized with the aid of a computer optimization program from only four simple linear gradient measurements. The effectiveness of a reliable retention prediction of solutes under any gradient profile using other gradient or isocratic data was also examined.

Nature of the main contaminant in the drug primaquine diphosphate: SFC and SFC-MS methods of analysis.

The drug primaquine diphosphate is used for causative treatment of malaria. Using HPLC-MS and GC-MS, this research group was previously able to show that the main contaminant of primaquine is the positional isomer quinocide [I. Brondz, D. Mantzilas, U. Klein, D. Ekeberg, E. Hvattum, M.N. Lebedeva, F.S. Mikhailitsyn, G.D. Soulimanov, J. Roe, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 800 (2004) 211-223; I. Brondz, U. Klein, D. Ekeberg, D. Mantzilas, E. Hvattum, H. Schultz, F. S. Mikhailitsyn, Asian J. Chem. 17 (2005) 1678-1688]. Primaquine and quinocide are highly toxic substances which can have a number of side effects upon use in medical treatment. A standard for quinocide is not typically commercially available. In the present work, supercritical fluid chromatography-mass spectrometry (SFC-MS) with two different columns was used to achieve a shorter analysis time for the separation between the positional isomers quinocide and primaquine in primaquine diphosphate and to elucidate additional information about differences in their MS fragmentation. Unlike using HPLC-MS, it was possible to achieve the differential fragmentation of positional isomers at branching points using the SFC-MS technique. The desired short analysis time was achieved using SFC equipped with a Discovery HS F5 column and the differential fragmentation of positional isomers during SFC-MS provides information on the differences in the structure of these substances. Using a Chiralpak AD-H chiral column, it was possible to resolve the enantiomers in primaquine and separate quinocide from those enantiomers.

Development and validation of a stability-indicating reversed-phase high performance liquid chromatography method for NPC 1161C, a novel 8-aminoquinoline anti-malarial drug.

NPC 1161C (+/-8-[(4-amino-1-methylbutyl)amino-5-(3,4-dichlorophenoxy)-6-methoxy-4-methylquinoline succinate]) is a novel investigational antimalarial drug of interest for its in vivo oral potency, activity against blood and tissue stage parasites, favorable toxicity profile, long duration of action, and utility in both prophylaxis and treatment models. The pharmaceutical development of NPC 1161C warranted the availability of an assay for the detection and quantification of the drug and its separation from the impurities and degradation products. A simple and rapid stability-indicating reversed-phase HPLC method was developed and validated according to ICH guidelines. The method was found to be linear, precise and accurate. It also proved to be selective in the presence of impurities and degradation products during forced degradation studies. The method was found to be robust by factorial experimental design and was well within the recommended parameters of system suitability testing. Degradants of the drug during stress studies were also identified using high resolution mass spectrometry.

Effects of (+/-)-huprine Y and (+/-)-huprine Z, two new anticholinesterasic drugs, on muscarinic receptors.

The cholinergic profile of (+/-)-huprine Y and (+/-)-huprine Z on muscarinic receptors has been determined. Displacement of [3H]-pirenzepine and [3H]-QNB plus pirenzepine was performed in rat hippocampus. Both compounds showed a higher degree of affinity to M1 muscarinic receptors (P < 0.01) than to M2 muscarinic receptors. To determine the M1 agonist or antagonist role of the two huprines, studies of inositol phosphates (IP) production were performed. Both huprines significantly stimulated IP accumulation in a concentration-dependent manner. The reversion of this effect by different antagonists showed that M1 muscarinic receptors were activated by (+/-)-huprine Y and (+/-)-huprine Z, but some other mechanisms, such as alpha1-adrenoceptors or nicotinic receptors, were involved.