A high throughput dried DMSO Log D lipophilicity measurement based on 96-well shake-flask and atmospheric pressure photoionization mass spectrometry detection

A rapid throughput octanol–water lipophilicity measurement based on 96-well shake-flask and LC/UV/APPI/MS is described. The method utilizes central liquid storage where compounds are stored as 10 mM solutions in dimethyl sulfoxide (DMSO). The DMSO is subsequently removed to generate solid like material used for Log D measurement. The removal of DMSO minimizes the concern for potential DMSO cosolvent effect on the measured value. Sample preparation is automated using a liquid handling workstation with 96-well pipetter. Both octanol and buffer phases are quantified using state of the art ultra-high pressure HPLC coupled with a superficially diffused core reversed-phase column and an atmospheric pressure photo ionization mass spectrometer. The throughput of the method is two days for a batch of 96 compounds. The method has been validated using 72 literature compounds with diverse ionization and Log D values ranging from −2 to +6. The observed coefficient of determination r² is 0.9973.     

Improvement of the high-speed log D assay using an injection marker for the water plug aspiration/injection method

An improved method for the high-speed assay for log D (HSLogD) employing an injection marker is described. The critical process of HSLogD is automated sampling of the water phase, which prevents contamination from the octanol phase. However, deployment of HSLogD sometimes produces incorrect and inaccurate values compared with data from closely related methods due to low solubility of compounds or instrument problems. Therefore, careful manual inspection of data is needed to confirm the results obtained by this method. To solve this problem, compounds were screened for suitability as injection markers and 2-(4-methoxyphenoxy)-4,8-dimethylquinoline was the most suitable compound examined. Introduction of the injection marker into the process successfully eliminated the need for the tedious and time-consuming manual inspection process.     

Synthesis of oxaspiropiperidines as a strategy for lowering log D

The syntheses of four new spiropiperidines designed as polar analogs of methyl 2-(3-azaspiro[5.5]undecan-9-yl)acetate are described.     

First examples of tetracyclic triterpenoids with a D:B-friedobaccharane skeleton. A tentative biosynthetic route

Baruol (1) and Leonal (2), first examples of tetracyclic triterpenes possessing a D:B-friedobaccharane skeleton, were isolated from Maytenus blepharodes and M. chiapensis, respectively. Their structures were established by spectroscopic analysis, molecular modeling studies and biogenetic background. The implication of the D:B-friedobaccharenyl cation in the biosynthetic route of baccharane and shionane skeletons is discussed. Baruol exhibited β-glucuronidase inhibitory activity, a target in the search for hepatoprotective agents.     

A new HPLC-DAD-MS/MS method for the simultaneous determination of major compounds in the crude extract of Lychnophora salicifolia Mart. (Brazilian arnicão) leaves: Application to chemical variability evaluation

Lychnophora salicifolia Mart., which occurs in the Brazilian Cerrado in the states of Bahia and Minas Gerais as well as in the southeast of the state of Goiás, is the most widely distributed and also the most polymorphic species of the genus. This plant is popularly known to have anti-inflammatory and analgesic activities. In this work, we have studied the variation in terms of polar metabolites of ninety-three Lychnophora salicifolia Mart. specimens collected from different regions of the Brazilian Cerrado. Identification of the constituents of this mixture was carried out by analysis of the UV spectra and MS data after chromatographic separation. Twenty substances were identified, including chlorogenic acid derivatives, a flavonoid C-glucoside, and other sesquiterpenes. The analytical method was validated, and the reliability and credibility of the results was ensured for the purposes of this study. The concentration range required for analysis of content variability within the analyzed group of specimens was covered with appropriate values of limits of detection and quantitation, as well as satisfactory precision and recovery. A quantitative variability was observed among specimens collected from the same location, but on average they were similar from a chemical viewpoint. In relation to the study involving specimens from different locations, there were both qualitative and quantitative differences among plants collected from different regions of Brazil. Statistical analysis revealed that there is a correlation between geographical localization and polar metabolites profile for specimens collected from different locations. This is evidence that the pattern of metabolites concentration depends on the geographical distribution of the specimens.     

Bis-cephalezomines A-E from Cephalotaxus harringtonia var. nana

Five new dimeric Cephalotaxus alkaloids, bis-cephalezomines A-E (1-5), have been isolated from the leaves of Cephalotaxus harringtonia var. nana, and the structures and stereochemistry were elucidated on the basis of spectroscopic data including 2D NMR and FABMS/MS spectra, and chemical means.     

Characterization of lipid components of Melanorrhoea usitata lacquer sap

The lipid component of Melanorrhoea usitata lacquer sap isolated by acetone was analyzed and compared to synthesized ω-phenylalkylcatechols and ω-phenylalkylphenols. In addition, laccol and urushiol analogues synthesized in our laboratory were used as standard materials to analyze the lipid component of the Myanmar lacquer sap. The GC and GC/MS measurements confirmed the results of Kumanotani and Du that neither ω-phenylalkylcatechol nor ω-phenylalkylphenol exist in the lacquer saps from Rhus vernicifera and R. succedanea.     

Highly sensitive method for the determination of adenosine by LC-MS/MS-ESI: method validation and scope of application to a pharmacokinetic/pharmacodynamic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of adenosine in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electro‐spray ionization in the positive‐ion mode. The assay procedure involves extraction of adenosine and phenacetin (internal standard, IS) from rat plasma with a simple protein precipitation extraction process. The method was validated using rat plasma with extinguished adenosine endogenous levels. Chromatographic separation was achieved using a binary gradient using mobile phase A (acetonitrile) and B (0.2% formic acid in water) at a flow rate of 0.50 mL/min on an Atlantis dC₁₈ column with a total run time of 4.0 min. The MS/MS ion transitions monitored were 268 → 136 for adenosine and 180 → 110 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.48 ng/mL and the linearity range extended from 0.48 to 1210 ng/mL. The intra‐ and inter‐day precisions were in the ranges 2.32–12.7 and 4.01–9.40%, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Validation and application of a novel LC/MS/MS method for the determination of isoginkgetin in rat plasma

Isoginkgetin is a biflavonoid compound isolated from the leaf extracts of Ginkgo biloba. In this study, an liquid chromatography–tandem mass spectrometry (LC/MS/MS) with liquid–liquid extraction was developed and validated for the analysis of isoginkgetin in rat plasma. In the process of chromatographic separation, selected reaction monitoring transitions for isoginkgetin and IS were m/z 566.8 → 134.7 and m/z 430.8 → 269.3, respectively. The validation parameters including selectivity, linearity, LLOQ, accuracy, precision, matrix effect, stability and recovery were satisfactory. The intra‐ and inter‐batch precision (RSD) were <12.1% in plasma, while the accuracy (RE) was within ±14.3%. This method was employed in a pharmacokinetic study on rats after the intravenous administration of isoginkgetin.     

An in-tube SPME device for the selective determination of chlorophyll a in aquatic systems

We report a new device for the estimation of the content of chlorophyll a pigment in water samples as an indicator of water quality. The extraction of the pigment from water was also optimized. 10 mL of water was filtered through a nylon filter (45 μm pore size and 13 mm of diameter), after the chlorophylls were dissolved by immersing the filter in 1 mL of a low non-hazardous solvent as ethanol. An in-valve in-tube SPME device coupled to capillary liquid chromatography with diode array detection was designed. A capillary column of 70 cm in length (0.32 mm i.d. coated with 5% diphenyl-95% polydimethylsiloxane, 3 μm coating thickness) was used as the loop of the injection valve for preconcentration and a Zorbax SB C₁₈ (SiO₂-based) 150 mm × 0.5 mm i.d., 5 μm column (Agilent) was used as analytical column. The achieved detection limit was 0.05 μg L⁻¹ and the working range of concentrations was 0.1-1 μg L⁻¹. % RSD values between 2 and 11 were obtained. Chlorophyll a in several water matrices was determined with good results in presence of other pigments such as chlorophyll b, pheophytin a and pheophytin b.     

Banchromene and other secondary metabolites from the endophytic fungus Fusarium sp. obtained from Piper guineense inhibit the motility of phytopathogenic Plasmopara viticola zoospores

A new chromene, (S)-banchromene (1), together with seven known compounds, ergosterol, beauvericin (2), fusaproliferin (3), radicinin (4), poly(3-hydroxybutyric acid) (PHB, 5), N-methylpyrrolidone and an inseparable mixture of isochromene derivatives 6a, 6b, were isolated from a culture of Fusarium sp. strain CAMKT24b1, an endophytic fungus from the leaves and twigs of Piper guineense (Piperaceae). The structures of these metabolites were elucidated on the basis of their spectroscopic data; the absolute configuration of 1 was determined by ab initio-calculation of the optical rotation. In tests with the zoospores of the grapevine downy mildew pathogen Plasmopara viticola, compounds 1–4 showed moderate to high levels of motility-impairing activity at concentrations as low as 2.5 μg/mL. Compound 2 was the most active, exhibiting both motility-halting and lytic activities. Furthermore, compounds 2 and 3 displayed significant cytotoxic activity against brine shrimp larvae (Artemia salina) at 10 μg/mL. This is the first report on motility inhibitory and lytic activities of metabolites from an endophytic Fusarium species against the zoospores of the downy mildew pathogen P. viticola.     

LC‐MS/MS method for the determination of pitolisant: application to rat pharmacokinetic and brain penetration studies

A simple and sensitive LC‐MS/MS method was developed and validated for the quantitation of pitolisant, an H₃ receptor antagonist/inverse agonist. Acetonitrile protein precipitation technique was used to prepare rat blood and brain tissue homogenate samples by using aripiprazole as internal standard (IS). Chromatographic separation was performed by using Xbridge column (2.1 × 50 mm, 3.5 µm) with a gradient elution program. The mobile phase consists of ammonium formate (10 mm ) with 0.2% formic acid and acetonitrile. Multiple reaction monitoring mode was used in positive polarity with a transition of m/z 296.3 → 98.2 for the pitolisant and m/z 448.2 → 285.3 for the IS. The calibration curves were linear in the range of 0.1–100 ng/mL in both the blood and brain homogenate samples. This method was applied to quantify samples obtained from the pharmacokinetic and brain penetration studies in male wistar rats. Mean maximum concentration, area under the curve from zero to infinity and half‐life of the pitolisant were found to be 3.4 ± 1.7 ng/mL, 5 ± 4 ng h/mL and 1.9 ± 0.3 h, respectively, after a 3 mg/kg oral dose. The mean calculated concentrations in the brain were found to be 38, 60 and 52 ng/g at 0.5, 1 and 2 h, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous identification of glucosinolates and phenolic compounds in a representative collection of vegetable Brassica rapa

Brassica raparapa group is widely distributed and consumed in northwestern Spain. The consumption of Brassica vegetables has been related to human health due to their phytochemicals, such as glucosinolates and phenolic compounds that induce a variety of physiological functions including antioxidant activity, enzymes regulation and apoptosis control and the cell cycle. For first time in Brassica crops, intact glucosinolates and phenolic compounds were simultaneously identified and characterized. Twelve intact glucosinolates, belonging to the three chemical classes, and more than 30 phenolic compounds were found in B. rapa leaves and young shoots (turnip greens and turnip tops) by LC–UV photodiode array detection (PAD)–electrospray ionization (ESI). The main naturally occurring phenolic compounds identified were flavonoids and derivatives of hydroxycinnamic acids. The majority of the flavonoids were kaempferol, quercetin and isorhamnetin glycosylated and acylated with different hydroxycinnamic acids. Quantification of the main compounds by HPLC-PAD showed significant differences for most of compounds between plant organs. Total glucosinolate content value was 26.84 μmol g⁻¹ dw for turnip greens and 29.11 μmol g⁻¹ dw for turnip tops; gluconapin being the predominant glucosinolate (23.2 μmol g⁻¹ dw). Phenolic compounds were higher in turnip greens 51.71 μmol g⁻¹ dw than in turnip tops 38.99 μmol g⁻¹ dw, in which flavonols were always the major compounds.     

Lipophilicity assessment of basic drugs (log P(o/w) determination) by a chromatographic method

A previously reported chromatographic method to determine the 1-octanol/water partition coefficient (log P(o/w)) of organic compounds is used to estimate the hydrophobicity of bases, mainly commercial drugs with diverse chemical nature and pK(a) values higher than 9. For that reason, mobile phases buffered at high pH to avoid the ionization of the solutes and three different columns (Phenomenex Gemini NX, Waters XTerra RP-18 and Waters XTerra MS C(18)) with appropriate alkaline-resistant stationary phases have been used. Non-ionizable substances studied in previous works were also included in the set of compounds to evaluate the consistency of the method. The results showed that all the columns provide good estimations of the log P(o/w) for most of the compounds included in this study. The Gemini NX column has been selected to calculate log P(o/w) values of the set of studied drugs, and really good correlations between the determined log P(o/w) values and those considered as reference were obtained, proving the ability of the procedure for the lipophilicity assessment of bioactive compounds with very different structures and functionalities.     

Development and validation of LC–MS/MS method for amlexanox in rat plasma and its application in preclinical pharmacokinetics

Amlexanox, an anti-inflammatory and anti-allergic agent, has been widely used clinically for the treatment of canker sores, asthma, and allergic rhinitis. Recently, amlexanox has received considerable attention in curing nonalcoholic fatty liver diseases and hepatitis virus infection. Herein, we first established a sensitive high-performance liquid chromatography-tandem mass spectrum (LC–MS/MS) method for the determination of amlexanox in rat plasma. Propranolol was used as the internal standard (IS). Using a simple protein precipitation method, the amlexanox and IS were separated with Capcell Pak C18 column (2.0 × 50 mm, 5 μm) and eluted with water and acetonitrile each containing 0.1% formic acid using gradient elution condition at a flow rate of 0.4 mL·min⁻¹. Amlexanox and IS were detected by a triple quadrupole mass in multiple reactive monitoring (MRM) under the transitions of m/z 299.2 → 281.2 and m/z 259.9 → 116.1 with positive electrospray ionization, respectively. The calibration curves of amlexanox were established with the range of 50 to 2000 ng·mL⁻¹ (r² > 0.99). The validation method consisted of selectivity, accuracy, precision, carryover effect, matrix effect, recovery, dilution effect, and stability. The fully validated method was successfully applied to the pharmacokinetic study of amlexanox in Wistar rats.     

Validation of a sensitive LC/MS/MS method for the determination of zidovudine and lamivudine in human plasma

A sensitive LC/MS/MS assay for determining zidovudine (ZDV) and lamivudine (3TC) in human plasma was validated to support antiretroviral pharmacology research programs. After addition of stable labeled isotopic zidovudine (ZDV‐IS) and lamivudine (3TC‐IS) as internal standard, a solid‐phase extraction was performed with an Oasis HLB 1 cm³ cartridge, with recoveries of 92.3% for ZDV and 93.9% for 3TC. A Phenomonex Synergi Hydro‐RP (2.0 × 150 mm) reversed‐phase analytical column was utilized for chromatographic separation. The mobile phase consisted of an aqueous solution of 15% acetonitrile and 0.1% acetic acid. Detection was accomplished by ESI/MS/MS in the positive ion mode, monitoring 268/127, 271/130, 230/112 and 233/115 transitions, for ZDV, ZDV‐IS, 3TC and 3TC‐IS, respectively. The method was linear from 1 to 3000 ng/mL with a minimum quantifiable limit of 1 ng/mL when 100 μL of plasma was analyzed. Validation results demonstrated high accuracy (≤8.3% deviation) and high precision (≤10% CV) for the quality control samples. The method was also shown to be specific and reproducible. The value of the high sensitivity was demonstrated by quantitation of approximately 100 existing samples that had ZDV below the limit of quantitation using a previously validated, less sensitive HPLC‐UV method utilized in the laboratory. Copyright © 2011 John Wiley & Sons, Ltd.     

Selective immobilization of Acinetobacter junii on the natural zeolitized tuff in municipal wastewater

The immobilization of desired bacteria onto material was usually performed in synthetic media. The aim of this study was to test the immobilization of phosphate (P)-accumulating bacteria Acinetobacter junii onto natural zeolitized tuff (NZ) in the raw or sterilized municipal wastewater containing the common bacteria Escherichia coli and Enterococcus faecalis and the performance of immobilized A. junii in the same type of wastewater. In the sterilized wastewater which contained the mixture of A. junii, E. coli and E. faecalis, the A. junii was selectively immobilized onto NZ in significantly higher numbers than E. coli and E. faecalis. The A. junii added in the form of bioparticles to the wastewater containing E. coli and E. faecalis, multiplied and removed P from wastewater. The P removal from wastewater was a function of biomass of P-accumulating bacteria and not the amount of NZ or bioparticles used. The performance of A. junii was significantly better in membrane filtered than in autoclaved wastewater. The experiments that were performed in raw non sterilized wastewater showed that A. junii can be successfully immobilized onto NZ in competition with natively present heterotrophic bacteria, retain its metabolic activity and successfully remove P from such water, which makes this technology feasible from biotechnological aspect.     

Stereochemical investigation and total synthesis of inuloidin,a biologically active sesquiterpenoid from Heterotheca inuloides

The stereochemistry of inuloidin (1), which was a sesquiterpenoid that was characterized as a plant growth inhibitory substance from Heterotheca inuloides, was investigated. The modified Mosher's method coupled with a total synthetic study using osmium oxidation and Burgess dehydration as key steps were performed to clarify the stereochemistry of 1, which was determined to be a 2S,4R isomer.