Approach Study for Mass Balance of Pesticide Residues in Distillers' Stillage along with Distillate and Absence Verification of Pesticides in Distilled Spirits from Pilot-Scale of Distillation Column.

Herein, contaminants remaining in distillate and distillers' stillage were quantitatively measured after distillation. After rice bran powder was contaminated with 10 ppm of lead (Pb) and cadmium (Cd) or 0.02-1.27 ppm of five pesticides (terbufos, fenthion, iprobenfos, flutolanil, and ethoprophos) followed by fermentation, single-stage distillation was performed. In the obtained distillate, no Pb or Cd was found, as expected. However, when the pesticides were added as contaminants, trace-0.05 ppm of some pesticides were detected in the distillate, possibly due to the high vapor pressure (e.g., that of ethoprophos) and contamination amount (e.g., that of flutolanil, terbufos, and fenthion). In contrast, none of the contaminating pesticides were observed in the distilled spirits when a fermented liquefaction contaminated with 0.04-4 ppm of six pesticides (fenthion, terbufos, ethoprophos, iprobenfos, oxadiazon, and flutolanil) was distilled using a pilot-plant scale distillation column, indicating that the pesticides hardly migrate to the distilled spirits.

Development and validation of an UPLC-MS/MS method for the therapeutic drug monitoring of oral anti-hormonal drugs in oncology.

A liquid chromatography-mass spectrometry assay was developed and validated for simultaneous quantification of anti-hormonal compounds abiraterone, anastrozole, bicalutamide, Δ(4)-abiraterone (D4A), N-desmethyl enzalutamide, enzalutamide, Z-endoxifen, exemestane and letrozole for the purpose of therapeutic drug monitoring (TDM). Plasma samples were prepared with protein precipitation. Analyses were performed with a triple quadrupole mass spectrometer operating in the positive and negative ion-mode. The validated assay ranges from 2 to 200 ng/mL for abiraterone, 0.2-20 ng/mL for D4A, 10-200 ng/mL for anastrozole and letrozole, 1-20 ng/mL for Z-endoxifen, 1.88-37.5 ng/mL for exemestane and 1500-30,000 ng/mL for enzalutamide, N-desmethyl enzalutamide and bicalutamide. Due to low sensitivity for exemestane, the final extract of exemestane patient samples should be concentrated prior to injection and a larger sample volume should be prepared for exemestane patient samples and QC samples to obtain adequate sensitivity. Furthermore, we observed a batch-dependent stability for abiraterone in plasma at room temperature and therefore samples should be shipped on ice. This newly validated method has been successfully applied for routine TDM of anti-hormonal drugs in cancer patients.

Ultra-performance liquid chromatography tandem mass spectrometry for determination of Direct Acting Antiviral drugs in human liver fine needle aspirates.

An ultra-performance liquid chromatography with triple quadrupole mass spectrometry method was developed and validated for the determination of direct acting antiviral drug concentrations in human liver fine needle aspirates. Liver fine needle aspirate (FNA) biopsy samples were homogenized in acetonitrile to stabilize the analytes and precipitate protein. The acetonitrile supernatants were diluted with internal standards and mobile phase. Separation was achieved with a Waters Acquity BEH C18 column (50×2.1mm, 1.7um) with a gradient elution of 0.1% formic acid in water and acetonitrile. The total run time was 4.25min. Detection of analytes was achieved using electrospray ionization (positive mode) and triple quadrupole selected reaction monitoring. Standard curve concentrations ranged from 12.5 to 5000ng/mL for dasabuvir and the m1 metabolite of dasabuvir, 1.25 to 2500ng/mL for ombitasvir and ritonavir, and 5.00 to 5000ng/mL for paritaprevir. The intra- and inter-day accuracy and precision were less than 13.7% in low, medium, and high quality control samples. The validated method was applied to the analysis of a liver fine needle aspirate of a patient undergoing direct acting antiviral therapy for hepatitis C virus.

Selective androgen receptor modulators: comparative excretion study of bicalutamide in bovine urine and faeces.

Besides their development for therapeutic purposes, non-steroidal selective androgen receptor modulators (non-steroidal SARMs) are also known to impact growth-associated pathways as ligands of androgenic receptors (AR). They present a potential for abuse in sports and food-producing animals as an interesting alternative to anabolic androgenic steroids (AAS). These compounds are easily available and could therefore be (mis)used in livestock production as growth promoters. To prevent such practices, dedicated analytical strategies should be developed for specific and sensitive detection of these compounds in biological matrices. The present study focused on Bicalutamide, a non-steroidal SARM used in human treatment of non-metastatic prostate cancer because of its anti-androgenic activity exhibiting no anti-anabolic effects. To select the most appropriate matrix to be used for control purposes, different animal matrices (urine and faeces) have been investigated and SARM metabolism studied to highlight relevant metabolites of such treatments and establish associated detection time windows. The aim of this work was thus to compare the urinary and faecal eliminations of bicalutamide in a calf, and investigate phase I and II metabolites. The results in both matrices showed that bicalutamide was very rapidly and mainly excreted under its free form. The concentration levels were observed as higher in faeces (ppm) than urine (ppb); although both matrices were assessed as suitable for residue control. The metabolites found were consistent with hydroxylation (phase I reaction) combined or not with glucuronidation and sulfation (phase II reactions). Copyright © 2016 John Wiley & Sons, Ltd.

Analytical and semipreparative high performance liquid chromatography enantioseparation of bicalutamide and its chiral impurities on an immobilized polysaccharide-based chiral stationary phase.

Direct HPLC separation of enantiomers of Bicalutamide (BCT), a non-steroidal antiandrogen used for the treatment of prostate cancer, was performed by using the immobilized amylose-based Chiralpak IA chiral stationary phase (CSP). Enantioselective conditions were achieved using standard normal phase mixtures n-hexane-alcohol (ethanol or 2-propanol) and a "non-standard" mobile phase containing ethyl acetate (EA). The chromatographic behaviour of the IA CSP under these elution modes was evaluated and compared at different temperatures. The eluent mixture n-hexane-EA-ethanol 100-30-5 (v/v/v) and the column temperature of 40°C were identified as the best operational conditions to carry out semipreparative enantioseparations on a 1-cm I.D. IA column. Using this protocol, about 960mg of (R)-BCT, which is the enantiomer with the almost entire anti-androgenic activity of BCT, per day could be isolated. The analytical and semipreparative HPLC resolution of chiral impurities of BCT, and their empiric absolute configuration assignment by circular dichroism correlation method are also presented.

[Development of Determination Method of Ipfencarbazone in Agricultural Products, Livestock Products and Seafood by LC-MS/MS].

A method for the determination of ipfencarbazone in agricultural products, livestock products and seafood by LC-MS/MS was developed. Agricultural samples were extracted with acetone. An aliquot of crude extract was partitioned with n-hexane and sat. sodium chloride solution. Clean-up was performed using GC/PSA and C18 cartridges. In the case of livestock products and seafood, samples were extracted with a mixture of acetone and n-hexane, and the organic layer was collected. After acetonitrile-hexane partitioning, the extract was cleaned up using PAS and C18 cartridges. The gradient LC separation was performed on a C18 column with acetonitrile-water containing acetic acid as a mobile phase, and MS with positive ion electrospray ionization was used for detection. The average recoveries (n=5) of ipfencarbazone from 16 kinds of agricultural products, livestock products and seafood spiked at the MRLs or at the uniform limits (0.01 ppm) were 73-101%, and the relative standard deviations were 1.3-5.1%. The limit of quantitation of the developed method was 0.01 mg/kg for ipfencarbazone.

Validation of RP-HPLC Method for Simultaneous Quantification of Bicalutamide and Hesperetin in Polycaprolactone-Bicalutamide-Hesperetin-Chitosan Nanoparticles.

Bicalutamide is a non-steroidal anti-androgen drug used for the treatment of androgen-dependent prostate cancer. Hesperetin is a natural bioflavonoid that can be used in combination with bicalutamide to improve efficacy and decrease tolerance. The aim of the present work was to develop and validate a simple, sensitive, rapid reverse phase-high performance liquid chromatographic method for simultaneous estimation of bicalutamide and hesperetin. The validation parameters such as specificity, linearity, precision and accuracy, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2 (R1) guidelines. Chromatographic separation was achieved on Lichrocart(®) CN column (250 × 4 mm, 5 µm, MERCK) with isocratic elution. The retention times and detection wavelength, for hesperetin and bicalutamide were 4.28 min, 288 nm and 5.90 min, 270 nm respectively. The intra-day and inter-day assay precision and accuracy were found to be <2% over linearity of 50-2000 ng/mL with R(2) 0.999. LOD and LOQ, of bicalutamide and hesperetin was 14.70, 44.57 ng/mL and 16.11, 48.84 ng/mL, respectively. The method was successfully applied for encapsulation efficiency and drug release studies from bicalutamide and hesperetin loaded nanoparticles.

Environmental concentrations of anti-androgenic pharmaceuticals do not impact sexual disruption in fish alone or in combination with steroid oestrogens.

Sexual disruption in wild fish has been linked to the contamination of river systems with steroid oestrogens, including the pharmaceutical 17α-ethinylestradiol, originating from domestic wastewaters. As analytical chemistry has advanced, more compounds derived from the human use of pharmaceuticals have been identified in the environment and questions have arisen as to whether these additional pharmaceuticals may also impact sexual disruption in fish. Indeed, pharmaceutical anti-androgens have been shown to induce such effects under laboratory conditions. These are of particular interest since anti-androgenic biological activity has been identified in the aquatic environment and is potentially implicated in sexual disruption alone and in combination with steroid oestrogens. Consequently, predictive modelling was employed to determine the concentrations of two anti-androgenic human pharmaceuticals, bicalutamide and cyproterone acetate, in UK sewage effluents and river catchments and their combined impacts on sexual disruption were then assessed in two fish models. Crucially, fish were also exposed to the anti-androgens in combination with steroid oestrogens to determine whether they had any additional impact on oestrogen induced feminisation. Modelling predicted that the anti-androgenic pharmaceuticals were likely to be widespread in UK river catchments. However, their concentrations were not sufficient to induce significant responses in plasma vitellogenin concentrations, secondary sexual characteristics or gross indices in male fathead minnow or intersex in Japanese medaka alone or in combination with steroid oestrogens. However, environmentally relevant mixtures of oestrone, 17ß-oestradiol and 17α-ethinylestradiol did induce vitellogenin and intersex, supporting their role in sexual disruption in wild fish populations. Unexpectedly, a male dominated sex ratio (100% in controls) was induced in medaka and the potential cause and implications are briefly discussed, highlighting the potential of non-chemical modes of action on this endpoint.

Determination of isotianil in brown rice and soil using supercritical fluid extraction and gas chromatography/mass spectrometry.

Isotianil (3,4-dichloro-2'-cyano-1,2-thiazole-5-carboxanilide) is a new plant-activating pesticide. Usage of the pesticide was approved for rice fields in 2010 and its production increased 400 times (2 × 10(4) kg) in the next year. In this work, a method for determining isotianil in brown rice and rice field soil was investigated for the first time. Isotianil was extracted by supercritical fluid extraction and measured by gas chromatography/mass spectrometry. Isotianil was successfully analyzed with good recoveries (95.1-99.3%) even from soil samples with strong adsorption of pesticides.

Determination method of clomeprop and clomeprop acid in livestock and seafood products by LC-MS/MS.

A method using liquid chromatography with tandem mass spectrometry has been developed for the determination of clomeprop and its metabolite clomeprop acid in livestock and seafood products. Clomeprop and clomeprop acid were extracted with acetone-n-hexane mixture under acidic conditions, and were defatted by liquid-liquid separation using acetonitrile and n-hexane, followed cleanup with SAX and PSA cartridges. The average recoveries from 10 kinds of food (bovine muscle, bovine fat, bovine liver, milk, yellowtail, salmon, eel, fresh water clam, egg and honey) spiked at the level of the MRLs or at uniform limits (0.01 ppm) were in the range of 81-97% for clomeprop and 93-101% for clomeprop acid. Repeatability was in the range of 2.1-14% for clomeprop and 1.3-4.0% for clomeprop acid. The quantitation limits were 0.002 mg/kg for clomeprop and 0.00154 mg/kg (0.002 mg/kg as clomeprop) for clomeprop acid.

Screening for metabolically stable aryl-propionamide-derived selective androgen receptor modulators for doping control purposes.

Anabolic agents have been among the most frequently detected drugs in amateur and professional sport. A novel class of therapeutics presumably complementing anabolic steroids in the near future includes so-called selective androgen receptor modulators (SARMs) that have been under clinical investigations for several years. Although not yet commercially available, their potential for misuse in sports is high. Four aryl-propionamide-derived SARMs were synthesized in order to establish a fast and robust screening procedure using liquid chromatography/electrospray ionization tandem mass spectrometry. Synthesized compounds were characterized by high-resolution/high-accuracy mass analysis employing a linear ion trap-Orbitrap hybrid mass spectrometer while routine analyses were conducted on a triple-quadrupole mass spectrometer. Characteristic product ions obtained by collision-induced dissociation were found at m/z 289 and 261 as well as m/z 269 and 241 representing the bisubstituted aniline residues of selected model compounds. Assay validation was performed regarding lower limit of detection (1 ng/mL), recovery (85-105%), intraday precision (7.6-11.6%) and interday precision (9.9-14.4%), and precursor ion scan experiments on diagnostic product ions enabled the detection of a structurally related compound at 50 ng/mL.

Multidimensional chemical genetic analysis of diversity-oriented synthesis-derived deacetylase inhibitors using cell-based assays.

Systematic chemical genetics aims to explore the space representing interactions between small molecules and biological systems. Beyond measuring binding interactions and enzyme inhibition, measuring changes in the activity of proteins in intact signaling networks is necessary. Toward this end, we are partitioning chemical space into regions with different biological activities using a panel of cell-based assays and small molecule "chemical genetic modifiers." Herein, we report on the use of this methodology for the discovery of 617 small molecule inhibitors of histone deacetylases from a multidimensional screen of an encoded, diversity-oriented synthesis library. Following decoding of chemical tags and resynthesis, we demonstrate the selectivity of one inhibitory molecule (tubacin) toward alpha-tubulin deacetylation and another (histacin) toward histone deacetylation. These small molecules will facilitate dissecting the role of acetylation in a variety of cell biological processes.

Epidemiologic evidence for a new class of compounds associated with toxic oil syndrome.

Toxic oil syndrome appeared in epidemic form in Spain in 1981. Epidemiologic studies have demonstrated that illness was caused by consumption of rapeseed oil that had been denatured with aniline. Chemical analyses of oil specimens conducted in conjunction with epidemiologic studies have established that consumption of specific oils containing fatty acid anilide contaminants was associated with increased risk for disease. New chemical analytic methods identified a family of compounds, the di-fatty acid esters of phenylamino propane-diol, and one of these compounds, the 1,2-di-oleyl ester of 3-(N-phenylamino)-1,2-propanediol (DPAP), has been found to be more strongly associated with disease status than the fatty acid anilides. We found the odds ratio for exposure to DPAP (OR = 26.4, 95% CI = 6.4-76.3) is much higher than the odds ratio for exposure to oleyl anilide (OR = 4.1, 95% CI = 2.2-7.8), implying that exposure to DPAP was a more relevant risk factor for development of toxic oil syndrome than exposure to oleyl anilide. In this paper, we review and present analyses of data from multiple studies of the possible etiologic role of DPAP in toxic oil syndrome. The presence of DPAP in oil collected from affected and unaffected households was a more specific correlate of case relatedness than was the presence of fatty acid anilides, and it was equally sensitive. Moreover, DPAP was found in oil from the only refinery whose oil was clearly associated with illness.

Toxic oil syndrome: traceback of the toxic oil and evidence for a point source epidemic.

Rapeseed oil denatured with aniline was the vehicle of the causal agent of the toxic oil syndrome (TOS) epidemic that occurred in Spain in 1981. Although the precise aetiologic agent remains unknown, researchers established that increasing concentrations of oleyl anilide and other fatty acid anilides were associated with an increased risk for disease. To examine the hypothesis that 5-litre plastic containers of rapeseed oil associated with TOS, and which contained oleyl anilide had a characteristic shape, we measured fatty acid, sterol and fatty acid anilide levels in oil from containers of different shapes. We identified 1673 bottles of oil that had been collected during the Spanish Government's oil exchange programme and linked these bottles to people with TOS as reported in the official government census of patients with TOS. Although rapeseed oil (identified by the presence of brassicasterol) was found in 798 (47.7%) of the 1673 bottles examined, contamination with fatty acid anilide occurred in only 329 (19.6%) of the 1673 bottles and 319 (97%) of the 329 were oil containers of the shape sold by RAELCA, an oil company in Madrid. The first aniline-denatured oil that RAELCA had purchased to be refined specifically for distribution was refined at the ITH refinery of Seville, and this oil has been most directly associated with the epidemic. Previous work has shown that the only toxic oil linked to a specific refinery was that associated with rapeseed oil from the ITH refinery in Seville, and the epidemic began shortly after this oil was delivered to RAELCA for retail sale. On the basis of these findings, we conclude that oil refined by ITH and distributed by RAELCA was the principal, and probably the only, oil responsible for the TOS epidemic. Information about the history and treatment of this oil may yield important clues towards identifying the aetiologic agent of TOS.

Characterisation and quantitation of a selenol intermediate in the reaction of ebselen with thiols.

The reaction of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) with thiols was investigated with particular attention to the formation of an ebselen selenol intermediate. The selenol intermediate could be trapped in a mixture of ebselen and thiols with 1-chloro-2,4-dinitrobenzene and the resulting product displayed unique spectral characteristics. The reaction of authentic, synthesised ebselen selenol with 1-chloro-2,4-dinitrobenzene (CDNB) was shown to give rise to the same compound (2,4-dinitrophenyl (N-phenyl-2-carboxamido phenyl) selenide as characterized by light spectroscopy, NMR, IR and elemental analysis. The determination of the absorbtion coefficient at 400 nm (E = 7.5 mM-1 cm-1) and the initial rate constant of the reaction (1.4 +/- 0.3 mM-1 min-1) allows for the convenient quantification of ebselen selenol concentrations by initial rate measurements after addition of CDNB. The choice of 400 nm to monitor the reaction excludes the interference of other intermediates in the reaction of ebselen with thiols as well as the reaction of the thiols with CDNB. When the assay is applied to typical incubation conditions used for investigating the glutathione peroxidase-like activity of ebselen it was shown that as much as 10-20% of ebselen is in the selenol form. If a stronger reductant (dithiothreitol) is used 60% is in the selenol form. These data could also be confirmed by the direct determination of ebselen selenol by UV spectroscopy, due to its peak absorption at 370 nm (E = 2 mM-1 cm-1). In conclusion, this investigation demonstrates, for the first time, the identity and quantity of ebselen selenol in the reaction of ebselen with thiols and also describes a convenient assay for its quantification. These observations allow further possibilities for investigation of the molecular species responsible for the antioxidant and peroxidase activities of ebselen.

Rapid chromatographic analysis of fatty acid anilides suspected of causing toxic oil syndrome.

A rapid method for isolation and determination of fatty acid anilides of palmitic, stearic, oleic, linoleic, linoleic, and erucic acids from oil samples was developed. Corn oil samples mixed with the fatty acid anilides were diluted with petroleum ether, passed through silica gel Sep-Pak cartridges, and washed with petroleum ether. The fatty acid anilides were eluted with petroleum ether-diethyl ether (19:1, v/v), dried under a vacuum, separated, and quantitated by reversed-phase thin-layer and high-performance liquid chromatographic methods. The recovery was between 79 and 97% for the anilides from the fortified oil sample. This method can be used for the identification and quantitation of the fatty acid anilides found in adulterated oil samples.

Chemical correlates of pathogenicity of oils related to the toxic oil syndrome epidemic in Spain.

The identity of the etiologic agent that caused the 1981 epidemic of toxic oil syndrome in Spain has not been established, and toxicologic study of oil specimens from the outbreak has been hampered by uncertainty about which oils were actually capable of causing illness. To identify chemical characteristics associated with pathogenicity, the authors compared specimens collected during the Spanish government's oil recall program in June and July 1981 from affected and unaffected households in the two contiguous towns of Alcorcón and Leganés (Madrid Province). Oils were blind-coded for laboratory analysis, and personnel with no knowledge of the laboratory results determined whether illness was present in a family. Contamination with free aniline and oleyl, linoleyl, and palmityl anilides was strikingly more frequent and extensive in oils collected from the case (affected) families. There was a clear-cut dose-response effect, with increasing concentrations of aniline and anilides associated with increasing risk of illness. Differences in fatty acid and sterol compositions among oils indicated more rapeseed oil admixture in the case group, but these indicators of rapeseed oil admixture did not contribute significantly to risk after the degree of aniline/anilide contamination had been taken into account. The authors conclude that the presence of relatively high levels of aniline and fatty acid anilides in oil specimens collected during the epidemic in the two towns studied indicates a high probability of the current or prior presence of the etiologic agent of toxic oil syndrome. Although these data do not necessarily indicate that any of the compounds measured actually caused the illness, further toxicologic work should concentrate on oils with substantial concentrations of the marker compounds.

Determination of oxidation products of N-phenyllinoleamide: Spanish toxic oil syndrome studies.

The early identification of fatty acid anilides in suspect oils directed attention to their possible role in the Spanish toxic oil syndrome. These anilides or their oxidized derivatives could have been spontaneously formed during the handling and/or storage of the oil. The exact cause of the intoxication is still unknown but free radical and peroxidative mechanisms have been implicated in its etiology. Epoxy-hydroxylated derivatives from linoleic acid anilide were obtained using a model of accelerated oxidation. Their mass spectral patterns agree with the trimethylsilyl ethers of N-phenyl-9,10-epoxy-11-hydroxy-12-octadecenamide and N-phenyl-12,13-epoxy-11-hydroxy-9-octadecenamide, respectively. These compounds were also identified is rapeseed oil samples supplemented with N-phenyl-linoleamide and submitted to the reported accelerated oxidation method.

1-Phenyl-5-vinyl-2-imidazolidinethione, a proposed causative agent of Spanish toxic oil syndrome: synthesis, and identification in one of a group of case-associated oil samples.

A method is described for the synthesis and characterization of N-(2-hydroxy-3-butenyl)-N'-phenylthiourea, and its cyclization product, 1-phenyl-5-vinyl-2-imidazolidinethione (PVIZT). Fourteen coded oil samples associated with toxic oil syndrome cases in Spain were examined by gas chromatography-electron impact mass spectrometry for the presence of PVIZT. Although these samples were obtained from households where cases of toxic oil syndrome had been recorded, they differed extremely with regard to their anilide and sulphur contents. In one sample PVIZT was detected at an estimated concentration of 1 mg/kg.