Urine Extracts Research Articles

In vivo metabolism of the designer anabolic steroid hemapolin in the thoroughbred horse.

Hemapolin (2α,3α-epithio-17α-methyl-5α-androstan-17β-ol) is a designer steroid that is an ingredient in several "dietary" and "nutritional" supplements available online. As an unusual chemical modification to the steroid A-ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC-MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α-methyl-5α-androst-2-en-17β-ol), reduced and dihydroxylated madol (17α-methyl-5α-androstane-2β,3α,17β-triol), and the isomeric enone metabolites 17β-hydroxy-17α-methyl-5α-androst-3-en-2-one and 17β-hydroxy-17α-methyl-5α-androst-2-en-4-one, were detected and confirmed in equine urine extracts by comparison with a library of synthetically derived reference materials. A number of additional madol derivatives derived from hydroxylation, dihydroxylation, and trihydroxylation were also detected but not fully identified by this approach. A yeast cell-based androgen receptor bioassay of available reference materials showed that hemapolin and many of the metabolites identified by this study were potent activators of the equine androgen receptor. This study reveals the metabolites resulting from the equine administration of the androgen hemapolin that can be incorporated into routine GC-MS antidoping screening and confirmation protocols to detect the illicit use of this agent in equine sports.

Effects of exogenous auxin on stem cutting growth of tea (Camellia sinensis)

Root growth rate of a stem cutting was affected by the auxin levels absorbed by the stem cutting. Level of endogenous auxin was insufficient to stimulate the root growth, so that exogenous auxin should be applied. As synthetic auxin was highly expensive, natural auxin sources gained more attention recently to be used. This study was aimed to determine the best source of exogenous auxin triggering the stem cutting growth of tea (Camellia sinensis). This research was conducted using a completely randomized design with four replicates of each treatment. Source of exogenous auxin used in this study consisted of both synthetic (IBA and Rootone-F™) and natural (goat urine and shallot tuber extract) types. These exogenous auxins were applied into the tea stem cutting before planting through dipping method. Growth parameters related to root, shoot and leaf development were observed. Data were evaluated statistically using one-way anova. Application of 6.000 ppm IBA enhanced the rooting percentage and leaf area. Increase of root length was found in the stem cutting treated with 10% goat urine. These results suggested that each type of exogenous auxin stimulated the root growth in a different way. Therefore, proportional combination of these auxin sources might provide a greater support on root growth of tea stem cutting.

Investigating the Antimicrobial Activities of the Biosynthesized Selenium Nanoparticles and Its Statistical Analysis

The present investigation concentrates on the green combination of biosynthesized selenium nanoparticles utilizing aqueous extract of cow urine as a green technique with no external chemicals, reagents, or surfactants. The combination of parameters, for example, the sodium selenite precursor concentration, or the concentration of cow urine extract, time of synthesis, and pH on the particle size of combined selenium particles, was explored and enhanced. The participation of the factors in controlling the particle size of reduced SeNPs was quantitatively evaluated by means of analysis of variance (ANOVA). The UV-Vis spectroscopy was utilized to screen the selenium nanoparticle development followed by electron microscopy (SEM and TEM), DLS, AFM, and EDAX. The optimized nanoparticles were subjected to antibacterial activity against Gram-positive and Gram-negative cultures, out of which the highest activity was observed for Klebsiella sp. The biosynthesized and optimized selenium nanoparticles can be further used for various pharmaceutical or industrial applications.

Presence of benznidazole conjugated metabolites in urine identified by β-glucuronidase treatment

Chagas disease is a serious public health problem in Latin America and, due to migration, in other non-endemic regions. Benznidazole (BNZ) is first choice drug in pediatric therapeutics. However, little is known regarding its metabolism in humans. The aim of the study was to isolate and identify products of human BZN metabolism in urine samples obtained from a pediatric Chagas patient and a healthy adult volunteer both treated with BZN. Urine samples were collected after dose of BNZ. Urine was treated with β-glucuronidase followed by an extraction procedure under two different pH conditions and a HPLC/UV and MS/MS identification of BZN and its metabolites. BZN (m/z 260.09847) was identified in all urine extracts. Peaks from each extracted chromatograms were selected for MS and MS/MS identification. Three compounds structurally related to BZN were identified: BZN-Na+ (m/z 283.08009), N-amine-BZN (m/z 230.12307) and N-hydroxi-amine-BZN (m/z 246.11702). BNZ-Na+ was identified in all extracts, but N-amine-BZN and N-hydroxi-amine-BZN were only observed in those extracts treated with β-glucuronidase. This is the first experimental report showing elimination of BZN N-reduced metabolites in urine. As they were released after treatment with β-glucuronidase it can be suggested that glucuronization plays a role in BNZ metabolism and renal elimination.

Streamlined MRM method transfer between instruments assisted with HRMS matching and retention-time prediction

Multiple reaction monitoring (MRM) mode using liquid-chromatography tandem mass spectrometry (e.g., LC-QqQ-MS/MS) has been extensively employed in the small molecule analysis with trace levels in complex samples owing to its high sensitivity. However, most of the reported MRM methods are developed using authentic standards, which are often costly yet not readily available. To address this question, a practical platform for the MRM method transfer between different LC-QqQ-MS/MS instruments, assisted by the high-resolution mass spectrometry (LC-HRMS) and retention time (RT) prediction, has been developed in this study. The reported platform can take advantage of both the high sensitivity of LC-MRM method and ion transition pairs from the previous publications. LC-HRMS can provide the accurate mass measurement of the compounds, though high-quality MS/MS fragments are usually difficult to obtain for chemicals at trace levels. Retention time matching and peaks matching between both instrumental platforms rule out isobaric candidates. With an additional retention time prediction filter from quantitative structure retention relationship (QSRR) model based on random forest feature selection (Pearson r2 = 0.63), identification of small molecules is achieved at a high confidence level without using authentic standards. The developed platform has been validated with robustness by examining spiked environmental chemicals in sludge water samples, biological urine, and cell extracts.

Simultaneous determination of organophosphorus nerve agent markers in urine by IC-MS/MS using anion-exchange solid-phase extraction

In this study a comprehensive approach for determination of low molecular organophosphorus nerve agent markers – highly polar alkylphosphonic acids and much less polar alkyl methylphosphonic acids is presented. Accurate, sensitive and simultaneous determination of the nerve agent markers in human urine was performed by ion chromatography and tandem mass spectrometry using deuterated internal standards. Analysis of the urine extracts was conducted on an anion-exchanger based on poly(styrene-co-divinylbenzene) substrate with a high degree of crosslinking and a covalently-bonded branched functional layer. The use of this type of column allowed achieving high values of retention factors for alkylphosphonic acids and alkyl methylphosphonic acids due to combination of anion-exchange and hydrophobic interactions between the analytes and the stationary phase of the column. Prior to the analysis, the urine samples were purified using anion-exchange cartridges for solid-phase extraction, and high recovery values were achieved for each analyte. The developed IC-MS/MS technique was validated for linearity, limit of detection, limit of quantification, precision and accuracy using two LC-MS/MS instruments. The proposed approach was successfully tested on the urine samples, provided by the Organisation for the Prohibition of Chemical Weapons in the frame of the 4th Biomedical Proficiency Test.

Characterization and tentative identification of new flunitrazepam metabolites in authentic human urine specimens using liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS).

Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat severe insomnia. In our recent study, FNZ metabolic profiles were investigated carefully. Six authentic human urine samples were purified using solid phase extraction (SPE) without enzymatic hydrolysis, and urine extracts were then analyzed by liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS), using the full scan positive ion mode and targeted MS/MS (ddms2) technique to make accurate mass measurements. There were 25 metabolites, including 13 phase I and 12 phase II metabolites, which were detected and tentatively identified by LC-QE-HF-MS. In addition, nine previously unreported phase II glucuronide conjugates and four phase I metabolites are reported here for the first time. Eight metabolic pathways, including N-reduction and O-reduction, N-glucuronidation, O-glucuronidation, mono-hydroxylation and di-hydroxylation, demethylation, acetylation, and combinations, were implicated in this work, and 2-O-reduction together with dihydroxylation were two novel metabolic pathways for FNZ that were identified tentatively. Although 7-amino FNZ is widely considered to be the primary metabolite, a previously unreported metabolites (M12) can also serve as a potential biomarker for FNZ misuse.

Use of ammonium sulfite as a post-column derivatization reagent for rapid detection and quantification of aldehydes by LC-MS

Derivatization is frequently used in liquid chromatography–mass spectrometry (LC-MS) to improve the ionization characteristics of analytes, and facilitate their structural elucidation and quantification. However, the off-line derivatization process usually requires more analysis time. Therefore, the present work develops a novel LC/post-column derivatization (PCD)/MS (LC-PCD-MS) method for rapid detection of aldehydes in complex samples. The PCD method is based on a bisulfite addition reaction with aldehydes that can be easily conducted by mixing the LC elute with an (NH4)2SO3 solution via a tee. The resulting derivatives readily produce diagnostic HSO3− ions under collision induced dissociation (CID) in the negative ion mode of MS, even at an extremely low CID energy such as 1 eV, which can significantly improve the detection selectivity of aldehydes and reduce the occurrence of false positives. The limits of detection (LODs) of hexanal and heptanal were 3 nM and 2 nM, respectively. Moreover, good linearity in the concentration range of 0.01–1 μM for hexanal and heptanal was obtained with the determination coefficient (R2) being greater than 0.996 and the intra- and inter-day relative standard deviations (RSDs) were lower than 13.5% and 17.9%, respectively, indicating the acceptable reproducibility of the method. The LC-PCD-MS method was successfully applied for the rapid quantification of aldehydes in human urine and solvent extracts of traditional Chinese medicines (TCMs). Furthermore, a precursor ion scan strategy based on the diagnostic HSO3− ion was also developed and applied for the preliminary screening of aldehydes in complex samples of cinnamon and lemongrass.

Cypate and Cypate-Glucosamine as Near-Infrared Fluorescent Probes for In Vivo Tumor Imaging.

Near-infrared (NIR) imaging is a promising technique for use as a noninvasive and sensitive diagnostic tool. Although the NIR fluorescently labeled glucose analog glucosamine (cypate-glucosamine) has applications in preclinical imaging, the transport pathways and fate of this probe in tissues remain unaddressed. Here, we have synthesized and characterized cypate and cypate-glucosamine conjugate (cy-2-glu), and investigated the probable transport pathways of these probes in vitro and in vivo. We compared uptake of the probes in the presence and absence of excess d-glucose, "saturated cypate" and palmitic acid in two normal-cancer cell line pairs: lung cancer (A549)-normal (MRC9) and prostate cancer (DU145)-normal (BPH). Breast cancer (MDA-MB-231) and liver cancer (HepG2) cell lines were also examined. Results support use of the glucose transport pathway by cy-2-glu and fatty acid transport pathway by cypate. Mass spectrometry data on the in vitro extracts revealed deamidation of cy-2-glu in prostate and liver cells, suggesting release of glucosamine. In vivo biodistribution studies in mice engrafted with breast tumors showed a distinct accumulation of cy-2-glu in liver and tumors, and to a lesser extent in kidneys and spleen. A negligible accumulation of cypate alone in tumors was observed. Analysis of urine extracts revealed renal excretion of the cy-2-glu probe in the form of free cypate, indicating deamidation of cy-2-glu in tissues. Thus, investigation of the metabolic pathways used by NIR probes such as cy-2-glu advances their use in the detection and monitoring of tumor progression in preclinical animal studies.

DQF J-RES NMR: Suppressing the singlet signals for improving the J-RES spectra from complex mixtures.

Two-dimensional J-RESolved spectroscopy (J-RES) finds routine use in metabolomics for reducing signal overlap as it separates chemical shift and multiplet information along two frequency axes. However, only magnitude mode of the experiment is practical which prevents exploitation of its full resolving power. Tailing from high-intensity metabolite peaks often obscure nearby low-intensity metabolite peaks which leads to ambiguity in assignment of metabolites. Absorptive mode J-RES spectroscopy offers better-resolving power but comes at the cost of either sensitivity or complicated post-processing. Quite often for certain complex mixtures such as bio-fluids some components of the mixture display intense singlet signals which dominate the whole spectrum resulting in less reliable detection of weaker metabolite signals. Multi-frequency presaturation could suppress these intense singlets but will also remove the useful weaker multiplet peaks which are either totally eclipsed with the intense singlets or very close in frequency. We show that by using a double quantum filter (DQF) in magnitude mode J-RES technique, the intensity of the strong singlet metabolite peaks can be reduced relative to the intensity of the sparsely present multiplet metabolite signals. This approach leads to the identification of many weak intensity multiplet peaks which are otherwise undetected due to their overlap with intense singlet peaks in regular J-RES as well as 1D 1H spectra. Although the improved intensity of most of the weaker peaks relative to the strong singlet peaks is observed, some multiplets can disappear due to the delay-dependent modulation of the signals by the DQF. A few DQF J-RES spectra recorded with different DQF delays, therefore, produce better assignment when analyzed together. The technique is demonstrated on a mixture of eight compounds, human urine, and plant extract samples.

Efficacy of organic products against Fusarium oxysporum f. sp. ciceris

Organic formulations viz., cow urine, beejamrit, panchgavya, vermiwash, biosol and composts viz., himcompost, vermicompost, FYM, CPP and NADEP were evaluated against Fusarium oxysporum f. sp. ciceris under in vitro conditions. Among composts, the extracts of CPP (cow pat pit) at 25 per cent concentration yielded maximum mycelial inhibition of 52.2 per cent followed by NADEP with 50.3 per cent. Among organic formulations, extract of cow urine at 10 per cent concentration yielded maximum mycelial inhibition of 72.9 per cent followed by panchgavya with 62.9 per cent. Hence, cow pat pit (CPP) and cow urine at 10 per cent concentrations can be used as soil amendment and seed dresser, respectively for eco-friendly management of Fusarium wilt of chickpea.

Characterization and epitope identification of the T cell response in non-allergic individuals exposed to mouse allergen

BackgroundExposure to airborne allergens is a frequent trigger of respiratory allergy and asthma in atopic individuals. While allergic patients suffer hypersensitivity reactions to these allergens, non-allergic individuals do not exhibit clinical symptoms despite environmental exposure to these ubiquitous allergen sources. The aim of this study was to characterize T cell responses in non-allergic laboratory workers, who are heavily exposed to mice allergens (Exposed Non-Allergics, ENA) and compare this data to previously published T cell responses measured in mouse (MO)-allergic patients. METHODS: Peripheral mononuclear cells (PBMC) from ENA subjects were expanded for 2 weeks in vitro with mouse urine extract and screened for IFNγ and IL-5 cytokine production in response to mouse antigen-derived peptides by ELISPOT. Ex vivo T cell reactivity in the ENA cohort was performed after 6hr stimulation with peptide pools by intracellular staining of CD154. ResultsVigorous responses were detected, associated with 147 epitopes derived from 16 mouse antigens. As expected, responses in ENA subjects were somewhat lower than those observed in MO-allergics for both responder frequency and overall response magnitude. While responses in allergics were polarized towards IL-5 production and associated with low IFNγ production, ENA responses were not polarized. The composition of targeted antigens and epitopes was overall similar between the two cohorts, with the majority of T cell reactivity directed against Mus m 1 and other major urinary proteins. However, kappa-casein precursor and odorant binding protein Ib were more abundantly recognized in MO-allergics compared to ENA subjects. Additionally, T cell responses against oligopeptides derived from the low molecular weight fraction of mouse urine were also assessed. Interestingly, no difference in the response frequency, magnitude or polarization between MO-allergic and ENA individuals was observed. Finally, assessment of ex vivo T cell activation also revealed T cell reactivity in the ENA cohort, with a non-significant trend for lower responses compared to MO-allergics. ConclusionExposure to mouse induces potent T cell responses in non-allergic individuals, targeting similar epitopes as seen in allergic patients.

Effect of Biopesticids in Controlling Leaf Curl Virus of Chilli

The experiment was conducted in the field laboratory of Spices Research Center, BARI, Shibganj, Bogra, Bangladesh during Rabi season of 2014-15 to find out the effect of biopesticides in controlling vector as well as leaf curl virus of chilli. The maximum infected plants (26.85%) were recorded in untreated control and the minimum infected plants (14.75%) were in Malathion (0.2%) which was statistically identical to all other biopesticides except cow urine (1:2). The highest disease reduction (45.13%) over untreated control was obtained from Malathion (0.2%) followed by cow urine (1:1) and neem leaf extract (1:2), and the lowest (26.37%) was obtained from cow milk (1:2). The highest yield (18.33 t/ha) was recorded in Malathion (0.2%) and the lowest (11.28 t/ha) was recorded in untreated control. All the biopesticides resulted least disease incidence and more yield compared to control. Among the biopesticides, cow urine (1:1), cow milk (1:1) and neem extract (1:2) showed comparatively better results against control. So, Melathion or biopesticides, cow urine (1:1), cow milk (1:1) and neem leaf extract (1:2) may be sprayed for controlling vector as well as leaf curl virus and increasing yield of chilli.

Phosphonate-Substituted Ruthenium(II) Bipyridyl Derivative as a Photoelectrochemical Probe for Sensitive and Selective Detection of Mercury(II) in Biofluids.

A ruthenium(II) bipyridyl derivative photoelectrochemical probe, Ru-1, is synthesized and coupled with TiO2 nanoparticles (Ru-1/TiO2) for the specific recognition and highly sensitive photoelectrochemical (PEC) detection of Hg2+ in a series of biofluids. The probe is designed with a chromophore, a thiocyanate recognition unit, a π-conjugated photoelectron-transfer pathway, and a phosphonate anchor. TiO2 nanoparticles with strong affinity to phosphonate and suitable conduction band energy are used as intermediate layers to increase the Ru-1 adsorption amount and amplify the photocurrent response. Under irradiation, the Ru-1/TiO2/fluorine-doped tin oxide (FTO), with strong visible light-harvesting capacity, aqueous stability, and efficient photoelectron transfer, shows a high and stable photocurrent response. In the presence of Hg2+, however, the specific Hg2+ and NCS coordination changes the photophysical properties of Ru-1, imposing the probe with a wider band gap, a weaker absorbance, and a poorer photoelectron and hole separation efficiency, thus resulting in a significant photocurrent decrease. On the basis of the Hg2+-induced photocurrent change, the Ru-1/TiO2/FTO shows good selectivity and high sensitivity toward the PEC detection of Hg2+, with wide linear ranges from 10-12 to 10-7 and 10-7 to 10-3 g/mL, and a low limit of detection of 0.63 pg/mL. The PEC probe is recyclable and accurate for selective detection of Hg2+ in urine, serum, and cell extracts. The whole analysis can be completed within 15 min. These good analytical performances indicate that the PEC method might have great potential for the onsite detection of small molecules in biosystems.

Hexafluoroisopropanol-alkyl carboxylic acid high-density supramolecular solvent based dispersive liquid-liquid microextraction of steroid sex hormones in human urine

A hexafluroisopropanol (HFIP)-alkyl carboxylic acid high-density supramolecular solvent (SUPRAS) was proposed and combined with dispersive liquid-liquid microextraction (DLLME) for extraction and preconcentration of steroid sex hormones in human urine prior to analysis by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Octanoic acid was used as extraction agent and HFIP, with the properties of high density, strong hydrogen bond donor and high hydrophobicity, was used as dispersing agent of DLLME as well as coacervation-inducing agent and density-regulating agent of octanoic acid. HFIP-octanoic acid SUPRAS was spontaneously formed in the process of DLLME and located in the bottom of a urine solution due to its density higher than water. Thus, the SUPRAS phase at trace volume was easy to be collected after centrifugation using normal centrifuge tubes. Five hormones (four estrogens and progesterone) were chosen as analytes. Urine hydrolysis time, extraction conditions and derivatization conditions for estrogens were optimized. Under the optimal conditions, only 0.07 mL of organic solvent (HFIP) was consumed. An external standard calibration method was used for quantification, and a linear relationship was obtained with correlation coefficients higher than 0.9957 and p values for linear trend less than 0.0001. Limits of detection of the method based on a signal-to-noise ratio of 3 were 0.01-0.10 μg L−1. The recoveries ranged from 82.7% to 120.2%, and intra-day and inter-day relative standard deviations of the method were less than 15%. The matrix effects of 84.0%–105.7% showed no urine matrix interferences with MS detection, which was mainly due to the restricted access property of HFIP-octanoic acid SUPRAS with reverse micelle aggregates. The absence of matrix effects contributed to the reliable quantitation by external calibration, avoiding the use of expensive isotopically labeled internal standards. The novel SUPRAS-based DLLME method, coupled with HPLC-MS/MS, was applied for determination of target hormones in real urine.

A new, sensitive and versatile assay for quantitative determination of α-fluoro-β-alanine (AFBA) in human urine by using the reversed-phase ultrahigh performance-tandem mass spectrometry (rp-UHPLC–MS/MS) system

A method for the quantitation of α-fluoro-β-alanine (AFBA), the main metabolite of capecitabine (Cape) and 5-fluoruracil (5-FU), is described. Among antineoplastic drugs (ADs), 5-FU and Cape (the new oral prodrug) are the most commonly applied drugs in cancer therapy.The main objective of this study was to develop a reliable method that would be easy to run on a reversed-phase UHPLC system coupled to tandem mass spectrometry. AFBA was derivatized with Sanger's reagent to ensure complete yield of a stable 2,4 dinitrophenil-α-fluoro-β-alanine derivative. This method was based on the use of a mixed-mode anion exchange solid phase extraction enabling urinary extracts to be clear of endogenous interferences affecting quantitative results. The assay was validated in human urine according to FDA criteria with the use of a labeled internal standard (β-alanine-d4) to minimize experimental error.Good accuracy and precision were demonstrated by determining spiked urine QC samples in four consecutive days. The recovery of AFBA was between 70.0 and 82.6%, with a matrix effect that was 12.8%–18.5%. The lower limit of quantitation (LOQ) was 0.5 ng/mL with a coefficient of variation of 5.3%.This assay was successfully applied to determine the levels of this metabolite in a large number of urine samples taken from personnel who were occupationally exposed to ADs.

Intestinal Metabolism and Bioaccumulation of Sucralose In Adipose Tissue In The Rat

ABSTRACTThe aim of this study was to (1) determine if the organochlorine artificial sweetener sucralose is metabolized in rat intestine with repeated dosing and (2) examine whether sucralose might bioaccumulate in rat adipose tissue. Sucralose was administered to 10 rats by gavage daily for 40 days at an average dosage of 80.4 mg/kg/day. The dosages were within the range utilized in historical toxicology studies submitted for regulatory approval in North America, Europe, and Asia. Feces and urine were collected individually from each animal for every 24-hr period during the 40-day dosing period. Analysis of the urine and fecal extracts by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) revealed two new biotransformation products that have not previously been reported. These two metabolites are both acetylated forms of sucralose that are less polar and hence more lipophilic than sucralose itself. These metabolites were present in urine and feces throughout the sucralose dosing period and still detected at low levels in the urine 11 days after discontinuation of sucralose administration and 6 days after sucralose was no longer detected in the urine or feces. The finding of acetylated sucralose metabolites in urine and feces do not support early metabolism studies, on which regulatory approval was based, that claimed ingested sucralose is excreted unchanged (i.e. not metabolized). The historical metabolic studies apparently failed to detect these metabolites in part because investigators used a methanol fraction from feces for analysis along with thin layer chromatography and a low-resolution linear radioactivity analyzer. Further, sucralose was found in adipose tissue in rats two weeks after cessation of the 40-day feeding period even though this compound had disappeared from the urine and feces. Thus, depuration of sucralose which accumulated in fatty tissue requires an extended period of time after discontinuation of chemical ingestion. These new findings of metabolism of sucralose in the gastrointestinal tract (GIT) and its accumulation in adipose tissue were not part of the original regulatory decision process for this agent and indicate that it now may be time to revisit the safety and regulatory status of this organochlorine artificial sweetener

Preparation and characterization of surface-modified Fe3O4 magnetic nanoparticles for extraction of flutamide in biological samples using HPLC

Flutamide (FLU) loaded onto functionalized magnetite iron oxide nanoparticles were prepared and characterized with scanning electron microscopy, elemental analysis, thermogravimetric analysis, Fourier transform infrared spectroscopy, and energy dispersive spectroscopy techniques. In addition, the effects of parameters of pH, temperature, contact time, and concentration on the adsorption capacity of nanoparticles were evaluated and optimized. The adsorption data were fitted to the Langmuir isotherm and the relevant parameters were inferred. Findings of the study suggested that N-vinylcaprolactam-allylimidazole copolymer coated MNPs are promising carrier for FLU delivery. The solid phase extraction technique was investigated for biological samples. Urine extraction recovery obtained about 91%.

Method selectivity evaluation using the co-feature ratio in LC/MS metabolomics: Comparison of HILIC stationary phase performance for the analysis of plasma, urine and cell extracts

Evaluation of the chromatographic separation in metabolomics studies has primarily been done using preselected sets of standards or by counting the number of detected features. An alternative approach is to calculate each feature’s co-feature ratio, which is a combined selectivity measurement for the separation (i.e. extent of co-elution) and the MS-signal (i.e. adduct formation and in-source fragmentation). The aim of this study was to demonstrate how the selectivity of different HILIC stationary phases can be evaluated using the co-feature ratio approach.The study was based on three sample types; plasma, urine and cell extracts. Samples were analyzed on an UHPLC-ESI-Q-ToF system using an amide, a bare silica and a sulfobetaine stationary phase. For each feature, a co-feature ratio was calculated and used for multivariate analysis of the selectivity differences between the three stationary phases.Unsupervised PCA models indicated that the co-feature ratios were highly dependent on type of stationary phase. For several metabolites a 15–30 fold difference in the co-feature ratio were observed between the stationary phases. Observed selectivity differences related primarily to the retention patterns of unwanted matrix components such as inorganic salts (detected as salt clusters), glycerophospholipids, and polyethylene glycols. These matrix components affected the signal intensity of co-eluting metabolites by interfering with the ionization efficiency and/or their adduct formation. Furthermore, the retention pattern of these matrix components had huge influence on the number of detected features.The co-feature ratio approach has successfully been applied for evaluation of the selectivity performance of three HILIC stationary phases. The co-feature ratio could therefore be used in metabolomics for developing selective methods fit for their purpose, thereby avoiding generic analytical approaches, which are often biased, as type and amount of interfering matrix components are metabolome dependent.

The pharmacokinetics and metabolism of diclofenac in chimeric humanized and murinized FRG mice.

The pharmacokinetics of diclofenac were investigated following single oral doses of 10mg/kg to chimeric liver humanized and murinized FRG and C57BL/6 mice. In addition, the metabolism and excretion were investigated in chimeric liver humanized and murinized FRG mice. Diclofenac reached maximum blood concentrations of 2.43 ± 0.9µg/mL (n = 3) at 0.25h post-dose with an AUCinf of 3.67µgh/mL and an effective half-life of 0.86h (n = 2). In the murinized animals, maximum blood concentrations were determined as 3.86 ± 2.31µg/mL at 0.25h post-dose with an AUCinf of 4.94 ± 2.93µg h/mL and a half-life of 0.52 ± 0.03h (n = 3). In C57BL/6J mice, mean peak blood concentrations of 2.31 ± 0.53µg/mL were seen 0.25h post-dose with a mean AUCinf of 2.10 ± 0.49µgh/mL and a half-life of 0.51 ± 0.49h (n = 3). Analysis of blood indicated only trace quantities of drug-related material in chimeric humanized and murinized FRG mice. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles in humanized mice were different to those of both murinized and wild-type animals, e.g., a higher proportion of the dose was detected in the form of acyl glucuronide metabolites and much reduced amounts as taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57BL/6J mice and humans revealed a greater, though not complete, match between chimeric humanized mice and humans, such that the liver humanized FRG model may represent a model for assessing the biotransformation of such compounds in humans.