Analysis of oxonic acid, uric acid, creatine, allantoin, xanthine and hypoxanthine in poultry litter by reverse phase HPLC

Summary A separation method has been developed to extract organic compounds from poultry manure and litter and subsequently analyze these extracts using reverse phase high-pressure liquid chromatography. Specifically, the method may be used to quantify oxonic acid, allantoin, creatine, uric acid, xanthine and hypoxanthine in poultry manure samples. In a representative sample of fresh poultry manure, oxonic acid, allantoin, creatine, uric acid and xanthine were present at concentrations of 2.4, 2.7, 3.9, 270 and 0.8 mg/g dry manure, respectively. Litter samples stored for eight or sixteen weeks showed the presence of hypoxanthine but no longer contained detectable quantities of creatine. Allantoic acid and urea, both previously shown to be present in the litter samples, could not be distinguished by the chromatographic technique. Orotic acid and uracil, although capable of being separated by the method, were not detected in any poultry litter samples.     

Determination of six indolic compounds, including melatonin, in rat pineal using high-performance liquid chromatography with serial fluorimetric-electrochemical detection.

A reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, N-acetylserotonin, tryptophan, 5-hydroxytryptamine (serotonin) and melatonin in rat pineal using a buffered aqueous eluent containing acetonitrile and methanol as organic modifiers and an ion-pairing agent to assist in controlling the retention of compounds containing an amine group. Serial fluorimetric-electrochemical detection provided additional assurance of compound identity. Analyte preparation simply involved the sonication of pineals in dilute perchloric acid containing an antioxidant and a chelating agent, followed by centrifugation to clarify. The method simplifies the determination of this range of indolic compounds, which normally would require at least two separate runs with different eluents. Detection limits for melatonin were 60 and 135 pg for fluorimetric and electrochemical detection, respectively. (This represented the "worst case" considering the levels and detection limits of all compounds present.) Using flow programming and a flow-rate varying between 1 and 1.5 ml/min, the analysis time was 27.5 min, which made the determination of ten samples in a working day possible.     

Capillary zone electrophoretic determination of phenolic compounds in chess (Bromus inermis L.) plant extracts

A simple CZE method for quantification of phenolic compounds (vanillin, cinnamic, sinapic, chlorogenic, syringic, ferulic, benzoic, p-coumaric, vanillic, p-hydroxybenzoic, rosmarinic, caffeic, gallic and protocatechuic acids) in less than 10 min using 20 mM sodium tetraborate (pH 9.2) with 5% v/v methanol as a BGE and with UV detection at 254 nm is described. The LODs (3 S/N) ranged between 0.02 and 0.12 microg/ mL. Repeatabilities (RSDs) were 0.66-1.8 and 1.56-4.23% for migration times and peak areas (n = 5), respectively. The method was applied to the determination of phenolic compounds in chess (Bromus inermis L.) after Soxhlet extraction and purification of the crude extracts with SPE procedures. The results compared well with those obtained by liquid chromatographic method. B. inermis was found as a suitable model plant containing a broad spectrum of phenolic compounds in easily detectable concentrations and as a potential source of antioxidants.     

Preparation and Properties of a Micro Enzyme Sensor for Creatine

Abstract

A micro enzyme sensor using creatine amidinohydrolase (EC 3. 5. 3. 3) and sarcosine oxidase (EC 1. 5. 3. 1) was developed for measuring creatine. The sensor consisted of platinum and silver wires fixed in a glass tubing with epoxy resin adhesive, and creatine amidinohydrolase and sarcosine oxidase immobilized on their tip by cross-linking method. The calibration curve for measurement of creatine, the effects of pH, temperature, the amount of flavin adenine dinucleotide and compounds on the sensitivity, and the life-time of the sensor were examined. Under optimal conditions, the response of the sensor was linear in the concontration range of 10 μ M ～ 0.65 mM creatine.     

Anthocyanins HPLC-DAD and MS Characterization,Total Phenolics,and Antioxidant Activity of Some Berries Extracts

Extracts of indigenous wild blackberries, mulberries, bilberries, and blackthorns were analyzed for anthocyanin composition, anthocyanin content, total phenolics, and antioxidant capacity. Anthocyanins extraction with acidified methanol in ultrasonic condition (59 kHz, 60 min., 25°C) was carried out. The extracts were analyzed by high-performance liquid chromatography (HPLC) using a Dionex Ultimate 3000 apparatus equipped with photodiode array detector for qualitative characterization of the anthocyanins. The chromatograms revealed the presence of a large number of anthocyanins in fruits extracts: blackberries, 4 compounds; mulberries, 3 compounds; bilberries, 18 compounds; and blackthorns, 5 compounds. The most abundant anthocyanins were cyanidin-3-glucoside in blackberry, mulberry, and bilberry, and cyanidin-3-rutinoside in blackthorn extract. Structural information about anthocyanins was obtained by using a mass spectrometric method based on fully automated chip-nanoelectrospray ionization (nanoESI) high capacity ion trap (HCT). Anthocyanin content was quantified by the pH differential method and total phenolics were determined by Folin-Ciocalteu method. A Jasco V 530 UV-VIS spectrophotometer was used for absorbance measurements. The free radical scavenging activity of the berries extracts was performed by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The reduction of DPPH was followed by a spectrophotometric method. Also, a correlation of the antioxidant capacities of the extracts with their anthocyanin content and total phenolics was attempted.     

Separation and measurement of clofibroyl coenzyme A and clofibric acid in rat liver after clofibrate adminstration by reversed-phase high-performance liquid chromatography with photodiode array detection

A method to identify and quantitate clofibric acid and clofibroyl coenzyme A (CoA) products in rat liver was developed using reversed-phase high-performance liquid chromatography. The system was developed with baseline separation of clofibroyl-CoA from clofibric acid using isocratic elution, with a mobile phase consisting of 52% methanol and 28 mM potassium phosphate buffer (pH 4.2). With this high methanol concentration, the large amount of UV-absorbing materials present in the liver extracts were eluted earlier than the investigated compounds. Clofibroyl-CoA has a characteristic absorbance spectrum with distinct peaks at 260 and 230 nm, while clofibric acid showed only a distinct peak at 230 nm. Using an on-line photodiode array detector, the spectra could be recorded during analysis without interrupting the flow of the mobile phase. This spectral analysis identification possibilities and evaluation of the purity of the chromatographic peaks. In a perchloric extract of rat liver, the recovery of clofibric acid and clofibroyl-CoA added to the liver extract ranged from 70 to 80%. A linear relationship was observed between clofibric acid and clofibroyl-CoA concentration and the area of their peaks in the chromatogram. The detection limit of the method was lower than 5 pmol for both compounds when the absorbance was recorded at 230 nm. The method could be used without modification for the estimation of clofibroyl-CoA and clofibric acid in biological extracts.     

Liquid chromatography/electrospray ionization tandem mass spectrometry profiling of compounds from the infusion of Byrsonima fagifolia Niedenzu

A rapid analytical approach suitable to achieve a comprehensive characterization of the compounds present in the infusion prepared from the leaves of Byrsonima fagifolia Niedenzu (Malpighiaceae), a Brazilian plant used as an infusion to treat gastric disorders, was developed. The method was based on high-performance liquid chromatography coupled to electrospray negative ionisation multistage ion trap mass spectrometry (HPLC/ESI-ITMSn).The main ions in the ESI-ITMS spectra were attributed to a quinic acid core containing from one to five galloyl units. Quercetin derivatives containing one and two sugar moieties as well as galloyl esterification were also detected. These results indicated that HPLC/ESI-ITMSn is easily applicable to infusions of this plant and allows the rapid and direct identification of these compounds in crude plant extracts.     

Determination of creatine and phosphocreatine in muscle biopsy samples by capillary electrophoresis with contactless conductivity detection

A capillary electrophoresis method with contactless conductivity detection was evaluated as a new approach for quantification of creatine and phosphocreatine in human quadriceps femoris biopsy samples. The running buffers employed consisted of 1 M acetic acid at a pH of 2.3 for the determination of creatine and 50 mM 3-(N-morpholino)propanesulfonic acid/30 mM histidine at a pH of 6.4 for the determination of phosphocreatine in the centrifuged muscle extracts. The limits of detection for creatine and phosphocreatine were found to be 2.5 and 1.0 μM, respectively. Creatine and phosphocreatine were determined in six human muscle biopsy samples and the results were found comparable to those of a standard enzymatic assay. The procedures developed for creatine and phosphocreatine also allow the determination of creatinine as well as adenosine diphosphate and adenosine triphosphate.     

Bioactive compounds, RP-HPLC analysis of phenolics, and antioxidant activity of some Portuguese shrub species extracts

In the ecosystem of Serra Da Estrela, some plant species have the potential to be used as raw material for extraction of bioactive products. The goal of this work was to determine the phenolic, flavonoid, tannin and alkaloid contents of the methanolic extracts of some shrubs (Echinospartum ibericum, Pterospartum tridentatum, Juniperus communis, Ruscus aculeatus, Rubus ulmifolius, Hakea sericea, Cytisus multiflorus, Crataegus monogyna, Erica arborea and Ipomoea acuminata), and then to correlate the phenolic compounds and flavonoids with the antioxidant activity of each extract. The Folin-Ciocalteu's method was used for the determination of total phenols, and tannins were then precipitated with polyvinylpolypyrrolidone (PVPP); a colorimetric method with aluminum chloride was used for the determination of flavonoids, and a Dragendorff's reagent method was used for total alkaloid estimation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and beta-carotene bleaching tests were used to assess the antioxidant activity of extracts. The identification of phenolic compounds present in extracts was performed using RP-HPLC. A positive linear correlation between antioxidant activity index and total phenolic content of methanolic extracts was observed. The RP-HPLC procedure showed that the most common compounds were ferulic and ellagic acids and quercetin. Most of the studied shrubs have significant antioxidant properties that are probably due to the existence of phenolic compounds in the extracts. It is noteworthy to emphasize that for Echinospartum ibericum, Hakea sericea and Ipomoea acuminata, to the best of our knowledge, no phytochemical studies have been undertaken nor their use in traditional medicine been described.     

Pharmacokinetics,hepatic disposition,and heart tissue distribution of 14 compounds in rat after oral administration of Qi-Li-Qiang-Xin capsule via ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry

In the present study, a specific and sensitive approach using ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry was developed and validated for the quantitative analysis of 14 constituents in rat plasma, liver, and heart. The method was fully validated and successfully applied to pharmacokinetic, hepatic disposition, and heart tissue distribution studies of 14 compounds after the oral administration of Qi-Li-Qiang-Xin capsule. Ginsenoside Rb1, alisol A, astragaloside IV, and periplocymarin were found to be highly exposed in rat plasma, while toxic components such as hypaconitine, mesaconitine, and periplocin had low circulation levels in vivo. Moreover, sinapine thiocyanate, neoline, formononetin, calycosin, and alisol A exhibited significant liver first-pass effects. Notably, high levels of alisol A, periplocymarin, benzoylmesaconine, and benzoylhypaconine were observed in the heart. Based on high exposure and appropriate pharmacokinetic features in the systemic plasma and heart, astragaloside IV, ginsenoside Rb1, periplocymarin, benzoylmesaconine, benzoylhypaconine, and alisol A can be considered as the main potentially effective components. Ultimately, the results provide relevant information for discovery of effective substances, as well as further anti-heart failure action mechanism investigations of Qi-Li-Qiang-Xin capsule.     

High-performance liquid chromatographic detection of hydroxylated benzoic acids as an indirect measure of hydroxyl radical in heart: its possible link with the myocardial reperfusion injury.

The present report describes a method suitable for the indirect assay of hydroxyl radical (OH.), which is likely to be produced during reperfusion of ischemic myocardium. Isolated rat heart perfused by the Langendorff technique was subjected to 30 min of ischemia, followed by 30 min of reperfusion. Salicylic acid (2 mM) was added to the perfusion circuit to trap any OH. radical generated during the experiment. 2,5- and 2,3-dihydroxybenzoic acids (hydroxylated products of salicylic acid) were identified by authentic standards as well as by pure OH.-generating system using high-performance liquid chromatography with electrochemical detection. In addition to serving as a chemical trap for the detection of OH., salicylate attenuated myocardial reperfusion injury as evidenced by reduced formation of creatine kinase, decreased lipid peroxidation, and improved myocardial contractile functions during reperfusion. These results thus provide direct evidence for the presence of OH. in heart and link it to the myocardial reperfusion injury.     

Optimization strategy and validation of one chromatographic method as approach to determine the phenolic compounds from different sources

We have designed a novel working strategy to optimize a unique chromatographic method consisting of diode array detection for the analysis of the most representative phenolic compounds from different food sources. The simultaneous inclusion of standard phenolic compounds, phenolic compounds isolated from food sources and representative real extracts as an ultimate test in analysis has allowed to establish, for the first time, a unique liquid gradient to serve as an excellent medium for the investigation of phenolics in samples from different food sources. Under the optimized conditions, 21 commercially available phenolic compounds and 25 commercially unavailable phenolic structures were analyzed in less than 30 min. The chromatographic method was designed as an alternative for the provisional identification of these compounds before their full characterization. The optimized chromatographic method was carefully validated for precision and accuracy. A high reproducibility in the retention time (<2%), peak area and calibration slope (<5%) as well as recoveries higher than 95% were obtained in all cases. Consequently, the currently described method was successfully employed to study the phenolic compounds in the most representative food samples.     

5-Dodecanolide,a Compound Isolated from Pig Lard,Presents Powerful Anti-Inflammatory Properties

Background: Pork lard (PL) is traditionally used as an anti-inflammatory agent. We propose to demonstrate the anti-inflammatory properties of PL, and elucidate which compounds could be responsible for the anti-inflammatory effects. Methods: The anti-inflammatory effects of PL were tested in a rat model of zymosan-induced hind paw inflammation. Further, the hydroalcoholic extract from PL was obtained, the composition analyzed, and the anti-inflammatory activity of the extracts and isolated components assayed using immune cells stimulated with lipopolysaccharide (LPS). Results: Applying the ointment on the inflamed rat feet reduced the foot diameter, foot weight, and activities of antioxidant enzymes and inflammatory markers of circulating neutrophils. The main components of the hydroalcoholic extract were 5-dodecanolide, oleamide, hexadecanoic acid, 9-octadecenoic acid, hexadecanamide, and resolvin D1. Conclusions: PL reduces the immune response in an animal model stimulated with zymosan. Hydroalcoholic PL extract and its components (5-Dodecanolide, Oleamide, and Resolvin D1) exerted an anti-inflammatory effect on LPS-stimulated neutrophils and peripheral mononuclear cells reducing the capability to produce TNFα, as well as the activities of antioxidant and pro-inflammatory enzymes. These effects are attributable to 5-dodecanolide, although the effects of this compound alone do not reach the magnitude of the anti-inflammatory effects observed by the complete hydroalcoholic extract.     

Microwave-assisted synthesis and anti-inflammatory activity evaluation of some novel α-aminophosphonates

An expeditious green synthetic approach was developed for the synthesis of α-aminophosphonates in good yields through one-pot three component reaction (Kabachnik-Fields reaction) of equimolar quantities of N-(4-amino-2-phenoxy phenyl)methanesulfonamide, diethylphosphite and various aldehydes under conventional as well as microwave irradiation methods. The newly synthesized compounds were characterized by NMR (³¹P, ¹H, and ¹³C), Mass, IR and C, H, N analyses. The synthesized compounds were screened for their anti-inflammatory activity using rat paw edema method. Most of the compounds from the series showed good anti-inflammatory activity when compared with standard drug. Especially the compounds 5d bearing 4-hydroxy-3-nitrophenyl moiety, 5e bearing 3-bromo-4-fluorophenyl moiety, 5g incorporated with 2,4-dichlorophenyl moiety and 5f containing 4-chlorophenyl moiety exhibiting edema inhibition of 91.01% to 85.39% after 4 h of carrageenan injection while the other compounds displayed inhibition ≥75%.     

Mass fragmentographic determination of 5-hydroxytryptamine and 5-hydroxyindole-3-acetic acid in brain tissue using deuterated internal standards.

A mass fragmentographic method for the determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in the same extract of rat brain tissue is decribed. Deuterium-labelled analogues were used as internal standards. 5-HT and 5-HIAA were separated by solvent extraction and pentafluoropropionyl derivatives were prepared for the mass fragmentographic analysis. Multiple ion analysis confirmed the identity of 5-HT and 5-HIAA in the rat brain. At the mass numbers used routinely for the determination of 5-HT and 5-HIAA in the rat brain. At the mass numbers used routinely for the determination of 5-HT and 5-HIAA, the experimental error was below 3% (calculated from mean values of 0.05 and 0.24 nmole, respectively). The recovery of the authentic compounds added to brain extracts was more than 95%. The levels of 5-HT and 5-HIAA in the rat brain were 2.95 +/- 0.16 and 0.64 +/- 0.18 nmole/g, respectively. More than 100 samples could be analyzed within 3 days. The presence of 5-hydroxytryptophol in rat brain was also investigated, but none could be detected either as a conjugate or as the free alcohol.     

Antimicrobial,DPPH scavenging and tyrosinase inhibitory activities of Thymus vulgaris,Helichrysum arenarium and Rosa damascena mill. ethanol extracts by using TLC bioautography and chemical screening methods

Antimicrobial, DPPH scavenging and tyrosinase inhibitory activities of Thymus vulgaris, Helichrysum arenarium and Rosa damascena Mill. ethanol extracts by using TLC bioautography and chemical screening methods. The ethanol extracts of Thymus vulgaris (Tv), Helichrysum arenarium (Ha) and Rosa damascena Mill. (Rm) (red) were screened for their antimicrobial, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and tyrosinase inhibitory activities. The test microorganisms included bacteria of Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). Thin Layer Chromatography (TLC) - bioautography, disk diffusion and well diffusion methods were used for the antimicrobial activity assays. Rosa damascena Mill. extract was effective against E. coli and all plant extracts showed antimicrobial activity against S. aureus. The phenolic acids in the structure of the extracts were also identified by LC-MS analysis. Human blood agar well diffusion method and TLC-DPPH assays were used to identify the hemolytic and antioxidant activity of plant extracts, respectively, along with 10 compounds including phenolic acids as a standard. Among these compounds, caffeic acid (Rf = 0.68) was detected in all extracts while vanillic acid (Rf = 0.75), and gallic acid (Rf = 0.51) was found in Tv extract. Kojic acid (Rf = 0.36), on the other hand, was detected in Rm extract as a tyrosinase inhibitor. All plant extracts presented tyrosinase inhibitory activities on TLC-bioautography assay.     

Separation and characterization of antioxidants from Spirulina platensis microalga combining pressurized liquid extraction, TLC, and HPLC-DAD

A new procedure has been developed to separate and characterize antioxidant compounds from Spirulina platensis microalga based on the combination of pressurized liquid extraction (PLE) and different chromatographic procedures, such as TLC, at preparative scale, and HPLC with a diode array detector (DAD). Different solvents were tested for PLE extraction of antioxidants from S. platensis microalga. An optimized PLE process using ethanol (generally recognized as safe, GRAS) as extraction solvent has been obtained that provides natural extracts with high yields and good antioxidant properties. TLC analysis of this ethanolic extract obtained at 115 degrees C for 15 min was carried out and the silica layer was stained with a DPPH (diphenyl-pycril-hydrazyl) radical solution to determine the antioxidant activity of different chromatographic bands. Next, these colored bands were collected for their subsequent analysis by HPLC-DAD, revealing that the compounds with the most important antioxidant activity present in Spirulina extracts were carotenoids, as well as phenolic compounds and degradation products of chlorophylls.     

Simultaneous determination of ginkgolides A,B, C and bilobalide by LC‐MS/MS and its application to a pharmacokinetic study in rats

The study of pharmacokinetics of Ginkgo biloba extracts in Traditional Chinese Medicine was relatively recent. In this study, a simple, quick and sensitive LC‐MS/MS analytical method was developed for the determination of ginkgolides A, B, C and bilobalide in rat plasma. The analytes were completely separated from the endogenous compounds on an Agilent Zorbax Eclipse plus C₁₈ column (50 mm × 3.0 mm, 1.8 µm) using an isocratic elution. The single‐run analysis time was as short as 5.0 min. Sample preparation for protein removal was accomplished used a simple methanol precipitation method, after SPE showing a simultaneous extraction and cleanup of extracts allowing for a direct analysis. Extraction recoveries in rat plasma for ginkgolides A, B, C and bilobalide ranged from 75.6% to 89.0%. The calibration curves were determined over the ranges 0.5–20,000 ng/mL for ginkgolides A, B, C and bilobalide respectively. The lower limits of quantification (LLOQ) of the analytes were 0.5 ng/mL. Inter‐day and intra‐day precision and accuracy were below 15% and between 85 and 115%, respectively. Finally, the developed method was successfully applied to a pharmacokinetic study following oral administration of the Ginkgo biloba extracts to the male ICR rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Extraction,Encapsulation into Lipid Vesicular Systems,and Biological Activity of Rosa canina L. Bioactive Compounds for Dermocosmetic Use

Valorization of wild plants to obtain botanical ingredients could be a strategy for sustainable production of cosmetics. This study aimed to select the rosehip extract containing the greatest amounts of bioactive compounds and to encapsulate it in vesicular systems capable of protecting their own antioxidant activity. Chemical analysis of Rosa canina L. extracts was performed by LC-DAD-MS/MS and ¹H-NMR and vitamins, phenolic compounds, sugars, and organic acids were detected as the main compounds of the extracts. Liposomes, prepared by the film hydration method, together with hyalurosomes and ethosomes, obtained by the ethanol injection method, were characterized in terms of vesicle size, polydispersity index, entrapment efficiency, zeta potential, in vitro release and biocompatibility on WS1 fibroblasts. Among all types of vesicular systems, ethosomes proved to be the most promising nanocarriers showing nanometric size (196 ± 1 nm), narrow polydispersity (0.20 ± 0.02), good entrapment efficiency (92.30 ± 0.02%), and negative zeta potential (−37.36 ± 0.55 mV). Moreover, ethosomes showed good stability over time, a slow release of polyphenols compared with free extract, and they were not cytotoxic. In conclusion, ethosomes could be innovative carriers for the encapsulation of rosehip extract.     

Two new chroman derivations from the endophytic Penicillium sp. DCS523

Strain DCS523 was isolated from the branch tissue of Daphniphyllum longeracemosum and determined to be a Penicillium sp. according to the ITS sequence analysis. The extracts from the PDA solid fermentation media of Penicillium sp. DCS523 were purified to give two new chroman derivatives as well as six known compounds. Based on their spectral data the new compounds were identified as (Z)-6-acetyl- 3-(1,2-dihydroxypropylidene)-5-hydroxy-8-methylchroman-2-one and 6-acetyl-2α,5- dihydroxy-2-(2-hydroxypropyl)- 3α,8-dimethylchroman, respectively.