Phytochemical profile of petals from black Dahlia pinnata by flow injection analysis-electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry.

INTRODUCTION: Dahlia pinnata Cav. is a flower native to Mexico that has many applications; in particular, its petals have been used for ornamental, food, and medicinal purposes, for example to treat skin rashes and skin cracks. It has been reported that the medicinal properties of plants are generally related to the phytochemical constituents they possess. However, there are few studies on black D. pinnata. OBJECTIVES: The present study was aimed at qualitatively and quantitatively determining the phytochemical profile of petals from black D. pinnata. METHODOLOGY: Phytochemicals from Dahlia petals were extracted by consecutive maceration (hexane, dichloromethane, and methanol); then, the extracts were analyzed through colorimetric assays and UV-Vis spectroscopy for qualitative identification and quantification of phytochemical compounds, respectively. The methanolic extract was analyzed by flow injection analysis-electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (FIA-ESI-FTICR-MS) in negative and positive mode. RESULTS: Quantitative phytochemical profiling of the methanolic extract by UV-Vis spectroscopy indicated high contents of phenolic compounds (34.35 ± 3.59 mg EQ/g plant) and sugars (23.91 ± 1.99 mg EQ/g plant), while the qualitative profiling by FIA-ESI-FTICR-MS allowed the tentative identification of several flavonoids and phenolic acids. Kaempferol-3-rutinoside, pelargonidin-3-(6″-malonylglucoside)-5-glucoside, rutin, kaempferol-3-(2″,3″-diacetyl-4″-p-coumaroylrhamnoside), and myricetin-3-(2‴-galloylrhamnoside) were the main compounds detected. CONCLUSION: The results expand our knowledge of the phytochemical constituents of petals from black D. pinnata.

Fragmentation of Cannabinoids by Flow Injection Analysis Tandem Mass Spectrometry (FIA-MS/MS).

BACKGROUND: The analysis of plant material from Cannabis sativa L. has long been targeted on its main psychologically active metabolite, Δ9-tetrahydrocannabinol (THC). In addition to the diverse plant composition and medicinal interest in several cannabinoids, these compounds may also be related to the different characteristics of samples sold illegally. Currently, it is indisputable that other cannabinoids should also be considered in cannabis assays. Mass spectrometry has been used to identify and characterize substances in the most different scenarios, and knowing the analyte fragmentation profile is essential for characterizing samples of diverse origin. OBJECTIVE: In this work, flow injection analysis-tandem mass spectrometry with electrospray ionization (FIA-ESI-MS/MS) in positive and negative modes was used to evaluate the fragmentation profiles of eight cannabinoids commonly found in cannabis samples: THC, tetrahydrocannabinolic acid, Δ8-tetrahydrocannabinol, cannabidiol, cannabidiolic acid, cannabigerol, cannabigerolic acid and cannabinol. METHODS: By exploring the fragmentation data from mass spectrometry, the samples were classified using a chemometric model of partial least squares discriminant analysis (PLS-DA). RESULTS: When ESI in negative mode is used with adequate collision energies, it is possible to identify differences in the fragmentation of isomers. Based on that, chemometric tools were employed to classify different samples. The PLS-DA applied to FIA-ESI-MS/MS data yielded satisfactory classification. CONCLUSION: Thus, the results presented can be applied as a preliminary tool in the analysis of unknown samples, guiding more accurate investigations in terms of chemical composition. HIGHLIGHTS: This study of the cannabinoid fragmentation pattern by flow injection MS showed that cannabinoids can be distinguished by their fragmentation spectra after negative electrospray ionization. Multivariate data analysis (PLS-DA) allowed classification of different cannabis samples.

Selenium inorganic speciation in beers using MSFIA-HG-AFS system after multivariate optimization.

In this work, the use of a multisyringe flow injection analysis coupled to hydride generation atomic fluorescence spectrometry (MSFIA-HG-AFS) for inorganic selenium chemical speciation was proposed. A Doehlert design was applied to optimize the experimental conditions for hydride generation (NaBH4 and HCl concentrations). The limits of quantification (LoQ) obtained were 0.07 µg L-1, for total inorganic Se, and 0.08 µg L-1, for Se(IV). Accuracy and precision of the proposed analytical method were evaluated through analysis of standard reference material and addition and recovery tests. The optimized method was applied to analyses of eight samples of beer, produced in Spain, obtaining concentrations for Se(IV) (<0.08 - 0.46 ± 0.01 µg L-1), total inorganic Se (0.47 ± 0.01 - 3.04 ± 0.62 µg L-1) and Se(VI) (0.06 ± 0.01 - 3.00 ± 0.59 µg L-1). The proposed analytical method was accurate, precise and sensitivity for determination of selenium species in beer samples.

Automatic multicommuted flow systems applied in sample treatment for radionuclide determination in biological and environmental analysis.

The presence of artificial and natural radioactivity in the environment is currently a topic of great relevance and ecological interest, even in human health issue, due to the increase of different anthropogenic activities. The use of multicommuted flow analysis techniques (e.g. Multi-Syringe Flow Injection Analysis - MSFIA, Lab-On-Valve - LOV and Lab-In-Syringe - LIS) has allowed the automation of radiochemical procedures to separate and preconcentrate radionuclides in environmental and biological samples. In comparison with the manual approach commonly used in routine analysis for radioactivity monitoring, the automation has enabled the development of highly reproducible methodologies with a great analysis frequency. Moreover, during the analytical procedure, the intervention of the analyst is drastically reduced, minimizing the radiological risk. The automation also offers significant advantages such as minimum consumption of time and reagents, reducing the cost and the generation of waste, contributing to the green chemistry. In this review, several multicommuted flow analysis techniques (MSFIA, LOV and LIS) reported in the last decade applied for the development of automatic sample treatment methodologies, used to separate, preconcentrate and quantify 90Sr, 99Tc, natural U and 226Ra in biological and environmental samples are described and critically compared.

Determination of total and bioavailable As and Sb in children's paints using the MSFIA system coupled to HG-AFS.

In this study, the use of hydride generation atomic fluorescence spectrometry (HG-AFS) coupled with the multi-syringe flow injection analysis (MSFIA) has been proposed in the application of the Doehlert design to optimise the determination of As and Sb in gouache and tempera children's paints. The determination of the total and bioavailable As and Sb in paint samples from various brands and colours was also investigated. The limits of quantification (LOQ) obtained for the determination of As and Sb were 14.0 and 8.6 ng g-1, respectively. The accuracy and precision of the method were evaluated through recovery tests (by the analyte addition method) at three levels for both elements, and by the analysis of certified reference materials of clay (CRM 052, Loamy Clay 1) and river water (SLRS-4). Twenty paint samples, manufactured in China, Italy, Spain and Brazil, were analysed. The concentrations of As varied between below LOQ (<14 ng g-1) and 136.0 ± 1.1 ng g-1 (average value of 101.0 ng g-1, n = 10), and Sb between below LOQ (<8.6 ng g-1) and 74.0 ± 5.4 ng g-1 (average value of 21.7 ng g-1, n = 17). The children's paint samples presented As and Sb concentrations that were below the maximum values established by the National Institute of Metrology, Quality and Technology (INMETRO), as well as by the European legislation Directive 2009/48/EC. Based on the obtained results for the total As and Sb concentrations, eight samples were selected to evaluate the migration or bioavailability of As and Sb after solubilisation in HCl solution. The obtained results showed that for the eight analysed samples, the concentration of both elements after solubilisation in HCl solution were below the LOQ (<16.2 ng g-1 for As and <7.1 ng g-1 for Sb). These values correspond to the limits of analytical concentrations of As and Sb established by INMETRO and the Brazilian Association of Technical Standards (ABNT). The determined concentrations of As and Sb guarantee the safety (with regards to these elements) for children when using the analysed paint samples since they do not cause any health risk. The analytical method for the determination of the total and bioavailable As and Sb in children's paints was efficient, accurate and precise.

Direct analysis of ascorbic acid in food beverage samples by flow injection analysis using reduced graphene oxide sensor.

In this paper, a simple, sensitive and precise electroanalytical method was developed using flow injection analysis (FIA) with amperometric detection and reduced graphene oxide sensor for ascorbic acid determination in samples of multivitamin beverages, milk, fermented milk, and milk chocolate. The advantages of this sensor include a potential displacement of 450 mV and a 2-fold peak current increase for electrochemical oxidation of ascorbic acid, which resulted in a highly sensitive method. No interference of sample matrix was observed, avoiding solvent extraction procedures (samples were only diluted). The FIA allowed a high analytical frequency, approximately 96 injections per hour, together with adequate detection limit of 4.7 µmol L-1. Good precision (RSD < 7%) and accuracy (recoveries between 91 and 108%) evidenced the robustness of the method. The method was compared with ultra-fast liquid chromatography (UFLC) obtaining statistically similar results (95% confidence level). The ascorbic acid content in samples varied from 0.065 to 2.53 mmol L-1.

Rapid Analysis in Continuous-Flow Electrochemical Paper-Based Analytical Devices.

A simple and low-cost continuous-flow (CF) electrochemical paper-based analytical device (ePAD) coupled with thermoplastic electrodes (TPEs) was developed. The fast, continuous flow combined with flow injection analysis was made possible by adding two inlet reservoirs to the same paper-based hollow channel flowing over detection electrodes, terminating in a fan-shaped pumping reservoir. The upstream inlet reservoir was filled with buffer and provided constant flow through the device. Sample injections were performed by adding 2 µL of the sample to the downstream sample inlet. Differences in flow resistance resulted in sample plugs displacing buffer as the solution flowed over the working electrodes. The electrodes were fabricated by mixing carbon black and polycaprolactone (50% w/w). CF-TPE-ePADs were characterized with chronoamperometry using ferrocenylmethyl trimethylammonium as the electrochemical probe. Optimized flow rates and injection volumes gave analysis times roughly an order of magnitude faster than those of previously reported flow injection analysis ePADs. To demonstrate applicability, the CF-TPE-ePADs were used to quantify caffeic acid in three different tea samples. The proposed method had a linear range from 10 to 500 µmol L-1 and limits of detection and quantification of 2.5 and 8.3 µmol L-1, respectively. Our approach is promising for fabricating simple, inexpensive, yet high-performance, flow injection analysis devices using paper substrates and easy-to-make electrodes that do not require external mechanical pumping systems or complicated valves.

Fast-response flow-based method for evaluating 131I from biological and hospital waste samples exploiting liquid scintillation detection.

This paper presents a fast and automatic flow-based method to extract 131I from biological samples and hospital waste, previous to liquid scintillation detection. 131I is a radionuclide extensively used in Nuclear Medicine due to their beta and gamma disintegrations, whereby hospitals have to manage the associated waste generation. The automatic developed system is based on Lab-On-Valve (LOV) flow-technique exploiting Cl-resin (135 mg per extraction). This methodology allows performing sample extractions and measurements on the same day, since the extraction frequency takes 1.4-4 h-1, depending on the analysed sample volume, plus up to 2 h of measurement for each vial. 131I is retained as iodine ion and eluted with sodium sulphide 0.2 mol L-1. The maximum sample volume that can be preconcentrated is 20 mL, reaching an extraction efficiency of 85 ±â€¯5%. The minimum detectable activity (MDA) is 0.05 Bq, showing a precision of 7% RSD (n = 5). Both, biological samples (urine and saliva) and hospital waste samples can be satisfactorily analysed by the proposed system, obtaining recoveries between 90 and 110%. The developed method is then suitable to implement in hospitals, improving the surveillance of the 131I environmental release.

Development of new analytical method for preconcentration/speciation of inorganic antimony in bottled mineral water using FIA-HG AAS system and SiO2/Al2O3/SnO2 ternary oxide.

In the present paper, a new analytical preconcentration/speciation method for antimony species determination in bottled mineral water samples using the SiO2/Al2O3/SnO2 adsorbent was developed. The method is based on selective adsorption of Sb(III) ions by SiO2/Al2O3/SnO2 under a wide pH range (2.5-7.5). Total antimony was determined with previous sample treatment using 0.1% (w/v) l-cysteine and the concentration of Sb(V) species was determined by the difference between total and Sb(III). The proposed method provided an analytical curve ranging from 0.50 to 5.00 µg L-1 (r = 0.999), limit of detection (LD) of 0.17 µg L-1 and preconcentration factor (PF) of 136-fold. The method exhibited tolerance to different metal ions and the accuracy was attested from addition and recovery tests (95.2-106.0%) in bottled mineral water samples using 2.0% (w/v) l-cysteine, as well as by analysis of certified material. Only Sb(III) species were determined in mineral water (0.54-1.04 µg L-1).

Increased Clinical Sensitivity and Specificity of Plasma Protein N-Glycan Profiling for Diagnosing Congenital Disorders of Glycosylation by Use of Flow Injection-Electrospray Ionization-Quadrupole Time-of-Flight Mass Spectrometry.

BACKGROUND: Congenital disorders of glycosylation (CDG) represent 1 of the largest groups of metabolic disorders with >130 subtypes identified to date. The majority of CDG subtypes are disorders of N-linked glycosylation, in which carbohydrate residues, namely, N-glycans, are posttranslationally linked to asparagine molecules in peptides. To improve the diagnostic capability for CDG, we developed and validated a plasma N-glycan assay using flow injection-electrospray ionization-quadrupole time-of-flight mass spectrometry. METHODS: After PNGase F digestion of plasma glycoproteins, N-glycans were linked to a quinolone using a transient amine group at the reducing end, isolated by a hydrophilic interaction chromatography column, and then identified by accurate mass and quantified using a stable isotope-labeled glycopeptide as the internal standard. RESULTS: This assay differed from other N-glycan profiling methods because it was free of any contamination from circulating free glycans and was semiquantitative. The low end of the detection range tested was at 63 nmol/L for disialo-biantennary N-glycan. The majority of N-glycans in normal plasma had <1% abundance. Abnormal N-glycan profiles from 19 patients with known diagnoses of 11 different CDG subtypes were generated, some of which had previously been reported to have normal N-linked protein glycosylation by carbohydrate-deficient transferrin analysis. CONCLUSIONS: The clinical specificity and sensitivity of N-glycan analysis was much improved with this method. Additional CDGs can be diagnosed that would be missed by carbohydrate-deficient transferrin analysis. The assay provides novel biomarkers with diagnostic and potentially therapeutic significance.

Poly glycidyl methacrylate-co-ethylene dimethacrylate porous monolith as a versatile platform for the development of separations and solid-phase extractions in sequential injection analyzers.

We demonstrated that the porous structure and the reactivity of the epoxy group in the poly glycidyl methacrylate-co-ethylene dimethacrylate monolith can be a platform for the development of separation and extraction methods based on sequential injection analysis. The epoxy group was functionalized to produce monoliths affording complexing and ion exchange properties. Derivatization with iminodiacetate and sodium sulfite produced weak and strong cation exchangers, respectively. Derivatization with ethylenediamine produced a weak anion exchanger, and the treatment of the ethylenediamine-modified monolith with chloroacetate produced another weak cation exchanger. All the monoliths also worked as chelating sorbents. The columns were prepared inside 50 × 2.01 mm id fused-silica lined stainless steel tubing and exhibited permeabilities between 0.76 and 4.92 × 10-13 m2 , which enabled the application of flow rates between 5 and 15 µL/s by the syringe pumps used in sequential injection analyzers. These columns separated proteins by cation or anion exchange in a sequential injection chromatograph in both synthetic mixtures and in egg white. Additionally, the online solid-phase extraction of copper ions was demonstrated in a sequential injection analyzer with the same columns. Postcolumn derivatization with ethylenediamine and spectrophotometric detection was used for the copper detection.

3D printing for electroanalysis: From multiuse electrochemical cells to sensors.

This work presents potential applications of low-cost fused deposition modeling 3D-printers to fabricate multiuse 3D-printed electrochemical cells for flow or batch measurements as well as the 3D-printing of electrochemical sensing platforms. Electrochemical cells and sensors were printed with acrylonitrile butadiene styrene (ABS) and conductive graphene-doped polylactic acid (G-PLA) filaments, respectively. The overall printing operation time and estimated cost per cell were 6 h and $ 6.00, respectively, while the sensors were printed within minutes (16 sensor strips of 1 × 2 cm in 10 min at a cost of $ 1.00 each sensor). The cell performance is demonstrated for the amperometric detection of tert-butylhydroquinone, dipyrone, dopamine and diclofenac by flow-injection analysis (FIA) and batch-injection analysis (BIA) using different working electrodes, including the proposed 3D-printed sensor, which presented comparable electroanalytical performance with other carbon-based electrodes (LOD of 0.1 µmol L-1 for dopamine). Raman spectroscopy and scanning electron microscopy of the 3D-printed sensor indicated the presence of graphene nanoribbons within the polymeric matrix. Electrochemical impedance spectroscopy and heterogeneous electron transfer constants (k0) for the redox probe Ru(NH3)6+3 revealed that a glassy-carbon electrode presented faster electron transfer rates than the 3D-printed sensor; however, the latter presented lower LOD values for dopamine and catechol probably due to oxygenated functional groups at the G-PLA surface.

High-throughput simultaneous quantitation of multi-analytes in tobacco by flow injection coupled to high-resolution mass spectrometry.

The high-throughput screening by flow injection coupled to high-resolution mass spectrometry (HTS-FIA-HRMS) is a powerful technique that enables the identification of several types of samples in a short period of time, either with qualitative or quantitative purposes. Sensory attributes of tobacco are affected by its chemical composition, and it is very important to quantify multi-analytes in a high-throughput methodology. HTS-FIA-HRMS coupled to multivariate analysis was used to create calibration models for 27 analytes, or group of compounds, of tobacco sensory interest. The models were validated by different approaches, including permutation test to avoid overfitting, evaluation of the equipment repeatability by control samples, reproducibility comparison of results from two different equipment and analysts, and with a blind test analysis. All tests demonstrated a good response to the proposed method. No statistical difference between the errors of both equipment was observed, with less than 7% error from the control samples, and a blind test error between 5.96% and 20.10%. The partial least squares (O-PLS) regression models were applied to 815 samples, and a principal component analysis (PCA) was performed from the predicted concentration values, aiming at the non-supervised classification based on tobacco type. We expect that this proposed methodology shows not only the applicability in tobacco samples, but also demonstrates a guideline to an efficient performance of multi-analytes target analysis using the flow injection mass spectrometry with reliable and robust validation steps.

Innovative Approaches for Estimating the Levels of Tobacco-Specific Nitrosamines in Cured Tobacco Samples.

Tobacco-specific nitrosamines (TSNAs), mainly the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are known carcinogens. Part of the NNK found in smoke is provided from matrix-bound NNK, and its determination is extremely relevant. However, the reference extraction procedure of matrix-bound NNK is time-consuming and labor-intensive and has a limited analytical capacity. Three different methodologies were proposed to predict matrix-bound NNK: simple linear regression (LR) with soluble NNK; multiple linear regression (MLR) considering soluble NNK and characteristic parameters of the samples; and orthogonal partial least-squares (O-PLS) regression using high-throughput screening by flow injection analysis coupled to high-resolution mass spectrometry (HTS-FIA-HRMS) data. Simple linear regression showed a high influence of matrix and leaf origin. Although an existing linearity trend has been observed ( R2 = 0.62) for the global model, higher correlation values were achieved for matrix and country segregation models. Multiple linear regression predicted matrix-bound NNK with more satisfactory efficiency than simple linear regression models. The coefficients of determination were 0.87 and 0.94 for flue-cured Virginia and air-cured Burley, respectively. However, this method has a limited application, since previous information about the sample is required. The proposed method based on HTS-FIA-HRMS and O-PLS has shown the most suitable performance in the prediction of matrix-bound NNK, with errors comparable to the reference method, and a higher throughput. In addition, this approach allows to determine other soluble nitrosamines, namely N'-nitrosoanatabine, N'-nitrosoanabasine, and N-nitrosonornicotine, with relative percentage errors between 5.25 and 11.98%. Therefore, the third approach is the best method for a large number of cured tobacco for accuracy in determination of TSNAs.

Fast and reliable BIA/amperometric quantification of acetylcysteine using a nanostructured double hydroxide sensor.

This study reports the preparation and characterization of nickel/lead hydroxide nanoparticles used to construct electrochemical sensors, which were investigated for amperometric quantification of N-acetylcysteine (NAC). The newly synthesised material presents good uniformity, with the lead (II) ions homogenously incorporated into the alpha nickel hydroxide crystal structure, confirmed by X-ray diffraction, transmission electron microscopy and X-ray photoelectron spectroscopy analyses. Films of nanoparticles (3 nm in size) were prepared on conductive fluorine-doped tin oxide-coated glass slides and used connected to a specially built batch injection analysis (BIA) cell with a capacity of only 4 mL and the electrode positioned in the bottom. To attain optimal analytical performance, the main parameters for BIA measurements (volume injected, different velocities of injection and best distance of the pipette from the electrode) were evaluated, as was the working potential, to determine the optimal conditions. Linear responses were obtained for the concentration range from 20 to 220 µmol L-1, and the limits of detection (3σ/slope) and quantification (10σ/slope) were calculated as 0.23 µmol L-1 and 0.70 µmol L-1, respectively. The new NAC sensor does not exhibit a memory effect and has enormous potential utility in the quantitative determination of N-acetylcysteine in drugs. The results of the analysis of NAC obtained using BIA presented good concordance with those obtained by chromatography. The analytical frequency attained using BIA (120 analysis h-1) compares very favourably with the one obtained using chromatography (6 analysis h-1).

Evaluation of the activity of ß-glucosidase immobilized on polydimethylsiloxane (PDMS) with a microfluidic flow injection analyzer with embedded optical fibers.

ß-glucosidase from almonds was immobilized on a polydimethylsiloxane (PDMS) microdevice by covalent chain using 3-aminopropyltrietoxysilane and glutaraldehyde. Enzymatic activity was evaluated using p-nitro-phenyl-ß-D-glucopyranoside dissolved in a 0.01 M pH 5.0 phosphate solution at 45 °C measuring the reaction product (p-nitrophenol) at 410 nm. The microdevice consisted of two parts: the one part where the enzymatic reaction was carried out and a second part where pH was adjusted at 10, with NaOH. The reaction product was measured at the microchip exit using two optical fibers which were aligned facing each other with a gap of 7 mm, between both tips using guides located perpendicular to the flow outlet. A water bath was used to carry out the enzymatic reaction on the microdevice at 45 °C. The enzymatic surface of the PDMS microdevice was 1.15 cm2 and the immobilized ß-glucosidase amount on the microdevice was of 1.17 µg/cm2. The calculated kinetics parameters were: Km 2.5 mM; Vmax 2.2 mM/min; Kcat 908.3/min and Kcat/Km 363.3/mM min. The immobilized enzyme is very stable decreasing only 5% the first 15 days; on the 30th day, the activity was 69%, regarding the initial activity.

A flow injection procedure using Layered Double Hydroxide for on line pre-concentration of fluoride.

This work showed a flow system designed with solenoid valves for preconcentration of fluoride using SPADNS method in water samples. The analyte was preconcentrated in a mini-column coated with Layered Double Hydroxides (LDH) used as adsorbent. Then, the fluoride ions were eluted with 0.5molL-1 sodium hydroxide and determined by spectrophotometry. The variables that affect the system such adsorbent mass, type of eluent, solutions flow rate, reagent concentration and pH effect were critically evaluated. Under optimized conditions, the detection limit, coefficient of variation, linear range and preconcentration factor were estimated at 15µgL-1 (99.7% confidence level), 0.8% (500µgL-1, n = 10), 50-500µgL-1 and 10, respectively. The accuracy of the method was evaluated by analysis of ALPHA APS 1076 (Simulated Rain Water) certified material, the values were not significantly different at a 95% level of confidence. The method was applied for fluoride determination in water samples and the levels found were below the maximum values established by Brazilian environmental and health legislations.

Selective and simultaneous determination of indigo carmine and allura red in candy samples at the nano-concentration range by flow injection analysis with multiple pulse amperometric detection.

A novel, unique electroanalytical method was developed for the simultaneous quantification of the dyes indigo carmine (IC) and allura red (AR) in candies by coupling flow injection analysis and multiple pulse amperometry with a cathodically pretreated boron-doped diamond electrode, using 0.30 mol L-1 H2SO4 as supporting electrolyte. A dual-potential waveform was employed, causing the electrooxidation of either IC solely or IC and AR simultaneously. Thence, subtraction of current signals was used to quantify IC and AR in the concentration ranges of 70.0-1000 nmol L-1 and 40.0-770 nmol L-1, with limits of detection of 40.0 nmol L-1 and 7.0 nmol L-1, respectively. The proposed method, which permits up to 153 determinations per hour with good precision, was successfully applied in the quantification of these dyes in samples of commercial candies; their obtained contents were similar (at a 95% confidence level) to those from a comparative HPLC method.

Effects of in ovo injection of lysine and methionine into fertile broiler (parent stock) eggs on hatchability, growth performance, caecum microbiota, and ileum histomorphology

We investigated the effect of in ovo injection of lysine, methionine, or their mixture into fertile broiler eggs on hatchability, chick weight, growth performance, inner organ development, caecum total aerobic bacteria, E. coli, coliforms, Enterobactericaea, and ileal histomorphology of broilers. Three hundred fertile eggs obtained from 60-week-old Ross 308 broiler breeders were used. Before this study, a preliminary study was conducted to determine the optimal amino acid dose. Optimum amino acid dose was determined as 2 mg/0.2 mL. Before replacing hatching machine, eggs were weighed individually and numbered. On day 16 of incubation, these eggs were allocated to treatment groups: negative control (no injection), positive control (distilled water injection 0.2 mL), lysine (2 mg/0.2 mL), methionine (2 mg/0.2 mL), and lysine + methionine (1 + 1 mg/0.2 mL). The hatching window lasted 32 h. During hatching, chicks hatched in the first hour and the last 6 h were discarded from the study to ensure equal hatching time. After hatching, 120 one-day-old healthy chicks were divided into five treatment groups with three replicates, each including eight birds, for 21 days. These chicks were given a starter diet (3080 kcal/kg metabolizable energy and 22% crude protein) during the trial. The results showed that in ovo injection of lysine, methionine, and lysine + methionine did not affect relative chick weight, livability, growth performance, caecum microbiota, and ileal villi length and thickness. Lysine injection increased hatchability compared with the negative control and methionine-injected groups. Gastrointestinal weight increased in lysine-injected group compared with the negative control group. The in ovo injection of 2 mg/0.2 mL lysine have a positive effect on the hatchability of fertile eggs.(AU)

A flow-based procedure exploiting the lab-in-syringe approach for the determination of ester content in biodiesel and diesel/biodiesel blends.

The ester content is an important parameter to be monitored in biodiesel for evaluation of the transesterification reaction yield and for assessing the purity of the final product. This is also a relevant quality parameter in diesel/biodiesel blends to avoid frauds, because legislation establishes a minimum amount of biodiesel to be added to diesel. The official method EN14103 requires the addition of an alternative internal standard (methyl nonadecanoate) for analysis of biodiesel from bovine tallow because the methyl heptadecanoate is found in high amounts in this product. In this work, it is proposed a fast, simple, practical, and environmental friendly flow-based spectrophotometric procedure, which exploits the formation of the violet complex between Fe(III) and the hydroxamate generated by the reactions of the alkyl esters with hydroxylamine. All involved steps are carried out inside the syringe pump of a sequential injection analyzer (lab-in-syringe approach). A single phase is attained by using ethanol as mediator solvent between the organic sample and aqueous soluble reagents. Linear responses for biodiesel samples and diesel/biodiesel blends were obtained from 4-99%(v/v) to 2.0-40%(v/v) methyl esters, described by the equations: A = 0.342 + 0.00305C (r = 0.997) and A = 0.174 + 0.00503C (r = 0.999), respectively. The analytical curve can be obtained by in-line dilution of a methyl linoleate stock solution. For biodiesel samples, the coefficient of variation (n = 10), limit of detection (99.7% confidence level), and sampling rate were estimated at 0.8%, 0.36%(v/v), and 15h-1, respectively, whereas the corresponding values for the blend samples were 0.20%, 0.03%(v/v), and 12h-1, respectively. The procedure consumes only 860µg of hydroxylamine, 366µg of Fe2(SO4)3·H2O, and 2.0mL ethanol and generates ca. 3.0mL of residue per determination. The results agreed with those obtained by official methods EN14103/2011 e EN14078, at the 95% confidence level.