Validation of an immunoassay method for the determination of traces of carbaryl in vegetable and fruit extracts by liquid chromatography with photodiode array and mass spectrometric detection

A competitive enzyme-linked immunosorbent assay (ELISA) method for carbaryl quantitation in crop extracts was validated by liquid chromatography (LC) with diode array detection (DAD). For this purpose, six crops (banana, carrot, green bean, orange, peach and potato) were chosen for recovery and reproducibility studies. The general sample preparation included extraction with methanol followed by liquid-liquid partitioning and clean-up on Celite-charcoal adsorbent column of the vegetable extracts. ELISA samples consisted of a diluted LC extract in assay phosphate buffer (pH 7.5). The potential effect of methanol in these samples was evaluated. It was observed that a maximum content of 10% methanol present in the assay buffer could be tolerated without expressive losses in the ELISA performance. Under these conditions, a IC50 similar to 1.48 mu g l(-1) was obtained. A minimum matrix effect with a 1:50 dilution of the methanolic extracts in assay buffer was noticed, except for green bean samples that inhibited completely the assay. For the vegetable extracts, the ELISA sensitivities varied from 3.9 to 5.7 mu g l(-1), and good recoveries (82-96%) with R.S.D.s ranging from 5.7 to 12.1% were found. An excellent correlation between the LC-DAD and ELISA techniques was obtained. The confirmation of the carbaryl in less concentrated samples was achieved by LC-mass spectrometry interfaced with atmospheric pressure chemical ionisation. The [M + H](+)= 202 and [M + H-57](+)=145 ions, equivalent to the protonated molecular and l-naphthol ions, respectively, were used to carbaryl identification in these samples. (C) 1998 Elsevier B.V. B.V. All rights reserved.

Rapid determination of zinc in foods by flow injection analysis with flame AAS using gradient calibration method

A calibration method was developed using flow injection analysis (FI) with a Gradient Calibration Method (GCM). The method allows the rapid determination of zinc In foods (approximately 30 min) after treatment with concentrated sulphuric acid and 30% hydrogen peroxide, and analysis with flame atomic absorption spectrometry (FAAS). The method provides analytical results with a relative standard deviation of about 2% and requires less time than by conventional FI calibration. The electronic selection of different segments along the gradient and monitoring of the technique covers wide concentration ranges while maintaining the inherent high precision of flow injection analysis. Concentrations, flow rates, and flow times of the reagents were optimized in order to obtain best accuracy and precision. Flow rates of 10 mL/min were selected for zinc. In addition, the system enables electronic dilution and calibration where a multipoint curve can be constructed using a single sample injection.

Uranium-lead dating method at the Pará-Iso isotope geology laboratory, UFPA, Belém – Brazil

ABSTRACT: Analytical procedures for U-Pb isotope dilution analyses at the Pará-Iso isotope geology laboratory of the Federal University of Pará (UFPA) are described in detail. The procedures are applied to zircon, titanite, rutile, apatite, columbite-tantalite and whole rock. Reagent preparation and chemical processing are done in clean-room conditions. Samples are dissolved using Teflon™ microcapsules in steel jacket Teflon™ Parr Instrument™ bomb or Teflon™ screw cap containers. U and Pb are separated using anion exchange AG 1x8 resin columns. Typical blanks for mineral sample amounts of 0.01 to 1.0 mg are less than 1 pg U and 20-30 pg Pb. Isotope analysis of the U and Pb from the same filament are carried out using a Finnigan MAT 262 mass-spectrometer in static and dynamic modes. The current analytical level is demonstrated on analyses of international standard zircon 91500 with three different ²³⁵U-²⁰⁵Pb and ²³⁵U-²⁰⁸Pb isotope tracers and whole rock standards. Results of analyses of two zircon samples are also presented.

Profiles of steroid hormones in canine X-Linked muscular dystrophy via stable isotope dilution LC-MS/MS

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Isotope analysis method (δ¹³C) in clarified apple juice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Isotopic analysis method (δ13C) in apple-flavoured soft drinks

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na2SO4, (NH4)(2)SO4, MgSO4, LiSO4, Na2HPO4, sodium phosphate buffer (pH 7.0), NaH2PO4, K2HPO4, potassium phosphate buffer (pH 7.0), KH2PO4, C6H8O7, Na3C6HSO7, KCHO2, NaCHO2, NaCO3, NaHCO3, C2H4O2, sodium acetate buffer (pH 4.5), and NaC2H3O2] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents. (C) 2011 Elsevier Inc. All rights reserved.

Development of a rapid column-switching LC-MS/MS method for the quantification of THCCOOH and THCCOOH-glucuronide in whole blood for assessing cannabis consumption frequency

The concentration of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in whole blood is used as a parameter for assessing the consumption behavior of cannabis consumers. The blood level of THCCOOH-glucuronide might provide additional information about the frequency of cannabis use. To verify this assumption, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and direct quantification of free and glucuronidated THCCOOH in human whole blood was newly developed. The method comprised protein precipitation, followed by injection of the processed sample onto a trapping column and subsequent gradient elution to an analytical column for separation and detection. The total LC run time was 4.5 min. Detection of the analytes was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was fully validated by evaluating the following parameters: linearity, lower limit of quantification, accuracy and imprecision, selectivity, extraction efficiency, matrix effect, carry-over, dilution integrity, analyte stability, and re-injection reproducibility. All acceptance criteria were analyzed and the predefined criteria met. Linearity ranged from 5.0 to 500 μg/L for both analytes. The method was successfully applied to whole blood samples from a large collective of cannabis consumers, demonstrating its applicability in the forensic field.

Development and evaluation of the polymerase chain reaction (PCR) method for diagnosis of spring viremia of carp virus (SVCV)

Aquaculture has been expanded rapidly to become a major commercial and food-producing sector worldwide in recent decade. In parallel, viral diseases rapidly spread among farms causing enormous economic losses. The accurate detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Spring Viraemia of Carp Virus (SVCV) is a very severe pathogen of carp fishes in different parts of the world and is categorized as a reportable listed disease in the annual published list of World Organization for animal Health (OIE). The objective of this study was to develop and evaluate RT- PCR test for detecting SVC virus and also the sensitivity and specificity of this test. A semi nested RT- PCR was designed using combination of three primers: two external (SVCF , SVCR) and one internal (SVCS) primers which based on conserved region of G gen. The specificity of designed primers (only external ones) by examination on Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV) was confirmed. For optimizing of the PCR test, primer concentration, primer annealing temperature, cycle number and Mgcl2 concentration were surveyed. Also for validity test, prevention of false negative and Assurance of its accuracy, a competitive internal control (mimic) designed and its suitable concentration was defined. Evaluation of the sensitivity of designed test were conducted first by comparing the different commercially available RNA isolation guidelines, two guidelines: isotiocyanate phenol–chloroform based protocols (RNX–Plus Iran, Iq2000 kit Taiwan ) and two column based protocols (Cinna pure RNA Iran , high pure viral RNA kit, Roche Germany ). The results indicated that the column based protocols (Roche method and Cinna pure), yield 36.77 ng/μl and 16/47 ng/μl RNA concentration respectively, which were significantly higher than other protocols(P<0.05). Then for evaluation of extracted RNA sensitivity, Serial dilution of SVCV strain 56.70 grown in EPC (1.9×105 TCID50/ml) was examined To compare sensitivity. Extracted RNA from serial dilution with stone's primers and commercial IQ-2000 kit were examined simultaneously. The result indicated that designed semi- nested RT- PCR was able to recognize SVC virus to 10-4 dilution and stone's primer recognize to 10-3 dilution whereas Iq-2000 commercial kit did not recognized in any dilution. In high virus titer in designed test two DNA band (462 bp and 266 bp) produced, and by decreasing virus titer 462 bp was omitted. In low virus titer or lack of virus, just DNA band (mimic) 729 bp can propagate. After designing and optimizing PCR test, a total of 400 suspected cultured Cyprinus carpio with high mortality from 4 aquaculture zone of Khuzestan province were collected and tested for SVCV during 2012- 2013 using developed PCR method and IQ- 2000. The results indicated that SVC virus was not observed in samples using both methods.

The effect of Physiological NaCl Dilution levels and Storage Duration to Sperm Motility and Abnormality in Muscovy Duck

The aim of this research are to know the effects of physiological NaCl dilution levels and storage duration on sperm motility and abnormality of  Muscovy duck. Materials which used in this research were semen that collected of 1  - 1,5 years of four Muscovy duck. Semen were treated by physiological NaCl dilution levels nol , three, six and nine and storage duration ( t0 = 0 minute t1 =45minutes and t2=90 minute). Replication was three tapping periode . The method of this research was laboratory experimental with Randomized Complete Blok Design (RBD) in Split Plot Design (SPD)  pattern ,as main plot is Physiological NaCl dilation levels and as sub plot is storage duration. Result of the experiment showed that physiological NaCl dilution levels was very significantly influences on sperm motility (p<0,01) and significantly influences and sperm abnormality (p<0,05) . The storage duration was very significantly influences and sperm motility and abnormality (p<0,01) .Orthogonal duration polynomial assayed showed that the effect of physiological NaCl dilution levels to sperm motility  had a linier regression line with the a equation as Y =69.94 - 2.65 X,  r=0,49 R2=0.24 with Y presented to sperm motility and X represented to physiological NaCl dilution levels  sperm and effect of storage duration to sperm motility had a linier regression line with the equation as Y =74.24-0.35 X, r =0.73 R2=0.53 with Y represented to sperm motility and X represented to storage duration . The effect of physiological NaCl dilution levels to sperm abnormality had a linier regression line with the equation as Y =5.61+0.34X, r=0.52 R2=0.27 with Y represented to sperm to abnormality and X represented to regression line with the equation as Y =6.74+0.01X, r=0.24R2=0.060 with Y represented to sperm abnormality and X represented to storage duration. It is can be concluded that a higher physiological NaCl dilation levels and storage duration caused sperm to motility decreased and sperm abnormality increased. (Animal Production 3(2): 45-52 (2001)Key Word: sperm Muscovy duck.

An approximate method for the solution of an influence function foil rolling model

Fleck and Johnson (Int. J. Mech. Sci. 29 (1987) 507) and Fleck et al. (Proc. Inst. Mech. Eng. 206 (1992) 119) have developed foil rolling models which allow for large deformations in the roll profile, including the possibility that the rolls flatten completely. However, these models require computationally expensive iterative solution techniques. A new approach to the approximate solution of the Fleck et al. (1992) Influence Function Model has been developed using both analytic and approximation techniques. The numerical difficulties arising from solving an integral equation in the flattened region have been reduced by applying an Inverse Hilbert Transform to get an analytic expression for the pressure. The method described in this paper is applicable to cases where there is or there is not a flat region.