Analytical strategies for the early quality and safety assurance in the global feed chain. Approaches for nitrogen adulterants in soybean meal and mineral and transformer oils in vegetable oils.

In the past decade, several major food safety crises originated from problems with feed. Consequently, there is an urgent need for early detection of fraudulent adulteration and contamination in the feed chain. Strategies are presented for two specific cases, viz. adulterations of (i) soybean meal with melamine and other types of adulterants/contaminants and (ii) vegetable oils with mineral oil, transformer oil or other oils. These strategies comprise screening at the feed mill or port of entry with non-destructive spectroscopic methods (NIRS and Raman), followed by post-screening and confirmation in the laboratory with MS-based methods. The spectroscopic techniques are suitable for on-site and on-line applications. Currently they are suited to detect fraudulent adulteration at relatively high levels but not to detect low level contamination. The potential use of the strategies for non-targeted analysis is demonstrated.

Chemical mass closure and source-specific composition of atmospheric particles

It has been known for decades that particles can cause adverse health effects as they are deposited within the respiratory system. Atmospheric aerosol particles influence climate by scattering solar radiation but aerosol particles act also as the nuclei around which cloud droplets form. The principal objectives of this thesis were to investigate the chemical composition and the sources of fine particles in different environments (traffic, urban background, remote) as well as during some specific air pollution situations. Quantifying the climate and health effects of atmospheric aerosols is not possible without detailed information of the aerosol chemical composition. Aerosol measurements were carried out at nine sites in six countries (Finland, Germany, Czech, Netherlands, Greece and Italy). Several different instruments were used in order to measure both the particulate matter (PM) mass and its chemical composition. In the off-line measurements the samples were collected first on a substrate or filter and gravimetric and chemical analysis were conducted in the laboratory. In the on-line measurements the sampling and analysis were either a combined procedure or performed successively within the same instrument. Results from the impactor samples were analyzed by the statistical methods. This thesis comprises also a work where a method for the determination carbonaceous matter size distribution by using a multistage impactor was developed. It was found that the chemistry of PM has usually strong spatial, temporal and size-dependent variability. In the Finnish sites most of the fine PM consisted of organic matter. However, in Greece sulfate dominated the fine PM and in Italy nitrate made the largest contribution to the fine PM. Regarding the size-dependent chemical composition, organic components were likely to be enriched in smaller particles than inorganic ions. Data analysis showed that organic carbon (OC) had four major sources in Helsinki. Secondary production was the major source in Helsinki during spring, summer and fall, whereas in winter biomass combustion dominated OC. The significant impact of biomass combustion on OC concentrations was also observed in the measurements performed in Central Europe. In this thesis aerosol samples were collected mainly by the conventional filter and impactor methods which suffered from the long integration time. However, by filter and impactor measurements chemical mass closure was achieved accurately, and a simple filter sampling was found to be useful in order to explain the sources of PM on the seasonal basis. The online instruments gave additional information related to the temporal variations of the sources and the atmospheric mixing conditions.

Comprehensive Two-Dimensional Gas Chromatography: Instrumental and Methodological Development

Comprehensive two-dimensional gas chromatography (GC×GC) offers enhanced separation efficiency, reliability in qualitative and quantitative analysis, capability to detect low quantities, and information on the whole sample and its components. These features are essential in the analysis of complex samples, in which the number of compounds may be large or the analytes of interest are present at trace level. This study involved the development of instrumentation, data analysis programs and methodologies for GC×GC and their application in studies on qualitative and quantitative aspects of GC×GC analysis. Environmental samples were used as model samples. Instrumental development comprised the construction of three versions of a semi-rotating cryogenic modulator in which modulation was based on two-step cryogenic trapping with continuously flowing carbon dioxide as coolant. Two-step trapping was achieved by rotating the nozzle spraying the carbon dioxide with a motor. The fastest rotation and highest modulation frequency were achieved with a permanent magnetic motor, and modulation was most accurate when the motor was controlled with a microcontroller containing a quartz crystal. Heated wire resistors were unnecessary for the desorption step when liquid carbon dioxide was used as coolant. With use of the modulators developed in this study, the narrowest peaks were 75 ms at base. Three data analysis programs were developed allowing basic, comparison and identification operations. Basic operations enabled the visualisation of two-dimensional plots and the determination of retention times, peak heights and volumes. The overlaying feature in the comparison program allowed easy comparison of 2D plots. An automated identification procedure based on mass spectra and retention parameters allowed the qualitative analysis of data obtained by GC×GC and time-of-flight mass spectrometry. In the methodological development, sample preparation (extraction and clean-up) and GC×GC methods were developed for the analysis of atmospheric aerosol and sediment samples. Dynamic sonication assisted extraction was well suited for atmospheric aerosols collected on a filter. A clean-up procedure utilising normal phase liquid chromatography with ultra violet detection worked well in the removal of aliphatic hydrocarbons from a sediment extract. GC×GC with flame ionisation detection or quadrupole mass spectrometry provided good reliability in the qualitative analysis of target analytes. However, GC×GC with time-of-flight mass spectrometry was needed in the analysis of unknowns. The automated identification procedure that was developed was efficient in the analysis of large data files, but manual search and analyst knowledge are invaluable as well. Quantitative analysis was examined in terms of calibration procedures and the effect of matrix compounds on GC×GC separation. In addition to calibration in GC×GC with summed peak areas or peak volumes, simplified area calibration based on normal GC signal can be used to quantify compounds in samples analysed by GC×GC so long as certain qualitative and quantitative prerequisites are met. In a study of the effect of matrix compounds on GC×GC separation, it was shown that quality of the separation of PAHs is not significantly disturbed by the amount of matrix and quantitativeness suffers only slightly in the presence of matrix and when the amount of target compounds is low. The benefits of GC×GC in the analysis of complex samples easily overcome some minor drawbacks of the technique. The developed instrumentation and methodologies performed well for environmental samples, but they could also be applied for other complex samples.

Deriving structural information from non-coding ribonucleic acids by mass spectrometry

The purpose of this study is to describe the development of application of mass spectrometry for the structural analyses of non-coding ribonucleic acids during past decade. Mass spectrometric methods are compared of traditional gel electrophoretic methods, the characteristics of performance of mass spectrometric, analyses are studied and the future trends of mass spectrometry of ribonucleic acids are discussed. Non-coding ribonucleic acids are short polymeric biomolecules which are not translated to proteins, but which may affect the gene expression in all organisms. Regulatory ribonucleic acids act through transient interactions with key molecules in signal transduction pathways. Interactions are mediated through specific secondary and tertiary structures. Posttranscriptional modifications in the structures of molecules may introduce new properties to the organism, such as adaptation to environmental changes or development of resistance to antibiotics. In the scope of this study, the structural studies include i) determination of the sequence of nucleobases in the polymer chain, ii) characterisation and localisation of posttranscriptional modifications in nucleobases and in the backbone structure, iii) identification of ribonucleic acid-binding molecules and iv) probing of higher order structures in the ribonucleic acid molecule. Bacteria, archaea, viruses and HeLa cancer cells have been used as target organisms. Synthesised ribonucleic acids consisting of structural regions of interest have been frequently used. Electrospray ionisation (ESI) and matrix-assisted laser desorption ionisation (MALDI) have been used for ionisation of ribonucleic analytes. Ammonium acetate and 2-propanol are common solvents for ESI. Trihydroxyacetophenone is the optimal MALDI matrix for ionisation of ribonucleic acids and peptides. Ammonium salts are used in ESI buffers and MALDI matrices as additives to remove cation adducts. Reverse phase high performance liquid chromatography has been used for desalting and fractionation of analytes either off-line of on-line, coupled with ESI source. Triethylamine and triethylammonium bicarbonate are used as ion pair reagents almost exclusively. Fourier transform ion cyclotron resonance analyser using ESI coupled with liquid chromatography is the platform of choice for all forms of structural analyses. Time-of-flight (TOF) analyser using MALDI may offer sensitive, easy-to-use and economical solution for simple sequencing of longer oligonucleotides and analyses of analyte mixtures without prior fractionation. Special analysis software is used for computer-aided interpretation of mass spectra. With mass spectrometry, sequences of 20-30 nucleotides of length may be determined unambiguously. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Sequencing in conjunction with other structural studies enables accurate localisation and characterisation of posttranscriptional modifications and identification of nucleobases and amino acids at the sites of interaction. High throughput screening methods for RNA-binding ligands have been developed. Probing of the higher order structures has provided supportive data for computer-generated three dimensional models of viral pseudoknots. In conclusion. mass spectrometric methods are well suited for structural analyses of small species of ribonucleic acids, such as short non-coding ribonucleic acids in the molecular size region of 20-30 nucleotides. Structural information not attainable with other methods of analyses, such as nuclear magnetic resonance and X-ray crystallography, may be obtained with the use of mass spectrometry. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Ligand screening may be used in the search of possible new therapeutic agents. Demanding assay design and challenging interpretation of data requires multidisclipinary knowledge. The implement of mass spectrometry to structural studies of ribonucleic acids is probably most efficiently conducted in specialist groups consisting of researchers from various fields of science.

A novel algesic peptide derived from skin secretions of the frog Amolops loloensis

Several algesic agents including bradykinin and tachykinin have been identified from skin secretions of amphibians. They may act in defensive roles against aggressors. In this study, a novel peptide named Amolos with an amino acid sequence of FLPIVGAKL an

A novel bradykinin-like peptide from skin secretions of the frog, Rana nigrovittata

A bradykinin-like peptide has been isolated from the skin secretions of the frog Rana nigrovittata. This peptide was named ranakinin-N. Its primary structure, RAEAVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Ranakinin-N is composed of 13 amino acid residues and is related to the bradykinin identified from the skin secretions of Odorrana schmackeri, which is composed of 9 amino acid residues. Ranakinin-N was found to exert concentration-dependent contractile effects on isolated guinea pig ileum. cDNA sequence encoding the precursor of ranakinin-N was isolated from a skin cDNA library of R. nigrovittata. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that the deficiency of a 15-nucleotide fragment (agaatgatcagacgc in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in the absence of a dibasic site for trypsin-like proteinases and an unusual -AEVA- insertion in the N-terminal part of ranakinin-N. The -AEAV- insertion resulted in neutral net charge at the N-terminus of ranakinin-N. Ranakinin-N is the first reported bradykinin-like peptide with a neutral net charge at the N-terminus. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Phantom mutation hotspots in human mitochondrial DNA

Phantom mutations are systematic artifacts generated in the course of the sequencing process. Contra common belief these artificial mutations are nearly ubiquitous in sequencing results, albeit at frequencies that may vary dramatically. The amount of artifacts depends not only on the sort of automated sequencer and sequencing chemistry employed, but also on other lab-specific factors. An experimental study executed on four samples under various combinations of sequencing conditions revealed a number of phantom mutations occurring at the same sites of mitochondrial DNA (mtDNA) repeatedly. To confirm these and identify further hotspots for artifacts, > 5000 mtDNA electropherograms were screened for artificial patterns. Further, > 30000 published hypervariable segment 1 sequences were compared at potential hotspots for phantom mutations, especially for variation at positions 16085 and 16197. Resequencing of several samples confirmed the artificial nature of these and other polymorphisms in the original publications. Single-strand sequencing, as typically executed in medical and anthropological studies, is thus highly vulnerable to this kind of artifacts. In particular, phantom mutation hotspots could easily lead to misidentification of somatic mutations and to misinterpretations in all kinds of clinical mtDNA studies.

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 mu g L-1. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (I) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future. (C) 2009 Published by Elsevier B.V.

Rapid quantification of total microcystins in cyanobacterial samples by periodate-permanganate oxidation and reversed-phase liquid chromatography

Microcystins (MCs) comprise a family of more than 80 related cyclic hepatotoxic heptapeptides. Oxidation of MCs causes cleavage of the chemically unique C-20 beta-amino acid (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) amino to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB), which has been exploited to enable analysis of the entire family. In the present study, the reaction conditions (e.g. concentration of the reactants. temperature and pH) used in the production of MMPB by oxidation of cyanobacterial samples with permanganate-periodate were optimized through a series of well-controlled batch experiments. The oxidation product (MMPB) was then directly analyzed by high-performance liquid chromatography with diode array detection. The results of this study provided insight into the influence of reaction conditions on the yield of MMPB. Specifically, the optimal conditions, including a high dose of permanganate (>= 50 mM) in saturated periodate solution at ambient temperature under alkaline conditions (pH similar to 9) over 1-4 h were proposed, as indicated by a MMPB yield of greater than 85%. The technique developed here was applied to determine the total concentration of MCs in cyanobacterial bloom samples, and indicated that the MMPB technique was a highly sensitive and accurate method of quantifying total MCs. Additionally, these results will aid in development of a highly effective analytical method for detection of MMPB as an oxidation product for evaluation of total MCs in a wide range of environmental sample matrices, including natural waters, soils (sediments) and animal tissues. (C) 2009 Elsevier B.V. All rights reserved.

Extraction of genomic DNA using a new amino silica monolithic column

A new amino silica monolithic column was developed for DNA extraction in a miniaturized format. The monolithic column was prepared in situ by polymerization of tetraethoxysilane (TEOS) and N-(beta-aminoethyl)-gamma-aminopropylmethyldimethoxysilane (AEAPMDMS). DNA was loaded in 50 mM tris(hydroxylmethyl)aminomethane-EDTA buffer at pH 7.0 and eluted with 300 mM potassium phosphate solution at pH 10.0. Under optimal condition, a 6.0-cm monolithic column provided a capacity of 56 ng DNA with an extraction efficiency of 71 +/- 5.2% (X +/- RSD). When the amino silica monolithic column was applied to extract genomic DNA from the whole blood of crucian carp, an extraction efficiency of 52 +/- 5.6% (X +/- SD) was obtained by three extractions. Since the chaotropic-based sample loading and organic solvent wash steps were avoided in this procedure, the purified DNA was suitable for downstream processes such as PCR. This amino silica monolithic column was demonstrated to allow rapid and efficient DNA purification in microscale.

Quantitative Determination of Microcystins in Rat Plasma by LC-ESI Tandem MS

A rapid and sensitive method was developed and validated for the determination of MCYST (microcystin)-RR, -LR, and [Dha(7)] MCYST-LR in rat plasma by liquid chromatography-tandem mass spectrometry. The analytes were extracted from rat plasma by protein precipitation, followed by solid-phase extraction. Liquid chromatography with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MCYST-RR, -LR, and [Dha(7)] MCYST-LR in rat plasma. The recoveries for each analyte in rat plasma ranged from 70.8 to 88.7%. The calibration curve was linear within the range from 0.005 to 1.25 mu g mL(-1). The limit of detection were 1.4, 1.0, 0.6 ng mL(-1) for MCYST-RR, -LR, and [Dha(7)] MCYST-LR. The overall precision was determined on three different days. The values for within- and between-day precision in rat plasma were within 15%. This method was applied to the identification and quantification of microcystins in rat plasma with acute exposure of microcystins via intravenous injection.

Macrocyclic polyamine-modified poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith for capillary electrochromatography

1,4,10,13,16-Pentaazatricycloheneicosane-9,17-dione (macrocyclic polyamine)-modified polymer-based monolithic column for CEC was prepared by ring opening reaction of epoxide groups from poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolith with macrocyclic polyamine. Conditions such as reaction time and concentration of macrocyclic polyamine for the modification reaction were optimized to generate substantial EOF and enough chromatographic interactions. Anodic EOF was observed in the pH range of 2.0-8.0 studied due to the protonation of macrcyclic polyamine at the surface of the monolith. Morphology of the monolithic column was examined by SEM and the incorporation of macrocyclic polyamine to the poly(GMA-co-EDMA) monolith was characterized by infrared (IR) spectra. Successful separation of inorganic anions, isomeric benzenediols, and benzoic acid derivatives on the monolithic column was achieved for CEC. In addition to hydrophobic interaction, hydrogen bonding and electrostatic interaction played a significant role in the separation process.

Application of methyl parathion hydrolase (MPH) as a labeling enzyme

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 mu L hemolymph from an infected shrimp and differentiated it from a healthy shrimp.

Detection of polybrominated diphenyl ethers in tilapia (Oreochromis mossambicus) from O'ahu, Hawai'i

Polybrominated diphenyl ethers (PBDEs) have been detected for the first time at a range from 231.58 to 685.61 ng g(-1) lipid weight in the muscles of tilapia ( Oreochromis mossambicus) collected from O'ahu, an island of the geographically isolated Hawaiian archipelago.

A novel chemiluminescent immunoassay for microcystin (MC) detection based on gold nanoparticles label and its application to MC analysis in aquatic environmental samples

A novel chemiluminescent immunoassay method based on gold nanoparticles was developed for the detection of microcystins (MCs). The immunoassay included three main steps: indirect competitive immunoreaction, oxidative dissolution of gold nanoparticles, and indirect determination for MCs with Au3+-catalysed luminol chemiluminesent system. The method has a wide working range (0.05-10 mu g L-1, r(2) = 0.9914), the limit of detection was determined to be 0.024 mu g L-1, which is much lower than the World Health Organization's proposed guidelines (1 mu g L-1) for drinking-water. The proposed method was applied to MC analysis in natural water and fish tissue samples, and most results in the proposed method were in agreement with the conventional indirect competitive enzyme-linked immunosorbent assay method, which indicated that the new chemiluminescent immunoassay was sensitive, reliable, and suitable for MC analysis in natural water and fish tissue samples.