Food Safety Testing: Rapid Molecular Methods for Chemical and Biological Hazards

The central goal of food safety policy in the European Union (EU) is to protect consumer health by guaranteeing a high level of food safety throughout the food chain. This goal can in part be achieved by testing foodstuffs for the presence of various chemical and biological hazards. The aim of this study was to facilitate food safety testing by providing rapid and user-friendly methods for the detection of particular food-related hazards. Heterogeneous competitive time-resolved fluoroimmunoassays were developed for the detection of selected veterinary residues, that is coccidiostat residues, in eggs and chicken liver. After a simplified sample preparation procedure, the immunoassays were performed either in manual format with dissociation-enhanced measurement or in automated format with pre-dried assay reagents and surface measurement. Although the assays were primarily designed for screening purposes providing only qualitative results, they could also be used in a quantitative mode. All the developed assays had good performance characteristics enabling reliable screening of samples at concentration levels required by the authorities. A novel polymerase chain reaction (PCR)-based assay system was developed for the detection of Salmonella spp. in food. The sample preparation included a short non-selective pre-enrichment step, after which the target cells were collected with immunomagnetic beads and applied to PCR reaction vessels containing all the reagents required for the assay in dry form. The homogeneous PCR assay was performed with a novel instrument platform, GenomEra^™, and the qualitative assay results were automatically interpreted based on end-point time-resolved fluorescence measurements and cut-off values. The assay was validated using various food matrices spiked with sub-lethally injured Salmonella cells at levels of 1-10 colony forming units (CFU)/25 g of food. The main advantage of the system was the exceptionally short time to result; the entire process starting from the pre-enrichment and ending with the PCR result could be completed in eight hours. In conclusion, molecular methods using state-of-the-art assay techniques were developed for food safety testing. The combination of time-resolved fluorescence detection and ready-to-use reagents enabled sensitive assays easily amenable to automation. Consequently, together with the simplified sample preparation, these methods could prove to be applicable in routine testing.

A simplified procedure for determination of clofentezine residues in fruits by liquid chromatography with UV detection

A rapid and sensitive method is described for the determination of clofentezine residues in apple, papaya, mango and orange. The procedure is based on the extraction of the sample with a hexane:ethyl acetate mixture (1:1, v/v) and liquid chromatographic analysis using UV detection. Mean recoveries from 4 replicates of fortified fruit samples ranged from 81% to 96%, with coefficients of variation from 8.9% to 12.5%. The detection and quantification limits of the method were of 0.05 and 0.1 mg kg-1, respectively.

Genetic Basis and Diagnostics of Extended-Spectrum Beta-Lactamases among Enterobacteriaceae in Finland

Since the introduction of antibiotic agents, the amount and prevalence of Beta-lactam resistant enterobacteria has become an increasing problem. Many enterobacteria are opportunistic pathogens that easily acquire resistance mechanisms and genes, which make the situation menacing. These bacteria have acquired resistance and can hydrolyse extended spectrum cephalosporins and penicillins by producing enzymes called extended-spectrum Beta-lactamases (ESBLs). ESBL-producing bacteria are most commonly found in the gastro-intestinal tract of colonised patients. These resistant strains can be found in both health-care associated and community-acquired isolates. The detection and treatment of infections caused by bacteria producing ESBLs are problematic. This study investigated the genetic basis of extended-spectrum Beta-lactamases in Enterobacteriaceae, especially in Escherichia coli and Klebsiella pneumoniae isolates. A total of 994 Finnish Enterobacteriaceae strains, collected at 26 hospital laboratories, during 2000 and 2007 were analysed. For the genetic basis studies, PCR, sequencing and pyrosequencing methods were optimised. In addition, international standard methods, the agar dilution and disk diffusion methods were performed for the resistance studies, and the susceptibility of these strains was tested for antimicrobial agents that are used for treating patients. The genetic analysis showed that bla_CTX-M was the most prevalent gene among the E. coli isolates, while blaS_HV-12 was the most common Beta-lactamase gene in K. pneumoniae. The susceptibility testing results showed that about 60% of the strains were multidrug resistant. The prevalence of ESBL-producing isolates in Finland has been increasing since 2000. However, the situation in Finland is still much better than in many other European countries.

Reversed phase HPLC determination of tamoxifen in dog plasma and its pharmaco-kinetics after a single oral dose administration

The analytical method developed to evaluate tamoxifen in dog plasma samples was precise, accurate, robust and linear in the range of 5-200 ng/mL. The limits of detection and quantification were 0.981 ng/mL and 2.97 ng/mL, respectively. Besides, the intra-day precision and accuracy variations were 8.78 and 10.16%, respectively. Tamoxifen concentrations were analyzed by combined reversed phase liquid chromatography and UV detection (lambda=280 nm). The study was conducted using an open randomized 2-period crossover balanced design with a 1-week washout period between the doses. This simple, rapid and selective method is suitable for pharmacokinetic, bioavailability and bioequivalence studies.

Hospital doctors' views and concerns about pharmacovigilance

Purpose: The aim of the study was to evaluate the opinions and concerns of hospital doctors about adverse drug reactions (ADRs) and pharmacovigilance. Methods: A qualitative study was undertaken using focus groups in sessions on pharmacovigilance activities conducted in thirteen clinical services of a tertiary university hospital. A total of 296 physicians participated in these sessions by giving their opinions or expressing their doubts about ADR and pharmacovigilance activities which were recorded by different observers and subsequently analysed. Results: Doctors remarked on: a) the importance, concern, frequency and specific types of ADRs that were observed in clinical practice; b) problems of clinical decision making related to the suspected ADRs; c) methods for improving detection and reporting ADRs; d) monitoring of specific ADRs or ADRs caused by specific drugs; e) and measures to prevent and minimize the risk of ADRs. Physicians expressed doubts related to: a) the basic concepts of ADRs; b) the methods of ADR identification and evaluation; c) the objectives and procedures of pharmacovigilance programmes; d) and the impact of pharmacovigilance activities. Conclusions: Hospital doctors believe that ADRs are a matter for concern in their daily clinical practice, and monitoring ADRs as well as measures for preventing the risk of ADRs are needed. Nevertheless, doctors have doubts about what an ADR is, the accuracy of diagnostic methods, the development of pharmacovigilance activities and their impact on clinical practice. Pharmacovigilance should be better explained through a continuous feedback and close relationship with hospital doctors.

Performance characteristics of bioassay UV-spectrophotometry and high perfomance liquid chromatographic determination of gatifloxacin in tablets

The microbiological bioassay, UV-spectrophotometry and HPLC methods for assaying gatifloxacin in tablets were compared. Validation parameters such as linearity, precision, accuracy, limit of detection and limit of quantitation were determined. Beer's law was obeyed in the ranges 4.0-14.0 μg/mL for HPLC and UV-spectrophotometric method, and 4.0-16.0 μg/mL for bioassay. All methods were reliable within acceptable limits for antibiotic pharmaceutical preparations being accurate, precise and reproducible. The bioassay and HPLC are more specific than UV-spectrophotometric analysis. The application of each method as a routine analysis should be investigated considering cost, simplicity, equipment, solvents, speed, and application to large or small workloads.

Method validation and stability study of quercetin in topical emulsions

This study validated a high performance liquid chromatography (HPLC) method for the quantitative evaluation of quercetin in topical emulsions. The method was linear within 0.05 - 200 μg/mL range with a correlation coefficient of 0.9997, and without interference in the quercetin peak. The detection and quantitation limits were 18 and 29 ng/mL, respectively. The intra- and inter-assay precisions presented R.S.D. values lower than 2%. An average of 93% and 94% of quercetin was recovered for non-ionic and anionic emulsions, respectively. The raw material and anionic emulsion, but not non-ionic emulsion, were stable in all storage conditions for one year. The method reported is a fast and reliable HPLC technique useful for quercetin determination in topical emulsions.

Development and validation of a GC-FID assay for determination of fluvastatin in pharmaceutical preparations

A gas chromatographic method has been developed for the assay of fluvastatin sodium (FLU). FLU was silylated with N,O-bis(trimethylsilyl)trifluoroacetamide-1% trimethylchlorosilane at 90 ºC for 30 min and analysed in a DB-1 column by capillary gas chromatograph with a flame ionization detector. The method was validated. The assay was linear over the concentration range at 10.0 to 50.0 µg mL-1. The limit of detection and the limit of quantitation were 1.0 and 3.0 µg mL-1, respectively. The recoveries of FLU derivatives were in the range of 99.25-99.80%. In inter-day and intra-day analysis, the values of relative standard deviation (%) and the relative mean error (%) were found between 0.20-0.80% and -0.20-0.75%, respectively. The developed method was succesfully applied to analyze the FLU content in tablet formulation. The results were statistically compared with those obtained by the official method, and no significant difference was found between the two methods. Therefore, it can be recommended for the quality control assay of FLU in pharmaceutical industry.

Development and validation of UV spectrophotometric method for determination of levofloxacin in pharmaceutical dosage forms

The objective of this research was to develop and validate an alternative analytical method for quantitative determination of levofloxacin in tablets and injection preparations. The calibration curves were linear over a concentration range from 3.0 to 8.0 μg mL-1. The relative standard deviation was below 1.0% for both formulations and average recovery was 101.42 ± 0.45% and 100.34 ± 0.85% for tablets and injection formulations, respectively. The limit of detection and limit of quantitation were 0.08 and 0.25 μg mL-1, respectively. It was concluded that the developed method is suitable for the quality control of levofloxacin in pharmaceuticals formulations.

MSPD procedure combined with GC-MS for the determination of procymidone, bifenthrin, malathion and pirimicarb in honey

A method based on matrix solid-phase dispersion and gas chromatography-mass spectrometry to determine procymidone, malathion, bifenthrin and pirimicarb in honey is described. The best results were obtained using 1.0 g of honey, 1.0 g of silica-gel as dispersant sorbent and acetonitrile as eluting solvent. The method was validated by fortified honey samples at three concentration levels (0.2, 0.5 to 1.0 mg kg-1). Average recoveries (n=7) ranged from 54 to 84%, with relative standard deviations between 3.7 and 8.5%. Detection and quantification limits attained by the developed method ranged from 0.02 to 0.08 mg kg-1 and 0.07 to 0.25 mg kg-1 for the honey, respectively.

Space–Time Block Codes and the Complexity of Sphere Decoding

In wireless communications the transmitted signals may be affected by noise. The receiver must decode the received message, which can be mathematically modelled as a search for the closest lattice point to a given vector. This problem is known to be NP-hard in general, but for communications applications there exist algorithms that, for a certain range of system parameters, offer polynomial expected complexity. The purpose of the thesis is to study the sphere decoding algorithm introduced in the article On Maximum-Likelihood Detection and the Search for the Closest Lattice Point, which was published by M.O. Damen, H. El Gamal and G. Caire in 2003. We concentrate especially on its computational complexity when used in space–time coding. Computer simulations are used to study how different system parameters affect the computational complexity of the algorithm. The aim is to find ways to improve the algorithm from the complexity point of view. The main contribution of the thesis is the construction of two new modifications to the sphere decoding algorithm, which are shown to perform faster than the original algorithm within a range of system parameters.

Comparison of USEPA 3050B and ISO 14869-1: 2001 digestion methods for sediment analysis by using FAAS and ICP-OES quantification techniques

A study of the partial USEPA 3050B and total ISO 14869-1:2001 digestion methods of sediments was performed. USEPA 3050B was recommended as the simpler method with less operational risk. However, the extraction ability of the method should be taken in account for the best environmental interpretation of the results. FAAS was used to quantify metal concentrations in sediment solutions. The alternative use of ICP-OES quantification should be conditioned by a previous detailed investigation and eventual correction of the matrix effect. For the first time, the EID method was employed for the detection and correction of the matrix effect in sediment ICP-OES analysis. Finally, some considerations were made about the level of metal contamination in the area under study.

Development and validation of a method for the analysis of Ochratoxin A in roasted coffee by liquid chromatography/electrospray-mass spectrometry in Tandem (LC/ESI-MS/MS)

A method using LC/ESI-MS/MS for the quantitative analysis of Ochratoxin A in roasted coffee was described. Linearity was demonstrated (r = 0.9175). The limits of detection and quantification were 1.0 and 3.0 ng g-1, respectively. Trueness, repeatability and intermediate precision values were 89.0-108.8%; 2.4-13.7%; 12.5-17.8%, respectively. To the best of our knowledge, this is the first report in which Ochratoxin A in roasted coffee is analysed by LC/ESI-MS/MS, contributing to the field of mycotoxin analysis, and it will be used for future production of Certified Reference Material.

Performance characteristics of high performance liquid chromatography, first order derivative UV spectrophotometry and bioassay for fluconazole determination in capsules

The bioassay, first order derivative UV spectrophotometry and chromatographic methods for assaying fluconazole capsules were compared. They have shown great advantages over the earlier published methods. Using the first order derivative, the UV spectrophotometry method does not suffer interference of excipients. Validation parameters such as linearity, precision, accuracy, limit of detection and limit of quantitation were determined. All methods were linear and reliable within acceptable limits for antibiotic pharmaceutical preparations being accurate, precise and reproducible. The application of each method as a routine analysis should be investigated considering cost, simplicity, equipment, solvents, speed, and application to large or small workloads.

Indirect spectrophotometric method for determination of captopril using Cr(VI) and diphenylcarbazide

A spectrophotometric method for the indirect determination of captopril (CP) in pharmaceutical formulations is proposed. The proposed procedure is based on the oxidation of captopril by potassium dichromate and the determination excess oxidant on the basis of its reaction with diphenylcarbazide (DPC). Under the optimum conditions, a good linear relationship (r = 0.9997) was obtained in the range of 0.08-3.5 µg mL-1. The assay limits of detection and quantitation were 0.024 and 0.08 µg mL-1, respectively. The results obtained for captopril determination in pharmaceuticals using the proposed method and those obtained with the US Pharmacopoeia method were in good agreement at the 95% confidence level.