Importance of REP values when comparing the CALUX bioassay results with chemoanalyses results Example with spiked vegetable oils

Differences between chemical activated luciferase gene expression (CALUX) bioassay and chemoanalyses results are observed.This paper shows that calculations of the TEQ values using REP values instead of WHO TEF values give different results. The REP values do affect the results obtained by the CALUX technique. These differences are more marked for the dioxin like PCB compounds (CALUX TEQ values are lower than WHO TEQ values) than for the dioxin compounds (CALUX TEQ values are higher than WHO TEQ values).The CALUX results were compared with the concentrations of the congeners’ spiked into the oil.     

Importance of REP values when comparing the CALUX bioassay results with chemoanalyses results: Example with spiked vegetable oils

Differences between chemical activated luciferase gene expression (CALUX) bioassay and chemoanalyses results are observed.

This paper shows that calculations of the TEQ values using REP values instead of WHO TEF values give different results. The REP values do affect the results obtained by the CALUX technique. These differences are more marked for the dioxin like PCB compounds (CALUX TEQ values are lower than WHO TEQ values) than for the dioxin compounds (CALUX TEQ values are higher than WHO TEQ values).

The CALUX results were compared with the concentrations of the congeners’ spiked into the oil.     

TEQ-value determinations of animal feed; emphasis on the CALUX bioassay validation

Polyhalogenated aromatic hydrocarbons, such as polychlorinated dibenzo-p-dioxins are a large and diverse group of environmental pollutants. Their tendency to accumulate in the food chain and their toxicity make monitoring necessary. The reference analysis method is laborious and very expensive, therefore cheap and rapid bioassays have been developed. The chemical-activated luciferase bioassay (CALUX) bioassay uses a recombinant cell line, which responds to dioxins and dioxin-like molecules with Ah receptor (AhR)-dependent induction of firefly luciferase in a dose related response. The CALUX was tested for its use in the screening of feed. Aliquots of 20 g of enriched feed were extracted with a toluene:methanol mixture (20:4 v/v) and extracts were defatted on 33% H₂SO₄ silica columns and purified on carbon columns. Only the dioxin and furan fraction was analysed, the PCB fraction was discarded. The precision of the method is acceptable and in compliance with an R.S.D. <30% as suggested for cell-based bioassays in the Commission Directive 2002/70/EC of July 2002. The results evidence good agreement between TEQ-values obtained by either CALUX or GC–HRMS. The method is now routinely in use for a feed screening programme designed by the Federal Agency for the Safety of the Food chain. Approximately, 25 samples are analysed weekly. From the obtained results approximately 10% was confirmed by GC–HRMS. The false positive ratio is 1% and no false negatives were found, making the use of the CALUX technology advantageous.     

Considerations for potency equivalent calculations in the Ah receptor-based CALUX bioassay: normalization of superinduction results for improved sample potency estimation

The chemically activated luciferase expression (CALUX) system is a mechanistically based recombinant luciferase reporter gene cell bioassay used in combination with chemical extraction and clean-up methods for the detection and relative quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like halogenated aromatic hydrocarbons in a wide variety of sample matrices. While sample extracts containing complex mixtures of chemicals can produce a variety of distinct concentration-dependent luciferase induction responses in CALUX cells, these effects are produced through a common mechanism of action (i.e. the Ah receptor (AhR)) allowing normalization of results and sample potency determination. Here we describe the diversity in CALUX response to PCDD/Fs from sediment and soil extracts and not only report the occurrence of superinduction of the CALUX bioassay, but we describe a mechanistically based approach for normalization of superinduction data that results in a more accurate estimation of the relative potency of such sample extracts.     

Enhancing the response of CALUX and CAFLUX cell bioassays for quantitative detection of dioxin-like compounds

Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications.Over the past 10 years,reporter gene assays have been widely accepted and used for analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like compounds in various types of matrices,such as biological,environmental,food and feed samples,given that high-resolution instrumental analysis techniques ...     

Validation of the CALUX bioassay for PCDD/F analyses in human blood plasma and comparison with GC-HRMS

Following the dioxin crisis of 1999, several studies were conducted to assess the impact of this crisis on the dioxin body burden in the Belgian population. The Scientific Institute of Public Health identified a population from whom plasma samples were available and from whom, during the follow up survey, plasma samples were obtained in 2000. In total, 496 samples were collected for GC-HRMS and CALUX analyses to verify statistical assessment conclusions. This study was seen as an opportunity to validate the CALUX bioassay for biological sample analysis and to compare toxic equivalency (TEQ) values obtained by the reference GC-HRMS technique and by the screening method. This article focuses on the validation results of the CALUX bioassay for the analyses of the dioxin fractions of blood plasma. The sample preparation is based on a liquid–liquid extraction, followed by an acid silica in series with an activated carbon clean-up. A good recovery (82%) and reproducibility (coefficient of variation less than 25%) were found for this method. Based on 341 plasma samples, a significant correlation was established between the bioassay and chemical method (R = 0.64). However, a proportional systematic error was observed when the results obtained with the CALUX bioassay were regressed with the results from the GC-HRMS analyses. The limit of quantification (LOQ) used to calculate TEQ values from the GC-HRMS determinations, the use of the relative potency values instead of the toxic equivalent factor and the potential of CALUX bioassay to measure all compounds with affinity for the AhR may partly explain this proportional systematic error. Nevertheless, the present results suggest that the CALUX bioassay could be a promising valid screening method for human blood plasma analyses.     

Study of the interference problems of dioxin-like chemicals with the bio-analytical method CALUX

The reporter gene expression method CALUX has proven to be a very valuable screening technique for assessing toxic equivalents of dioxins and dioxin-like compounds, because it detects all AhR ligands in a variety of sample matrices. However, the exact meaning of the CALUX response is difficult to evaluate for complex mixtures mainly since not all AhR ligands are known and since antagonistic or synergistic effects occurs.

In this paper, non-additive effects on the CALUX response of dioxins were investigated for a limited number of dioxin-like compounds in concentration ranges that are 10²–10⁸ times higher than that of 2,3,7,8-TCDD. Antagonistic effects are detected for three Aroclors (1242, 1254, 1260), Halowax 1014 (PCN), HCB and PBB 169. The ratios, Aroclor/dioxin, Halowax/dioxin and HCB/dioxin, needed to observe an antagonistic effect are 10 000, 5000 and 50 000, respectively. No significant deviation from additivity was observed for Aroclor 5442 (PCT) and PBB 77 in the concentration range investigated.

Two clean-up procedures have also been tested: in some cases the non-additive effects disappeared or were strongly reduced. Using only an acidic silica column, the classical dioxin-like compounds investigated here (PCB, PCT, PBB, PCN, HCB) as well as the dioxins are collected and analyzed altogether in one fraction. Consequently, no major alteration of the non-additive effects is expected. An acidic silica column combined with an activated carbon column allows the separation of PCDD/F and dioxin-like PCB in two different fractions, PBB 169 is completely eluted in the dioxin fraction and PBB 77 is distributed between the PCB and dioxin fraction. HCB is completely separated from the PCDD/F fraction.     

A review of analytical methods for the determination of aminoglycoside and macrolide residues in food matrices

The development of antibiotic resistance in bacteria has been attributed to the overuse of antimicrobials in human medicine. Another route by which humans are exposed to antibiotics is through the animal foods we eat. In modern agricultural practice, veterinary drugs are being used on a large scale, administered for treating infection or prophylactically to prevent infection. Hence, there is pressure on analytical scientists to detect and confirm the presence of antimicrobials in foods of animal origin.The aminoglycosides and macrolides are two families of antibiotics, each with important applications in veterinary medicine. These antibiotics are widely used in the treatment of bacterial disease, e.g., aminoglycosides for mastitis and macrolides for enteric infections. They have also been used as feed additives for growth promotion. As a result, legislation has been laid down by the European commission in which member states must meet strict criteria for monitoring residues (including antimicrobials). Testing for low levels of aminoglycosides and macrolides in foods is a priority and hence the development of fast, reliable, sensitive methods for their extraction and subsequent analysis is of great interest.This paper reviews analytical methods for both extracting and determining these classes of antibiotics in various food matrices focusing in particular on the last 10 years. Extraction and clean-up methods such as deproteinisation, and solid-phase extraction are described. Various screening methods are also covered including thin layer chromatography (TLC), enzyme immunoassay, capillary electrophoresis (CE) and microbiological assays. Finally, liquid chromatography (LC) methods are discussed which are combined with mass spectrometry (MS) when sensitivity requirements are stringent.     

A multivariate multianalyte screening method for sulfonamides in milk based on front-face fluorescence spectroscopy

Screening methods are used to detect the presence of a substance or class of substances at the level of interest and are specifically designed to avoid false compliant results. They should allow the running of a high number of samples per day at a low cost under routine conditions. In this work, a rapid and simple method for the screening of six sulfonamides (sulfadiazine, SD; sulfamerazine, SMR; sulfamethazine, SMT; sulfachloropyridazine, SCP; sulfathiazole, STZ and sulfamethoxazole, SMO) in milk samples is proposed and assessed according to the criteria required by the European Regulation, Decision 2002/657/EC. The method is based on modelling front-face fluorescence emission spectra by means of partial least squares class modelling (PLS-CM). The milk samples are pre-treated with a single easy step of derivatization with fluorescamine.After confirming that the method has equal analytical sensitivity for all the six sulfonamides, it is established that the multivariate analytical sensitivity at 100 μg L⁻¹ is 37.5 μg L⁻¹ when analysing a mixture of six sulfonamides added to different brands of milk and measured in different days. In addition, the method is applied to samples from 11 commercial brands of milk. For β = 0.05, threshold value established by the Decision 2002/657/EC for this method, the probability of false non-compliance, α, is equal to 0.17, allowing the suitable screening of these six sulfonamides.     

Effective monitoring for ractopamine residues in samples of animal origin by SPR biosensor and mass spectrometry

Ractopamine (RCT) is a member of the β-2-agonist (β-agonist) family. It is licensed for use as an animal growth promoter in more than 20 countries worldwide, including the United States and Canada, but is either not licensed or prohibited by over 150 others, including those within the European Union. The issue of the use of RCT in livestock bound for human consumption has risen to prominence recently following the decision by The People's Republic of China to ban the import of pork from a number of processing plants after finding traces of RCT in shipments from the U.S.A.In order to monitor for the illegal use of such compounds within Europe, there is a requirement to have a robust and reliable testing scheme capable of the detection of low concentrations of RCT. In the present study an optical biosensor screening assay was developed. The developed assay was compared with a liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) confirmatory procedure. These methods were used to study the ability to detect RCT in pigs following treatment. Both testing procedures were capable of detecting low μg kg⁻¹ concentrations of the drug in urine and liver. Liver was found to be a less suitable sample matrix, with RCT residue levels being undetectable after 5 days withdrawal of the drug. Urine samples however still contained detectable RCT residues several weeks after withdrawal. The correlation (as measured by r²) between the biosensor and LC-MS/MS methods was 0.99 and 0.97 for urine and liver samples, respectively.It is concluded that testing regimes based on RCT analysis in liver are less likely to detect illegal administration of the drug than those based on urine analysis. Urine samples provide an excellent matrix for the detection of RCT residues for an extended period post withdrawal.     

Screening of antibiotics in surface and wastewater samples by ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry

The potential of ultra-high-pressure liquid chromatography (UHPLC) coupled to hybrid quadrupole time-of-flight mass spectrometry (QTOF–MS) for the screening and confirmation of antibiotics in water samples is illustrated in this paper. UHPLC presents several advantages over conventional liquid chromatography as it generates narrow peaks (increasing peak height and improving sensitivity) and reduces chromatographic runs. Regarding QTOF–MS, its increased mass resolution, high sensitivity in full-spectrum acquisition mode and mass accuracy, in both MS and MS/MS modes, make this technique ideal for the detection and reliable confirmation of organic contaminants in environmental samples. After a solid-phase extraction using Oasis HLB cartridges, UHPLC–QTOF–MS has been applied in this work to several types of water samples (surface water and influent and effluent wastewaters). Several antibiotics were found in the samples, such as ofloxacin, ciprofloxacin, clarythromycin or erythromycin, among others. Moreover, the full spectrum data provided by TOF–MS acquisition has enabled searching for many other pharmaceuticals that could be present in the samples in a “post-target” way. This approach has allowed the detection and confirmation of paracetamol, omeprazole and codeine, among others. UPLC–QTOF–MS has been shown as an attractive and efficient hyphenated technique for the rapid detection and confirmation of pharmaceuticals in water with very little sample handling.     

Detection limits and quantitation limits in the view of international harmonization and the consequences for analytical laboratories

 In the literature and in daily work there exist many different models to describe detection limits and quantitation limits in the chemical measurement process (CMP). It is still an open question whether the evaluation of detection and quantitation limits should be an one-off process carried out during the validation of a CMP, or whether it should be a continuous process. Because of these reasons many laboratories have difficulties to interprete such values. It is necessary that the official standardization bodies should do some efforts in the international harmonization of the special standards concerning detection and quantitation limits. Received: 3 November 1997 · Accepted: 25 January 1998     

A view of uncertainty at the bench analytical level

 The problem with which analytical laboratories are confronted, after traceability of their results has been demonstrated, is correctly estimating their uncertainty– to which traceability is also to some extent subject. While the general principles for calculating the uncertainty of physical measurements are applicable to chemical metrology, some refinements are needed, especially careful selection and planning the level at which uncertainty will be estimated by each laboratory in accordance with its capacity and required demands. Depending on the particular decision to be made, the mechanism to be used to estimate the uncertainty varies markedly; also, the rigour of the estimation increases with increasing stringency of the demands. This paper describes the primary sources of uncertainty in chemical metrology and discusses different approaches to its estimation in relation to the type of analytical laboratory concerned. The view presented tries to be close to the bench analytical level, in order to be practical and flexible for laboratories, although it could sometimes be considered slightly heterodox. Received: 25 March 1997 · Accepted: 20 September 1997     

Direct screening of tetracyclines in water and bovine milk using room temperature phosphorescence detection

A fast and simple flow-through optosensor was designed and characterized for the direct screening of four tetracycline (TCC) antibiotics (tetracycline, oxytetracycline, chlortetracycline and doxycycline) in water and bovine milk samples. The proposed optosensor provides rapid binary yes/no overall responses, being appropriate for the screening of this family of antibiotics above or below a pre-set concentration threshold.The experimental set-up is based on a flow-injection manifold coupled on-line to a phosphorescence detector. Aliquots of the samples are pretreated with Eu(III) to form room temperature phosphorescent metal chelates and injected in the flow manifold. Those chelates are then on-line retained on a conventional flow-cell (packed with polymeric Amberlite XAD-4 particles) which is placed inside the cell holder of the phosphorimeter. After the emission is registered, the antibiotic-metal complexes are eluted from the packed resin with 1 M HCl (for milk samples a second regeneration step, using methanol, should be performed). A sample throughput of about 20 samples per hour was obtained. Optimum experimental conditions include a pH 9, a Eu(III) concentration of 2 × 10⁻⁴ M and 8 mM sodium sulphite as chemical deoxygenant. The phosphorescence emitted by the europium-TCC complexes was measured at 394 and 617 nm for excitation and emission wavelengths, respectively.The unreliability region, given by the probability of false positives and false negatives, respectively (set at 5% in both cases) was in the range between 0.2 and 11.6 nМ for detection of tetracyclines in water samples (at a cut-off level of 4 nM) and in the range between 165 and 238 nM for detection of tetracyclines in milk (cut-off level fixed at the normative EU level of 200 nM). Finally, the applicability of the proposed screening optosensor was tested for the reliable control of tetracyclines in contaminated and uncontaminated water and milk samples.     

Tutorial on estimating the limit of detection using LC-MS analysis,part I: Theoretical review

A large body of literature exists on the limit of detection (LOD), but there is still a lot of confusion about this important validation parameter. This confusion mainly stems from its statistically complex background. The goal of this two-part tutorial is to discuss and clarify the topic of LOD for practitioners. The two main conclusions of this tutorial are: (1) the choice of how to estimate LOD should be based on the purpose of the analytical method that is being validated (e.g. considerable effort should not be made to estimate LOD for a method that is not used for detecting traces in the vicinity of LOD), and (2) LOD estimates are strongly dependent on different assumptions and the approach used, and therefore caution must be exercised when using the estimate or when comparing different estimates.     

Tutorial on estimating the limit of detection using LC-MS analysis,part II: Practical aspects

In part II of this tutorial, the investigated approaches of estimating the limit of detection (LOD) are applied to experimental data from LC-MS measurements. Important practical aspects specific to LC-MS and related to LOD are reviewed. The results of different tests of estimating linearity and scedasticity are compared. LOD estimates obtained with different approaches (for both simple characterization of the analysis method and accurate interpretation of the results) are applied to the data and the obtained values are compared. As a conclusion, a decision tree is proposed for estimating LOD for analytical methods using the LC-MS technique.     

Rapid determination of sulfonamides in milk samples using fluorescence spectroscopy and class modeling with n-way partial least squares

In this paper, a methodology to evaluate the probability of false non-compliance and false compliance for screening methods, which give first or second-order multivariate signals is proposed. For this task 120 samples of 6 different kinds of milk have been measured by excitation-emission fluorescence. The samples have been spiked with different amounts of three sulfonamides (sulfadiazine, sulfamerazine and sulfamethazine). These substances have been classified in group B1 (veterinary medicines and contaminants) of annex I of Directive 96/23/EC. The European Union (Commission Regulation EC no. 281/96) has set the maximum residue level (MRL) of total sulfonamides at 100 μg kg⁻¹ in muscle, liver, kidney and milk.The work shows that excitation-emission fluorescence together with the partial least squares class modeling (PLS-CM) procedure may be a suitable and cheap screening method for the total amount of sulfonamides in milk. Three models, PLS-CM, have been built, for the emission and excitation spectra (first-order signals) and for the excitation-emission matrices (second-order signals). In all the cases it reaches probabilities of false compliance below 5% as required by Decision 2002/657/EC.With the same flourescence signals, the total quantity of sulfonamide was calibrated using 2-PLS, 3-PLS and PARAFAC regressions. Using this quantitative approach, the capability of detection, CCβ, around the MRL has been estimated between 114.3 and 115.1 μg kg⁻¹ for a probability of false non-compliance and false compliance equal to 5%.     

Optimization of analytical conditions and validation of a fluorescence method for the determination of sulfadiazine in milk

This paper describes optimization and validation of a method for sulfadiazine determination in milk samples based on sulfadiazine derivatization with fluorescamine followed by excitation–emission (fluorescence) measurement. For both the optimization and the validation, a comparison between zero-order and first-order signals has been made, showing the advantages of using first-order signals. In the optimization the effects of the temperature of the derivatization reaction, the amount of fluorescamine and the derivatization time on the instrumental signal (maximum intensity or the net analyte signal) are studied by a factorial experimental design, with the optimal values of these factors which give the highest signal being 22 °C for the reaction temperature, 50 μl fluorescamine and 20 min of derivatization time. The validation of the method under the optimal experimental conditions shows that the analytical method is fit-for-purpose, with values of the capability of detection (CCβ) of 4.3 μg l⁻¹ at a sulfadiazine concentration of zero and with probabilities of a false positive and a false negative of 5%. Around the permitted limit (established for the sulfonamides at 100 μg l⁻¹), CCβ is 112 μg l⁻¹. The precision, as the intermediate reproducibility, was established as 1.2 and 3.3 μg l⁻¹ around 0 and 100 μg l⁻¹, respectively. In the application to milk samples spiked with sulfadiazine a mean recovery of around 90% was obtained with a standard deviation of about 8% (14 samples of different concentrations).     

Rapid screening detection of fluoroquinolone residues in milk based on turn-on fluorescence of terbium coordination polymer nanosheets

Trace fluoroquinolone residues in milk could be detected based on turn-on fluorescence of AMP/Tb CPNSs. It might provide a new platform for the rapid detection of antibiotic pollutants with the advantages of simple sample pretreatment processes and excellent selectivity.     

Electrochemical sensor for the detection and presumptive identification of quinolone and tetracycline residues in milk

A novel application of an electrochemical biosensor is here employed as analytical method for the detection and presumptive identification of antimicrobial drug residues in milk. The measurement was based on carbon dioxide production rate in relation to inhibition of microbial grow (Escherichia coli ATCC 11303). In this pilot study quinolone and tetracycline residues have been taken into consideration because use of these last in livestock production has been identified as area of particular concern. The experimental approach and analytical method developed appear adequate for the purpose, and compared to older screening methods as, for example, the microbial inhibition assays and immunoassays, offers the advantages of (i) very short analysis time (about 120 min); (ii) smaller sample amount (approximately 0.5 mL); (iii) no sample treatment (iv) good precision; and (v) the possibility of following, in a continuous manner, the inhibition process. Moreover, sensitivity of electrochemical biosensor system is resulted very high considering that for all quinolones and tetracyclines investigated it has been possible detect a residue concentration below or equal to 25 μg L⁻¹. Under this point of view, it must be considered that the maximum residue limits fixed by UE for quinolones and tetracyclines in milk are, at present, all higher of this concentration.