Supercritical fluid chromatography coupled to high-resolution tandem mass spectrometry: an innovative one-run method for the comprehensive assessment of chocolate quality and authenticity.

To assess chocolate quality and authenticity comprehensively, a combination of various analytical procedures is involved, thereby making the process time-consuming and costly. Thus, we investigated the potential of ultra-high performance supercritical fluid chromatography coupled to quadrupole-time of flight mass spectrometry (UHPSFC-QTOF-MS) as an alternative to "classic" methods. By combining hexane and aqueous extracts from sequential extraction, a single 8-min analytical run enabled us (i) to determine cocoa butter equivalents (CBEs) and milk fat content based on the detection of selected triacylglycerols, (ii) to calculate dry non-fat cocoa solids based on determined theobromine and caffeine content, and (iii) to profile contained sugars. To obtain the most comprehensive information about sample composition, the MS method comprised a full MS scan for non-target screening and several time-scheduled targeted MS/MS functions ("parallel reaction monitoring") optimized according to the possible concentration ranges of the analytes. For 40 different chocolate samples, our results and those obtained by using standard methods (LC-UV for non-fat cocoa solids, and GC-FID for CBEs) were in good agreement. Compared to the conventional approach for chocolate quality and authenticity control, the presented SFC-MS method is a fast, cost-effective, and efficient alternative, and only samples suspicious for the presence of CBE should be referred to the standard GC-FID method for exact CBE quantification. In the study, also some challenges offered by SFC-MS have been addressed.

ELISA kit for peanut protein determination: collaborative study.

A collaborative study in 10 laboratories was performed to validate an ELISA method developed for the quantitative determination of peanut protein in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. This kit does not produce any false-positive results or cross-reactivity with a broad range of peanut-free food matrixes. All participants obtained the peanut ELISA kit with standard operational procedures, a list of samples, the samples, and a protocol for recording test results. The study included 15 food samples. Three food matrix samples of zero peanut content showed peanut protein content lower than the first standard (0.10 mg/kg). Three samples with peanut declared as an ingredient revealed peanut protein content outside the calibration curve (absorbance was above the highest standard) in all laboratories, and three samples had the peanut content reported either above the highest standard or within the calibration curve, depending on the laboratory. Six samples with peanut declared as an ingredient gave the peanut protein content within the calibration curve. Only these six samples, together with a positive control sample (CS2), were used for statistical evaluation. The statistical tests (Cochran, Grubbs, and Mandel) and analysis of variance were used for the evaluation of the collaborative study results. Repeatability and reproducibility limits, as well as an LOQ (LOQcollaborative 0.22 mg peanut proteins/kg) and an LOD (LODcollaborative 0.07 mg peanut proteinslkg) for the kit were calculated.

ELISA kit for mustard protein determination: interlaboratory study.

An interlaboratory study in 12 laboratories was performed to prove the validation of the ELISA method developed for the quantitative determination of mustard protein in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. This kit did not produce any false-positive results or cross-reactivity with in-house validation for a broad range of food matrixes with no detectable mustard protein. All participants obtained the Mustard ELISA kit with standard operational procedures, a list of samples, samples, and a protocol for recording test results. The study included 15 food samples and two spiked samples. Seven food matrix samples of zero mustard content and four samples with mustard declared as an ingredient showed mustard protein content lower than that of the first standard (0.42 mg/kg). Four samples with mustard declared as an ingredient revealed mustard protein content above 12.5 mg/kg (the highest standard). The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as an LOQ (1.8 mg mustard proteins/kg) and LOD (0.5 mg mustard proteins/kg), for the kit were calculated.

ELISA kit for determination of egg white proteins: interlaboratory study.

An interlaboratory study was conducted in 11 laboratories to validate an ELISA method developed for the quantitative determination of egg white proteins (EWPs) in foods. The ELISA kit used for this study is based on sheep polyclonal antibody. It does not produce any false-positive results or cross-reactivity in a broad food matrix range with zero EWP content. All participants obtained the Egg ELISA Kit-native with standard operational procedure and the list of samples, as well as the samples and a protocol for recording test results. The study included 10 food samples. Four samples of food matrix with zero EWP content showed EWP content lower than the first standard (EWP content 0.5 mg/kg). One sample of food matrix with zero EWP content revealed EWP content higher than standard 3 (1.5 mg EWP/kg). Five food samples containing EWP as an ingredient tested positive and one negative. The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as LOQ (1.4 mg EWP/kg) and LOD (0.43 mg EWP/kg), were calculated for the kit.

ELISA kit for casein determination: interlaboratory study.

An interlaboratory study was performed in eight laboratories to validate an ELISA method developed for quantitative determination of casein in foods. The ELISA kit used is based on rabbit polyclonal antibodies. The kit is quite specific; no false-positive results or cross-reactivities were obtained for a broad range of food matrixes with zero content of milk proteins. All participants in the study received the casein kit, which included a standard operating procedure, a list of the samples, the samples, and a protocol for recording test results. The study included nine food samples: wheat flour, buckwheat flour, instant potato purée with milk, instant coffee with sugar and cream, a mixture for fancy bread, salami, liver paté, chocolate muesli with nuts, and a mixture for gluten-free bread. Three food samples with zero content of milk proteins showed a casein content lower than the lowest casein standard (1.0 mg CAS/kg) in most laboratories and measurements (64%). In 98% of the cases, the casein content was lower than the estimated LOQ. Two food samples with no dairy ingredient declared on the ingredient list contained casein levels higher than the second casein standard (3.0 mg CAS/kg) and the third standard (10.0 mg CAS/kg), respectively. Four food samples containing milk as an ingredient tested positive, and three showed casein contents higher than the highest standard (30.0 mg CAS/kg). The statistical tests (Cochran, Dixon) and analysis of variance were used for evaluation of the interlaboratory study results. Repeatability and reproducibility limits as well as LOQ (1.8 mg CAS/kg) and LOD (0.5 mg CAS/kg) for the kit were calculated from the results of the interlaboratory study.

Enzyme-linked immunosorbent assay kit for beta-lactoglobulin determination: interlaboratory study.

An interlaboratory study was performed in six laboratories to prove the validation of the ELISA method developed for quantitative determination of beta-lactoglobulin (BLG) in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. In-house validation of the kit did not produce false-positive results or cross-reactivity in a broad range of food matrixes containing no milk proteins. All participants obtained the BLG kit with a standard operational procedure, the list of the samples, samples, and a protocol for recording test results. The study included 14 food samples (extruded breakfast cereals, bread, two soy desserts, butter, chicken ham, chicken meat, wheat flour, long grain rice, jelly, two whey drinks, crackers, and bitter chocolate) and six spiked samples (two rice, two wheat flour, and two chicken meat). Nine samples of food matrixes containing no milk proteins showed BLG content lower than the first standard (0.15 mg/kg). Two samples of food matrixes with no milk proteins revealed BLG content higher than standard 3 (1.5 mg/100 g) and standard 4 (5.0 mg/100 g). Three food samples containing milk were tested as positive, and all spiked samples were evaluated as positive. The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as LOQ (0.22 mg BLG/kg) and LOD (0.07 mg BLG/kg), for the kit were calculated.

Analysis of phosphodiesterase type 5 inhibitors as possible adulterants of botanical-based dietary supplements: extensive survey of preparations available at the Czech market.

Popularity of natural-based preparations supporting the sexual potency significantly increased in recent years, which also led to the increase of illegal use of phosphodiesterase type 5 inhibitor (PDE-5) in sexual performance enhancement products. In this study, a rapid U-HPLC‒HRMS/MS method has been developed to simultaneously determine 59 PDE-5 inhibitors and their analogues. Within the development of sensitive method for analysis of 59 PDE-5 inhibitors and their analogues, both sample preparation procedure, as well as separation / detection conditions have been optimized. Extraction efficiency of particular extraction solvents, influence of different mobile phase additives on target analytes separation, as well as impact of various settings of mass analyzer on sensitivity of detection were examined. Data were collected in the 'full MS/data dependent MS/MS' acquisition mode (full MS-dd-MS/MS). Before the U-HPLC‒HRMS/MS method was used for analysis of real samples, proper validation had been conducted. The precision of the method expressed as the relative standard deviation (RSD) was ≤4.2% and ≤5.2% at spiking concentrations 5 µg/g and 0.25 µg/g, respectively. The limits of quantification were in the range 0.25 - 0.05 µg/g and the recovery ranged between 71 and 90%. The optimized method was successfully applied for analysis of 64 real samples, and 10 of them were proved to contain both registered or unregistered synthetic PDE-5 inhibitors. Additionally, the acquired U-HPLC‒HRMS/MS fingerprints were demonstrated to serve as an efficient tool for revealing of other type of possible fraud in products labeling. Retrospective mining of markers of herbs declared on dietary supplements packaging allowed to assess the trueness / untruth in the declaration of medical herbs composition.

In plastico: laboratory material newness affects growth and reproduction of Daphnia magna reared in 50-ml polypropylene tubes.

Plastic laboratory materials are found to affect vital parameters of the waterflea Daphnia magna. The main responsible factor is defined as "newness" of the materials. Juvenile D. magna were raised individually in; a) new laboratory-standard 50 ml polypropylene tubes, and; b) identical tubes which had been washed and aerated for several weeks. Newness had significant effects on growth and fecundity of D. magna. New tubes caused delayed maturation, reduced reproduction and reduced growth when compared to washed and re-used tubes of the same commercial brand. The findings indicate that newness of tubes has inhibiting or toxic effects on D. magna. Often laboratory plastics are intended for single-use due to sterility demands. Newness might be an important confounding factor in research results and should not be disregarded. Disposable plastic utensils may come with a seemingly ignored cost and induce adverse effects in biological test-organisms and systems. The presented findings accentuate continued need for general awareness concerning confounding factors stemming from material laboratory environment. Based on the present findings the authors suggest that plastics intended for use in sensitive research may need to be washed and aerated prior to use.

Gluten determination by gliadin enzyme-linked immunosorbent assay kit: interlaboratory study.

An interlaboratory study with 10 participants was performed to obtain validation and performance data for an enzyme-linked immunosorbent assay (ELISA) kit developed for quantitative gluten determination in foods. The ELISA kit used for this study is based on 2 monoclonal and 1 polyclonal antibody developed by Immunotech, a Beckman Coulter Co. This kit did not show any false positive results or cross-reactivity with oat, rice, maize, and buckwheat. The gliadin standard from the Working Group on Prolamin Analysis and Toxicity was included in the kit as reference material for calibration. All participants obtained a gliadin ELISA kit with Standard Operational Procedure and a form for recording test results. The study included 13 samples labeled as "gluten-free" and 2 samples spiked by wheat flour. Seven samples had gliadin content below the limit of quantitation (LOQ) of the method, and 1 sample exceeded the highest calibration level. Gliadin content in the range from 10 to 157 mg/kg (1st day) and from 11 to 183 mg/kg (2nd day) was found in 7 samples (including 2 spiked samples). Results of these samples were used for further statistical analysis and evaluation. The Cochran, Dixon, and Mandel statistical tests were applied for detection of outliers. The LOQ of the kit was estimated.

Deoxynivalenol and its conjugates in beer: a critical assessment of data obtained by enzyme-linked immunosorbent assay and liquid chromatography coupled to tandem mass spectrometry.

Enzyme-linked immunosorbent assays (ELISAs) are often employed for the control of deoxynivalenol (DON) in barley and other intermediates involved in beer production chain. Because of the occurrence of high levels of DON-3-glucoside (DON-3-Glc) in malt and beer that have been reported for the first time in our earlier study, research focused on the accuracy of DON determination by immunoassays in cereal-based matrices has been initiated. DON-3-Glc was strongly cross-reacting in all examined commercial ELISA test kits (Ridascreen) DON (R-Biopharm), Veratox 5/5 DON) (Neogen Corporation), Deoxynivalenol EIA (Euro-Diagnostica), and AgraQuant) DON Assay 0.25/5.0 Test Kit (Romer Labs). The highest overestimation in beer analysis, up to 1000%, when taking the DON content determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) as a reference method, was obtained by AgraQuant assay. Besides of DON-3-Glc and 3- and 15-acetyldeoxynivalenol (ADONs), also other, not known yet, matrix components contributed to false positive results. Similar phenomenon, although in a lesser extent due to lower content of these substances, was observed for using ELISA in the analysis of wheat. The relationship between a way of sample handling and DON overestimation was demonstrated; higher ELISA response was measured in an aqueous extract compared to that prepared by acetonitrile-water (84:16, v/v). Most of cross-reacting co-extracts were removed by MycoSep# 226 cartridge, what leads us to the hypothesis on the presence of currently unknown cross-reactive species.