A new green approach to L-histidine and ß-alanine analysis in dietary supplements using rapid and simple contactless conductivity detection integrated with high-resolution glass-microchip electrophoresis.

The analysis of dietary supplements is far less regulated than pharmaceuticals, leading to potential quality issues. Considering their positive effect, many athletes consume supplements containing L-histidine and ß-alanine. A new microfluidic method for the determination of L-histidine and ß-alanine in dietary supplement formulations has been developed. For the first time, capacitively coupled contactless conductivity detection was employed for the microchip electrophoresis of amino acids in real samples. A linear relationship between detector response and concentration was observed in the range of 10-100 µmol L-1 for L-histidine (R2 = 0.9968) and ß-alanine (R2 = 0.9954), while achieved limits of detection (3 × S/N ratio) were 4.2 µmol L-1 and 5.2 µmol L-1, respectively. The accuracy of the method was confirmed using recovery experiments as well as CE-UV-VIS and HPLC-UV-VIS techniques. The developed method allows unambiguous identification of amino acids in native form without chemical derivatization and with the possibility of simultaneous analysis of amino acids with metal cations.

Electrophoretic Determination of L-Carnosine in Health Supplements Using an Integrated Lab-on-a-Chip Platform with Contactless Conductivity Detection.

The health supplement industry is one of the fastest growing industries in the world, but there is a lack of suitable analytical methods for the determination of active compounds in health supplements such as peptides. The present work describes an implementation of contactless conductivity detection on microchip technology as a new strategy for the electrophoretic determination of L-carnosine in complex health supplement formulations without pre-concentration and derivatization steps. The best results were obtained in the case of +1.00 kV applied for 20 s for injection and +2.75 kV applied for 260 s for the separation step. Under the selected conditions, a linear detector response of 5 × 10-6 to 5 × 10-5 M was achieved. L-carnosine retention time was 61 s. The excellent reproducibility of both migration time and detector response confirmed the high precision of the method. The applicability of the method was demonstrated by the determination of L-carnosine in three different samples of health supplements. The recoveries ranged from 91 to 105%. Subsequent analysis of the samples by CE-UV-VIS and HPLC-DAD confirmed the accuracy of the obtained results.

The 1,3-Dioctadecyl-1H-imidazol-3-ium Based Potentiometric Surfactant Sensor for Detecting Cationic Surfactants in Commercial Products.

A low-cost and fast potentiometric surfactant sensor for cationic surfactants, based on the new ion-pair 1,3-dioctadecyl-1H-imidazol-3-ium-tetraphenylborate (DODI-TPB), is presented. The new cationic surfactant DODI-Br was synthesized and characterized by NMR, LC-MS, and elemental analysis, and was used for synthesis of the DODI-TPB ionophore. The DODI-TPB surfactant sensor was obtained by implementation of the ionophore in PVC. The sensor showed excellent response characteristics with near-Nernstian slopes to the cationic surfactants DMIC, CPC, CTAB, and Hyamine 1622. The highest voltage responses were obtained for DMIC and CPC (58.7 mV/decade of activity). DMIC had the lowest detection limit (0.9 × 10-6 M) and the broadest useful linear concentration range (1.8 × 10-6 to 1.0 × 10-4 M). An interference study showed remarkable stability. Potentiometric titration curves for the titration of cationic surfactants (DMIC, CPC, CTAB, and Hyamine 1622), with DDS and TPB used as titrants, showed sigmoidal curves with well-defined inflexion points and a broad signal change. The standard addition method was successfully applied with recovery rates from 98.9 to 101.2 at two concentrations. The amount of cationic surfactant found in disinfectants and antiseptics was in good agreement with the referent two-phase titration method and the surfactant sensor on the market. This new surfactant sensor represents a low-cost alternative to existing methods for cationic surfactant detection.

Fungi and their secondary metabolites in water-damaged indoors after a major flood event in eastern Croatia.

In winter and summer of 2016 and 2017, airborne fungi and house dust were collected in indoors of the village Gunja, which had been flooded, and the control village Gornji Stupnik (Croatia) in order to explore variations of fungal indoor levels, particularly Aspergilli section Nidulantes series Versicolores, as well as fungal metabolites in dust. Levels of airborne Aspergilli (Versicolores) were three times as high in winter and summer in Gunja than in the control village, while dustborne isolates were equally present in both locations. Sequencing of the calmodulin gene region revealed that among Aspergilli (Versicolores), A. jensenii and A. creber were dominant and together with A. puulaauensis, A. tennesseensis and A. venenatus produced sterigmatocystin and 5-methoxysterigmatocystin (HPLC coupled with mass spectrometry); A. amoenus, A. fructus, A. griseoaurantiacus, A. pepii, and A. protuberus produced sterigmatocystin but not 5-methoxysterigmatocystin; A. sydowii did not produce any of these toxins. A total of 75 metabolites related to Penicillium (29), Aspergillus (22), Fusarium (10), Alternaria (5), Stachybotrys (2), and other fungi (7) were detected in dust by liquid chromatography-tandem mass spectrometry. The majority of metabolites including sterigmatocystin and 5-methoxysterigmatocystin exhibited a higher prevalence in winter in Gunja.

Direct Potentiometric Study of Cationic and Nonionic Surfactants in Disinfectants and Personal Care Products by New Surfactant Sensor Based on 1,3-Dihexadecyl-1H-benzo[d]imidazol-3-ium.

A novel, simple, low-cost, and user-friendly potentiometric surfactant sensor based on the new 1,3-dihexadecyl-1H-benzo[d]imidazol-3-ium-tetraphenylborate (DHBI-TPB) ion-pair for the detection of cationic surfactants in personal care products and disinfectants is presented here. The new cationic surfactant DHBI-Br was successfully synthesized and characterized by nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectrometry, liquid chromatography-mass spectrometry (LC-MS) and elemental analysis and was further employed for DHBI-TPB ion-pair preparation. The sensor gave excellent response characteristics for CTAB, CPC and Hyamine with a Nernstian slope (57.1 to 59.1 mV/decade) whereas the lowest limit of detection (LOD) value was measured for CTAB (0.3 × 10-6 M). The sensor exhibited a fast dynamic response to dodecyl sulfate (DDS) and TPB. High sensor performances stayed intact regardless of the employment of inorganic and organic cations and in a broad pH range (2-11). Titration of cationic and etoxylated (EO)-nonionic surfactant (NSs) (in Ba2+) mixtures with TPB revealed the first inflexion point for a cationic surfactant and the second for an EO-nonionic surfactant. The increased concentration of EO-nonionic surfactants and the number of EO groups had a negative influence on titration curves and signal change. The sensor was successfully applied for the quantification of technical-grade cationic surfactants and in 12 personal care products and disinfectants. The results showed good agreement with the measurements obtained by a commercial surfactant sensor and by a two-phase titration. A good recovery for the standard addition method (98-102%) was observed.

Potentiometric Surfactant Sensor Based on 1,3-Dihexadecyl-1H-benzo[d]imidazol-3-ium for Anionic Surfactants in Detergents and Household Care Products.

A 1,3-dihexadecyl-1H-benzo[d]imidazol-3-ium-tetraphenylborate (DHBI-TPB) ion-pair implemented in DHBI-TPB surfactant sensor was used for the potentiometric quantification of anionic surfactants in detergents and commercial household care products. The DHBI-TPB ion-pair was characterized by FTIR spectroscopy and computational analysis which revealed a crucial contribution of the C-H∙∙∙π contacts for the optimal complex formation. The DHBI-TPB sensor potentiometric response showed excellent analytical properties and Nernstian slope for SDS (60.1 mV/decade) with LOD 3.2 × 10-7 M; and DBS (58.4 mV/decade) with LOD 6.1 × 10-7 M was obtained. The sensor possesses exceptional resistance to different organic and inorganic interferences in broad pH (2-10) range. DMIC used as a titrant demonstrated superior analytical performances for potentiometric titrations of SDS, compared to other tested cationic surfactants (DMIC > CTAB > CPC > Hyamine 1622). The combination of DHBI-TPB sensor and DMIC was successfully employed to perform titrations of the highly soluble alkane sulfonate homologues. Nonionic surfactants (increased concentration and number of EO groups) had a negative impact on anionic surfactant titration curves and a signal change. The DHBI-TPB sensor was effectively employed for the determination of technical grade anionic surfactants presenting the recoveries from 99.5 to 101.3%. The sensor was applied on twelve powered samples as well as liquid-gel and handwashing home care detergents containing anionic surfactants. The obtained results showed good agreement compared to the outcomes measured by ISE surfactant sensor and a two-phase titration method. The developed DHBI-TPB surfactant sensor could be used for quality control in industry and has great potential in environmental monitoring.

Application of Spectrophotometric Fingerprint in Cluster Analysis for Starch Origin Determination.

The botanical origin of starch is of importance in industrial applications and food processing because it may influence the properties of the final product. Current microscopic methods are time-consuming. Starch consists of an origin-dependent amylose/amylopectin ratio. Triiodide ions bind characteristically to the amylose and amylopectin depending on the botanical origin of the starch. The absorbance of the starch-triiodide complex was measured for: wheat, potato, corn, rye, barley, rice, tapioca and unknown origin starch; and within the different cultivars. Each starch sample had specific parameters: starch-triiodide complex peak wavelength maximum (λ max/nm), maximum absorbance change at λ max (ΔA) and λ max shift towards the unknown origin starch sample values. The visible absorption spectra (500-800 nm) for each starch sample were used as a unique fingerprint, and then elaborated by cluster analysis. The cluster analysis managed to distinguish data of two clusters, a cereal type cluster and a potato/tapioca/rice starch cluster. The cereal subclusters extensively distinguished wheat/barley/rye starches from corn starches. Data for cultivars were mostly in good agreement within the same subclaster. The proposed method that combines cluster analysis and visible absorbance data for starch-triiodide complex was able to distinguish starch of different botanical origins and cultivars within the same species. This method is simpler and more convenient than standard time-consuming methods.

The Fusarium metabolite culmorin suppresses the in vitro glucuronidation of deoxynivalenol.

Glucuronidation is a major phase II conjugation pathway in mammals, playing an important role in the detoxification and biotransformation of xenobiotics including mycotoxins such as deoxynivalenol (DON). Culmorin (CUL), a potentially co-occurring Fusarium metabolite, was recently found to inhibit the corresponding detoxification reaction in plants, namely DON-glucoside formation, raising the question whether CUL might affect also the mammalian counterpart. Using cell-free conditions, CUL when present equimolar (67 µM) or in fivefold excess, suppressed DON glucuronidation by human liver microsomes, reducing the formation of DON-15-glucuronide by 15 and 50%, and DON-3-glucuronide by 30 and 50%, respectively. Substantial inhibitory effects on DON glucuronidation up to 100% were found using the human recombinant uridine 5'-diphospho-glucuronosyltransferases (UGT) 2B4 and 2B7, applying a tenfold excess of CUL (100 µM). In addition, we observed the formation of a novel metabolite of CUL, CUL-11-glucuronide, identified for the first time in vitro as well as in vivo in piglet and human urine samples. Despite the observed potency of CUL to inhibit glucuronidation, no significant synergistic toxicity on cell viability was observed in combinations of CUL (0.1-100 µM) and DON (0.01-10 µM) in HT-29 and HepG2 cells, presumably reflecting the limited capacity of the tested cell lines for DON glucuronidation. However, in humans, glucuronidation is known to represent the main detoxification pathway for DON. The present results, including the identification of CUL-11-glucuronide in urine samples of piglets and humans, underline the necessity of further studies on the relevance of CUL as a potentially co-occurring modulator of DON toxicokinetics in vivo.

Simultaneous Determination of Acrylamide and Hydroxymethylfurfural in Extruded Products by LC-MS/MS Method.

The aim of this study was to develop a liquid chromatography/tandem mass spectrometry LC-MS/MS method for the simultaneous determination of acrylamide and hydroxymethylfurfural (HMF) in corn snack products enriched with food industry by-products: brewer's spent grain (BSG), sugar beet pulp (SBP) and apple pomace (AP). Development of the method included the study of different sources for ionization, different mobile phases, different extraction conditions as well as different methods of sample preparation. Finally, the single LC-MS/MS method was developed for the analysis of both analytes in one step with a duration of 20 min using a simple single-step extraction. The method with apparent recoveries of 91.4 and 90.4 for acrylamide and HMF, respectively, was applied for the analysis of non-extruded and extruded samples. The obtained results shown that the acrylamide content was

Chemical Diversity of Codium bursa (Olivi) C. Agardh Headspace Compounds, Volatiles, Fatty Acids and Insight into Its Antifungal Activity.

The focus of present study is on Codium bursa collected from the Adriatic Sea. C. bursa volatiles were identified by gas chromatography and mass spectrometry (GC-FID; GC-MS) after headspace solid-phase microextraction (HS-SPME), hydrodistillation (HD), and supercritical CO2 extraction (SC-CO2). The headspace composition of dried (HS-D) and fresh (HS-F) C. bursa was remarkably different. Dimethyl sulfide, the major HS-F compound was present in HS-D only as a minor constituent and heptadecane percentage was raised in HS-D. The distillate of fresh C. bursa contained heptadecane and docosane among the major compounds. After air-drying, a significantly different composition of the volatile oil was obtained with (E)-phytol as the predominant compound. It was also found in SC-CO2 extract of freeze-dried C. bursa (FD-CB) as the major constituent. Loliolide (3.51%) was only identified in SC-CO2 extract. Fatty acids were determined from FD-CB after derivatisation as methyl esters by GC-FID. The most dominant acids were palmitic (25.4%), oleic (36.5%), linoleic (11.6%), and stearic (9.0%). FD-CB H2O extract exhibited better antifungal effects against Fusarium spp., while dimethyl sulfoxide (DMSO) extract was better for the inhibition of Penicillium expansum, Aspergillus flavus, and Rhizophus spp. The extracts showed relatively good antifungal activity, especially against P. expansum (for DMSO extract MIC50 was at 50 µg/mL).

Polymorphisms of Toll-like receptors 2 and 4 in chronically infected hepatitis C patients from north-east Croatia.

Chronic infection with hepatitis C virus (HCV) is caused by an inadequate immune response. Experimental data suggest that the impaired activation of Toll-like receptors (TLRs) 2 and 4 contributes to chronic infection. We assessed the distribution of three single-nucleotide polymorphisms (SNPs) in the TLR2 (Arg753Gln) and TLR4 (Asp299Gly/Thr399Ile) genes in individuals from north-east Croatia and their effect on the outcome of antiviral therapy. The study consisted of 60 chronically infected patients and 40 healthy subjects. TLR polymorphisms were determined by the PCR-based melting curve analysis. HCV genotyping was performed using the Linear Array Hepatitis C Virus Genotyping Test. Thirty-three patients were treated with standard interferon and ribavirin therapy, and their viral load was evaluated at weeks 28 and 53 after the beginning of therapy. The majority of chronic infections were caused by genotype 1 (77%), followed by genotypes 3 (15%) and 4 (7%). Patients with genotype 1 had higher viral loads than patients infected with other genotypes (P = 0.0428). Healthy individuals and patients with chronic infection had similar frequencies of TLR2-Arg753Gln and TLR4-Asp299Gly/Thr399Ile SNPs. Heterozygous and homozygous TLR4-Asp299Gly/Thr399Ile polymorphisms correlated with higher viral loads and delayed responses to antiviral therapy. We have provided the first evidence that TLR4 polymorphisms influence the success of antiviral therapy in our region. This suggests that therapeutic strategies should be adjusted not only according to HCV genotype but also to individual TLR polymorphism(s).

Deoxynivalenol-sulfates: identification and quantification of novel conjugated (masked) mycotoxins in wheat.

We report the identification of deoxynivalenol-3-sulfate and deoxynivalenol-15-sulfate as two novel metabolites of the trichothecene mycotoxin deoxynivalenol in wheat. Wheat ears which were either artificially infected with Fusarium graminearum or directly treated with the major Fusarium toxin deoxynivalenol (DON) were sampled 96 h after treatment. Reference standards, which have been chemically synthesized and confirmed by NMR, were used to establish a liquid chromatography-electrospray ionization (LC-ESI)-MS/MS-based "dilute and shoot" method for the detection, unambiguous identification, and quantification of both sulfate conjugates in wheat extracts. Using this approach, detection limits of 0.003 mg/kg for deoxynivalenol-3-sulfate and 0.002 mg/kg for deoxynivalenol-15-sulfate were achieved. Matrix-matched calibration was used for the quantification of DON-sulfates in the investigated samples. In DON-treated samples, DON-3-sulfate was detected in the range of 0.29-1.4 mg/kg fresh weight while DON-15-sulfate concentrations were significantly lower (range 0.015-0.061 mg/kg fresh weight). In Fusarium-infected wheat samples, DON-3-sulfate was the only detected sulfate conjugate (range 0.022-0.059 mg/kg fresh weight). These results clearly demonstrate the potential of wheat to form sulfate conjugates of DON. In order to test whether sulfation is a detoxification reaction in planta, we determined the ability of the sulfated DON derivatives to inhibit in vitro protein synthesis of wheat ribosomes. The results demonstrate that both DON-sulfates can be regarded as detoxification products. DON-15-sulfate was about 44× less inhibitory than the native toxin, and no toxicity was observed for DON-3-sulfate in the tested range.

Design and synthesis of some new 1,3,4-thiadiazines with coumarin moieties and their antioxidative and antifungal activity.

A series of newly disubstituted (compounds 4a,b) and trisubstituted 1,3,4-thiadiazines 5a-l with various substituents was prepared utilizing different thiosemicarbazides and 3-α-bromoacetylcoumarins as starting compounds. The structures of the synthesized 1,3,4-thiadiazines are elucidated and confirmed utilizing the corresponding analytical and spectroscopic data. All of the new thiadiazine derivatives were tested for their antioxidant activity, employing different antioxidant assays (DPPH scavenging activity, iron chelating activity, power reducing activity). Compounds 5b, 5f, 5j and 4b were proven to be the best DPPH radical scavengers, while compounds 5h and 5j have shown the best iron chelating activity. Thiadiazine derivatives were also tested on their antifungal activity against four mycotoxicogenic fungi, Aspergillus flavus, A. ochraceus, Fusarium graminearum and F. verticillioides. The best antifungal against A. flavus was proven to be compound 5e, while compounds 4a and 5c were the best antifungals on A. ochraceus, and compound 5g showed the best antifungal activity on F. verticillioides.

Prevalence and genotype distribution of high-risk human papillomavirus (HR HPV) in male genital samples of Osijek-Baranja County.

This is a first cross-sectional study on the prevalence and distribution of HPV infection in asymptomatic, heterosexual men from Osijek-Baranja County, Croatia. Between 2009 and 2011, 330 men tested for sexually transmitted diseases (STDs) were recruited. Their genital swabs were tested for high-risk HPV (HR HPV) infection by the AMPLICOR HPV test and further genotyped by the Linear Array HPV Genotyping Test (both by Roche). Infection with a single HR HPV was detected in almost one third of men (39%) whereas multiple HPV types, in more than a half of HR HPV-positive men (61%). The highest HR HPV prevalence was detected in those younger than 20 (37.5%) and lowest in 31-35 year old men (27.8%). The most common genotypes were HPV 6 (24%), 16 (17.8%), 51 (9%), 52 (6%), 35, 55, 66, 84 (each 5%), 31, 62 (each 4%), 39, 58, 59, 83 (each 2.5%), and finally 56, 18, 53, and 54 (each 1.3%). Having more than one sexual partner per year was significantly associated with HR HPV infection in age group between 26 and 30 years (p = 0.001). Due to the high prevalence of HR HPV infection in men of this County and its risk of transmission to women, we recommend more public awareness about this particular STD and initiating vaccination programs of young men and women.

A Comparative Analysis of Radical Scavenging, Antifungal and Enzyme Inhibition Activity of 3'-8″-Biflavones and Their Monomeric Subunits.

Biflavonoids are dimeric forms of flavonoids that have recently gained importance as an effective new scaffold for drug discovery. In particular, 3'-8″-biflavones exhibit antiviral and antimicrobial activity and are promising molecules for the treatment of neurodegenerative and metabolic diseases as well as cancer therapies. In the present study, we directly compared 3'-8″-biflavones (amentoflavone, bilobetin, ginkgetin, isoginkgetin, and sciadopitysin) and their monomeric subunits (apigenin, genkwanin, and acacetin) and evaluated their radical scavenging activity (with DPPH), antifungal activity against mycotoxigenic fungi (Alternaria alternata, Aspergillus flavus, Aspergillus ochraceus, Fusarium graminearum, and Fusarium verticillioides), and inhibitory activity on enzymes (acetylcholinesterase, tyrosinase, α-amylase, and α-glucosidase). All the tested compounds showed weak radical scavenging activity, while antifungal activity strongly depended on the tested concentration and fungal species. Biflavonoids, especially ginkgetin and isoginkgetin, proved to be potent acetylcholinesterase inhibitors, whereas monomeric flavonoids showed higher tyrosinase inhibitory activity than the tested 3'-8″-biflavones. Amentoflavone proved to be a potent α-amylase and α-glucosidase inhibitor, and in general, 3'-8″-biflavones showed a stronger inhibitory potential on these enzymes than their monomeric subunits. Thus, we can conclude that 3'-8″-dimerization enhanced acetylcholinesterase, α-amylase, and α-glucosidase activities, but the activity also depends on the number of hydroxyl and methoxy groups in the structure of the compound.

Metabolic Profiling Identifies Changes in the Winter Wheat Grains Following Fusarium Treatment at Two Locations in Croatia.

Fusarium head blight (FHB) is one of the most dangerous diseases of winter wheat, resulting in reduced grain yield and quality, and production of mycotoxins by the Fusarium fungi. In the present study, changes in the grain metabolomics of winter wheat samples infected with Fusarium spp. and corresponding non-infected samples from two locations in Croatia were investigated by GC-MS. A Mann-Whitney test revealed that 24 metabolites detected were significantly separated between Fusarium-inoculated and non-infected samples during the variety by treatment interactions. The results confirmed that in grains of six FHB-resistant varieties, ten metabolites were identified as possible resistance-related metabolites. These metabolites included heptadecanoic acid, 9-(Z)-hexadecenoic acid, sophorose, and secolaganin in grains of FHB-resistant varieties at the Osijek location, as well as 2-methylaminomethyltartronic acid, maleamic acid, 4-hydroxyphenylacetonitrile, 1,4-lactonearabinonic acid, secolaganin, and alanine in grains of FHB-resistant varieties at the Tovarnik location. Moreover, on the PCA bi-plot, FHB-susceptible wheat varieties were closer to glycyl proline, decanoic acid, and lactic acid dimer that could have affected other metabolites, and thus, suppressed resistance to FHB. Although defense reactions were genetically conditioned and variety specific, resulting metabolomics changes may give insight into defense-related pathways that could be manipulated to engineer plants with improved resistance to the pathogen.

How different microfilters affect the recovery of eleven EU-regulated mycotoxins.

Microfiltration is a common step in liquid chromatography - tandem mass spectrometry (LC-MS), a method of choice in determining several mycotoxins in a solution at once. However, microfiltration may entail filter-analyte interactions that can affect the accuracy of the procedure, and underestimate exposure. The aim of our study was to assess how five different membrane materials for syringe filters (nylon, polytetrafluoroethylene, polyethersulphone, mixed cellulose ester, and cellulose acetate) affect microfiltration and recovery of EU-regulated mycotoxins, including aflatoxins B1, B2, G1, and G2, deoxynivalenol, fumonisins B1 and B2, zearalenone, T-2 and HT-2 toxins, and ochratoxin A. Polytetrafluoroethylene filters turned out to least affect microfiltration through mycotoxin loss, followed by more commonly used nylon filters, whereas the remaining three filter membrane materials had such a negative effect on recoveries that we found them incompatible with the procedure. Our findings clearly suggest that it is important to select a proper filter type that suits analyte properties and solution composition and to discard the first few filtrate drops to ensure the accuracy of the analytical procedure.

Development and Validation of an UHPLC-MS/MS Method for the Simultaneous Determination of 11 EU-Regulated Mycotoxins in Selected Cereals.

The availability of reliable sensitive multi-analyte methods for unambiguous determination of mycotoxins is crucial for ensuring food and feed safety, considering their adverse health effects and (co-)occurrence in various foods. Accordingly, a multi-mycotoxin confirmatory method for simultaneous determination of 11 mycotoxins regulated in cereals within the European Union (EU) using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was developed and in-house validated to fit the EU legislation requirements for analytical methods. A simple sample preparation was based on a solid−liquid extraction using a solvent mixture acetonitrile/water/formic acid (79/20/1, v/v/v) and a dilution of raw extract using water/acetonitrile/formic acid (79/20/1, v/v/v) before instrumental analysis. Average recoveries in all three validated cereal crop types (maize, wheat, and barley), spiked at multiple levels, were found acceptable for all analytes when matrix-matched calibration was used, ranging from 63.2% to 111.2% and also showing very good repeatability, with relative standard deviations below 20%. Matrix effect (SSE) evaluation revealed maize as the most complex of the three analyzed cereal matrices, with strong SSE (<50% and >150%) recorded for all 11 analyzed mycotoxins. An additional method verification was performed through successful participation in proficiency testing schemes, with the achieved z-scores generally in the acceptable range of −2 ≤ z ≤ 2. The obtained validation results demonstrated the suitability of the developed confirmatory multi-mycotoxin UHPLC-MS/MS method based on a dilute-and-shoot principle for the simultaneous determination of low concentrations of 11 EU-regulated mycotoxins in cereals, including aflatoxins B1, B2, G1 and G2, deoxynivalenol, fumonisins B1 and B2, zearalenone, T-2 and HT-2 toxins, and ochratoxin A.

Regulated Mycotoxin Occurrence and Co-Occurrence in Croatian Cereals.

A total of 209 samples of various cereal crops (maize, wheat, barley, rye and oats) grown in Croatian fields during 2016 and 2017 were collected to analyze and determine the occurrence and co-occurrence of EU regulated mycotoxins in cereals (AFB1, AFB2, AFG1, AFG2, DON, FB1, FB2, ZEA, T-2, HT-2 and OTA). The analysis, performed by a validated confirmatory LC-MS/MS method based on a dilute and shoot principle, highlighted Fusarium mycotoxins as the main contaminants, often co-occurring in samples from both years (50.0% in 2016 and 33.7% in 2017). DON was found to be the most frequent mycotoxin, present in 72.5% of the 2016 samples and 32.6% of the 2017 samples, while maize proved to be the most contaminated cereal type of both years with FUM as the most abundant mycotoxins, with an average concentration of 1180 µg/kg. Moderate temperatures with periods of high humidity favored the accumulation of DON in wheat samples instead of other Fusarium mycotoxins, while similar conditions favored maize contamination with FUM. A total of 8.3% of all the 2016 harvest samples and 7.9% of the 2017 harvest samples were assessed as non-compliant, containing mycotoxins in concentrations higher than the levels set by the EU legislation for food.

The Occurrence of Five Unregulated Mycotoxins Most Important for Traditional Dry-Cured Meat Products.

This study investigated the occurrence of 5 unregulated mycotoxins in a total of 250 traditional dry-cured meat products sampled in 2020 and 2021 in five Croatian regions (eastern, northern, central, western, and southern). Aflatoxin B1 (AFB1), ochratoxin A (OTA), sterigmatocystin (STC), citrinin (CIT), and cyclopiazonic acid (CPA) concentrations were related to the geographical region of the product's origin and to local weather. The results revealed the contamination of 27% of samples, namely, STC in 4% of samples in concentrations of up to 3.93 µg/kg, OTA in 10% of samples in concentrations of up to 4.81 µg/kg, and CPA in 13% of samples in concentrations of up to 335.5 µg/kg. No AFB1 or CIT contamination was seen. Although no statistically significant differences in concentrations of individual mycotoxins across the production regions were found, differences in mycotoxin occurrence were revealed. The eastern and western regions, with moderate climate, delivered the largest number of contaminated samples, while the southern region, often compared with subtropics, delivered the smallest, so that the determined mycotoxins were probably mainly produced by the Penicillium rather than the Aspergillus species. Due to the interaction of various factors that may affect mycotoxin biosynthesis during production, the detected concentrations cannot be related solely to the weather.