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Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by the fungi of the Fusarium genera, and is a contaminant of cereals and plant products. ZEN and its metabolites are considered endocrine disruptors, and could have various toxic effects on animals and humans. In recent years, there has been a significant demographic increase in wild boar (Sus scrofa) in many mountainous and hilly areas of Italy, including the Campania region, mainly due to global climate change. The wild boar can be defined as a generalist and omnivorous species capable of varying its diet; therefore, it can play a role as an environmental bioindicator towards contaminants such as mycotoxins. This study was conducted to evaluate, for the first time, the concentrations of ZEN and its metabolites in the liver, kidney, and muscle of 82 wild boars shot in their habitat by hunters with hunting permits in different localities of Avellino province (Campania region, Southern Italy) from 2021 to 2022. The samples were collected and analyzed with an SPE clean-up and high-pressure liquid chromatography method with fluorescence detection. The results indicated that ZEN and α-Zearalenol were present in most of the samples, suggesting that a plan to monitor these mycoestrogens is essential to achieve the goals of “One Health”.

Bisphenols (BPs) are a class of endocrine disruptors widely existing in the environment. They have a great impact on human health owing to their environmental endocrine disrupting effects, chronic toxicity, neurotoxicity, cytotoxicity and genetic toxicity. In this paper, an on-line packed fiber solid phase extraction (PFSPE) coupling with column-switching HPLC-FLD determination method was developed for the determination of eight BPs in drinking water. The poly (dibenzo-18-crown-6-ether)/polystyrene composite nanofibers (PDB18C6/PS) were prepared by electrospinning and used as an adsorbent for the on-line PFSPE column. The on-line PFSPE-HPLC equipment contained a dual ternary pump and a switching valve to enable enrichment, purification, and analysis directly in the system. The results showed that the proposed on-line PFSPE-HPLC-FLD method realized the simultaneous separation and detection of eight BPs: BPF, BPE, BPA, BPB, BPAF, BPAP, BPC and BPZ. The curves of the target analytes were prepared with good correlation coefficient values (r2 > 0.998) in the range of 50–1000 pg/mL. The limit of detection (S/N = 3) was 20 pg/mL, the limit of quantitation (S/N = 10) is 50 pg/mL. The recoveries of eight BPs were 94.8–127.3%, and the intra-day precisions (RSD) were less than 10%. The PFSPE column made of the PDB18C6/PS composite nanofibers has stable properties and can be reused at least 200 times. In the detection of drinking water samples, BPZ was detected in nearly 80% of drinking water samples, and BPA, BPAP, BPF and BPAF were also detected in some water samples. This high level of integration and automation was achieved in pretreatment of eight BPs from water samples. The proposed simple, rapid, and practical method has been successfully applied to the detection of eight BPs in drinking water, which can provide powerful technical support for drinking water quality and safety monitoring.

Paralytic Shellfish Toxins (PSTs) are marine biotoxins, primarily produced by dinoflagellates of the genera Gymnodinium spp., Alexandrium spp. They can accumulate in shellfish and, through the food chain, be assimilated by humans, giving rise to Paralytic Shellfish Poisoning. The maximum permitted level for PSTs in bivalves is 800 μg STX·2HCl eqv/kg (Reg. EC N° 853/2004). Until recently, the reference analytical method was the Mouse Bioassay, but Reg. EU N° 1709/2021 entered into force on 13 October 2021 and identified in the Standard EN14526:2017 or in any other internationally recognized validated method not entailing the use of live animals as official methods. Then the official control laboratories had urgently to fulfill the new requests, face out the Mouse Bioassay and implement instrumental analytical methods. The “EURLMB SOP for the analysis of PSTs by pre-column HPLC-FLD according to OMA AOAC 2005.06” also introduced a simplified semiquantitative approach to discriminate samples above and below the regulatory limit. The aim of the present paper is to present a new presence/absence test with a cut-off at 600 μg STX·2HCl eqv/kg enabling the fast discrimination of samples with very low PSTs levels from those to be submitted to the full quantitative confirmatory EN14526:2017 method. The method was implemented, avoiding the use of a large number of certified reference standards and long quantification procedures, resulting in an efficient, economical screening instrument available for official control laboratories. The protocol was fully validated, obtaining good performances in terms of repeatability (

Paprika is a widely consumed spice in the world and its authentication has gained interest considering the increase in adulteration cases in recent years. In this study, second-order fingerprints acquired by liquid chromatography with fluorescence detection (HPLC-FLD) were first used to detect and quantify adulteration levels of Chinese paprika samples. Six different adulteration cases, involving paprika production region, cultivar, or both, were investigated by pairs. Two strategies were employed to reduce the data matrices: (1) chromatographic fingerprints collected at specific wavelengths and (2) fusion of the mean data profiles in both spectral and time dimensions. Afterward, the fingerprint data with different data orders were analyzed using partial least squares (PLS) and n-way partial least squares (N-PLS) regression models, respectively. For most adulteration cases, N-PLS based on second-order fingerprints provided the overall best quantitation results with cross-validation and prediction errors lower than 2.27% and 20.28%, respectively, for external validation sets with 15–85% adulteration levels. To conclude, second-order HPLC-FLD fingerprints coupled with chemometrics can be a promising screening technique to assess paprika quality and authenticity in the control and prevention of food frauds.

Polysaccharides are abundant in natural resources and perform numerous physiological functions. Polysaccharide structures often lack chromophore groups; thus, current analytical methods cannot distinguish polysaccharide metabolites in the body or polysaccharide prototypes in biological samples. Thus, the measurement of polysaccharides in blood, bodily fluid, and cell-culture medium is difficult. Our early-stage research resulted in the isolation of two homogeneous polysaccharides from Pseudostellaria heterophylla, PHP0.5MSC-F and PHPH-1-2, which have anti-hyperglycemia and insulin resistance improvement effects for type 2 diabetes. In this study, the reducing terminal sugars of PHP0.5MSC-F and PHPH-1-2 were labeled with 2-aminobenzamide (2-AB) to prepare novel fluorescent probes for HPLC-coupled fluorescence detection (HPLC-FLD). Quantitative analysis was performed in reference to T40, and the detection limit for PHP0.5MSC-F was found to be 8.84 μg/mL with a linear range of 29.45–683.28 μg/mL. In reference to T70, the detection limit for PHPH-1-2 was found to be 13.89 μg/mL with a linear range of 46.29–462.76 μg/mL. This method was used to measure the bidirectional transport of polysaccharides across caco-2 cells from apical to basolateral (AP→BL) or from basolateral to apical (BL→AP) directions and to evaluate the polysaccharide bioavailability. The drug absorption capacity was determined based on the apparent permeability coefficient (Papp), and the Papp values for the two polysaccharides were found to be greater than 1 × 10−6 cm/s, which suggests easy absorption. Regarding bidirectional transport, the AP→BL Papp values were greater than the BL→AP values; thus, PHP0.5MSC-F and PHPH-1-2 mainly underwent passive transference. The two membrane permeable polysaccharides were not P-gp efflux transporter substrates. The absorption mechanism of PHP0.5MSC-F complies with passive diffusion under a concentration gradient, whereas PHPH-1-2 mainly utilizes a clathrin-mediated endocytic pathway to enter caco-2 cells. This innovative HPLC-FLD method can help to track polysaccharide internalization in vitro and in vivo to facilitate cellular uptake and biodistribution exploration.

Most cans intended to come into contact with food require the application of an internal coating made from synthetic polymers. The most widely used type of coating are epoxy resins based on bisphenol A diglycidyl ether (BADGE). It has been found that some components like cyclo-di-BADGE (CdB) and other BADGE derivatives can migrate into food. In this study, the polymeric coating of forty-eight cans was assessed using an infrared spectrometer with attenuated total reflectance. The food samples were extracted, and a targeted analysis was carried out to quantify CdB using liquid chromatography with fluorescence detection (HPLC-FLD). The first estimation of the exposure of the adult population was estimated by combining the concentration of CdB in the samples and the Spanish consumption data. In addition, a non-targeted screening by LC-MS was performed in the food sample extracts for the identification of other BADGE derivatives. Twenty samples were positive for the presence of CdB reaching concentrations of 2623 µg/kg. However, the tolerable daily intake of 1.5 µg/kg bw/day recommended for chemical compounds with high toxicological risk was not exceeded. A total of 18 epoxy oligomers could be tentatively identified in the food extracts being BADGE.H2O.BuEtOH the derivative with the highest incidence.

Dispersive liquid–liquid microextraction (DLLME) was optimized for the simultaneous extraction of aflatoxins (AFB1, AFB2, AFG1, and AFG2) from powdered senna leaves and pods. Detection was performed using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and pre-column derivatization. The parameters affecting the DLLME extraction efficiency were evaluated. Chloroform (200 µL) was used as an extraction solvent, 500 µL of distilled water was used as a dispersive solvent, and the extraction was performed at pH 5.6 with no salt added. The optimized method was validated using leaves and pods according to the European Commission guidelines. The linear range for all aflatoxins was 2–50 µg/kg, with values for regression coefficients of determination exceeding 0.995. The recoveries of spiked senna leaves and pods were in the ranges of 91.77–108.71% and 83.50–102.73%, respectively. The RSD values for intra-day and inter-day precisions were in the ranges of 2.30–7.93% and 3.13–10.59%, respectively. The limits of detection and quantification varied in the ranges of 0.70–1.27 µg/kg and 2.13–3.84 µg/kg, respectively. The validated method was successfully applied for the quantification of aflatoxins in 60 real samples of dried senna leaves and pods.

This study was supported by project RTI2018-099835-A-I00 financed by MCIN/AEI/10.13039/501100011033/FEDER “Una manera de hacer Europa”, and by the project Proyectos I + D + i del Programa Operativo FEDER 2020 cod. B-AGR-506-UGR20., Funding for open access charge: Universidad de Granada / CBUA, Avocado peel is one of the main by-products of avocado processing and is considered a promising source of phenolic compounds with various bioactivities. The drying step is essential for its storage at the industrial level, and it is the first step in the strategy of transforming by-products into functional ingredients. Therefore, this research evaluates the effect of the convective air-drying of avocado peels at three different temperatures (40, 60 and 80 ◦C) and airflows (0, 0.8 and 1.6 m/s) on the flavan-3-ols content and antioxidant activity. Moreover, the mathematical modelling of its drying kinetic was developed. A decrease in the flavan-3-ol and antioxidant content was found with increasing temperatures. However, a high impact of the airflow reducing the drying time and limiting the decrease in interesting compounds was found. Among the tested mathematical models, the Page model reported the highest values of R2 (from 0.9907 to 0.9973) and the lowest errors for most of the tem- peratures and airflows tested. However, at 80 ◦C with airflow, the Lewis model seemed to fit better (R2 = 0.9982). Finally, the drying conditions that showed the lowest decrease in procyanidin and antioxidants were 40 ◦C and an airflow of 1.6 m/s for 105 min., MCIN/AEI/FEDER "Una manera de hacer Europa" RTI2018-099835-A-I00, Project Proyectos I + D + i del Programa Operativo FEDER 2020 B-AGR-506-UGR20

Graphical abstract, Highlights • B[a]A, Chry, B[b]F and B[a]P were quantified in infusions of 12 yerba mate brands with large inter-brand variability. • Infusions prepared as habitually consumed in Uruguay contained about 40 % of combined PAH in mate. • Probability of Margin of Exposure (B[a]P + Chry) > (B[a]A + Chry + B[b]F + B[a]P). • The probability of B[a]P Margin of Exposure, The aim of this study was to assess the risk of exposure to polycyclic aromatic hydrocarbons (PAH) from yerba mate infusions in Uruguay using the margin of exposure approach (MOE) and a probabilistic method (Monte Carlo simulation). Servings/day, portion size, weekly frequency of mate consumption and body weight were the factors considered. The amount in infusions of benz[a]pyrene (B[a]P), PAH2 (sum of chrysene and B[a]P), and PAH4 (sum of benz[a]anthracene, chrysene, benz[b]fluoranthene and B[a]P) were used as markers of PAH exposure. Total content of PAH in infusions had large inter-brand variability (48–54 %) with significant differences among brands. PAH content in infusions prepared as habitually consumed was about 40 % of total content. The probability of occurrence of MOE

Flavanols are a class of polyphenols that have become the focus of much research because of their medicinal properties. For this paper, a new method to determine polyphenols in nutraceutical samples was developed, based on solid-liquid extraction and further analysis by liquid chromatography. Analytes were recovered by ultrasound-assisted extraction, using a mixture of methanol/water/hydrochloric acid (70:29:1, v:v:v) as the solvent. Reversed-phase high-performance liquid chromatography with ultraviolet/visible (HPLC-UV/Vis) and fluorescence detection (HPLC-FLD) were developed to determine the content of the principal flavanols. The HPLC-FLD method showed better selectivity and sensitivity, so it was selected for further studies. The analytical parameters established with FLD were satisfactory in terms of linearity (R2 ≥ 0.993), repeatability (RSD% ≥ 2.2), and limits of detection of 0.1 mg L−1 or below. The nutraceutical samples were analyzed by HPLC-FLD to determine the compositional profiles of flavanols, then they were subsequently treated using chemometric methods, such as principal component analysis (PCA). Results showed that the study of compositional profiles from the selected flavanols was sufficient for description and discrimination purposes. The cranberry samples were the richest in flavanols, procyanidin A2 being the principal flavanol, and artichoke samples the least rich.

Antibacterial substances such as sulfonamides are widely used in veterinary medicine to treat many bacterial diseases. After their administration to animals, up to 90% of the initial dose of the antibiotic is excreted in the feces and/or urine, which can be applied to farmland as natural or organic fertilizers. In this work, an analytical method was developed with the use of HPLC-FLD for the detection and quantification of five sulfonamides (sulfaguanidine, sulfadiazine, sulfamerazine, sulamethazine and sulfamethoxazol) in poultry and pig feces, slurry and digestates. The method was validated according to EU requirements (Commission Decision 2002/657/EC and VICH GL49). Linearity, decision limit, detection capability, detection and quantification limits, recovery, precision, and selectivity were determined, and adequate results were obtained. Using the HPLC-FLD method for all analyzed matrices, recoveries were satisfactory (77.00–121.16%), with repeatability and reproducibility in the range of 4.36–17.34% to 7.94–18.55%, respectively. Decision limit (CCα) and detection capability (CCβ) were 33.87–67.63 and 53.36–92.00 µg/kg, respectively, and limit of detection (LOD) and limit of quantification (LOQ) were 13.53–23.30 and 26.02–40.38 µg/kg, respectively, depending on the analyte. The forty-four samples of natural and organic fertilizers were analyzed, and four samples showed sulfamethoxazole in the amount from range 158 to 11,070 µg/kg. The application of antibiotics including sulfonamides for farming animals is widespread and may lead to the development of antibiotic resistance and other environmental effects.

The food-borne pathogen Campylobacter jejuni produces autoinducer-2 (AI-2) as an interspecies signalling molecule. AI-2 can trigger enhanced colonisation and biofilm formation, and this poses a serious risk to public health. To date, this communication system of C. jejuni is only partially understood, as detection and quantifi- cation of such autoinducer signalling molecules in complex media is hard to achieve. We have developed a whole-cell Vibrio harveyi-based biosensor assay to accurately quantify and follow production of AI-2 by C. jejuni 81–176 in a defined growth medium and in a model food system. Several V. harveyi strains were tested, but the most sensitive bioluminescent response to C. jejuni AI-2 was achieved with V. harveyi MM30, likely due to its ability to self-amplify the response to AI-2. The AI-2 concentrations measured by this biosensor were confirmed using an independent analytical method, HPLC-FLD, which we introduced for Campylobacter analytics for the first time. The AI-2 concentration produced by C. jejuni 81–176 in the model food system was ~5-fold that in the defined growth medium, at the same cell density. Together with the linear increments in AI-2 concentrations with cell density, this suggests that in C. jejuni, AI-2 represents a metabolic by-product rather than a true quorum- sensing molecule. This biosensor method is highly sensitive, as shown by the reduction in the limit of detection (by a factor of 100) compared to HPLC-FLD, and it enables quantification of AI-2 in complex matrices, such as food, which will help to improve the quality and safety of food production.

In sports, curcumin, a substance derived from the rhizome of Curcuma longa (turmeric) plant with antioxidant effect 8 times greater than vitamin E, has attracted the attention of scientists because of its potent antioxidant action, since in athletes subjected to intense exercise the—endogenous mechanisms of neutralization of reactive species are saturated. However, the pharmacokinetic characteristics of curcumin do not favor its medicinal use due to its low absorption, accelerated metabolism and rapid systemic elimination. Thus, the determination of plasma levels in supplemented patients is a crucial step in their pharmacodynamic evaluation. Therefore, the objective of this work was to develop and validate an analytical method by HPLC-FLD for curcumin evaluation in plasma of supplemented athletes. Luna column (C18; 150 × 4 mm; 3 µm), acetonitrile: acetic acid pH 3.2 (45:55 to 60:40) as mobile phase, flow rate of 1 mL min−1, excitation at 429/285 nm and emission at 529 nm and injection of 10 µL were the chromatographic conditions used. Plasma samples were extracted using ethylacetate and methanol (95: 5, 500 µL) and estradiol (30 µg mL−1) as internal standard, with subsequent stirring (3 min) and centrifugation (8 min) (triple extraction). The organic fraction was evaporated under N2 (20 min) and the dried residue reconstituted in acetonitrile. The method was linear between 44 and 261 ng mL−1, showing intra-day (2.05.6%) and inter-day (4.0–5.1%) precision with accuracy and selectiveness (curcumin tR = 8.7 min and internal standard tR = 13.9 min with relative recovery of 83.2%). So, it can be successfully used for curcumin evaluation in plasma samples from supplemented athletes, as well as being an alternative and advantageous method to UV–Vis and MS/MS in bioavailability studies.

The accumulation of antibiotics by plants is a currently concern associated to yield reduction and transference of antibiotic residues along the food web. Maize is a staple food in many parts of the world; it is also considered an important ingredient in animal nutrition. Considering the lack of validated analytical methods for the analysis of ciprofloxacin (CIPRO) and enrofloxacin (ENRO) in the shoot of Zea mays maize, we developed a reliable reversed-phase high-performance liquid chromatography method with fluorescence detection (HPLC-FLD). This method was validated for linearity, matrix effects, precision, accuracy, limits of quantification (LOQ) and detection (LOD), and robustness. The analytical curves were linear with coefficient of determination (R2) of 0.9907 for CIPRO and 0.9962 for ENRO. The LOD values were 16.65 and 6.57 μg kg−1 for CIPRO and ENRO, respectively, whereas LOQ values were 50.44 μg kg−1 (CIPRO) and 19.92 μg kg−1 (ENRO). HPLC-FLD also displayed good precision and accuracy. Therefore, the proposed method can be considered a reliable and useful tool for the analysis of ciprofloxacin and enrofloxacin in the shoot of maize.

Kuru incirlerde görülen en önemli kimyasal tehlikeler aflatoksinler (AFs) ve okratoksin A (OTA)'dır. Bu araştırmada, ülkemizde tüketime sunulan kuru incirlerde AFs ve OTA varlığı ve miktarı floresans dedektörlü yüksek performanslı sıvı kromatografisi (HPLC-FLD) yöntemi ile belirlenmiştir. Ayrıca, yetişkin bireylerin kuru incir tüketimi yoluyla AFs ve OTA'ya maruz kalma miktarları hesaplanmış ve risk değerlendirmesi yapılmıştır. Araştırma kapsamında, Nisan-Haziran 2019 tarihleri arasında Çorum, İstanbul, Ankara ve İzmir'deki çeşitli satış noktalarından rastgele satın alınan 100 incir örneği (300'er g) analiz edilmiştir. Kullanılan analiz yöntemi valide edilmiş olup, Avrupa Birliği (AB) tarafından belirlenen metot validasyon parametrelerini karşıladığı görülmüştür. Aflatoksin B1 (AFB1), aflatoksin B2 (AFB2), aflatoksin G1 (AFG1), aflatoksin G2 (AFG2) ve OTA'nın ölçüm limitleri (LOQs) sırasıyla 0,277; 0,247; 0,307; 0,253 ve 0,437 µg kg-1 olarak bulunmuştur. Kuru incir örneklerinin 86'sında AFs tespit edilemezken, 14'ünde saptanan toplam AFs miktarı 0,258-12,6 µg kg-1 arasında değişiklik göstermiştir. AFs tipleri arasında AFB1 en sık rastlanan olup, örneklerde 0,258-11,92 µg kg-1 arasında değişen miktarlarda saptanmıştır. Kuru incir örneklerinin yalnızca 2'sinde tespit edilen AFB1 ve toplam AFs miktarları, AB ve Türk Gıda Kodeksi (TGK) Bulaşanlar Yönetmeliği'nde yer alan maksimum limit (ML) değerlerinin üzerinde bulunmuştur. Bununla birlikte, kuru incir örneklerinin 7'sinde AFB2 (0,095-0,675 µg kg-1), 2'sinde AFG1 (0,153 ve 2,427 µg kg-1) ve yalnızca 1'inde AFG2 (0,121 µg kg-1) bulunmuştur. OTA ise kuru incir örneklerinin 8'inde 0,151-1,723 µg kg-1 arasında değişen konsantrasyonlarda saptanmıştır. Ülkemizde yaşayan yetişkin bireylerin kuru incir tüketimi yoluyla AFB1'e, toplam AFs'ye ve OTA'ya ortalama alt sınır ve üst sınır maruz kalma miktarları sırasıyla 0,0039-0,0046; 0,0044-0,0058; 0,0005-0,0017 ng kg-1 v.a. gün-1 olarak hesaplanmıştır. Kişi başı kuru incir tüketim miktarına (0,685 g gün-1) göre hesaplanan maruz kalma sınırı (MOE) değerleri AFB1 için sağlık açısından riskli bir durum oluşturmazken, günde 1 adet kuru incir tüketim senaryosuna (20 g gün-1) göre örneklerde saptanan AFB1 miktarının sağlık endişesine yol açma potansiyeli taşıyabileceği değerlendirilmektedir. Diğer yandan, OTA için hem kişi başı hem de günde 1 adet kuru incir tüketim senaryosuna göre MOE değerleri 10.000'in oldukça üzerinde bulunmuş olup, OTA'ya maruz kalma düzeyinin yetişkinlerde ciddi sağlık endişesi oluşturmadığı tespit edilmiştir. The most important chemical hazards in dried figs are aflatoxins (AFs) and ochratoxin A (OTA). In this study, the presence and amounts of AFs and OTA in dried figs consumed in Turkey were determined by fluorescence detector coupled with high-performance liquid chromatography (HPLC-FLD). In addition, the exposure of adults to AFs and OTA through the consumption of dried figs and risk assessment were determined. Between April-June 2019, a total of 100 dried fig samples (300 g) collected randomly from different retail stores in Corum, Istanbul, Ankara and Izmir were analysed. The validated method fulfilled the requirements of validation parameters of the European Union (EU). The limit of quantifications (LOQs) of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and OTA were 0.277, 0.247, 0.307, 0.253 and 0.437 µg kg-1, respectively. While AFs were not detected in 86 out of 100 dried fig samples, AFs were positive in 14 samples at levels ranging from 0.258 to 12.6 µg kg-1. Among the AFs, AFB1 was the most frequently occurring toxin, with levels ranging from 0.258 to 11.92 µg kg-1. Only two dried fig samples exceeded the both EU and Turkish Food Codex (TGK) maximum limits (MLs) for AFB1 and total AFs. Moreover, AFB2, AFG1 and AFG2 were detected in seven (0.095-0.675 µg kg-1), two (0.153 and 2.427 µg kg-1) and only one dried fig sample (0.121 µg kg-1), respectively. OTA was recorded in eight out of 100 dried fig samples at concentrations varying from 0.151 to 1.723 µg kg-1. In the Turkish adults, the mean exposure to AFB1, total AFs and OTA ranged from 0.0039 to 0.0046, 0.0044 to 0.0058 and from 0.0005 to 0.0017 ng kg-1 b.w. day-1 at lower bound and upper bound, respectively. While the margin of exposure (MOE) value for AFB1 was not a health concern according to per capita consumption, the AFB1 levels recorded in dried fig samples raised a potential health concern in the consumption scenario one dried fig per day (20 g day-1) for Turkish adults. However, the MOE values for OTA were considerably higher than 10 000 in both scenarios. Thus, the exposure to OTA from the consumption of dried figs does not a health concern for adults. İÇİNDEKİLER Sayfa ÖZET -- iv ABSTRACT -- vi TEŞEKKÜR -- viii İÇİNDEKİLER -- x TABLOLAR DİZİNİ -- xii ŞEKİLLER DİZİNİ -- xiii RESİMLER DİZİNİ -- xiv SİMGELER VE KISALTMALAR -- xv GİRİŞ -- 1 1. BÖLÜM LİTERATÜR ÖZETİ 1.1. İncir -- 3 1.2. Kuru İncir -- 5 1.3. Küfler Hakkında Genel Bilgi -- 10 1.4. Mikotoksinler -- 11 1.5. AFs -- 15 1.5.1. AFs’nin kimyasal yapıları ve özellikleri -- 16 1.5.2. AFs’nin sağlık üzerine etkileri -- 17 1.5.3. AFs yasal limitleri -- 18 1.6. OTA -- 19 1.6.1. OTA’nın kimyasal yapısı ve özellikleri -- 19 1.6.2. OTA’nın sağlık üzerine etkileri -- 20 1.6.3. OTA yasal limitleri -- 21 xi Sayfa 1.7. Kuru İncirlerde AFs ve OTA Kontaminasyonu Konusunda Yapılan Çalışmalar -- 22 2. BÖLÜM MATERYAL VE YÖNTEM 2.1. Materyal -- 25 2.1.1. Kuru incir -- 25 2.1.2. Kimyasal maddeler -- 26 2.1.3. PBS -- 26 2.1.4. AFs standardı -- 26 2.1.5. OTA standardı -- 26 2.1.6. Immunoaffinite kolon (IAC) -- 27 2.2. Yöntem -- 27 2.2.1. Ekstraksiyon -- 27 2.2.2. HPLC-FLD analizi -- 27 2.2.3. Metot validasyonu -- 29 2.2.4. Maruz kalma düzeyi hesaplaması -- 30 3. BÖLÜM ARAŞTIRMA BULGULARI VE TARTIŞMA 3.1. Metot Validasyon Parametrelerinin Değerlendirilmesi -- 32 3.2. Kuru İncirlerde Aflatoksin Varlığı/Miktarı -- 36 3.3. Kuru İncirlerde OTA Varlığı/Miktarı -- 37 3.4. AFs ve OTA’ya Maruz Kalma Değerlendirmesi -- 39 SONUÇ VE ÖNERİLER -- 41 KAYNAKÇA -- 43

ANALYTICAL METHODS FOR DETERMINATION OF MACROCYCLIC LACTONES IN DIFFERENT TYPES OF MATRICES. A REVIEW. The macrocyclic lactones (ML) are drugs widely used to combat endo- and ectoparasites in livestock, pets, crops, etc. Since marketing of ivermectin, the number of available ML increased considerably and, consequently, a wide variety of analytical methodologies for determination of MLs in multiple types of matrices have been published. Most of these methodologies have already been reviewed before so this paper is about those most recent and/or those have not been previously considered. In this review the focus is on biological, alimentary, and environmental matrices emphasizing on the most important techniques employed on sample preparation. Also, the liquid chromatography with of fluorescence and mass spectrometry detectors is discussed as the most used instrumental techniques for identification of MLs. Finally, a brief review is made on the importance of method validation and some strategies for its implementation.

Spices are a popular ingredient in cuisine worldwide but can pose a health risk as they are prone to fungal infestation and mycotoxin contamination. The purpose of this study was to evaluate ochratoxin A (OTA) in 54 single-kind traditional and less traditional spices, each of which was purchased in six samples of different batches (324 samples in total) at the Czech market during 2019–2020. The HPLC-FLD method with pre-treatment by immunoaffinity columns was employed to determine OTA. The limits of detection and quantification were 0.03 ng g−1 and 0.10 ng g−1, respectively. A total of 101 (31%) samples of 19 spice kinds were positive at concentrations ranging from 0.11–38.46 ng g−1. Only turmeric was contaminated with an OTA level exceeding the European Union limits. However, most spices have no regulation, thus further extensive monitoring of various mycotoxins in various kinds of spices is necessary. Chilli and black pepper are the most studied spices for OTA contamination, however, many other kinds of spice can also be highly contaminated, but studies on them are less common, rare, or have not yet been performed. The uniqueness of this study lies in the wide range of spice types studied for the presence of OTA on the Czech market.

In order to meet the analytical requirements of the European Medicines Agency (EMA), a new HPLC-FLD method was successfully developed using dansyl chloride for the derivatization and determination of the genotoxic impurities N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA) in Lisinopril API and its final product. Samples’ pretreatment includes liquid–liquid microextraction, denitrosation, and derivatization steps. To optimize the process, the parameters contributing to high sensitivity and yielding reliable results were thoroughly studied and optimized using one-factor-at-a-time and experimental design approaches. The analytes were pre-column derivatized with Dansyl-Cl and analyzed by HPLC-fluorescence (λem/λem = 340/530) using a C18 column and a mixture of phosphate buffer (pH = 2.8; 20 mM)/acetonitrile 55:45 v/v as the mobile phase. The six-level concentration calibration was shown to be linear, with R equal to 0.9995 for both analytes. The limit of detection (LOD) was satisfactory and equal to 4.7 and 0.04 ng/mL for NDMA and NDEA, respectively. Precision was less than 13.4% in all cases, and the average recoveries were equal to 109.2 and 98.1% for NDMA and NDEA, respectively. The proposed procedure is relatively easy, rapid, and suitable for the determination of the two nitrosamines in routine analysis tests.

In September and November 2016, eight marine sampling sites along the coast of the southeastern Gulf of Mexico were monitored for the presence of lipophilic and hydrophilic toxins. Water temperature, salinity, hydrogen potential, dissolved oxygen saturation, inorganic nutrients and phytoplankton abundance were also determined. Two samples filtered through glass fiber filters were used for the extraction and analysis of paralytic shellfish toxins (PSTs) by lateral flow immunochromatography (IFL), HPLC with post-column oxidation and fluorescent detection (FLD) and UHPLC coupled to tandem mass spectrometry (UHPLC-MS/MS). Elevated nutrient contents were associated with the sites of rainwater discharge or those near anthropogenic activities. A predominance of the dinoflagellate Pyrodinium bahamense was found with abundances of up to 104 cells L−1. Identification of the dinoflagellate was corroborated by light and scanning electron microscopy. Samples for toxins were positive by IFL, and the analogs NeoSTX and STX were identified and quantified by HPLC-FLD and UHPLC-MS/MS, with a total PST concentration of 6.5 pg cell−1. This study is the first report that confirms the presence of PSTs in P. bahamense in Mexican waters of the Gulf of Mexico.

Sialic acids are postulated to improve cognitive abilities. This study aimed to evaluate the effects of sialic acid on behavior when administered in a free form as N-acetylneuraminic acid (Neu5Ac) to pregnant mothers or rat pups. The experiment involved 40 male 21-day-old rat pups and 20 15-day-pregnant rats that were randomized into four Neu5Ac treated groups: 0 (control), or 10, 20, and 40 mg/kg. Morris water maze test and shuttle box test were performed on the rat pups and maternal Neu5Ac-supplemented offspring on day 100 to evaluate their cognitive performance. The Neu5Ac levels in the cerebral cortex and hippocampus were tested with high-performance liquid chromatography-fluorescence detection (HPLC-FLD). We found that the maternal Neu5Ac-supplemented offspring showed better cognitive performance, less escape latency in the Morris water maze test, and less electric shock time shuttle box test, compared with the untreated control. In the meantime, the Neu5Ac level in the cerebral cortex and hippocampus of the offspring was higher in the Neu5Ac treatment group than that in the untreated control group. However, no significant differences were observed between rat pups in the treated and the untreated control groups in terms of cognitive performance and Neu5Ac content in the cerebral cortex and hippocampus. Maternal Neu5Ac supplementation during pregnancy could effectively promote the brain Neu5Ac content of the offspring and enhance their cognitive performance, but Neu5Ac had no such effect on rat pups while directly supplemented with Neu5Ac.

Analytical methods using the fluorescence properties of bisphenols (BPA, BPF and BPS) and their complexes with β-cyclodextrin and methyl-β-cyclodextrin were developed. The methods were applied for the analysis of thermal paper and canned food. Their performance was compared with a standard HPLC approach with a diode array and fluorescence detections. For comparison purposes, basic validation parameters (linear range, limit of detection, sensitivity, precision) were evaluated. It was concluded the developed methods facilitate fast and cost-effective determination of three bisphenol species in liquid samples, similar to the HPLC performance. They are also environmentally friendly. BPA, BPF and BPS can be routinely determined with the presented approach.

Cytostatics are toxic pharmaceuticals, whose presence in surfaces puts healthcare workers at risk. These drugs might also end up in hospital effluents (HWW), potentially damaging aquatic ecosystems. Bicalutamide is a cytostatic extensively consumed worldwide, but few analytical methods exist for its quantification and most of them require advanced techniques, such as liquid chromatography mass spectrometry (LC-MS), which are very complex and expensive for large monitoring studies. Therefore, a simple but reliable multi-matrix high performance liquid chromatographic method, with fluorescence detection, was developed and validated to rapidly screen abnormal concentrations of bicalutamide in HWW and relevant contamination levels of bicalutamide in indoor surfaces (&gt, 100 pg/cm2), prior to confirmation by LC-MS. The method presents good linearity and relatively low method detection limits (HWW: 0.14 ng/mL, surfaces: 0.28 pg/cm2). Global uncertainty was below 20% for concentrations higher than 25 ng/mL (HWW) and 50 pg/cm2 (surfaces), global uncertainty was little affected by the matrix. Therefore, a multi-matrix assessment could be achieved with this method, thus contributing to a holistic quantification of bicalutamide along the cytostatic circuit. Bicalutamide was not detected in any of the grab samples from a Portuguese hospital, but an enlarged sampling is required to conclude about its occurrence and exposure risks.

Mycotoxins are bioaccumulative contaminants impacting animals and humans. The simultaneous detection of frequent active exposures and accumulated mycotoxin level (s) in exposed organisms would be the most ideal to enable appropriate actions. However, few methods are available for the purpose, and there is a demand for dedicated, sensitive, reliable, and practical assays. To demonstrate the issue, mice were exposed to a relevant agent Ochratoxin A (OTA), and accumulated OTA was measured by fine-tuned commercial assays. Quantitative high-performance liquid chromatography with fluorescence detection, enzyme-linked immunosorbent assay, and flow cytometry assays have been developed/modified using reagents available as commercial products when appropriate. Assays were performed on excised samples, and results were compared. Accumulated OTA could be detected and quantified; positive correlations (between applied doses of exposure and accumulated OTA levels and the results from assays) were found. Dedicated assays could be developed, which provided comparable results. The presence and accumulation of OTA following even a short exposure could be quantitatively detected. The assays performed similarly, but HPLC had the greatest sensitivity. Blood contained higher levels of OTA than liver and kidney. We demonstrate that specific but flexible and practical assays should be used for specific/local purposes, to measure the exposure itself and accumulation in blood or organs.

Vito Verardo thanks the Spanish Ministry of Economy and Competitiveness (MINECO) for the "Ramon y Cajal" contract (RYC-2015-18795)., Hawthorn leaves are a rich source of phenolic compounds that possess beneficial activities for human health. Ultrasonic-assisted extraction (UAE) is an extraction technique frequently used for the isolation of phenolic compounds in plants. Thus, in this study, a Box–Behnken design was used to optimize UAE conditions such as the percentage of acetone, the extraction time and solvent-to-solid ratio (v/w) in order to obtain the maximum content of total compounds by Folin– Ciocalteu and the maximum in vitro antioxidant activity by DPPH, ABTS and FRAP assays in Crataegus monogyna leaves. The optimum conditions to obtain the highest total phenolic content and antioxidant activities were 50% acetone, 55 min and 1/1000 (w/v). A total of 30 phenolic compounds were identified and quantified in C. monogyna leaf extract obtained at these optimum UAE conditions. HPLC-MS allows the identification and quantification of 19 phenolic compounds and NP-HPLC-FLD analyses showed the presence of 11 proanthocyanidins. According to the results, the most concentrated phenolic compounds in C. monogyna leaf extract obtained at optimum UAE conditions were phenolic acid derivatives such as protocatechuic acid-glucoside, dihydroxy benzoic acid pentoside and chlorogenic acid, flavones such as 2”-O-rhamnosyl-C-hexosyl-apigenin, flavonols such as hyperoside and isoquercetin and proanthocyanidins such as monomer and dimer. As a result, the optimized UAE conditions could be used to obtain an extract of C. monogyna leaves enriched with phenolic compounds., Spanish Ministry of Economy and Competitiveness (MINECO) for the "Ramon y Cajal" contract RYC-2015-18795

This survey describes the occurrence and levels of AFs and OTA in chocolate products consumed in Turkey. A total of 130 samples, including bitter chocolate, milk chocolate and chocolate wafers were analysed for these mycotoxins by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). The values of recovery (81–92%) and precision (RSD < 9%) fulfilled the requirements of EC Regulation No. 401/2006. OTA was the most prevalent mycotoxin, with an incidence of 46.7% in bitter chocolate, 22.8% in milk chocolate and 17.4% in chocolate wafers, ranging from 0.18 to 0.75 ?g kg?1. AFs were detected in 13.3% of bitter chocolate, in 19.6% of milk chocolate and in 8.7% of chocolate wafers, in concentrations ranging from 0.15 to 2.04 ?g kg?1. © 2019, © 2019 Taylor & Francis Group, LLC. 2-s2.0-85064006280 PubMed: 30961454

Aflatoxins (AFS) are toxic and carcinogenic fungal metabolites. Aflatoxin B1 is the most toxic and has been classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC). Samples of imported rice were analyzed for their AFS content. Finley ground rice subsamples were extracted with water/methanol (100:150 v/v) followed by purification with Immunoaffinity columns (IAC). AFS purified from extracts were determined with RP-HPLC-FLD using post column electrochemical derivatization with a Kobra Cell. Concentrations of aflatoxin B1 and total AFS in test rice samples were ≤0.123 and ≤2.58 µg/kg, respectively. Tween 80 improved recoveries (86 and 106%) of aflatoxin B1 and aflatoxin G1 from brown rice. Recoveries of Aflatoxin B2 and aflatoxin G2 were substantially reduced (non-detected to 27%) by Tween 80 used in IAC cleanup of brown rice extracts. Visible dense growth of Aspergillus parasiticus (food isolate) occurred at 25 °C but higher aflatoxin B1amounts (23.9–39.3 µg/kg) accumulated when the mold grew at 37 °C in rice seeds stored for three weeks. It could be concluded that levels of aflatoxin B1 and total AFS in rice samples were within the permissible amounts of the EU and other international legislations. Keywords: Rice, Brown rice, Saudi Arabia, Aflatoxins, Aflatoxin B1, HPLC-FLD, Aspergillus parasiticus, Tween 80

Polycyclic aromatic hydrocarbons (PAHs) represent an important contaminant group in a variety of food products. Since PAHs are commonly present in meat products, this study aimed to determine PAH content in four types of Croatian dry-cured hams (Dalmatian, Drniš, Krk and Istrian) produced by four different processing methods. Determination and quantification of PAHs were performed by High performance liquid chromatography with fluorescence detection (HPLC-FLD). Out of 15 investigated PAHs, 13 compounds were detected. The total average benzo(a)pyrene (BaP) and PAH4 levels obtained from dry-cured hams ranged from 0.05-0.10 µg/kg and 0.41-0.67 µg/kg, respectively. Even though Krk and Istrian dry-cured ham manufacturing processes do not include the smoking phase, no significant differences were found between investigated dry-cured hams in terms of BaP, PAH4, PAH8 and PAH 15 contents. The presence of detected PAHs in non-smoked dry-cured hams could be a result of the addition of spices in the salting phase. BaP and PAH4 contents found in dry-cured ham samples did not exceed the currently legal levels according to the European legislation.

Polycyclic aromatic hydrocarbons (PAHs) are produced during incomplete combustion of organic matter. Many of them are likely to be carcinogenic and cause mutations. In this study, the PAH4 (benzo[a]pyrene (BaP), benz[a]anthracene (BaA), chrysene (CHR), benzo[b]fluoranthene (BbF)) content in deep-fat fried pork was evaluated according to temperature and time, and a risk assessment was conducted. The high performance liquid chromatography-fluorescence detection (HPLC-FLD) method for PAH4 analysis was validated by determining linearity (R2), recovery, limit of detection (LOD), and limit of quantitation (LOQ). The linearity was R2 ≥ 0.99. The PAH4 level was dependent on the temperature, time, and nature of the edible oil. Before heat treatment, the PAH4 content of pork was 0.38 μg/kg. The PAH4 content of deep-fat fried pork ranged from 0.86 to 6.86 μg/kg according to temperature (160, 180, 200 °C) and time (3, 6, 9 min). Exposure to PAH4 via the consumption of deep-fat fried pork for different age groups among the Korean population was 0.01–0.89 μg-TEQBaP/kg/day, with the margin of exposure calculated as 7.88 × 104–5.22 × 106. The PAH4 content and risk of exposure increased proportionally with the heat treatment temperature and time. The survey provided important information in terms of evaluating the health risks that PAH compounds can cause in people’s diets due to the heat treatment of pork.

Summary The aim of this study was to investigate aflatoxin M1 (AFM1) transfer from naturally contaminated raw milk into cheese and whey, during the production of Livanjski cheese (hard cheese type). Raw milk samples were collected from 4 farms in Serbia. The samples were then tested for AFM1 content and were later used for hard cheese production. Four cheese samples were produced, and the matching whey samples were also collected. The milk samples included two samples containing AFM1 above the EU maximum level (0.077±0.009 and 0.118±0.008 µg/kg) and the other two samples below the maximum level of 0.05 µg/kg (0.021±0.002 and 0.034±0.004 µg/kg). Regarding AFM1 transfer into cheese from the milk samples containing AFM1 above the EU maximum level, the rate was approximately 4-fold (383% and 410%). On the other hand, in the cheese samples made from milk containing AFM1 below the EU maximum level, almost 10-fold levels of AFM1 content in milk were found (transfer rates of 934% and 961%). As for the whey samples, AFM1 levels were below the levels found in the milk samples (transfer rates of 78%, 74%, 68% and 57%). The difference in the transfer rates for the cheese made from the milk samples contaminated at different levels may indicate the possibility that the AFM1 transfer rate from milk into cheese depends on the content of this toxin in milk.

A total of 172 different salamis were purchased from farms and small salami factories located in four Italian regions (Piedmont, Veneto, Calabria, and Sicily) and analyzed for the presence of ochratoxin A (OTA). Analysis was performed by high-performance liquid chromatography coupled to a fluorimetric detector (HPLC-FLD). The detection limit (LOD) for the method used was 0.05μg/kg, while the quantitation limit (LOQ) was 0.20μg/kg; the average recovery rate was 89.1%. OTA was detected in 22 salamis, and 3 samples exceeded the Italian guidance value for OTA in pork meat (1μg/kg). In particular, what emerges from this research is the high percentage of spicy salamis among positive samples (68.2%, 15 out of 22), although spicy salamis are only 27.3% of the total number of samples collected and analyzed. Red chili pepper contaminated by OTA could be responsible for the presence of the mycotoxin in these spicy salamis. It follow that, also the control of some ingredients used in the manufacture of these meat products, like spices, should not be neglected.

Predmet ove disertacije bio je proučavanje uticaja herbicida sa ili bez primene folijarnog đubriva na sadržaj fitohemikalija u listu i zrnu četiri hibrida kukuruza šećerca (ZP504su, ZP355su, ZP553su i ZP515su) tokom dve godine, u cilju definisanja njihove tolerantnosti i karakterističnog odgovora. Sadržaj fitohemikalija određen je primenom visoko-efikasne tečne hromatografije sa ultravioletnim detektorom sa više dioda i fluorescentnim detektorom i visoko-efikasne jonske hromatografije sa pulsno-amperometrijskim detektorom. Sadržaj ukupnih fenolnih jedinjenja, glutationa i antioksidativna aktivnost određeni su primenom UV/VIS spektrofotometrije. Hromatografska razdvajanja su postignuta pri nižim protocima mobilnih faza uz smanjen utrošak organskih rastvarača podržavajući na taj način trendove zelene analitičke hemije. Dobra linearnost, tačnost, niska granica detekcije, direktno HPLC određivanje ekstrakata bez derivatizacije, skraćeno vreme ekstrakcije i trajanja analize omogućili su rutinsko korišćenje primenjenih hromatografskih metoda. Rezultati su pokazali da je uticaj tretmana bio izraženiji u zrnu, dok u listu uglavnom nije bio statistički značajan. Primena oba herbicida dovela je do statistički značajnog povećanja ukupnog sadržaja svih fitohemikalija u ZP553su na osnovu čega se on može definisati kao tolerantan genotip. Primenjeni tretmani izazvali su aktiviranje neenzimskih antioksidanasa kod ZP355su, a kod ZP515su aktiviranje metabolizma ugljenih hidrata. Rezultati su pokazali da se sadržaj karotenoida, vitamina E, fenolnih kiselina i šećera može koristiti kao jedinstven pokazatelj genotipa nakon primenjenih tretmana. Pokazano je da kontrolisana upotreba herbicida može dovesti do poboljšanja fitohemijskog profila i povećanja nutritivnog statusa kukuruza šećerca. The subject of this doctoral dissertation was the impact of herbicides with or without foliar fertilizer on phytochemicals content in leaf and grain in four hybrids of sweet maize (ZP504su, ZP355su, ZP553su, and ZP515su) during two years in order to define their tolerance and characteristic response. The content of phytochemicals was determined using high-performance liquid chromatography with an ultraviolet multi-diode detector and a fluorescent detector, and high-performance anion exchange chromatography with pulsed amperometric detection. The content of total phenolic compounds, glutathione and antioxidant activity was determined using UV / VIS spectrophotometry. Chromatographic separations were achieved at lower mobile phase flows with reduced consumption of organic solvents, thus supporting the trends of green analytical chemistry. Good linearity, accuracy, low limit of detection, direct HPLC determination of extracts without derivatization, shortened extraction time, and duration of analysis enabled the routine use of applied chromatographic methods. The results showed that the impact of the treatment was more conspicuous in the grain, while in the leaf it was generally not statistically significant. The application of both herbicides led to a statistically significant increase in the total content of all phytochemicals in ZP553su, based on which it can be defined as a tolerant genotype. The applied treatments caused the activation of non-enzymatic antioxidants in ZP355su, and in ZP515su the activation of carbohydrate metabolism. The results of this study indicated that the content of carotenoids, vitamin E, phenolic acids and sugars can be used as a characteristic phytochemical parameter of the genotype after the applied treatments. It has been shown that the controlled use of herbicides can lead to an improvement in the phytochemical profile and an increase in the nutritional status of sweet maize.

Coffee, one of the most popular drinks around the world, is also one of the beverages most susceptible of being adulterated. Untargeted high-performance liquid chromatography with ultraviolet and fluorescence detection (HPLC-UV-FLD) fingerprinting strategies in combination with chemometrics were employed for the authenticity assessment and fraud quantitation of adulterated coffees involving three different and common adulterants: chicory, barley, and flours. The methodologies were applied after a solid–liquid extraction procedure with a methanol:water 50:50 (v/v) solution as extracting solvent. Chromatographic fingerprints were obtained using a Kinetex® C18 reversed-phase column under gradient elution conditions using 0.1% formic acid aqueous solution and methanol as mobile phase components. The obtained coffee and adulterants extract HPLC-UV-FLD fingerprints were evaluated by partial least squares regression-discriminants analysis (PLS-DA) resulting to be excellent chemical descriptors for sample discrimination. One hundred percent classification rates for both PLS-DA calibration and prediction models were obtained. In addition, Arabica and Robusta coffee samples were adulterated with chicory, barley, and flours, and the obtained HPLC-UV-FLD fingerprints subjected to partial least squares (PLS) regression, demonstrating the feasibility of the proposed methodologies to assess coffee authenticity and to quantify adulteration levels (down to 15%), showing both calibration and prediction errors below 1.3% and 2.4%, respectively.

Atmospheric contamination of plant raw material with Polycyclic Aromatic Hydrocarbons (PAHs) helps explain their presence in edible vegetable oils. This study compared PAH contamination of Turkish olive fruits during their growing period on the tree and their respective oils from three different industrial sources (petroleum refinery, thermal power plant and heavy industry site). The method included liquid-liquid extraction solid-phase extraction for cleanup followed by HPLC with fluorescence detector. There were statistically significant differences between the three industrial sources in benzo[a]pyrene content, the sum of light, total PAHs and PAH4 (p, Turkish Ministry of Agriculture and Forestry [TAGEM/HSGYAD/15/A05/P01/76], The authors are grateful for financial support through the project of the Turkish Ministry of Agriculture and Forestry, (Grant number; TAGEM/HSGYAD/15/A05/P01/76).

A chromatographic method with the Analytical Quality by Design (AQbD) methodology is developed for the simultaneous determination by HPLC-FLD of ten PAHs (naphthalene, phenanthrene, anthracene, flu- oranthene, pyrene, chrysene, benzo[a]anthracene, perylene, benzo[b]fluoranthene, and benzo[a]pyrene), widely spread in the environment. The construction of the Method Operable Design Region (MODR) is conducted, for the first time, via the inversion of a multiresponse Partial Least Squares (PLS2) model, which is needed to maintain the correlations among the Critical Method Parameters (CMP), among the Critical Quality Attributes (CQA), and the covariance between one another. The five CMP considered were the composition of the mobile phase (water, methanol, acetonitrile), flow rate, and column temperature. The eight CQA were linked to resolution between peaks recorded in the same emission wavelength (greater than 1.4) and the total time (less than 15 minutes). By systematic use of experimental design and parallel coordinates plots to explore the Pareto optimal front obtained with the PLS2 model inversion, the computed MODR is formed by convex combinations of eight specific settings of Critical Method Parameters that have a mobile phase with percentages of water between 37 and 38 %, of methanol from 13 and 22 %, and of acetonitrile between 41 and 49 %, together with a flow rate between 1.47 and 1.50 mL min −1 , and column temperature between 41.9 and 44.0 °C in their adequate combinations. All the chromatographic peaks are well resolved, with total time varying between 12.96 and 15.66 min inside the estimated MODR and the analytical method is accurate with CC βbetween 0.9 and 7.0 μg L −1 with probability of both false positive and false negative equal to 0.05., Research projects CTQ2017-88894-R and BU052P20, financed by the Spanish Ministerio de Ciencia, Innovación y Universidades (AEI/FEDER, UE) and Consejería de Educación de la Junta de Castilla y León, both cofinanced with European Development Funds. M.M. Arce wishes to thank Junta de Castilla y León for her postdoctoral contract through BU052P20 project.

Polycyclische aromatische Kohlewasserstoffe (PAK) sind eine große Stoffgruppe, welche aufgrund einiger von der internationalen Agentur für Krebsforschung (IARC) als karzinogen eingestuften Vertreter immer wieder im Fokus steht. PAKs entstehen bei der Verbrennung von organischem Material bei hohen Temperaturen. Sie werden über die Nahrung und die Atmung in den Körper aufgenommen. Diese Arbeit beschäftigt sich mit der Analyse von PAKs in menschlichem Fett- und Lungengewebe. Ziel ist es herauszufinden, ob sich die Konzentration an PAKs oder die Karzinogenität der gefundenen PAKs bei gesundem und pathogenem Gewebe unterscheiden. Lungengewebe wurde hierfür zunächst lyophilisiert. Anschließend wurde das Gewebe mit Natriumsulfat verrieben und mit Hexan extrahiert. Das Extrakt wurde durch Ausschütteln mit Acetonitril und anschließender Festphasenextraktion gereinigt. Die Analyse der Proben erfolgte mittels Hochleistungsflüssigkeitschromatographie und Fluoreszenz Detektor (HPLC-FLD). In der Probe eines Patienten konnte Naphthalin und Pyren nachgewiesen werden. Fettgewebe wurde weitestgehend analog zu Lungengewebe aufgearbeitet. Die Proben wurden nicht lyophilisiert. Insgesamt wurden 19 Proben von elf verschiedenen Patienten analysiert. Keine der Proben von pathogenem Gewebe zeigte eine erhöhte Konzentration an PAKs im Vergleich zu gesundem Gewebe desselben Patienten. Es konnten keine PAKs nachgewiesen werden, welche von der IARC als karzinogen eingestuft werden. Es zeigte sich allerdings ein Trend wonach Patienten mit Tumor eine erhöhte Konzentration an PAKs aufwiesen. Lungengewebe vom Schwein wurde auf hydroxylierte PAKs und PAK Glucuronide untersucht. Die Proben wurden lyophilisiert und mit Acetonitril extrahiert. Anschließend wurde die Probe hydrolysiert. Die Analyse der Proben erfolgte mittels HPLC FLD. Bei einem Vergleich mit einer nicht hydrolysierten Probe wurden erhöhte Konzentrationen an 2 Hydroxynaphthalin und 9 Hydroxyphenanthren festgestellt. Dies deutet auf die Anwesenheit von PAK Glucuroniden hin. Diese Ergebnisse konnten allerdings nicht durch eine Analyse mittels HPLC-Massenspektrometrie bestätigt werden. Es wurde untersucht ob sich broncho alveoläre Lavage (BAL) als Alternative, für die nicht invasive Probenahme eignet, um PAKs im menschlichen Körper zu analysieren. 10 Proben BAL wurden auf PAKs untersucht. Die Proben wurden nach der Zugabe eines internen Standards direkt mittels HPLC-FLD analysiert. Naphthalin, Fluoren, Acenaphthen und Anthracen konnten in sehr geringen Mengen nachgewiesen werden. Aufgrund der polaren Eigenschaften von BAL scheint eine Anreicherung von PAKs unwahrscheinlich. Polycyclic aromatic hydrocarbons (PAH) form a large group of compounds, and some of them are classified as carcinogenic by the International Agency for Research on Cancer. They are formed during the combustion of organic compounds at high temperatures. The human body may take up PAHs via food and respiration. During this work human lung and fat tissue was analyzed to identify differences in concentration and carcinogenicity of the found PAHs in healthy and pathogenic tissue. In the first step lung tissue was lyophilized. Afterwards the sample was ground with sodium sulfate and subsequently extracted with hexane. The extract was cleaned by liquid liquid extraction with acetonitrile and solid phase extraction. The samples were analyzed using high performance liquid chromatography and fluorescence detection (HPLC-FLD). Naphthalene and pyrene were found in one human sample. Fat tissue samples were not lyophilized. Otherwise, they were prepared like lung tissue. A total of 19 samples from eleven different patients were available. None of the pathogenic samples showed higher concentrations than the healthy tissue of the same patient. No carcinogenic PAHs were found in any of the samples. There was a trend which showed that there were higher concentrations of PAHs in the tissue of patients which had a tumor. Hydroxylated PAHs and PAH glucuronides were analyzed in the lung tissue of a pig. The samples were lyophilized and subsequently extracted with acetonitrile. Afterwards the samples were hydrolyzed and analyzed using HPLC FLD. In comparison to a non hydrolyzed sample the analysis showed higher peaks of 2 hydroxynaphthalene and 9 hydroxyphenahtren. This indicates the presence of PAH glucuronides. However, the results could not be verified by HPLC and mass spectrometry. Bronchoalveolar lavage (BAL) was tested as a substitution for non invasive sampling to analyze PAHs in humans. Ten samples were available. After addition of an internal standard the samples were directly analyzed using HPLC FLD. Naphthalene, fluorene, acenaphthene and anthracene were found in very small concentrations. There is probably no enrichment of PAHs in BAL because of the difference in polar properties eingereicht von Sebastian Friedl Abweichender Titel laut Übersetzung der Verfasserin/des Verfassers Universität Linz, Masterarbeit, 2021

Major type of internal can coating used for food and beverages is made from epoxy resins, which contain among their components bisphenol A (BPA) or bisphenol A diglycidyl ether (BADGE). These components can be released and contaminate the food or beverage. There is no specific European legislation for coatings, but there is legislation on specific substances setting migration limits. Many investigations have paid attention to BPA due to its classification as endocrine disruptor, however, few studies are available concerning to other bisphenol analogues that have been used in the manufacture of these resins. To evaluate the presence of this family of compounds, ten cans of beverages were taken as study samples. Firstly, the type of coating was verified using an attenuated total reflectance-FTIR spectrometer to check the type of coating presents in most of the samples examined. A screening method was also performed to investigate potential volatiles from polymeric can coatings of beverages using Purge and Trap (P&T) technique coupled to gas chromatography with mass spectrometry detection (GC-MS). Moreover, a selective analytical method based on high performance liquid chromatography with fluorescence detection (HPLC-FLD) for the simultaneous identification and quantification of thirteen compounds including bisphenol analogues (BPA, BPB, BPC, BPE, BPF, BPG) and BADGEs (BADGE, BADGE.H2O, BADGE.2H2O, BADGE.HCl, BADGE.2HCl, BADGE.H2O.HCl, cyclo-di-BADGE) in the polymeric can coatings and in the beverage samples was applied. In addition, a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was optimized for confirmation purposes. The method showed an adequate linearity (R2 >0.9994) and low detection levels down to 5 µg/L. Cyclo-di-BADGE was detected in all extracts of polymeric coatings. The concentrations ranged from 0.004 to 0.60 mg/dm2. No detectable amounts of bisphenol related compounds were found in any of the beverage samples at levels that may pose a risk to human health, suggesting a low intake of bisphenols from beverages The study was financially supported by the Ministerio de Ciencia, Innovación y Universidades, by Fondo Europeo de Desarrollo Regional (FEDER), and by Agencia Estatal de Investigación Ref. No. PGC2018-094518-B-I00 “MIGRACOATING” (MINECO/FEDER, UE) SI

Bitkisel hammaddenin atmosferik yolla polisiklik aromatik hidrokarbonlarla kontaminasyonu bu bileşiklerin yenilebilir yağlardaki varlığının en önemli sebeplerinden birisidir. Bu çalışmada bu kontaminasyon yolu temel alınarak zeytin meyvelerinin ağaç üzerinde gelişimi sırasında üç farklı endüstriyel kaynaktan (Aliağa Petrol Rafinerisi, Yatağan Termik Santrali ve ağır sanayi kollarının bulunduğu Kemalpaşa Organize Sanayi Bölgesi) atmosfere salınan emisyonlara maruz kalan zeytin meyvelerinde ve onların yağlarında PAH bileşiklerinin miktarları ve PAH profilleri karşılaştırılmıştır. Ayrıca bu endüstriyel kaynaklardan üç farklı uzaklık (1, 5, 10 km) ve üç farklı doğrultuda alınan örneklerde PAH içeriğinin uzaklıkla nasıl değiştiği de araştırılmıştır. PAH bileşiklerinin zeytin meyvesinde ve yağlarında miktar tayini için sıvı-sıvı ekstraksiyonu, onu takiben temizleme için katı faz ekstraksiyon (SPE) ve floresans dedektörlü likit kromatografisi (HPLC-FLD) metodu kullanılmıştır. Örnek analizleri öncesi kullanılan metodun laboratuvar içi validasyon çalışmaları gerçekleştirilmiş ve metodun bu matrikslerde PAH tayini için mevzuatta belirtilen performans parametrelerini karşılayan güvenilir bir metot olduğu doğrulanmıştır. Üç farklı endüstriyel kaynak, zeytin meyvelerinde ve onların yağında Benzo[a]piren içeriği, hafif PAH toplamı, toplam PAH miktarı ve PAH4 toplamı miktarında istatistiksel olarak anlamlı düzeyde (p˂0.05), PAH profillerinde ise minör düzeyde farklılığa neden olmuştur. En yüksek PAH miktarları petrol rafinerisi emisyonlarına maruz kalan örneklerde tespit edilmiş olup kontaminasyon seviyesinin en düşük olduğu termik santral emisyonlarına maruz kalan örneklerdeki miktarların yaklaşık iki katı kadardır. Sonuç olarak, zeytinin yetiştirildiği bölgelerde endüstriyel kaynakların zeytin meyvesi ve yağlarında PAH kontaminasyonuna neden olduğu ancak yürürlükteki yasal düzenlemelerde belirtilen limitleri aşmadığı tespit edilmiştir. Zeytin meyvesi ve yağındaki PAH değerlerinden meyveden yağa geçen PAH miktarları % olarak hesaplanmış ve %23-73 arasında değiştiği görülmüştür. Meyveden yağa PAH geçiş oranlarındaki bu büyük farklılığın zeytin çeşidine ve dolaylı olarak yağ miktarına bağlı olduğu söylenebilir. Zeytinyağındaki PAH varlığının zeytinin dış kabuğunda birikim yapan PAH bileşiklerinin büyük oranda zeytinin iç dokularına ve yağ bünyesine geçmesinden veya zeytinden yağ elde etme işlemi prosesi sırasındaki kırma ve yoğurma işlemlerinde çapraz kontaminasyondan kaynaklandığı düşünülmektedir. Pirinanın kurutulması sırasında yapılan yanlış uygulamalar sebebiyle pirina yağlarında önemli bir sorun olan PAH bileşiklerinin rafinasyon kademelerinde nasıl değiştiği de bu çalışmanın diğer amacını oluşturmaktadır. Bu kapsamda ülkemizde pirina yağı işleyen bir firmadan temin edilen pirina yağı örneklerinde PAH bileşiklerinin etkili bir şekilde uzaklaştırıldığı ve miktarlarının yaklaşık %99-100 arasında azaltıldığı tespit edilmiştir. En etkili işlem basamakları ağartma (%99-100) ve deodorizasyon (%64,5-83) olarak saptanmıştır. Ağartma işleminin etkinliğinin %5 ağartma toprağına ilave olarak %1 düzeyinde aktif kömür kullanımına bağlı olduğu görülmüştür. Geçmiş literatürde yer alan bilgilerle uyumlu olarak deodorizasyon aşamasının daha çok hafif PAH'lar üzerinde, ağartma aşamasının ise daha çok ağır PAH'ları azaltmada daha etkili olduğu bu çalışma ile de doğrulanmıştır. Sonuç olarak bu çalışma, ülkemiz koşullarında uygulanan rafinasyon işleminin güvenilir son ürün sağlama konusunda başarılı olduğunu ortaya koymuş ve bu bilgilerin ülkemizde ilk kez dokümante edilmesine imkan sağlamıştır., Contamination of vegetable raw materials with polycyclic aromatic hydrocarbons in an atmospheric way is one of the most important reasons for the presence of these compounds in edible oils. In this project, basing on this contamination route, PAH contamination levels and profiles of olive fruits exposed to emissions from three different industrial sources (Aliağa Petroleum Refinery, Yatağan Thermal Power Plant and Kemalpaşa Heavy Industry Site) that were released to atmosphere during their growing period on the tree and in their respective oils were compared. In addition, in the samples taken from these industrial sources in three different distances (1, 5, 10 km) and three different directions, how the PAH content changes with distance was also investigated. The method used to quantificate the PAHs in samples, included liquid-liquid extraction, solid-phase extraction (SPE) for cleanup followed by HPLC with fluorescence detector (HPLC-FLD). Before sample analyzes, in-house validation studies of the method were carried out and it was confirmed that the method is a reliable method that meets the performance parameters specified in the legislation for PAH determination in these matrices. Three different industrial sources caused a statistically significant difference (p˂0.05) in the amount of Benzo[a]pyrene, light PAH total, total PAH amount and PAH4 total in olive fruits and their oil, and a minor difference in PAH profiles. The highest level of PAH compounds was measured in samples exposed to pollution from the petroleum refinery, nearly twice as high as samples exposed to the thermal power plant which showed the lowest contamination levels. As a result, it has been determined that industrial sources in the regions where olives are grown cause PAH contamination in olive fruits and their respective oils but do not exceed the limits specified in the current legal regulations. The PAH transfer ratio from fruits to oil was defined as the percentage ratio of PAH levels in fruits to those in the oils' and it varied between 23% and 73%. According to the research findings, it can be said that this variability in PAH transfer ratios may be correlated to the olive variety, particularly to the amount of fruit oil content. The presence of PAH in olive oil is thought to be caused by the PAH compounds accumulated in the outer skin of the olive transiting to the inner tissues and the oil body, or from cross contamination during the crushing and malaxation treatment during the olive oil production process. Another purpose of this project was to investigate how the PAH content changes during the refining stages of pomace, which is an important problem in pomace that arises during the drying process. For this reason, it has been determined that PAH compounds are effectively removed and their amounts are reduced by approximately 99-100% in the pomace oil samples supplied from a company operating in the refining of olive pomace oil in our country. The most effective process steps were determined as bleaching (99-100%) and deodorization (64.5-83%). It has been observed that the efficiency of the bleaching process depends on the use of 1% activated charcoal in addition to 5% bleaching earth. In accordance with the previous reports, it has been confirmed by this study that the deodorization stage is more effective on light PAHs and the bleaching stage is more effective in reducing heavy PAHs. As a result, this study has shown that the refining process applied in our country is successful in providing reliable final products and this output has been documented for the first time in our country.

Tea is a widely consumed drink in the world which is susceptible to undergoing adulterations to reduce manufacturing costs and rise financial benefits. The development of simple analytical methodologies to assess tea authenticity, as well as to detect and quantify frauds, is an important matter considering the rise of adulteration issues in recent years. In the present study, untargeted HPLC-UV and HPLC-FLD fingerprinting methods were employed to characterize, classify, and authenticate tea extracts belonging to different varieties (red, green, black, oolong, and white teas) by partial least squares-discriminant analysis (PLS-DA), as well as to detect and quantify adulteration frauds when chicory was used as the adulterant by partial least squares (PLS) regression, to ensure the authenticity and integrity of foodstuffs. Overall, PLS-DA showed a good classification and grouping of the tea samples according to the tea variety and, except for some white tea extracts, perfectly discriminated from the chicory ones. One hundred percent classification rates for the PLS-DA calibration models were achieved, except for green and oolong tea when HPLC-FLD fingerprints were employed, which showed classification rates of 96.43% and 95.45%, respectively. Good predictions were also accomplished, also showing, in almost all the cases, a 100% classification rate for prediction, with the exception of white tea and oolong tea when HPLC-UV fingerprints were employed that exhibited a classification rate of 77.78% and 88.89%, respectively. Good PLS results for chicory adulteration detection and quantitation were also accomplished, with calibration, cross-validation, and external validation errors beneath 1.4%, 6.4%, and 3.7%, respectively. Acceptable prediction errors (below 21.7%) were also observed, except for white tea extracts that showed higher errors which were attributed to the low sample variability available.

Ochratoxin A (OTA) is a mycotoxin that may be present in various food and feed of plant and animal origin, including chicken meat. In Italy, backyard poultry farming is rather widespread. Animals are raised for meat, eggs and for ornamental purpose, and they are often fed with home-made diets not subject to official controls. The purpose of this study was to evaluate exposure of ornamental and backyard chickens to OTA using biliary ochratoxin A as a biomarker. Therefore, bile samples, in addition to kidney, liver and muscle, were collected from 102 chickens reared in 16 farms located in 6 Italian regions. High-performance liquid chromatography method and fluorimetric detection (HPLC-FLD) analysis were carried out firstly on bile from all animals, and OTA was detected in 12 chickens (concentration range 3.83&ndash, 170.42 &micro, g/L). Subsequently, the kidneys of these chickens were also analysed, and the mycotoxin was not detected. The analytical detection limits (LODs) of OTA in bile and kidney were 2.1 &micro, g/L and 0.1 &micro, g/kg, respectively. In conclusion, these animals were exposed to OTA but their meat can be considered safe, given that this mycotoxin, if present, concentrates highest in kidneys. Biliary ochratoxin A confirms its use as a valid biomarker to assess exposure of poultry to OTA.

Bisphenol A (BPA) is one of the chemicals used to produce both polycarbonate plastics and epoxy resin coatings. Research has shown that small amounts of BPA can migrate into the foods and beverages enclosed in these types of containers. In this research, an analytical method based on high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed and validated for the determination of BPA in canned vegetables. The results were confirmed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was performed, to identify the coating material of each tin can. Nineteen cans of vegetables were taken as study samples (eleven samples from the Spanish market, and eight samples from the Portuguese market). Excellent linear correlation (r2 = 0.9999) was observed over the range of 0.01 to 0.25 mg/L. Limit of detection (LOD) and limit of quantification (LOQ) values were calculated to be 0.005 mg/kg and 0.01 mg/kg, respectively. Good recoveries, between 72% and 90% were obtained at three different levels of concentration (RSD% = 4.6). BPA was not detected in the samples. The proposed HPLC-FLD was found to be suitable for the determination of BPA in canned vegetables MF Vaz acknowledges FCT, through IDMEC, under LAETA, project UID/EMS/50022/2019. The work was supported by UIDB/00211/2020 with funding from FCT/MCTES through national funds. Fernanda Vilarinho acknowledges National Institute of Health Dr. Ricardo Jorge through the project “Development of methodologies for the evaluation of polymeric food packaging components and determination of their structural and mechanical properties” (2016 DAN 1289). Antía Lestido Cardama is grateful for her grant “Programa de axudas á etapa predoutoral” da Xunta de Galicia (Conselleriía de Cultura, Educación e Ordenación Universitaria). This research was cofounded by the “Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia,” reference number GRC 2019/030 SI

Histamine level determination in fish and fish products is important for human safety, fish quality and food industry. For this reason, a rapid, robust, and precise method is needed. To achieve this objective, an HPLC method with fluorescence detection was developed and validated. Histamine in fish samples was efficiently extracted with perchloric acid, and purified with ion-exchange solid-phase extraction cartridge. A pre-column derivatization was adopted with ortho-phthalaldehyde (OPA) in the presence of the reducing agent 2-mercaptoethanol, and the stability of the histamine-OPA derivatives was achieved with the acidification of the reaction medium. In terms of validation, besides the excellent linear correlations, satisfactory recoveries at all spiking levels ranging between 0 and 200 mg kg−1 were attained, with limit of detection calculated at 1.8 mg kg−1, whereas limit of quantification determined at 5 mg kg−1. The proposed method was successfully used in the analysis of reference materials and proficiency tests, and was found to be suitable, accurate, and rapid for detection and quantification of histamine in various fish samples.

Samples (n = 485) of raw (n = 394) or heat-treated (n = 91) milk of three different species (cow, n = 170; sheep, n = 133; donkey, n = 84), collected 2013–2016 in Western Sicily (Southern Italy), were analyzed for aflatoxin M1 (AFM1) by enzyme-linked immunosorbent assay (ELISA). Positive ELISA results were further analyzed by HPLC with fluorescence detection. Both methods had a detection limit for AFM1 in milk of 7 ng kg−1. ELISA yielded 12.9 and 5% positives in cows and sheep milk, respectively, all samples of donkey milk were negative. Levels of AFM1 were in most cases at 0.007–

This study presents the determination of aflatoxins B1, B2, G1, and G2 in rice, applying a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction followed by high-performance liquid chromatography-fluorescence detection (HPLC-FLD). The objective of the proposed method is a fast, inexpensive, and easy analysis of aflatoxins. The aflatoxins were separated on a C18 column by isocratic elution with water: methanol: acetonitrile (65 : 25 : 10, v/v/v), and detection was achieved at excitation/emission wavelengths of 360/450 nm, without derivatization. The calibration curves showed good linearity (R2 > 0.99), and the limits of detection and quantification were ≤ 6 and ≤ 8 µg·kg-1, respectively. Average intraday and interday recoveries were in the range 104 - 119 % and 104 - 113 % with RSD % ≤ 12 % for concentrations between 6 - 20 µg·kg-1. The proposed method was tested on rice samples sold on markets, of which none exhibited presence of aflatoxins.

Major types of internal can coatings used for food and beverages are made from synthetic polymers known as epoxy-based resins, mainly based on bisphenol A diglycidyl ether (BADGE). The migration of components from coatings to food is a concern for food safety. A multiresidue method was developed for the identification and quantification of six bisphenols, BADGE and its derivatives, and cyclo-di-BADGE in sixteen canned food samples based on HPLC-FLD. The method developed showed excellent validation data with an adequate linearity, low detection levels, good repeatability and acceptable recoveries. Confirmation of the obtained results was made by LC–MS/MS. The exposure of the adult population to these compounds through the consumption of canned food was assessed. In general, the results suggested a low dietary exposure to this type of compounds (0.003 to 0.985 μg/kg bw/day) with values lower than the established tolerable day intake (TDI). The highest mean concentration was observed for cyclo-di-BADGE in a sample of pickled mussels SI

peer-reviewed, In recent years climate changes recorded in temperate regions of Europe have led to aflatoxin (AF) contamination of maize. Thus, the aim of this study was to investigate the influence of weather conditions on levels of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) in 180 maize samples collected from the main maize-growing regions (Western Bačka, North Banat, South Banat and Central Serbia) in Serbia after harvest in 2015. The concentrations of AFs were determined by a validated HPLC method with post-column derivatisation and fluorescence detection (HPLC-FLD). The presence of AFB1, AFB2, AFG1 and AFG2 was detected in 57.2%, 13.9%, 5.6% and 2.8% of maize samples in the concentration ranges of 1.3–88.8 µg kg–1, 0.60–2.8 µg kg–1, 1.8–28.5 µg kg–1 and 2.1–7.5 µg kg–1 respectively. The recorded smaller amount of precipitation and especially higher air temperatures during the summer of 2015 were favourable for AF production, which resulted in 32.2% and 21.1% of samples being unsuitable for human consumption, since AFB1 and the sum of AFs concentrations were above 5.0 and 10.0 µg kg–1 respectively. Furthermore, the findings in this study indicate that the microclimate conditions in the investigated regions had a great influence on the contamination frequency of maize with AFs. The highest percentage of samples unsuitable for human consumption, considering both AFB1 and total AFs content were 72.5% and 51.5% respectively from Central Serbia, whilst the lowest percentages of 15.6% and 6.2% respectively were found in Western Bačka. These findings confirmed that maize should be continuously monitored in order to protect human and animal health from the harmful effects caused by AFs contamination., This paper is a result of the research conducted within Project III 46001 financed by the Ministry of Education, Science and Technological Development, Republic of Serbia.

Fatty acids (FAs) are basic components in plants. The pharmacological significance of FAs has attracted attentions of nutritionists and pharmaceutists. Sensitive and accurate detection of FAs is of great importance. In the present study, a pre-column derivatization and online mass spectrometry-based qualitative and quantitative analysis of FAs was developed. Nineteen main FAs were derivatized by 2-(7-methyl-1H-pyrazolo-[3,4-b]quinoline-1-yl)ethyl-4-methyl benzenesulfonate (NMP) and separated on reversed-phase Hypersil BDS C8 column with gradient elution. All FAs showed excellent linear responses with correlation coefficients more than 0.9996. The method obtained LOQs between 0.93 ng/mL and 5.64 ng/mL. FA derivatives were identified by both retention time and protonated molecular ion corresponding to m/z [M + H]+. A comparative study based on FA contents in peel and pulp, seeds and leaves of Nitraria tangutourum Bobr (NTB) from different geographical origins was performed with the established method. Results indicated that NTB were rich in FAs, and the types and contents of FAs varied among tissues. On the other hand, the same tissue of NTB from different geographical areas differed in the content, but not in type, of FAs.

Paralytic shellfish toxins (PST) and tetrodotoxin (TTX) are naturally-occurring toxins that may contaminate the food chain, inducing similar neurological symptoms in humans. They are co-extracted under the same conditions and thus their combined detection is desirable. Whilst PST are regulated and officially monitored in Europe, more data on TTX occurrence in bivalves and gastropods are needed before meaningful regulations can be established. In this study, we used three separate analytical methods - pre-column oxidation with liquid chromatography and fluorescence detection, ultrahigh performance hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry (MS/MS) and HILIC high resolution (HR) MS/MS - to investigate the presence of PST and TTX in seawater and shellfish (mussels, clams) collected in spring summer 2015 to 2017 in the Mediterranean Sea. Samples were collected at 10 sites in the Syracuse Bay (Sicily, Italy) in concomitance with a mixed bloom of Alexandrium minutum and A. pacificum. A very high PST contamination in mussels emerged, unprecedentedly found in Italy, with maximum total concentration of 10851 mu g saxitoxin equivalents per kg of shellfish tissue measured in 2016. In addition, for the first time TTX was detected in Italy in most of the analysed samples in the range 0.8-6.4 mu g TTX eq/kg. The recurring blooms of PST-producing species over the 3-year period, the high PST levels and the first finding of TTX in mussels from the Syracuse bay, suggest that monitoring programmes of PST and TTX in seafood should be activated in this geographical area. (C) 2018 Elsevier Ltd. All rights reserved.

Environmental pollution by petroleum derivatives is a very current topic. In particular, low concentration of this kind of pollutants can seriously compromise the life of animals and plants of aquatic ecosystems (Yu, 2017). The current legislation provides that bilgewater, wastewater produced by boats, can be discharged directly into the sea only if the total hydrocarbon concentration not exceds 15 mg L-1. In this work, new activated carbons were tested as adsorbent materials of oil / hydrocarbons from wastewater. Moreover, an instrumental technique able to quickly measure the required low hydrocarbons concentration is also proposed. The new activated carbons were obtained from bio-oil production waste, a biochar produced by pyrolysis of Posidonia oceanica, a marine plant widespread in the Mediterranean sea. The biochar has been characterized and adsorption experiments were carried out with the pristine biochar (not activated) and with two chemically activated biochars (BCB and BCA) by means of acid or alkali treatments. Moreover, a commercial activated carbon (Filtrasorb 400) has been used for comparison purpose. Synthetic bilge waters were prepared following reference standards (MEPC, 2003) containing DMA (distillate marine fuel) and SDS (sodium lauryl sulfate). Batch adsorption isotherms were carried out without ionic medium and at different concentrations of NaCl in order to evaluate the effect of salinity on the adsorption ability of adsorbent materials. The same adsorbents were tested by column experiments. In particular, a bench pilot system was built and breakthrough curves were obtained changing amount of adsorbent material in column, flow rate, initial DMA and surfactant concentrations. Several instrumental techniques (turbidimetry, TOC, HPLC-FLD) have been used to measure surfactant and hydrocarbon concentrations in experimental samples. The batch experimental data were fitted with the most used isotherm models (Langmuir, Freundlich, Sips) and important considerations were made on the breakthrough curves of column experiments.

Polycyclic aromatic hydrocarbons (PAHs) represent an important contaminant group in a variety of food products. Since PAHs are commonly present in meat products, this study aimed to determine PAH content in four types of Croatian dry-cured hams (Dalmatian, Drniš, Krk and Istrian) produced by four different processing methods. Determination and quantification of PAHs were performed by High performance liquid chromatography with fluorescence detection (HPLC-FLD). Out of 15 investigated PAHs, 13 compounds were detected. The total average benzo(a)pyrene (BaP) and PAH4 levels obtained from dry-cured hams ranged from 0.05-0.10 µg/kg and 0.41-0.67 µg/kg, respectively. Even though Krk and Istrian dry-cured ham manufacturing processes do not include the smoking phase, no significant differences were found between investigated dry-cured hams in terms of BaP, PAH4, PAH8 and PAH 15 contents. The presence of detected PAHs in non-smoked dry-cured hams could be a result of the addition of spices in the salting phase. BaP and PAH4 contents found in dry-cured ham samples did not exceed the currently legal levels according to the European legislation., Policiklički aromatski ugljikovodici (PAH-ovi) česti su kontaminanti u prehrambenim proizvodima. S obzirom da mesni proizvodi u značajnoj mjeri mogu biti kontaminirani PAH-ovima, cilj ovog rada bio je odrediti njihov udio u četiri vrste hrvatskih pršuta. Istraživanje je provedeno na Dalmatinskom, Drniškom, Krčkom i Istarskom pršutu, čiji se tehnološki procesi proizvodnje razlikuju. Analiza PAH spojeva provedena je primjenom visokoučinkovite tekućinske kromatografije sa flourescentnom detekcijom (HPLC-FLD). Od 15 istraživanih PAH spojeva, identificirano je i kvantificirano 13 spojeva. Prosječna koncentracija benz(a)pirena (BaP) iznosila je 0,05-0,10 µg/kg, dok su prosječne količine PAH4 spojeva iznosile 0,41-0,67 µg/kg. Unatoč tome što proces proizvodnje Krčkog i Istarskog pršuta ne uključuje fazu dimljenja, nisu određene statistički značajne razlike u udjelima BaP, PAH4, PAH8 i PAH15 spojeva između svih istraživanih pršuta. Kontaminacija PAH-ovima kod nedimljenih pršuta je mogla bi biti posljedica primjene začina u fazi soljenja. Koncentracije PAH spojeva određene u uzrocima hrvatskih vrsta pršuta nisu prekoračile dopuštene razine određene trenutno važećim zakonodavstvom Europske Unije., Polyzyklische aromatische Kohlenwasserstoffe (PAH) sind Kontaminate, die in Lebensmitteln häufig erscheinen. Da Fleischprodukte häufig mit polyzyklischen aromatischen Kohlenwasserstoffen kontaminiert sein können, ist das Ziel dieser Arbeit, ihren Anteil in vier Sorten des kroatischen Pršut zu untersuchen. Die Untersuchung wurde am Dalmatiniski, Drniški, Krčki und Istarski Pršut durchgeführt, deren Technologieverfahren sich unterscheiden. Die Analyse der polyzyklischen aromatischen Kohlenwasserstoffe wurde anhand der Hochleistungsflüssigkeitschromatograhpie mit Fluoreszentdetektor (HPLC-FLD) vorgenommen. Von den 15 untersuchten PAH-Verbindungen wurden 13 Verbindungen identifiziert und quantifiziert. Die durchschnittliche Konzentration von Benz(a)pyren betrug 0,05-0,10 µg/kg, während eine durchschnittliche Menge von PAH4 –Verbindungen 0,41-0,67 µg/kg ermittelt wurde. Obwohl das Herstellungsverfahren des Krčki und Istarski Pršut keine Räucherungsphase umfasst, wurden keine statistisch relevanten Unterschiede bei den Anteilen der BaP-, PAH4-, PAH- und PAH15-Verbindungen bei den untersuchten Pršut festgestellt. Die Kontaminierung mit polyzyklischen aromatischen Kohlenwasserstoffen bei nicht geräucherten Pršut könnte die Folge der Verwendung von Gewürzen in der Salzungsphase sein. Die Konzentration der PAH-Verbindungen, die bei den Proben der kroatischen Pršut -Sorten ermittelt wurde, überschreitet nicht die zulässigen Grenzwerte, die durch die aktuell gültige Gesetzgebung der Europäischen Union vorgeschrieben sind., Gli idrocarburi policiclici aromatici (PAH) sono frequenti agenti contaminanti nei prodotti alimentari. Poiché i prodotti a base di carne possono essere significativamente contaminati dagli PAH, questo studio aveva come scopo quello di accertare la loro presenza e la loro percentuale in quattro differenti tipi di prosciutto crudo croato. La ricerca è stata condotta su prosciutti crudi croati differenti (dalmata, di Drniš, dell’isola di Veglia e istriano) i cui processi tecnologici di produzione si differenziano l’uno dall’altro. L’analisi tendente ad accertare la presenza dei composti PAH è stata condotta con la tecnica della cromatografia liquida ad alta prestazione con rivelatori a fluorescenza (HPLC-FLD). Dei 15 PAH oggetto della ricerca, ne sono stati identificati e quantificati ben 13. È stata rilevata una concentrazione media di benzo[a]pirene (BaP) pari a 0,05-0,10 µg/kg, contro una concentrazione media di composti PAH4 pari a 0,41-0,67 µg/kg. Nonostante il fatto che il processo di produzione del prosciutto crudo dell’isola di Veglia e del prosciutto crudo istriano non preveda la fase dell’affumicamento, non sono state accertate differenze statisticamente significative nelle percentuali dei composti BaP, PAH4, PAH8 e PAH15 tra tutti i prosciutti esaminati. La presenza contaminante d’idrocarburi policiclici aromatici nei prosciutti crudi non affumicati potrebbe essere dovuta all’aggiunta di spezie nella fase della salatura. Le concentrazioni di PAH accertate nei campioni dei prosciutti crudi croati esaminati non hanno superato i livelli consentiti previsti dalla legislazione attualmente in vigore nell’Unione Europea., Hidrocarburos aromáticos policíclicos (HAP) son contaminantes frecuentes en los productos alimenticios. Tomando en cuenta que los productos cárnicos pueden estar contaminados en gran medida por los HAP, el fin de este trabajo fue determinar su contenido en cuatro tipos de los jamones croatas. La investigación fue hecha en los jamones de Dalmacia, Drniš, Krk e Istria, con los procesos tecnológicos de producción diferentes. El análisis de los compuestos HAP fue hecho por la cromatografía de líquidos de alta resolución con detección por fluorescencia (HPLC-FLD). Entre los 15 compuestos HAP analizados, 13 fueron identificados. La concentración media del benz(a)pireno (BaP) fue 0,05-0,10 µg/kg, mientras la cantidad media de los compuestos PAH4 fue 0,41-0,67 µg/kg. A pesar de que los procesos de producción de los jamones de Krk y de Istria no incluyen la fase del ahumado, no fueron determinadas las diferencias estadísticamente significativas en el contenido de los compuestos BaP, HAP4, HAP8 y HAP15 en todos los jamones analizados. La contaminación por los PAH de los jamones no ahumados pudo ser la consecuencia del uso de las especies en la fase de salazón. Las concentraciones de los compuestos HAP determinadas en las muestras de los jamones de Croacia no excedieron los niveles determinados por la legislación actual de la Unión Europea.

Today, the use of monoclonal antibodies in the treatment of a variety of diseases has made them a pioneering product in the biopharmaceutical market. In present thesis, analytical methods for bioequivalence studies of Trastuzumab, which is a monoclonal antibody in IgG structure, affects human epidermal growth factor, and used in breast cancer treatment are examined. For this purpose, three different methods were used. Initial studies included Trastuzumab analysis using HPLC-FLD system in serum samples. For total Trastuzumab determination, purification was performed from serum using a Protein G column and was analyzed by HPLC-FLD system. In the second part, considering the mechanism of action of Trastuzumab, it is examined by using electroanalytical methods whether Trastuzumab analysis can be performed by means of HER2 immobilized electrode surfaces. For this purpose, bare PGE, graphene oxide and multi-walled carbon nanotubes and their gold nanoparticle electrodes were used. After each modification, CV and EIS measurements were taken. The third part includes LC-MS/MS analysis of Trastuzumab on selected peptides after tryptic digestion. The m/z 543.2929 for the IYPTNGYTR signature peptide and m/z 402.3566 for the PTNGYTR fragment were selected and analyzed by the LC-Q-TOF-MS system. In HPLC-FLD studies, although the linearity was excellent (R=0.999) in the 5.0-120 µg/mL range, peak resolution was not sufficient. Furthermore, the selectivity of the method is limited due to multiple peak formation and poor resolution. Compared to other analytical methods, the working time is long and time consuming. When electroanalytical methods were evaluated, the best bonding was obtained with multi-walled carbon nanotubes according to the ΔRct values calculated according to the obtained data. This technique provides a great advantage to get immediate results compared to other methods. In addition, it has been found that the determination of Trastuzumab can be done by using HER2-Trastuzumab interaction for the first time electrochemically. In the LC-MS/MS studies, it was found that the quantitative analysis of Trastuzumab was possible using the LC-Q-TOF-MS system on the selected peptide., Günümüzde monoklonal antikorların çok çeşitli hastalıkların tedavisinde kullanılması, onları biyofarmasötik pazarında öncü bir ürün haline getirmiştir. Bu tez kapsamında IgG yapısında bir monoklonal antikor olan, insan epidermal büyüme faktörüne etki eden ve meme kanseri tedavisinde kullanılan Trastuzumab'ın biyoeşdeğerlik çalışmaları için analitik yöntemler incelenmiştir. Bu amaçla üç farklı yöntemden yararlanılmıştır. İlk çalışmalar, serum örneklerinde HPLC-FLD sistemi kullanılarak Trastuzumab analizini içermektedir. Toplam Trastuzumab tayini için Protein G kolan kullanılarak serumdan saflaştırma yapılmış ve HPLC-FLD sistemi ile analizlenmiştir. İkinci bölümde Trastuzumab'ın etki mekanizması dikkate alınarak HER2 immobilize edilen elektrot yüzeyleri vasıtasıyla Trastuzumab analizi yapılıp yapılamayacağı elektroanalitik yöntemler kullanılarak incelenmiştir. Bu amaçla yalın PGE, grafen oksit ve çok duvarlı karbon nanotüpler ve bunların altın nanopartiküllü elektrotları kullanılmıştır. Her modifikasyon işleminden sonra CV ve EIS ölçümleri alınmıştır. Üçüncü kısım ise, Trastuzumab'ın triptik bozundurma işlemi sonrasında seçilen peptitleri üzerinden LC-MS/MS analizini içermektedir. IYPTNGYTR işaretli peptidi için m/z 543.2929 ve PTNGYTR fragmenti için m/z 402.3566 iyonu seçilerek LC-Q-TOF-MS sistemi ile analizlenmiştir. HPLC-FLD çalışmalarında, doğrusallık 5.0-120 µg/mL aralığında mükemmel (R=0.999) olmasına rağmen, pik ayrımı yeterli değildir. Ayrıca yöntemin seçiciliği, çoklu tepe oluşumu ve zayıf çözünürlük nedeniyle sınırlıdır. Diğer analitik yöntemlere göre çalışma süresi uzun ve zaman alıcıdır. Elektroanalitik yöntemler değerlendirildiğinde elde edilen veriler doğrultusunda hesaplanan ΔRct değerlerine göre en iyi bağlanma çok duvarlı karbon nanotüpler ile elde edilmiştir. Bu teknik diğer yöntemlere göre anında sonuç almak için büyük bir avantaj sağlamaktadır. Ayrıca elektrokimyasal olarak ilk defa HER2-Trastuzumab etkileşimi kullanılarak Trastuzumab tayininin yapılabileceği görülmüştür. LC-MS/MS çalışmalarında, seçilen peptit üzerinden LC-Q-TOF-MS sistemi kullanılarak Trastuzumab'ın kantitatif analizinin mümkün olduğu bulunmuştur.