Recombinant therapeutic proteins produced in plants: towards engineering of human-type O-and N-glycosylation

Background and Purpose: Recombinant DNA technology has allowed expression of different heterologous proteins in many host systems, ranging from prokaryotic to eukaryotic organisms. Therapeutic properties of recombinant proteins are very often affected by the composition and heterogeneity of their glycans. Conventional expression systems for recombinant pharmaceuticals typically do not address this problem and result with products that contain a mixture of glycoforms that are neither identical to human glycans nor optimized for enhanced biological activity. Over the last decade plants have been developed as production platforms for recombinant proteins with pharmaceutical or industrial applications. Namely, plant expression systems contain very small differences in the post-translational modifications, mainly glycosylation, which can partly be overpowered by glycoengineering, whose goal is production of recombinant proteins with highly homogenous glycosylation that closely resembles the native system. This review attempts to present current accomplishments in the production of plant-derived glycoconjugates with humanized N- and O-glycans. Materials and Methods: Main goal of N-glycoengineering is to reduce or eliminate plant-specific N-glycans, and at the same time to introduce mammalian-specific N-glycans through the several approaches. The easiest way is to change intracellular targeting of plant-made recombinant proteins and to ensure their retention in the ER; next approach is to eliminate the addition of plant-specific glycans; while the final step is engineering the plant glycosylation pathway to introduce mammalian glycotransferases into plants with generation of biantennary and multi-antennary structures on complex N-glycans. Due to significant differences in O-glycosylation between humans and plants, different approaches to engineering of O-glycosylation have been taken. Besides having their typical O-glycoslyation on Hyp-residues, plants in general miss the machinery for production of mammalian-type O-glycosylation. Attempts have been made to mimic mammalian O-glycosylation in plants, specifically the mucin-type addition of GalNAc residues. Result: Efficient generation of bisected tetraantennary complex N-glycans without typical plant glycoepitopes on human erythropoietin (hEPO) and human transferrin (hTF) was obtained in Nicotiana benthamiana plants, thus demonstrating generation of recombinant proteins with human-type N-glycosylation at great uniformity. As for the O-glycosylation, attempts to produce mucin-type O-GalNAc and disialylated core 1 O-linked glycan structures on hEPO in N. benthamiana transgenic plants proved to be successful. Moreover, although small amounts of Hyp residues were found on recombinant EPO, no plant-specific O-glycans were detected, which demonstrates that plants are eligible candidates for production of recombinant therapeutics with fully humanized O- and N-glycans. Conclusion: Plants and methods of plant molecular farming offer a powerful expression platform for the production of a variety of recombinant proteins, which show similar, or even higher, biological activity then protein or native homologs in cultured mammalian cells currently used for large-scale production

FOOD SAFETY IS AN IMPORTANT PUBLIC HEALTH ISSUE: CHLORAMPHENICOL RESIDUES DETERMINATION BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LC-MS/MS) IN HONEY

Background: Honey is used for nutritional, medicinal and industrial purposes and antibiotic residues may harm its quality and constitute a danger to human health. The broad spectrum antibiotic chloramphenicol (CAP) was used for curative purposes in veterinary medicine, but is now forbidden in European Union (EU) because of its many serious side effects (e.g. aplastic anaemia, grey syndrome, severe bone marrow depression and hypersensitivity). The aim of this study was to facilitate analyses of the quality and safety of Croatian honey distributed to whole European Union market; an assessment that has not previously been made. Subjects and methods: CAP in honey was qualifying and quantifying by validated liquid chromatography tandem mass spectrometry with negative electrospray ionisation method (LC-MS/MS). The target antibiotic was separated on chromatographic column Zorbax SB C18 (150 mm × 2.1 mm, 3.5 μm) with a gradient elution using acetonitrile - 0.1% formic acid mobile phase at a flow rate of 0.3 mL/min, with column temperature 35 oC for CAP and 5D-CAP as internal standard. Homogenised honey samples were diluted with acetate buffer solution and extracted on Oasis Hydrophilic-Lipophilic-Balanced (HLB) sorbents. The method was used to analyse 280 domestic honey samples collected throughout Croatia between 2005.–2013. Results: Recoveries of the method for real (acacia, chestnut, linden and flower) honey samples were 102% with RSD 8.4%. The value CCα and CCß were 0.09 and 0.12 μg/kg, respectively. Results showed only three subsequent positive detections (1.1%) of CAP in honey. Conlusions: Analysed honey samples from Croatia showed good quality and safety what is the one of the main objective in consumer health policy in EU

Silver nanoparticles affect germination and photosynthesis in tobacco seedlings

Extensive commercialization of silver nanoparticles (AgNPs) raises the risk of their accumulation in the soil-plant system. Once released into the environment, AgNPs are prone to chemical transformations, which make it hard to determine whether their phytotoxic effects are purely NP-related or a consequence of released Ag+ ions. In this study the effects of 25, 50, 75, 100 and 150 μM AgNPs and AgNO3 on seed germination and early growth of tobacco (Nicotiana tabacum L.) seedlings were compared. Additionally, the effects on photosynthetic performance and pigment content were investigated. Germination rate and index values indicated delayed and slower germination in some AgNP treatments. Lower AgNP concentrations stimulated root growth, but induced a prominent reduction in fresh weight. By contrast, all AgNO3 concentrations inhibited root growth but only the higher ones decreased fresh weight. Obtained results imply that the observed AgNP toxicity could be ascribed to NP form and can be correlated with high AgNP stability in the solid medium. On the other hand, the majority of AgNP and AgNO3 treatments induced an increase in chlorophyll content that was accompanied by significantly lower values of relative electron transport rate and coefficient of photochemical quenching, implying an inhibition of the electron transport chain. A similar impact of AgNPs and AgNO3 on photosynthesis can be correlated with lower stability of AgNPs in a liquid medium, resulting in AgNP aggregation and dissolution of Ag+ ions

Rast i fotosinteza u vodene leće (Lemna minor L.) izložene kadmiju u kombinaciji s cinkom ili bakrom

Metals have a variety of negative outcomes on plants, essential components of any ecosystem. The effects of CdCl2 (5 μmol L-1), ZnCl2 (25 or 50 μmol L-1), and CuCl2 (2.5 or 5 μmol L-1) and combinations of CdCl2 with either ZnCl2 or CuCl2 on the growth, photosynthetic pigments, and photosystem II (PSII) efficiency of duckweed (Lemna minor L.) were investigated. All of the treatments caused growth inhibition and remarkable metal accumulation in plant tissue after 4 and 7 days. In the combined treatments, the accumulation of each metal applied was lesser in comparison to treatments with single metals. After 4 days, all of the treatments generally diminished chlorophyll a content and decreased the maximum quantum yield (Fv/Fm) and effective quantum yield (ΔF/F’m) of PSII. However, after 7 days of exposure to a combination of Cd and Zn, pigment content and PSII activity recovered to control levels. A higher concentration of Cu (5 μmol L-1) as well as Cd in combination with Cu had a prolonged inhibitory effect on photosynthetic features. Our results suggest that growth inhibition was due to the toxic effect of absolute metal quantity in plant tissue. Zn counteracted Cd uptake, as seen from the recovery of pigment content and PSII efficiency in plants exposed for 7 days to the Cd and Zn combination. Cu-induced oxidative stress led to a prolonged inhibitory effect in plants treated both with a higher concentration of Cu (5 μmol L-1) and simultaneously with Cd and Cu. Our findings could contribute to general knowledge on anthropogenic and environmental contaminants that endanger plant communities and significantly disrupt the sensitive balance of an ecosystem by influencing photosynthetic mechanisms.Izloženost metalima može izazvati različite štetne učinke u biljaka. Vodene leće izložili smo solima teških metala CdCl2 (5 μmol L 1), ZnCl2 (25 μmol L 1 ili 50 μmol L 1) i CuCl2 (2,5 μmol L 1 ili 5 μmol L 1) te kombinaciji CdCl2 sa svakom od navedenih koncentracija ZnCl2 i CuCl2. Rast biljaka, količina fotosintetskih pigmenata i učinkovitost fotosistema II (PSII) mjereni su nakon četiri i sedam dana tretmana. Utvrđeno je da su svi tretmani uzrokovali značajnu inhibiciju rasta te akumulaciju metala u biljci. U biljaka koje su bile izložene kombinacijama teških metala količina pojedinog metala u tkivu bila je niža u odnosu na količinu istog metala u biljaka izloženih samo tom metalu. Nakon četiri dana tretmana sva su tri metala, neovisno o tome jesu li bila primijenjena zasebno ili u kombinacijama, uzrokovala smanjenje količine klorofila a i pad vrijednosti maksimalnog (Fv/Fm) i efektivnog (ΔF/F’m) prinosa PSII. Međutim, u biljaka koje su bile istovremeno izlagane kadmiju i cinku, vrijednosti količine pigmenata i učinkovitost PSII vratile su se nakon sedam dana na kontrolnu razinu, a bakar u koncentraciji 5 μmol L 1 te kombinacija kadmija i bakra i dalje su imali inhibitorni učinak. Budući da smanjeno primanje pojedinog metala uočeno u biljaka izloženih kombiniranim tretmanima nije ublažilo inhibitorni učinak na rast, možemo zaključiti da je inhibicija rasta uzrokovana apsolutnom količinom metala primljenog u tkivo. Povećanje količine fotosintetskih pigmenata i učinkovitosti PSII nakon sedam dana tretmana kadmijem i cinkom upućuje na oporavak biljaka, što se može objasniti ublažavajućim djelovanjem cinka na učinak kadmija uslijed smanjenog primanja kadmija u biljku. Suprotno tome, dugotrajni inhibitorni učinak istovremenog tretmana biljaka kadmijem i bakrom te samim bakrom u koncentraciji 5 μmol L 1 može se objasniti oksidacijskim stresom uzrokovanim bakrom. Rezultati ovoga istraživanja pridonose saznanjima o štetnim učincima antropogenih i okolišnih onečišćivača, koji narušavanjem mehanizama fotosinteze ugrožavaju ne samo biljke i njihove zajednice nego i osjetljivu ravnotežu ekosustava

Procjena toksičnosti akrilamida pomoću standardiziranih biotestova

Acrylamide is a monomer widely used as an intermediate in the production of organic chemicals, e.g. polyacrylamides (PAMs). Since PAMs are low cost chemicals with applications in various industries and waste- and drinking water treatment, a certain amount of non-polymerised acrylamide is expected to end up in waterways. PAMs are non-toxic but acrylamide induces neurotoxic effects in humans and genotoxic, reproductive, and carcinogenic effects in laboratory animals. In order to evaluate the effect of acrylamide on freshwater organisms, bioassays were conducted on four species: algae Desmodesmus subspicatus and Pseudokirchneriella subcapitata, duckweed Lemna minor and water flea Daphnia magna according to ISO (International Organization for Standardisation) standardised methods. This approach ensures the evaluation of acrylamide toxicity on organisms with different levels of organisation and the comparability of results, and it examines the value of using a battery of low-cost standardised bioassays in the monitoring of pollution and contamination of aquatic ecosystems. These results showed that EC50 values were lower for Desmodesmus subspicatus and Pseudokirchneriella subcapitata than for Daphnia magna and Lemna minor, which suggests an increased sensitivity of algae to acrylamide. According to the toxic unit approach, the values estimated by the Lemna minor and Daphnia magna bioassays, classify acrylamide as slightly toxic (TU=0-1; Class 1). The results obtained from algal bioassays (Desmodesmus subspicatus and Pseudokirchneriella subcapitata) revealed the toxic effect of acrylamide (TU=1-10; Class 2) on these organisms.Akrilamid je monomer koji se koristi kao intermedijer u proizvodnji organskih kemikalija, npr. poliakrilamida (PAM-a). PAM se primjenjuje u različitim industrijama te u obradi otpadnih voda i tretmanu vode za piće, a prihvatljiv je i zbog niske cijene. Široka upotreba PAM-a u spomenute svrhe predstavlja rizik od toga da određena količina nepolimeriziranog akrilamida dospije u vodene ekosustave. PAM je netoksičan, ali akrilamid ima neurotoksične učinke u ljudi te je također dokazana njegova genotoksičnost, kancerogenost i štetan utjecaj na reprodukcijski sustav u laboratorijskih životinja. U cilju procjene toksičnosti akrilamida provedeni su biotestovi na četirima vrstama slatkovodnih organizama: na zelenim algama Desmodesmus subspicatus i Pseudokirchneriella subcapitata, vodenoj leći Lemna minor i vodenbuhi Daphnia magna. Biotestovi su provedeni prema standardiziranim ISO metodama, što osigurava procjenu toksičnosti akrilamida na organizme različitog stupnja organizacije, usporedivost rezultata i procjenu pogodnosti korištenja niza standardiziranih biotestova prihvatljive cijene za praćenje zagađenja i onečišćenja vodnih ekosustava. Rezultati su pokazali da su EC50 vrijednosti niže nakon izlaganja zelenih algi Desmodesmus subspicatus i Pseudokirchneriella subcapitata akrilamidu u odnosu na vrijednosti dobivene za vodenu leću Lemna minor i vodenbuhu Daphnia magna, što pokazuje veću osjetljivost algi. S obzirom na klasifikacijski sustav za izražavanje stupnja toksičnosti, a na temelju vrijednosti utvrđenih za vodenu leću Lemna minor i vodenbuhu Daphnia magna, akrilamid se može klasificirati kao slabo toksičan (TU=0-1; razred 1). Rezultati dobiveni testovima na algama (Desmodesmus subspicatus i Pseudokirchneriella subspicata) dokazuju toksičan učinak akrilamida na tim organizmima (TU=1-10; razred 2)

Flower honey as a potential bio-indicator of environmental pollution by pesticides in the Varaždin County

Cilj: U ovom istraživanju uzorci cvjetnoga meda korišteni su kao bio-pokazatelji onečišćenja okoliša pesticidima. Materijal i metode: Ukupno je prikupljeno 40 (4x10) uzoraka sa 4 različite prostorne lokacije (subregije), u ukupno 16 općina, od proizvođača meda u Varaždinskoj županiji, kao i 20 (2x10) uzoraka s dvije različite lokacije proizvođača u susjednoj županiji, registriranih kao "ekoloških proizvođači", a udaljenih od poljoprivrednih površina pod konvencionalnom proizvodnjom. Analize su provedene korištenjem vezanog sustava plinske kromatografije i spektrometrije masa (GCMS) i tekućinskom kromatografijom visoke djelotvornosti (HPLC). Rezultati: Identificirano je i kvantificirano 78 aktivnih sastojaka pesticide, koji su dio od ukupno cca 400 pesticida koji se analiziraju u voću i povrću, a za koje je laboratorij raspolagao analitičkom opremom koja i dijelom određuje i vrstu i broj pesticida koje se detektiraju. Rezultati analiza ukazali su na mogući uzročno posljedični odnos između konvencionalnih poljoprivrednih aktivnosti u Varaždinskoj županiji s identificiranim i kvantificiranim aktivnim sastojcima pesticida. Iz rezultata dobivenih na udjelima identificiranih i kvantificiranih ostataka pesticida, s obzirom na dopustivost, vidljivo je da je udio dopuštenih pesticida u uzorcima cvjetnoga meda iznosio 55,2%, dok je udio ilegalnih pesticida iznosio 44,8%. Analizirajući broj otkrivenih insekticida i fungicida, ukupno na sve 4 lokacije utvrđeno je da cvjetni med sadrži 13 različitih vrsta insekticida i 3 vrste fungicida. Zaključak: Rezultati i statistička analiza podataka pokazuju da je cvjetni med dobar bio-indikator onečišćenja okoliša pesticidima, te stoga ova studija može poslužiti kao osnova za znanstvenike i stručnjake koji se bave istraživanjem štetnih učinaka pesticida na okoliš i ljudsko zdravlje.Aim: In this research samples of floral honey were used as bio-indicators of environmental pollution by pesticides. Material and methods: A total of 40 (4x10) samples from bee farms were collected at 4 different locations (sub-regions) including 16 municipalities in the Varaždin County, as well as 20 (2x10) samples from two locations – bee-farms registered as "ecological producers" in the neighborhood County, both located far away from conventional agricultural production. Analyses were carried out using the gas chromatography joint system with mass spectrometry (GCMS) and high-performance liquid chromatography (HPLC). Results: Identification and quantification of 78 pesticide active ingredients were performed which are part of a total of approximately 400 pesticides that are usually analyzed in fruits and vegetables whereby the laboratory had available analytical equipment that also determined the number of detected pesticides. Due to identified and quantified active ingredients of pesticides, the results of analyses might indicate a possible cause-effect relationship with conventional agricultural activities in the Varaždin County. It was evident from the obtained results on the shares of identified and quantified pesticide residues, and with respect to admissibility, that the share of permitted pesticides in the samples of floral honey accounted for 55.2%, while the share of illegal pesticides accounted for 44.8%. Analyzing the number of detected insecticides and fungicides, on a total of all 4 locations (sub-regions), it was found that floral honey contained 13 different types of insecticides and 3 types of fungicides. Conclusion: The results and statistical data analysis showed that floral honey is a good bio-indicator of environmental pollution by pesticides, therefore this study can serve as a basis for scientists and experts engaged in the research of harmful effects of pesticides on the environment and human health

Učinak bakra na toksičnost i genotoksičnost kadmija u vodenoj leći (Lemna minor L.)

We investigated interactions between copper (in the concentrations of 2.5 μmol L-1 and 5 μmol L-1) and cadmium (5 μmol L-1) in common duckweed (Lemna minor L.) by exposing it to either metal or to their combinations for four or seven days. Their uptake increased with time, but it was lower in plants treated with combinations of metals than in plants treated with either metal given alone. In separate treatments, either metal increased malondialdehyde (MDA) level and catalase and peroxidase activity. Both induced DNA damage, but copper did it only after 7 days of treatment. On day 4, the combination of cadmium and 5 μmol L-1 copper additionally increased MDA as well as catalase and peroxidase activity. In contrast, on day 7, MDA dropped in plants treated with combinations of metals, and especially with 2.5 μmol L-1 copper plus cadmium. In these plants, catalase activity was higher than in copper treated plants. Peroxidase activity increased after treatment with cadmium and 2.5 μmol L-1 copper but decreased in plants treated with cadmium and 5 μmol L-1 copper. Compared to copper alone, combinations of metals enhanced DNA damage after 4 days of treatment but it dropped on day 7. In conclusion, either metal given alone was toxic/genotoxic and caused oxidative stress. On day 4 of combined treatment, the higher copper concentration was more toxic than either metal alone. In contrast, on day 7 of combined treatment, the lower copper concentration showed lower oxidative and DNA damage. These complex interactions can not be explained by simple antagonism and/or synergism. Further studies should go in that direction.U svrhu istraživanja interakcija između bakra kao esencijalnog elementa te kadmija kao neesencijalnog i toksičnog metala, vodenu leću Lemna minor L. uzgajali smo na podlogama s kadmijem (5 μmol L-1) odnosno s bakrom (2,5 μmol L-1 i 5 μmol L-1) te s njihovim kombinacijama. Unos metala u biljke povećavao se s trajanjem pokusa, a kod kombinacije metala u biljkama je izmjerena niža količina kadmija nego u onima uzgajanima samo na kadmiju. U biljkama tretiranim pojedinačnim metalom došlo je do povećanja sadržaja malondialdehida (MDA) te aktivnosti katalaze i peroksidaze u odnosu na kontrolne biljke. Također, primijećeno je oštećenje DNA iako kod bakra tek sedmog dana tretmana. Količina MDA i aktivnost obaju enzima dodatno se povećala na tretmanu kombinacijom kadmija i bakra (5 μmol L-1) nakon četvrtog dana pokusa, dok se količina MDA smanjila nakon sedmog dana kod kombinacije kadmija i 2,5 μmol L-1 bakra. U tim biljkama primijećena je i veća aktivnost katalaze, dok je aktivnost peroksidaze porasla na tretmanu kadmijem i 2,5 μmol L-1 bakrom, ali se smanjila na tretmanu kadmijem i 5 μmol L-1 bakrom. Oštećenje DNA koje je bilo veće kod kombinacije metala nakon četvrtog dana, osobito u usporedbi sa samim bakrom, smanjilo se nakon sedmog dana pokusa. Iz ovih rezultata može se zaključiti da su oba metala u istraživanim koncentracijama toksična i genotoksična za vodenu leću i da uzrokuju oksidacijski stres. Kadmij u kombinaciji s bakrom više koncentracije bio je toksičniji od pojedinačnih metala nakon četvrtog dana pokusa, dok su u biljaka tretiranih kombinacijom kadmija i bakra niže koncentracije toksični učinci bili manji. Budući da su primijećene interakcije vrlo kompleksne i ne uključuju samo antagonizam odnosno sinergizam potrebna su daljnja istraživanja

Water for all : Proceedings of the 7th international scientific and professional conference Water for all

The 7th International Scientific and Professional Conference Water for all is organized to honour the World Water Day by the Josip Juraj Strossmayer University of Osijek, European Hygienic Engineering & Design Group (EHEDG), Danube Parks, Croatian Food Agency, Croatian Water, Faculty of Food Technology Osijek, Faculty of Agriculture in Osijek, Faculty of Civil Engineering Osijek, Josip Juraj Strossmayer University of Osijek Department of Biology, Josip Juraj Strossmayer University of Osijek Department of Chemistry, Nature Park “Kopački rit”, Osijek- Baranja County, Public Health Institute of the Osijek- Baranja County and „Vodovod-Osijek“ -water supply company in Osijek. The topic of World Water Day 2017 was "Wastewater" emphasizing the importance and influence of wastewater treatments on global environment. The international scientific and professional conference Water for all is a gathering of scientists and experts in the field of water management, including chemists, biologists, civil and agriculture engineers, with a goal to remind people about the significance of fresh water and to promote an interdisciplinary approach and sustainability for fresh water resource management. The Conference has been held since 2011. About 300 scientists and engineers submitted 95 abstracts to the 7th International Scientific and Professional Conference Water for all, out of which 33 was presented orally and 62 as posters. 47 full papers were accepted by the Scientific Committee. 38 full papers became the part of the this Proceedings while 9 papers were accepted for publication in Croatian Journal of Food Science and Technology and Electronic Journal of the Faculty of Civil Engineering Osijek - e-GFOS

Kadmij i cink induciraju slične promjene u uzorku proteina i glikoproteina u klijancima i odraslim biljkama duhana (Nicotiana tabacum L.)

The effects of 10 μmol L-1 and 15 μmol L-1 cadmium (Cd), a nonessential toxic element and 25 μmol L-1 and 50 μmol L-1 zinc (Zn), an essential micronutrient, on proteins and glycoproteins of Nicotiana tabacum L. seedlings and plants were investigated after exposure to each metal alone or to their combinations. Changes in only few polypeptides related to heavy metal treatments were observed in tobacco seedlings and leaves of adult plants, while the greatest change in total soluble protein pattern was observed in plant roots. Differences between control and treated tobacco tissues were more pronounced in the glycoprotein pattern, which was analysed by application of different lectins. The majority of the detected glycoproteins in leaves and roots of adult plants can be considered as a result of enhanced glycosylation due to heavy metal stress. The difference in glycoproteins between Cd and Zn application on tobacco seedlings and adult plants could not be determined since enhanced glycosylation was noticed after treatment with either metal alone or in combination. Therefore, it can be concluded that both metals induced N- and Oglycosylation as a result of changed environmental conditions.Ispitivali smo učinke 10 μmol L-1 i 15 μmol L-1 kadmija (Cd), neesencijalnoga toksičnog elementa i 25 μmol L-1 i 50 μmol L-1 cinka (Zn), esencijalnog mikronutrijenta, na proteine i glikoproteine u klijancima i odraslim biljkama Nicotiana tabacum L. nakon izlaganja svakomu metalu posebno ili njihovim kombinacijama. Promjene kod nekoliko polipeptida koje su uočene u klijancima i listovima odraslih biljaka nisu bile brojne, dok su one najvažnije zabilježene u uzorku ukupnih topljivih proteina u korijenu biljke. Razlike između kontrole i tretiranog tkiva duhana bile su izraženije kod glikoproteina koji su analizirani primjenom različitih lektina. Većina glikoproteina uočenih u listovima i korijenu odraslih biljaka može se smatrati rezultatom povećane glikozilacije zbog stresa koji uzrokuju teški metali. Nije bilo moguće utvrditi razliku u glikoproteinima između tretiranja klijanaca i odraslih biljaka duhana kadmijem i cinkom jer je povećana glikozilacija utvrđena i nakon tretmana svakim metalom posebno i nakon tretmana njihovim kombinacijama. Stoga se može zaključiti da su, kao rezultat promijenjenih uvjeta u okolišu, oba metala potaknula N- i O-glikozilaciju

FOOD SAFETY IS AN IMPORTANT PUBLIC HEALTH ISSUE: CHLORAMPHENICOL RESIDUES DETERMINATION BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LC-MS/MS) IN HONEY

Background: Honey is used for nutritional, medicinal and industrial purposes and antibiotic residues may harm its quality and constitute a danger to human health. The broad spectrum antibiotic chloramphenicol (CAP) was used for curative purposes in veterinary medicine, but is now forbidden in European Union (EU) because of its many serious side effects (e.g. aplastic anaemia, grey syndrome, severe bone marrow depression and hypersensitivity). The aim of this study was to facilitate analyses of the quality and safety of Croatian honey distributed to whole European Union market; an assessment that has not previously been made. Subjects and methods: CAP in honey was qualifying and quantifying by validated liquid chromatography tandem mass spectrometry with negative electrospray ionisation method (LC-MS/MS). The target antibiotic was separated on chromatographic column Zorbax SB C18 (150 mm × 2.1 mm, 3.5 μm) with a gradient elution using acetonitrile - 0.1% formic acid mobile phase at a flow rate of 0.3 mL/min, with column temperature 35 oC for CAP and 5D-CAP as internal standard. Homogenised honey samples were diluted with acetate buffer solution and extracted on Oasis Hydrophilic-Lipophilic-Balanced (HLB) sorbents. The method was used to analyse 280 domestic honey samples collected throughout Croatia between 2005.–2013. Results: Recoveries of the method for real (acacia, chestnut, linden and flower) honey samples were 102% with RSD 8.4%. The value CCα and CCß were 0.09 and 0.12 μg/kg, respectively. Results showed only three subsequent positive detections (1.1%) of CAP in honey. Conlusions: Analysed honey samples from Croatia showed good quality and safety what is the one of the main objective in consumer health policy in EU