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The use of thermochromic ink is growing daily, but they contain pseudoestrogenic compounds that have a harmful effect on the human health. Common pseudoestrogenic compounds found in thermochromic ink are bisphenol A and benzophenone. Bisphenol A has a role of the colour developer and benzophenone is used as a photo- initiator in the UV-curing ink. Since they are registered as harmful and their use is limited, manufacturers are using new compounds whose use is not limited yet but also have a pseudoestrogenic effect. Therefore, the main goal of this study is to develop and validate an appropriate chromatographic method for monitoring 4, 4'- (4-methylpentane-2, 2-diyl)diphenol in various ink samples.[1, 2] Validated parameters were specificity, selectivity, linearity, precision, accuracy, quantitation limit and detection limit. Following validation, eleven samples of thermochromic ink were analyzed and 4, 4'-(4-methylpentane-2, 2- diyl)diphenol was found in nine of them. The mass fractions of 4, 4'-(4-methylpentane-2, 2- diyl)diphenol in the samples ranged from 0.12 % to 0.90 %. These results indicate that thermochromic ink that do not contain bisphenol A as a developer can contain a significant amount of bisphenol A related pseudoestrogenic compound. So, it could be concluded that thermochromic ink without bisphenol A may also present a health risk in the use of thermochromic ink for human but also for environment.

Termokromne boje su materijal čija je uporaba u porastu, a glavne komponente često su im pseudoestrogeni bisfenol A i benzofenon. Za identifikaciju i kvantifikaciju ovih spojeva u termokromnim bojama razvijena je tekućinskokromatografska metoda obrnutih faza s UV-detekcijom. Tri od analiziranih petnaest uzoraka boja sadržavali su bisfenol A s masenim udjelima oko 2 %, a dva uzorka benzofenon s masenim udjelima od 0,34 % i 0,66 %. Termokromna boja koja sadrži bisfenol A i koju je moguće tiskati u laboratorijskim uvjetima otisnuta je na različite vrste papira. Razvijena je tekućinskokromatografska metoda za određivanje bisfenola A u termokromnim otiscima na papiru ekstrakcijom metanolom. Maseni udio bisfenola A u papirima bio je od 0,126 mg g‒1 do 0,778 mg g‒1. Otisci termokromne boje koja sadrži bisfenol A podvrgnuti su anaerobnoj razgradnji u tlu čime su simulirani uvjeti razgradnje na deponiju otpada. Razvijena je tekućinskokromatografska metoda s UV-detekcijom kojom je određen maseni udio bisfenola A u tlu u vremenu od 14 do 150 dana razgradnje papira i procijenjena brzina njegova raspada. Tijekom anaerobne ragradnje maseni udio bisfenola A u termokromnim papirima smanjio se do vrijednosti bliske granici određivanja metode od 0,0025 mg g‒1. Maseni udio bisfenola A u tlu tijekom anaerobne razgradnje se povećavao i nakon 150 dana razgradnje bio je od 3,21 ng g‒1 do 35,1 ng g‒1. Thermochromic inks, materials increasingly used, may contain pseudo-estrogens bisphenol A and benzophenone as main ingredients. To identify and quantify these compounds, reversed-phase liquid chromatographic method with UV-detection was developed. Three out of 15 analyzed samples contained bisphenol A in mass fractions of about 2 %. Two samples contained benzophenone with mass fractions of 0,34 % and 0,66 %. Thermochromic ink that contains bisphenol A and was applicable in laboratory conditions was printed on various types of papers. Liquid chromatographic method for determination of bisphenol A extracted in methanol was developed. Mass fraction of bisphenol A in papers was from 0,126 mg g–1 to 0,788 mg g–1. Prints of thermochromic ink containing bisphenol A were subjected to anaerobic degradation in soil to simulate the conditions at a landfill site. Reversed-phase liquid chromatographic method with UV-detection was developed to determine mass fraction of bisphenol A in soil at time intervals from 14 to 150 days, and the rate of its degradation was estimated. During the anaerobic degradation, mass fraction of bisphenol A in papers decreased to value close to detection limit of the method of 0,0025 mg g–1. Mass fraction of bisphenol A in soil increased during the anaerobic degradation, and after 150 days ranged from 3,21 ng g–1 to 35,1 ng g–1.

Bisphenol A (BPA) has received a great deal of attention since 1996, when it was classified by the European Commission as a substance of external origin with a harmful effect on human health. Numerous toxicological and biochemical studies have confirmed that BPA has estrogenic properties through its agonistic effect toward the estrogenic receptor, and it was classified as endocrine disruptor. The effects of exposure to BPA can be particularly harmful to the fetus, infants and young children. Contact with BPA at that time may lead to irreversible changes appearing even after much delay. Exposure to BPA increases the risk of miscarriages, genetic defects (e.g. Down’s syndrome), breast and prostate cancer, diabetes, obesity and cardiovascular disease. Thermochromic printing inks, which are widely used in security printing, in thermal printing paper, as temperature indicators, and as a source of various effects in packaging and design often contain BPA as one of the main compounds present in mass fractions of up to several percent. This study presents the BPA determination during anaerobic degradation of thermochromic prints in soil, which simulated the conditions at a landfill site. Thermochromic ink that contains bisphenol A was printed on seven types of papers. The papers were subjected to anaerobic degradation in soil at time intervals from 14 to 150 days. Reversed-phase liquid chromatographic methods with UV-detection were developed to determine the mass fraction of bisphenol A in paper and in soil, and the rate of BPA degradation was estimated. During the anaerobic degradation, mass fraction of BPA in papers decreased from the initial values of 0.126– 0.788 mg g-1 to value close to detection limit of the method of 0.0025 mg g-1. Mass fraction of BPA in soil increased during the anaerobic degradation, and after 150 days ranged from 3.21 ng g-1 to 35.1 ng g-1.

More than 1055 active ingredients are currently registered as pesticide, which compose more that 16000 different pesticide products. Pesticide use raises a number of environmental and health concerns since they are toxic to living organisms. Over 98 % of sprayed insecticides and 95 % of herbicides reach a destination other than their target species, such as air, water and soil and it is necessary to monitor their presence in environment, food and water as well as their distribution, persistency and degradation path. Pesticides can cause a variety of acute and delayed adverse health effects from simple irritation of the skin and eyes to more severe effects such as affecting the nervous system, mimicking hormones causing reproductive problems, and causing cancer. Currently, the most used techniques for pesticide analyses are GC, GC-MS and LCMS, which allow simultaneous and sensitive determination of larger number of pesticides. However, these techniques are more suitable for non-polar pesticides than for smaller polar molecules, which are more difficult to handle regarding selectivity and detection. Articles that describe pesticide analysis using ion chromatography are quite rare and mostly not recent. Usual application is pesticide separation on anion-exchange column with carbonate mobile phase followed by conductivity or eventually UV detection. In the last few years new stationary phases were developed, detection techniques other than conductivity are commonly used and sensitivity is improved by involving new devices and miniaturization of ion chromatography. Thus, the aim of this work is to study the new possibilities of pesticide determination using combination of several stationary phases and detection techniques characteristic for ion chromatography. Representatives of pesticides were selected according to chemical structure, usage and toxicity. Phenoxy-pesticides are represented by 2, 4-D (2, 4-dichlorophenoxyacetic acid), very common pesticide in use more than 50 years, dicamba (3, 6-dichloro-2-methoxyibenzoic acid) which replaced on market very dangerous 2, 4, 5-T (2, 4, 5-trichlorophenoxyacetic acid) and moderately toxic chloramben (3-amino-2, 5-dichlorobenzoic acid). Sellection of pyridines are systematic pesticide picloram (4-amino-3, 5, 6-trichloro-2- pyridinecarboxyiic acid), triclopyr ((3, 5, 6-trichloro-2-pyridinil)oxyacetic acid)) which is banned in several countries and clopyralid (3, 6-dichloro-2-pyridinecarboxylic acid) with characteristic accumulation in vegetables. Chosen phosphoamines are one of most used pesticides glyphosate (N- (phosphomethyl)glycine), its metabolite AMPA (aminomethylphosphonic acid) and similar glufosinate ammonium (2-amino-4-(hydroxy-methyl- phosphoryl)butanoic acid). Triazolic pesticides are represented by carcinogenic amitrole (3-amino- 1, 2, 4-triazole) and taken from triazines are prometon, desmetrin and metabolite cyanuric acid. Possibilities of pesticide determination will be studied first on capillary anionexchange column with conductivity detection. Separation on microbore anion-exchanger with conductivity, UV and amperometric detection will be tested, as well as separation on cationexchange column with at least conductivity detection. Finally, separation on ion-exclusion and mixed mode stationary phases with adequate detection techniques will be examined.

Lecture is giving an overview through development and validation of cation-exchange chromatography methods for determination of protein impurity in protein pharmaceutical product. Active ingredient in pharmaceutical product Neupogen® is granulocyte-colony stimulating factor (G-CSF), a protein, which controls number of neutrophil. Its application is useful for patients suffering from congenital defects, bone marrow suppression, infections and cancer treated with cytotoxic chemotherapy, which can cause decrease of neutrophil number. This condition, known as neutropenia can lead to bacterial and secondary fungal infections causing serious health complications. Application of recombinant human granulocyte-colony stimulating factor (rhG-CSF) produced in Escherichia coli also known as Filgrastim ot by commercial name Neupogen® can decrease period of neutropenia or even prevent it. One of impurities that appears during synthesis of rhG-CSF in E. coli is formyl-methionine granulocyte-colony stimulating factor (f-Met rhG- CSF), N-terminally blocked isoform of G-CSF. Chromatographic method for determination of f-Met rhG-CSF was first described in 1992 based on cation exchange chromatography with UV absorbance detection. Results obtained for different combinations of stationary and mobile phase, detection technique and chromatographic condition will b presented. Optimal choice of stationary and mobile phase, chromatographic conditions and detection technique enabled fraction collecting without coeluting interferences of other rhG-CSF variants and identification by MALDI-TOF mass spectrometer, as well as validation of chosen method with results within acceptance criteria for pharmaceutical industry.

The lecture describes method development, validation, and the results of its application to protein samples with high chloride concentration. Sodium azide was used as preservative for preparative column during production. So it was necessary to prove its absence in pharmaceutical product. Suitable analytical technique for azide determination in pharmaceutical industry was ion chromatography with suppression of mobile phase conductivity.

U ovom diplomskom radu proučavan je utjecaj vodikovih veza na pekiranje molekula u kristalnoj strukturi kompleksa 4,4'-dipiridil-N, N'-dioksida s piromelitnom kiselinom (1,2,4,5-benzentetrakarboksilnom kiselinom).