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Soy, red clover, chaste tree, hop and flax have all been found to contain a wide range of phytoestrogenic compounds, and a large number of dietary supplements contain their extracts as principal ingredients. This study is aimed to evaluate the total polyphenolic content and antioxidant activity of phytoestrogen-containing food and formulated dietary supplements. The HPLC-DPPH method was applied for DPPH free radical scavenging activity testing of various phytoestrogen-containing samples. Polyphenol content and antioxidant activity in dietary supplements were higher than in functional food samples; multiple-botanical-source preparations showed higher polyphenol content and antioxidant activity than the mono-botanical counterparts. Furthermore, the correlation between polyphenol content and anti-oxidant activity was strongly statistically significant, so it might be concluded that antioxidant activity is proportional to the content of these secondary metabolites. The most striking batch-to-batch deviations were represented by one chaste berry-based product (RSD 41.3 %) and one red clover derived product (RSD 57.9 %). The results of this study contribute to a better understanding of the phenolic profile and antioxidant properties of phytoestrogen containing food and dietary supplements.

Since inflammatory bowel disease is a progressive disease, optimal treatment early in the disease course is paramount. Still, the use of conventional drugs is accompanied by side effects. The herbal therapies for gastrointestinal disorders are traditionally widespread in Asia, while in Western countries have been adopted in recent years. Andrographis paniculata is a herbaceous plant used in anti-inflammatory treatment. To improve its clinical application, an analytical method with the potential to define a relationship between dose and plasma concentration of active ingredients is prerequisite. Considering the increasing demand for eco-friendly bioanalytical methods, our research aimed to implement new sample preparation technique microextraction by packed sorbent for extraction of marker compounds of A. paniculata from plasma. This green procedure using polymeric sorbent based on the poly (styrene-divinylbenzene) allowed miniaturization of sample volume (100 µL), decrease in consumption of solvents, and high recovery (92–98%). A highly selective and sensitive reversed-phase ultrahigh-performance liquid chromatography method has been developed and validated. The newest greenness assessment software AGREE calculator was used for evaluation of the method’s environmental performance. It suggests that our method conforms to the eco-friendly conditions (score 0.8). The sample preparation was identified as weak point of the bioanalytical methods which highlights the importance of microextraction.

Adherence in chronic diseases is a major problem which can be combated by prescribing fixed-dose combinations in the therapy of the disease. Thus, a combination of azathioprine and folic acid in the treatment of inflammatory bowel disease is highly required, but prior to formulation development, chemical compatibility of the two drugs needs to be investigated. In this work, differential scanning calorimetry, isothermal stress testing, in vitro dissolution and forced degradation studies were utilized to investigate compatibility. Moreover, a stability-indicating HPLC-DAD method for the determination of parent drugs and five of their impurities was developed, validated and applied to the in-house sample. Compatibility testing revealed no noteworthy interactions of the two drug substances. Furthermore, forced degradation showed no substantial differences between the degradation profiles of each active pharmaceutical ingredient, their mixture and the in-house sample, further reinforcing the claim of compatibility. Lastly, the in-house sample was analyzed: it was shown to conform to the requirements of relevant regulatory documents for all the investigated analytes, demonstrating the method's viability for use in formulation and process development. Our results give way to the possibility of realization of said fixed-dose combination.

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

Sulfonylureas are the oldest class of oral antidiabetic medication used to treat type 2 diabetes. There are two generations of sulfonylureas, and both are equally effective in lowering glucose concentration in the blood. Still, they differ in pharmacological properties such as absorption, metabolism, elimination, and dosing. Therefore, modern and advanced methods are being researched daily to determine the pharmacological properties of the new potential drugs. One of the latest methods is biomimetic chromatography which uses columns that mimic biological conditions and computer (in silico) methods that predict various ADME (Adsorption, Distribution, Metabolism, Excretion) and QSPR (Quantitative Structure- Property Relationship) parameters. The present study was designed to gain insight into the ADME parameters such as lipophilicity, solubility, phospholipophilicity, protein plasma binding, oral bioavailability, distribution coefficient, gastrointestinal absorption, and compliance with Lipinski's "rule of five" of three sulfonylureas (N- methylcarbamoyl)-4- methylbenzenesulfoamide, (N- benzylcarbamoyl)-4- methylbenzenesulfonamide, and 1, 1’- trimethylenebis[3-(p-tolylsulfonyl)-urea] by using HPLC biomimetic chromatography and various computer programs for prediction of stated properties. More precisely, our study focused on determining the lipophilicity and phospholipopilicity of three sulfonylureas using C18 (octadecylsilane) chromatographic and Immobilized Artificial Membrane (IAM) columns. Chromatographic columns with human serum albumin (HSA) and α-1-acid glycoprotein (AGP) were used to characterize plasma proteins binding. Various free and commercially available programs were used to predict the ADME properties of stated compounds. For the parameters for which it was possible, the experimentally obtained values were correlated with the calculated ones. The experimental results showed that all three sulfonylureas are lipophilic, interact with phospholipids, and bind to HSA and AGP plasma proteins. Furthermore, a high coefficient of determination (R2>0.9) was obtained for most of the calculated parameters with experimentally determined parameters. Therefore, obtained calculated and experimental values' results would benefit further research of the analyzed sulfonylureas as potential drugs.

Tijekom posljednja tri desetljeća mikroekstrakcija čvrstom fazom donijela je značajan napredak u bioanalitičkim istraživanjima. Razlog tome su brojne modifikacije tehnike koje su omogućile njezinu primjenjivost za raznovrsne i složene biološke uzorke, ali i ekološku prihvatljivost. Tehnika se primjenjuje u kombinaciji s različitim analitičkim instrumentima među kojima se posebno ističu tekućinska i plinska kromatografija. Ovo pregledno istraživanje usmjereno je na najnovija dostignuća i načine primjene mikroekstrakcije čvrstom fazom. Nadalje, posebno su istaknuta nova, inovativna rješenja koja trenutačno nemaju komercijalnu primjenu., In last three decades, the solid-phase microextraction technology has brought significant progress in bioanalytical research due to the versatility of this fast and solvent-free approach to biological sample preparation. It has been widely used in combination with various analytical instrumentation, even if most of the work has been done by coupling the extraction technique with liquid or gas chromatography. This review focuses on the new developments and advances in solid-phase microextraction-based techniques. Furthermore, some interesting, new innovations that fail to go beyond academic research are also reported.

A micellar electrokinetic capillary chromatography (MEKC) method has been developed and validated for simultaneous analysis of febuxostat and its related substances in active pharmaceutical ingredients and pharmaceutical formulations. Analysis was performed in an uncoated fused silica capillary (34 cm effective length, 50 μm diameter). Method sensitivity was enhanced using an extended light path capillary, with an optical path 150 μm and the choice of 320 nm detector operat-ing wavelength due to lower background noise and higher selectivity. BGE consisted of 20 mM borate buffer pH 9.3 and 50 mM SDS and 2% acetonitrile. Analysis was performed at 30 kV separation voltage and 32 °C capillary temperature. The method was linear over the range 1.0–10.0 μg mL−1 for each impurity corresponding to reporting limits regarding the ICH Q3A guidelines. The method has proven to be selective, precise, accurate and robust. Proposed MEKC method has been successfully applied for the quantitative determination of febuxostat and purity assay of active pharmaceutical ingredient and tablet dosage forms.

Systematic development of a UHPLC method with UV detection was performed for simultaneous determination of aglycone (genistein, daidzein, biochanin A and formononetin) and glycoside (genistin, daidzin, sissotrin, ononin) forms of isoflavones. Method development was performed by the QbD approach on three different stationary phases using the DryLab software modelling package. Optimised chromatographic separation was achieved on a Zorbax RRHD Eclipse Plus C18 column with gradient elution in a 2.0 min run time. The resolutions between eight isoflavones were found to be greater than 2.0. The developed method was validated with respect to selectivity, LOD, LOQ, linearity, precision, accuracy, stability and robustness. The proposed method was successfully applied for the analysis of isoflavones present in dietary supplements also containing various other substances. Due to the complex matrices present in the dietary supplements, a 2D-UHPLC-MS method was employed for identification and selectivity confirmation of the developed method. Analysis of investigated dietary supplements revealed deviations between the measured and labelled content of isoflavones as well as high batch to batch variations. The obtained results emphasize the need for quality control of phytoestrogen dietary supplements to provide efficient and safe products.

A new method for determination of distribution coefficient of drugs azathioprine, 6-mercaptopurine and 6-thioguanine and nutrient folic acid used in the treatment of inflammatory bowel disease based on a miniaturized shake-flask and HPLC/DAD was developed. Special attention was made to the most commonly reported problems in the measurement of distribution coefficients using a shake-flask method such as mixing technique, speed and time, the temperature of experiment, type of buffer and its pH as well as n-octanol/buffer phase ratio. The concentration of compounds in the buffer is determined by HPLC directly from shake flasks or conventional 2-mL vials. The developed method was fully validated according to ICH guidelines. Furthermore, experimental data were successfully compared with lipophilicity and human intestinal absorption calculated by the use of four different theoretical approaches. The method shows potential for high-throughput measurements of a large number of compounds.

This work was carried out to develop a high- performance liquid chromatography tandem mass spectrometry method for simultaneous determination of 11 marker compounds in phytoestrogen-rich food and dietary supplements (raw materials and formulated products) prepared from various sources: soy, red clover, chastetree, hop, and flax. The method was fully validated and showed detection limits varying from 0.2 to 1.5 μg/mL, average recoveries ranging from 95 to 105%, and relative standard deviation values lower than 4.98%. Evidently lower amounts of phytoestrogens were found in food samples than in the dietary supplements. Calculations for the actual daily dose of total phytoestrogens ranged from 0.01 to 6.61 mg among the different products. All derived total phytoestrogen amounts turned out to be below the total content stated by manufacturers (less than 63% of the claimed specifications). Moreover, two samples did not contained phytoestrogens at all. The most eye-catching batch-tobatch deviations were represented by chasteberry-based products (relative standard deviation higher than 9.8%).

In the present study, various procedures have been compared for the determination of lipophilicity, hydrophobicity, and plasma protein binding of curcuminoids, boswellic acids, andrographolides, and piperine as biologically active ingredients of botanicals used in IBD treatment. Our results have shown that IAM-HPLC assay is the most suitable one for lipophilicity determination of all analytes regardless of their class and botanical source. HSA-HPAC and AGP-HPAC assays revealed that all investigated compounds have a higher affinity for HSA which is the most abundant protein in human plasma. The high affinity of biologically active compounds to all biological structures (phospholipids and proteins) admonishes that their small portion is available for therapeutic effects in IBD patients. Our experimental research is complemented by various theoretical approaches based on different algorithms for pharmacokinetic properties prediction. The similarities between experimental and calculated values were evaluated using PCA and CA as a statistical tool. The statistical analysis implies that plasma protein binding is a complex process, and theoretical approaches still cannot fully replace experimental ones.

A fast and simple supercritical fluid chromatography method for the simultaneous determination of two antituberculotic drugs, isoniazid and rifabutin, and their impurities from a new proposed fixed-dose combination (FDC) was developed. Isoniazid and rifabutin are used in antituberculotic therapy as single active substance medications. Although FDCs have proved beneficial in general, compatibility issues for some combinations have been reported. The development of a suitable analytical method was, therefore, necessary to assess the applicability of these two active substances for a new FDC formulation. Two active components and their related compounds were successfully separated in only 7 min using an Acquity UPC2™ Torus DEA, 130 °A, 1.7 mm, 3.0 x 100 mm column. Compressed CO2 was modified with a mixture of methanol–2- propanol–water–triethylamine, in ratio 70 : 28 : 2 : 0.1 (v/v/v/v), to obtain gradient elution. Quality-by-design principles were implemented to accomplish a reliable and robust analytical method. Modeling software (JMP®, version 13.2.1) was utilized for the Design of Experiments. Mathematical models for the relationship of column temperature, pressure, modifier composition and flow rate to five critical quality attributes were generated. A comprehensive method development approach facilitated detection of influencing factors and significant second-order terms indicating factors' interaction and nonlinear effects. The new proposed method was successfully validated for specificity, linearity, sensitivity, accuracy, precision, stability, filter study and robustness in compliance with internationally accepted guidelines. Acceptable validation results were obtained for both concentration level ranges (assay and impurities determination). Finally, the applicability of the method was tested for commercially available samples of both active substances.

In this work, a systematical compatibility investigation of 6-mercaptopurine and folic acid, two commonly used medications in the treatment of inflammatory bowel disease, for the needs of a fixed-dose combination development strategy is shown. Various techniques and approaches, such as differential scanning calorimetry, isothermal stress testing, attenuated total reflectance–Fourier-transform infrared spectroscopy, dissolution medium stability and forced degradation studies, were used to elucidate the possible interactions from different aspects. The results predominantly point to the absence of physicochemical interactions between the examined substances in a variety of possible conditions. However, the forced degradation of the blend of substances and excipients in basic conditions showed a drastic degradation of 6-mercaptopurine, signifying that attention needs to be directed to the careful selection of the excipients for the formulation. To sum up, our findings indicate that a fixed-dose combination of 6-mercaptopurine and folic acid could be produced using one formulation blend, immensely simplifying its manufacture.

Fixed-dose combinations have shown to be a great alternative to traditional polytherapy; however, development of such formulation requires thorough physicochemical compatibility investigation of active pharmaceutical ingredients to provide a stable, safe and therapeutically effective product. In this work, differential scanning calorimetry, X-ray powder diffraction, isothermal stress testing followed by Fourier-transform infrared spectroscopy and chromatographic analysis as well as dissolution studies were used for physicochemical compatibility investigation of folic acid and balsalazide or olsalazine. Balsalazide and olsalazine as well as their blend were successfully characterised regarding their physicochemical properties using the mentioned techniques. Differential scanning calorimetry gave ambiguous results due to premature degradation of balsalazide. On the contrary, other techniques have implied the absence of any chemical reactions or physical changes in prepared blends. Obtained result imply that folic acid is compatible with both balsalazide and olsalazine which goes in favour of developing proposed fixed-dose combinations. This work is licensed under a Creative Commons Attribution 4.0 International License.

Background: Ondansetron and paracetamol are often co-administrated to prevent and treat nausea and vomiting caused by anaesthesia and to control postoperative pain. In addition, ondansetron is used as the first-line antiemetic in paracetamol overdose. Therefore, a selective and sensitive method for their simultaneous analysis is of great importance. The electroanalytical methods are highly sensitive and offer many possibilities for new sensor platform design. However, at present, no electroanalytical method for simultaneous determination of these drugs has been proposed. Objective: The aim of this study was to develop a novel nanosensor for selective monitoring of ondansetron and paracetamol in pharmaceutical and biological samples without expensive and timeconsuming pretreatments. Methods: The graphitized multi-walled carbon nanotubes embedded in a cation exchange polymer matrix were selected, among various surface functionalizations evaluated, to design a novel sensor. Based on its excellent sensing performance, the first electroanalytical method was developed for the rapid concurrent determination of investigated drugs. Results: The scanning electron microscopy study showed an interlinked nanoporous network structure and a highly enlarged active surface. The developed sensor facilitated electron transfer in the oxidation of both drugs and tremendously enhanced the adsorption capacity for ondansetron, thus exhibiting a significant increase in drug responses and sensitivity. To obtain much sensitive response of investigated drugs, the effect of pH values of supporting electrolyte, dispersed nanomaterial amount, the cation exchange polymer concentration, drop-casting volume of nanocomposite suspension, accumulation potential and deposition time on the peak current was evaluated. The developed electroanalytical method was validated and the practical utility of the proposed nanosensor was tested. Conclusion: The developed sensor is a promising sensing platform with a fast response time for analysis of ondansetron and paracetamol at very different concentration levels found in their fixeddose combination and human serum sample after recommended daily doses showing its potential usage in pharmaceutical quality control and clinical research.

Mycotoxin-producing Aspergilli (Circumdati, Flavi, and Nigri), usually associated with contaminated food, may also cause respiratory disorders and are insufficiently studied in water-damaged indoor environments. Airborne (N = 71) and dust borne (N = 76) Aspergilli collected at post-flood and control locations in Croatia resulted in eleven different species based on their calmodulin marker: A. ochraceus, A. ostianus, A. pallidofulvus, A. sclerotiorum, and A. westerdijkiae (Circumdati), A. flavus (Flavi), and A. tubingensis, A. welwitschiae, A. niger, A. piperis, and A. uvarum (Nigri). Most of the airborne (73%) and dust borne (54%) isolates were found at post-flood locations, and the highest concentrations measured in indoor air (5720 colony-forming units (CFU)/m3) and dust (2.5 &times, 105 CFU/g) were up to twenty times higher than in the control locations. A. flavus dominated among airborne isolates (25%) at the unrepaired locations, while 56% of the dust borne Aspergilli were identified as A. tubingensis and A. welwitschiae. The ability of identified isolates to produce mycotoxins aflatoxin B1 (AFB1), fumonisin B2 (FB2), and ochratoxin A were assessed by LC-MS analysis. All ochratoxin A (OTA)-producing Circumdati belonged to A. westerdijkiae (13.7 &plusmn, 15.81 &micro, g/mL), in the section, FlaviA. flavus produced AFB1 (2.51 &plusmn, 5.31 &micro, g/mL), while A. welwitschiae and A. niger (section Nigri) produced FB2 (6.76 &plusmn, 13.51 &micro, g/mL and 11.24 &plusmn, 18.30 &micro, g/mL, respectively). Water damage dominantly supported the occurrence of aflatoxigenic A. flavus in indoor environments. Yet unresolved, the causal relationship of exposure to indoor Aspergilli and adverse health effects may support the significance of this research.

Inflammatory bowel disease is a common name for Crohn&rsquo, s disease and ulcerative colitis. These inflammatory states cause damage in the sidewalls of the gastrointestinal tract, resulting in malabsorption of food and vitamins. Folic acid (Vitamin B9) is often associated with inflammatory bowel diseases since reduced overall folate concentration in the human body may lead to the development of colorectal cancer and megaloblastic anaemia. However, its deficiency is easily compensated by taking an additional folic acid pill during regular therapy. At the moment, there are no studies that have examined the compatibility of folic acid with 5-aminosalicylate drugs used in the treatment of inflammatory bowel diseases. In this work, differential scanning calorimetry, forced degradation studies, isothermal stress testing and dissolution stability testing were used to determine the stability of folic acid and one of the most commonly used 5-aminosalicylates, mesalazine, when present in the same solution or blend. To monitor the assay of folic acid, mesalazine and nine of its related impurities, a single HPLC method was developed. Results of compatibility studies showed that no physicochemical interaction between mesalazine and folic acid occurs when combined, opening the path to the development of new formulations, such as a mesalazine/folic acid fixed-dose combination.

Medication adherence is an important factor in inflammatory bowel disease therapy, which includes regular supplementation of malabsorbed vitamins. Absorption of folic acid is limited due to the damaging of the gastrointestinal tract, which can increase the chances to develop megaloblastic anaemia and colorectal cancer. In this work, 5-aminosalicylates (mesalazine, balsalazide, sulfasalazine and olsalazine) and folic acid were characterized regarding their pharmacokinetic related properties (hydrophobicity, phospholipid and plasma protein binding) using the biomimetic chromatographic approach. Despite the high binding percentage of 5-aminosalicylates for human serum albumin (> 61.44%), results have shown that folic acid binding to human serum albumin protein is far greater (69.40%) compared to α1-acid-glycoprotein (3.45%). Frontal analysis and zonal elution studies were conducted to provide an insight into the binding of folic acid to human serum albumin and potential competition with 5-aminosalicylates. The analytical method for the simultaneous determination of assay in proposed fixed-dose combinations was developed and validated according to ICH Q2 (R1) and FDA method validation guidelines. Separation of all compounds was achieved within 16 min with satisfactory resolution (Rs > 3.67) using the XBridge Phenyl column (150 × 4.6 mm, 3.5 µm). High linearity (r > 0.9997) and precision (RSD

The compatibility of formulation components is crucial for safe and high-quality medicines. To detect the potential for incompatibility and to assess formulation stability, it is beneficial to conduct a compatibility study during the drug development phase. The therapy of tuberculosis normally consists of two or more medicines taken together. Consequently, different antituberculotic fixed-dose combination (FDC) formulations have been developed. Isoniazid is first-line medicine and present in several FDCs. Low bioavailability due to the active substances’ incompatibility in acidic medium was reported for some of these FDC forms. Rifabutin, also a first- line antituberculotic, is available in the market as a single component formulation. This study presents compatibility testing of these two active substances for a new FDC and evaluates the impact of the most common solid dosage forms’ excipients on the stability of two active substances. The potential for interaction between the formulation components was analyzed by the UHPLC method. One degradation product and one interaction product were observed and further characterized by high- resolution mass spectrometry. Still, significant degradation of two active substances, such as reported in marketed FDC formulations was not detected for this combination. The stability and drug delivery of the proposed combination were confirmed by the dissolution test in acidic medium.

Immunosuppressants azathioprine (AZA), 6- mercaptopurine (6-MP) and 6-thioguanine (6-TG) are one of the most common therapy choices for patients with inflammatory bowel disease (IBD). Malabsorption of many nutrients, including folic acid (FA), a vitamin whose inadequate intake is associated with megaloblastic anemia and possible increase of colorectal cancer risk, is often presented in these patients. So as to improve the adherence of patients, a fixed dose combination of one of the immunosuppressants and FA is proposed. The aim of this work was to determine the lipophilicity of the drugs in order to evaluate their pharmacokinetic properties, as well as potentially simplify the formulation process. LogD values were determined by traditional shake-flask procedure followed by HPLC measurements of aqueous phase (phosphate buffer pH 7.4) before and after equilibration with n- octanol. Furthermore, two sophisticated indirect approaches were used. TLC measurements were done using RP-C18 F254 silicagel plates, while HPLC measurements were done using various stationary phases octadecylsilyl, immobilized artificial membrane, human serum albumin and α-1 acid glycoprotein. Finally, in silico approximations of logP, logD and plasma protein binding were predicted by 14 on-line based platforms. Experimental results showed the highest lipophilicity for AZA (logD = 0.08), while 6-TG (logD = -0.29) and 6-MP (logD = -0.29) were slightly less lipophilic. FA varied from the most hydrophilic of the analytes to moderately lipophilic, depending on the method used. Significant correlations (R2 from 0.869 to 0.973) have been obtained between predicted and experimental values ; on-line platforms which showed the most prominent correlations were PreADMET, pkCSM, Chemicalize, ALOGPS, ADMETLab and Mcule. In this work various parameters describing pharmacokinetic properties have been determined experimentally and obtained data were correlated with values predicted by on-line platforms. Correlations obtained between these values imply that such platforms could potentially be used in screening of similar molecules in terms of physicochemical and pharmacokinetic properties, thus enhancing drug discovery and drug development effectiveness. This work has been supported in part by the Croatian Science Foundation under the project number [UIP-2017- 05-3949]. This work has been supported in part by the European Union from the European Social Fund.

Functional foods are foods that provide positive health effects apart from the provision of essential nutrients. Along with nutraceuticals, they represent the top trends in the food industry. Fruit wines are considered functional foods. When assessing the fruit wine quality, a wide range of descriptors are taken into consideration, namely physicochemical and sensorial properties of fruit wine. Furthermore, within the context of the new food products development (e.g. functional products), functional properties of fruit wines are also taken into consideration. Functional properties are determined by the content of the biologically active components, such as polyphenols, vitamins and micro- and macrominerals. It is also important to consider the food-safety issues regarding the fruit wines consummation, that is, the presence of pesticides, mycotoxins and biogenic amines in different fruit wines. This chapter aims to give an overview of various factors used to evaluate the quality and the functional properties of fruit wines.

The simultaneous administration of sulfasalazine and folic acid is regular practice in the therapy of inflammatory bowel diseases in order to maintain sufficient folate concentration in patients. Having multiple drugs in the therapy increases the possibility of patients failing adherence, thus unintentionally endangering their health. A fixed-dose combination of sulfasalazine and folic would simplify the classical polytherapeutic approach, however, the physicochemical compatibility investigation of two active pharmaceutical ingredients plays an important role in the development of such a product. In this work, various analytical tools were used to determine the physicochemical compatibility of sulfasalazine and folic acid. For the evaluation of chemical compatibility, infrared spectroscopy in combination with advanced statistical methods, such as the principal component analysis and cluster analysis, were used, whilst a simultaneous thermogravimetry/differential thermal analysis gave us an insight into the physical compatibility of two drugs. Isothermal stress testing, forced degradation and dissolution studies, followed by the analysis with a developed chromatographic method for the monitoring of folic acid, sulfasalazine and two of its related impurities, sulfapyridine and salicylic acid, gave us an insight into its chemical compatibility. The combination of the results obtained from the used techniques implies a satisfactory physicochemical compatibility between sulfasalazine and folic acid, which opens the path to the development of the proposed fixed-dose combination.

This study brings out the data on the content of 21 mineral and heavy metal in 15 blackberry wines made of conventionally and organically grown blackberries. The objective of this study was to classify the blackberry wine samples based on their mineral composition and the applied cultivation method of the starting raw material by using chemometric analysis. The metal content of Croatian blackberry wine samples was determined by AAS after dry ashing. The comparison between an organic and conventional group of investigated blackberry wines showed statistically significant difference in concentrations of Si and Li, where the organic group contained higher concentrations of these compounds. According to multivariate data analysis, the model based on the original metal content data set finally included seven original variables (K, Fe, Mn, Cu, Ba, Cd and Cr) and gave a satisfactory separation of two applied cultivation methods of the starting raw material.

Species assigned to Aspergillus section Flavi are food contaminants and may cause mycoses. Higher humidity may favour their occurrence in indoor environments and support the biosynthesis of aflatoxin B1 (AFB1), their toxic and cancerogenic metabolite. The aim of this study was to compare the frequencies of aflatoxin-producing and non-producing Aspergilli isolated from flood affected and control area in Croatia. Additionally, comet assay was employed to evaluate their genotoxic potential. Aspergilli of interest were isolated from air (N=60) and dust (N=60) samples collected during 2016 and 2017, two and three years after flood in Eastern Croatia. Identification of the isolates was based on morphology and the partial calmodulin sequences. Microextracts prepared from fungal cultures were analysed on AFB1 content by LC/MS. Genotoxicity of extracted Aspergilli was tested in human lung adenocarcinoma cell line A549 by alkaline comet- assay. Aspergilli (Flavi) dominated in the samples collected in flood-affected area (33/40) compared to the control area (3/40) and were more frequent in the airsamples (83%). AFB1 producing abilities were confirmed only for isolates from flood affected area (10/33). Genotoxic potential measured as tail intensity (% of DNA in comet tail) was slightly higher for AFB1-negative compared to AFB1-positive extract (0.57 ±0.94 and 0.14 ± 0.31, respectively). Flood may support the occurrence of Aspergilli from section Flavi in indoor environments. Inhalation of these mycoparticles may have a harmful effect on the respiratory system of the exposed people regardless of AFB1 presence. This work has been fully supported by Croatian Science Foundation under the project MycotoxA (IP-092014-5982).

First analytical method was developed for simultaneous determination of aminosalycilate drug mesalazine (5-ASA) and folic acid (FA) used as a supplement in treatment of inflammatory bowel diseases. Various types of functionalized multi-walled carbon nanotubes embedded in different kinds of polymer matrix were evaluated to design selective and sensitive electrochemical sensor for this purpose. Carbon nanotubes functionalized with amino groups and chitosan as biopolymer modifier exhibited an excellent electroanalytical performance due to strong adsorptive ability of ionized analytes and subtle electronic properties followed by well-separated oxidation peaks towards 5-ASA and FA at potential of 0.33 and 0.81 V, respectively. After 60 s preconcentration at deposited nanocomposite layer, the voltammetric response had linear relationship with 5-ASA concentration in the range of 1.25 × 10−7–8.0 × 10−6 M, while FA gave a good linear correlation in a broad range from 1.25 × 10−8 to 1.25 × 10−6 M. Applicability of proposed nanosensor for rapid and precise measurements of 5-ASA and FA in human serum samples and pharmaceutical products was demonstrated.

Background. Black Aspergilli are ubiquitous moulds which contaminate foodstuffs and occur in indoor environments. Recent studies showed that some species from the section produce several fumonisin isomers including FB2 that could contribute to irritant, allergic and toxic effects. Methodology. Black Aspergilli were isolated from the airsamples (N=60) and dust (N=60) collected during winter and summer of 2016 and 2017 in flood affected area and control area. Identification to the species level was based on the partial calmodulin sequences. FB2 producing abilities were tested by analysing prepared microextracts of an each isolate using LC/MS. Results and conclusion. In the flood affected area the highest concentration of airborne black Aspergilli was observed in winter period and their concentrations were 50 times higher than in control area (987 ± 1154 CFU/m3). Their concentration in dust was similar in flood affected and control area. 49/85 analysed black Aspergilli was from the samples collected in flood affected area. FB2 was detected in 4 extracted airborne and 11 dustborne black Aspergilli in concentrations 0.617 ± 0.582 µg/ml and 9.237 ± 15.092 µg/ml and thus may contribute to the harmful effects in conditions of increased concentration of black Aspergilli, especially in flood affected area.

Nepafenac is novel ophthalmic agents with a unique prodrug structure and superior corneal permeability to other currently available topical nonsteroidal anti-inflammatory drugs. A graphene nanoplatelets and carbon nanofibers modified glassy carbon electrode was developed and used as first electrochemical sensor for the determination of nepafenac. The effective combination of graphene and carbon nanofibers resulted in electrochemical sensor with large surface area, high sensitivity, good stability and selectivity. After method optimization, the square wave voltammograms, at a preconcentration time of 60 s, display two linear ranges of 4.0 × 10−6–1.5 × 10−5 M and 2.5 × 10−7–4.0 × 10−6 M for drug quantification with a detection limit of 6.3 × 10−8 M. The modified electrode was employed for fast assay of nepafenac in ophthalmic solution with satisfactory results and rapid run time of 66 s in comparison with previously reported analytical methods.

The red yeast rice formulated products, used worldwide as cholesterol-lowering dietary supplements, have the main active ingredient monacolin K which is actually lovastatin registered as the first statin drug. In this work, a rapid and simple square-wave voltammetric method was developed for determination of monacolin K based on the electrochemical reduction of the carbonyl group in lactone ring. Considering the pH dependence of current response and the interconversion of monacolin K to its acid form, the Britton-Robinson buffer of pH 5.0 was selected as a supporting electrolyte for analytical measurements. The net square-wave voltammetric response of monacolin K was shifted towards more negative potential values by increasing the concentration of analytes. The peak current was linearly proportional to monacolin K concentration in the range of 0.25–20 μg/mL. The fully validated method was successfully applied for fast screening and direct voltammetric determination of monacolin K in red yeast rice products formulated in various dosage forms. To evaluate the proposed voltammetric method, the results obtained were compared with those obtained by HPLC methods.

With the increase in the number of medicines patients have to take, there has been a rapid rise of fixed-dose combinations (FDCs) in the last two decades. Prior to FDC development, pharmacokinetic properties of active pharmaceutical ingredients (APIs) have to be evaluated, as well as methods for their determination developed. So as to increase patient compliance in inflammatory bowel disease, three novel FDCs of thiopurine immunosuppressants and folic acid are proposed, physico-chemical and pharmacokinetic properties such as hydrophobicity, lipophilicity and plasma protein binding of all APIs are evaluated. Moreover, experimental results of different properties are compared to those computed by various on-line prediction platforms so as to evaluate the viability of the in silico approach. A simultaneous method for their determination is developed, optimized, validated and applied to commercial tablet formulations. The method has shown to be fast, selective, accurate and precise, showing potential for reliable determination of API content in proposed FDCs during its development.

This paper presents lipophilicity and bio‐ mimetic property determination of 15 phytoestrogens, namely biochanin A, daidzein, formononetin, genistein, genistein‐ 4, 7‐dimethylether, prunetin, 3, 4, 7‐ trihydroxyisoflavon, 4, 6, 7‐trihydroxyisoflavon, 4, 6, 7‐trimethoxyisoflavon, daidzin, genistin, ononin, sissotrin, coumestrol and coumestrol dimethylether. High‐performance liquid chromatography with fast gradient elution and Caco‐2 cell line were used to determine the physicochemical properties of selected phytoestrogens. Lipophilicity was determined on octadecyl‐sylane stationary phase using pH2.0 and pH 7.4 buffers. Immobilized artificial membrane chromatography was used for prediction of interaction with biological membranes. Protein binding was measured on human serum albumin and α‐1‐acid‐glycoprotein (AGP) stationary phases. Caco‐2 assay was used as a gold standard for assessing in vitro permeability. The obtained results differentiate phytoestrogens according to their structure where aglycones show significantly higher lipophilicity, immobilized artificial membrane partitioning, AGP binding and Caco‐2 permeability compared with glucosides. However, human serum albumin binding was very high for all investigated compounds. Furthermore, a good correlation between experimentally obtained chromatographic parameters and in silico prediction was obtained for lipophilicity and human serum albumin binding, while the somewhat greater difference was obtained for AGP binding and Caco‐2 permeability.