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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.

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.

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%).

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.

Festival znanosti 2019: nastavnici i djelatnici Farmaceutsko-biokemijskog fakulteta Sveučilišta u Zagrebu održali 12 radionica, prezentacija i predavanja

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).

Sekcija farmaceuta juniora Hrvatskog farmaceutskog društva tradicionalno je četvrtu godinu zaredom, u suradnji s Udrugom studenata farmacije i medicinske biokemije Hrvatske (CPSA), obilježila Svjetski dan šećerne bolesti 14. studenoga 2017.

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.

A new headspace gas chromatographic method with flame ionization detection (HSS-GC-FID) was developed and validated for the determination of methanol as the main volatile impurity present in ethanolic pharmaceutical preparations. The use of static headspace sampling minimized the interference of other volatile matrix components and provided satisfactory results in purity assessment of different complex samples. The developed procedure revealed to be a rapid, sensitive, reproducible and accurate. The detection and quantification limits of methanol were 0.0003 and 0.0011% (v/v), respectively, and were sufficiently low to enable the estimation of organic volatile impurity according to the ICH guideline as well as the examination of methanol limit specified in European Pharmacopoeia for liquid pharmaceutical preparations. The proposed method was successfully applied to the analysis of diverse alcoholic herbal extracts and tinctures as well as ethanolic dermatological lotions.

Graphene nanoplatelets were dispersed in a Nafion matrix and used to modify a glassy carbon electrode for rapid determination of quetiapine (QTP) at the working potential of +990 mV (vs. Ag/AgCl). The material was deposited by drop-casting and characterized by scanning electron microscopy. Owing to the unique properties of graphene and the accumulation capability of the anionic polymer for the cationic target analyte, the modified electrode has a strongly improved limit of detection (22 nM) and works in the 100 nM to 10 μM concentration range. Low-cost quantification of QTP in urine samples without prior extraction was achieved after selective adsorption of QTP at the surface using differential pulse voltammetry. In comparison with HPLC method, the modified electrode allows simpler analysis of QTP in enteric-coated tablets that takes 40 s only.

Despite efforts by many dietary supplements’ manufactures to reduce or replace ethanol, many products containing ethanol in concentrations up to 70% are available on market. Furthermore, botanical dietary supplements can vary in metal content as a function of the environment, processing equipment and product containers. Therefore, the aim of study was to develop a new rapid and highly sensitive method for simultaneous determination of ethanol and its impurities in dietary supplements by sHSS-GC-FID technique. In addition, contamination with metals by GFAAS technique was evaluated. The proposed sHSS-GC-FID method was successfully applied for analysis of 93 samples containing various amounts of ethanol. It should be highlighted that the dramatic variation from manufacture’s claims was found in even one third of products. Furthermore, high amounts of ethanol were found in several products especially designed for children and in one product labeled as “alcohol-free”. Metal impurities were below the limits established by USP.

Adrenocortical carcinoma (ACC) is a rare malignancy with an incompletely understood pathogenesis and a poor prognosis. The adrenalytic activity of mitotane has made it the most important single drug in the treatment of ACC. Unfortunately, the exact mechanism of mitotane action is still unknown. It is believed that mitotane belongs to the class of drugs that require metabolic transformation by cytochrome P450 for therapeutic action; therefore determination of plasma levels of not only mitotane but also its metabolites would help in carrying out the treatment. The objective of this work was to develop and validate an SPE-HPLC method for simultaneous determination of mitotane and its metabolites in different biological fluids. The sample preparation consisted of a solid-phase extraction on a Discovery DSC(18) cartridge, while analysis of extracts was performed on a Symmetry C(18) column. The usefulness of the proposed method was confirmed by analysis of plasma, red cell and urine samples from patient chronically treated with 1.5 g of mitotane. The patient involved in this study had a high plasma concentration of mitotane and none of the investigated metabolites were found. In order to investigate whether the polymorphism of CYP2C9 and CYP2C19 enzymes could be related to the metabolism of mitotane, RT-PCR analysis was performed.

The electrochemical behavior of ondansetron was studied on the multi-walled carbon nanotubes/Nafion polymer composite modified glassy carbon electrode (MWCNTs–Nafion/GCE). The oxidation peak potential was shifted from 1.32 V to 1.18 V compared to the bare electrode indicating excellent electrocatalytic activity of immobilized film toward drug molecule. The modified electrode exhibited a remarkable enhancement effect on voltammetric response due to the synergistic effect of nanomaterial and cation-exchange polymer on the electron transfer rate, the effective electrode area and the accumulation capability. After optimizing the experimental parameters, adsorptive stripping procedure was used for the determination of ondansetron in pharmaceutical formulation. The results were satisfactory in comparison with those obtained by high-performance liquid chromatography. In addition, the MWCNTs–Nafion/GCE exhibited high selectivity in the voltammetric measurements of ondansetron and co-administrated drug morphine with potential difference of 430 mV. The response peak currents had linear relationship with drug concentration in the range of 1.0×10 −7 –5.0×10 −6 M and 1.0×10 −7 –4.0×10 −6 M with detection limits 3.1×10 −8 and 3.2×10 −8 M for ondansetron and morphine, respectively. The electrode was successfully applied for simultaneous electrochemical sensing of both drugs in human serum samples after selective accumulation at the electrode surface.

A new, fast-screening method for identification of various pharmacologically active ingredients in cholesterol-lowering dietary supplements using direct injection mass spectrometry was developed. The optimized method was proven to be useful for multitarget screening: 17 compounds of interest were identified, including monacolin K and its acid form, cynarin, green tea catechines, genistein, daidzein, guggulsterones, allicin and l- arginine. The method was successfully applied for the fingerprint analysis of dietary supplements containing only one or several cholesterol-lowering agents including lovastatin registered as a drug. According to the obtained results, it is apparent that cholesterol-lowering dietary supplements are poorly standardized, as two samples did not contain active ingredients (monacolin K, monacolin K acid form and cynarin). Moreover, the application of the proposed direct injection method to quality control of cholesterol-lowering dietary supplements may be proposed for the routine analysis of a large number of samples in the laboratories of regulatory agencies.

Red fermented rice is used worldwide by many patients as an alternative therapy for hyperlipidemia; however, the discovery of a toxic fermentation byproduct, citrinin, causes much controversy about the safety of red mold rice products. A new and fast high-performance liquid chromatography method was developed and validated for simultaneous determination of cholesterol-lowering compounds monacolin K (lovastatin), monacolin K hydroxy acid, and other monacolins present in red fermented rice as well as nephrotoxic mycotoxin citrinin in a single run using connected diode array and fluorescence and mass spectrometric detectors. The proposed method was successfully applied for the analysis of red fermented rice food samples and various dietary supplements also containing other natural lipid-lowering agents. The deviations between label content and levels of active compounds found in investigated samples as well as high batch-to-batch variation found in one product indicate that the regular quality control of red fermented rice products is of great importance.

Lovastatin is a main component of Monascus purpureus fermented red rice contributing to the lipid-lowering effect. Citrinin is a toxic fermentation by-product which can be found as a contaminant. An accurate, simple and rapid micellar electrokinetic capillary chromatographic method was developed for the first time for simultaneous determination of lovastatin present in lactone and hydroxy acid forms and citrinin in red rice products provided by different manufacturers and formulated in various dosage forms. Separation was achieved within only 2 min using 20 mM of phosphate buffer at pH 7.0 and 30 mM of sodium dodecyl sulphate at an applied voltage of 25 kV. Sensitivity crucial for detecting citrinin was enhanced by using an extended light path capillary. The results showed that the content of lovastatin and its acid form in dietary supplements were considerably different indicating the need for improved standardization in order to ensure efficiency and safety of these products.

Statins are the most effective drugs currently available for treating hypercholesterolemia, as they reduce the level of total and low-density lipoprotein cholesterol, triglycerides and apolipoprotein B in plasma of patients at risk of cardiovascular disease, even they are currently among the most widely prescribed drugs. They are specific and competitive inhibitors of 3-hydroxy-3-methlyglutaryl coenzyme A (HMG-CoA) reductase, which is a key enzyme that catalyzes the conversion of HMG-CoA to mevalonate. This is an early rate-limiting step in the cholesterol biosynthetic pathway. Statins include natural (lovastatin), semi-synthetic (simvastatin, and pravastatin) and synthetic compounds (fluvastatin, atorvastatin, rosuvastatin and pitavastatin). All statins share an HMG-like pharmacophore, which may be present in the active hydroxy acid form or in the inactive lactone form. Lovastatin and simvastatin are administered as lactone prodrugs and subsequently hydrolyzed to active metabolites, while other statins are formulated in β-hydroxy acid form. The interconversion between lactone and hydroxy acid forms of statin molecules is a highly pH dependent. Statin use has soared over the last decade, and is expected to increase further in the years ahead because high cholesterol and cardiovascular diseases are being diagnosed more frequently. For this reason, rapid and effective ways for the identification and determination of these drugs are highly required. The therapy with statins is considered for the long-term use and the purity evaluation of these drugs is of great significance. Bioanalytical methods are necessary for the clinical trials, for therapeutic drug monitoring and individual dosage scheme adjustment of statins. High performance liquid chromatography with various types of detection is the technique of choice in developing new methods for analysis of statin molecules. This chapter presents recent trends and advances in chromatographic and capillary electrophoretic methods for the determination of statin drugs in various fields of application.

Adrenocortical carcinoma (ACC) is a rare malignancy with an incompletely understood pathogenesis and a poor prognosis. The adrenalytic activity of mitotane has made it the most important single drug in the treatment of ACC. Unfortunately, the exact mechanism of mitotane action is still unknown. It is believed that mitotane belongs to the class of drugs that require metabolic transformation by cytochrome P450 for therapeutic action; therefore determination of plasma levels of not only mitotane but also its metabolites would help in carrying out the treatment. The objective of this work was to develop and validate an SPE-HPLC method for simultaneous determination of mitotane and its metabolites in different biological fluids. The sample preparation consisted of a solid-phase extraction on a Discovery DSC(18) cartridge, while analysis of extracts was performed on a Symmetry C(18) column. The usefulness of the proposed method was confirmed by analysis of plasma, red cell and urine samples from patient chronically treated with 1.5 g of mitotane. The patient involved in this study had a high plasma concentration of mitotane and none of the investigated metabolites were found. In order to investigate whether the polymorphism of CYP2C9 and CYP2C19 enzymes could be related to the metabolism of mitotane, RT-PCR analysis was performed.