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The contamination of botanical dietary supplements with pesticide and fungicide ethylene oxide (EO) is well-recognized problem worldwide. Contamination of botanicals with pathogenic bacteria is attributed in part to growing conditions and environment, sanitation and hygiene practices among harvest workers, and lack of good agricultural and manufacturing practices within some developing countries. Processing methods for botanicals include fumigation with EO, irradiation, and vacuum-assisted steam. Moreover, capsule shells as the most used delivery system of botanical dietary supplements can be sterilized with EO by manufacturers before releasing to user enterprises. [1, 2] Due to its mutagenic and carcinogenic properties, herbal dietary supplements need to comply with the maximum residue level established by regulatory agencies. [3, 4] It is important to ensure that the levels of residual EO and its by-product 2-chloroethanol (2-CE) obtained by the reaction of acetaldehyde and chloride ion pose a minimal risk to the patient. The aim of this research was the assessment of EOTotal expressed as EO equivalents as the sum of native EO and 2-CE in 53 samples of dietary supplements used in the treatment of inflammatory bowel disease (IBD). 28 samples were obtained from local pharmacies and health food stores and 26 were purchased from the Internet. The dietary supplements analyzed in this work were in multiple oral dosage forms including tablets (6), hard capsules (25), soft capsules (4), powders (16) and liquid extracts (2). The main botanical ingredients included turmeric, Indian frankincense and green chiretta. The samples were analyzed using GC-MS/MS (model TQ8050, Shimadzu). In 25 samples (43%) EO and 2-CE were quantified in the range from 0.02 to 3.29 mg/kg. 2 samples (1 hard capsule and 1 tablet) contained levels higher than 1 mg/kg. This study provides a reference for regulatory bodies to routinely control the quality of botanical dietary supplements and to prevent EO residues from jeopardizing patients’ health.

As the number of patients with inflammatory bowel disease (IBD) that use dietary supplements (DS) continues to increase in many countries, the importance of ensuring product quality is of scientific matter. Regulators, health professionals, and manufacturers often disagree on how much quality testing is necessary for DS. This is highlighted by the World Health Organization’s Strategy on Traditional Medicines 2014–2023 [1] where quality is seen as a basis of the DS sector. The United States Pharmacopeia (USP) is an independent, nonprofit, science-based organization whose mission is to improve human health through quality standards and tests for DS. [2] The aim of this research was to perform oral dosage testing based on USP protocols of botanical DS used in the treatment of IBD. 53 samples tested were in multiple oral dosage forms: tablets, hard capsules, soft capsules, powders, and liquid extracts. The following test were performed: Tapped Density , Loss on Drying , Tablet Friability , Tablet Breaking Force , Weight Variation , and Disintegration . The tests were performed using tapped density testing instrument (PT-DT300), tablet friability test instrument (PTF 100), and tablet disintegration tester (PTZ-S) by Pharma Test (Germany), tablet hardness tester (TBH 125) by Erweka (Germany) and moisture analyzer (DBS 60-3) obtained by Kern & Sohn GmbH (Germany). The lowest tapped density was found for turmeric (CI 25%) and the highest for green chiretta (CI 45%). The tablet breaking force was in the range of 27.7 to 248.5 N. One sample containing black pepper as a botanical ingredient did not meet the requirements of loss on the drying test (5.2%). All investigated tablets met the requirements of the friability test expressed in terms of allowable weight loss (≤0.61%). Investigated tablets and capsules met the requirements of the weight variation test expressed in terms of the allowable deviation from the average weight of the sample (≤7.7% tablets, ≤9.0% capsule). Only one sample did not meet the requirements of the disintegration test as none of investigated 6 tablets had not disintegrated under specified conditions. The challenges in DS science and its regulation provide new opportunities for scientists and regulators to work together both nationally and internationally, and to cooperate to improve public health. Assessments of the quality of DS are needed to provide the scientific information that regulators need.

Sphingosine-1 phosphate (S1P) receptor–modulating therapies from the new generation of oral small molecules represents a milestone in the evolution of the therapeutic armamentarium followed by two decades of monoclonal antibodies. S1P receptors have been proposed as a therapeutic target for IBD due to their involvement in the regulation of lymphocyte trafficking out of lymph nodes and into sites of inflammation in gastrointestinal tract. In 2021. ozanimod was the first S1P receptor modulator approved by the FDA and EC for the treatment of moderate-to-severe UC. With the introduction of S1P receptor modulators in the therapeutic landscape of IBD, more options are offered to patients and clinicians. Still, we are witnessing continuous progress in this field with the development of promising second-generation drug classes that are in the final stages of clinical study. The results of the phase 2 clinical trial of patients with UC published in 2019 indicated the efficiency of the new S1P receptor modulator etrasimod and were the basis for several ongoing phase 3 studies (ClinicalTrials.gov Id: NCT03996369, NCT04176588, NCT03945188, NCT04173273). The aim of this research is a development and validation of a green analytical method for quality control of etrasimod as an integral part of the preformulation stage crucial for the consistent efficacy of a drug product. The analysis was performed at Agilent 1260 UHPLC system using Phoroshell C18 (3.0×100 mm, 2.7 µm) column at 25°C. The mobile phase consisted of water and acetonitrile acidified with formic acid (0.1%) and delivered with a gradient system (flow: 1 mL/min). The quantitation was performed at 227 nm. Analytical GREEnness metric approach software was used for greenness score evaluation. The system suitability parameters were measured by injecting a standard solution (6 µg/mL) in six replicates (retention time: 3.66 min, peak area: 131.8 mAU, plate count: 31550, and symmetry: 1.0). The RSD values were less than 1%, thus leading to a high degree of accuracy of the system. The validation was performed according to ICH Q2(R1) guidelines. The peak purity was higher than 999. The high linearity (range: 2-10 µg/mL ; r=0.999) was observed. Precision was studied as repeatability with six analyses within the first day (RSD≤0.73%) and as intra-day precision with three analyses for three days (RSD≤0.82%). Accuracy was tested on three concentration levels to confirm the applicability of the method in the whole range (99.0-100.4%). The method showed an acceptable green score (0.8). The validated green method represents useful support for preformulation studies essential for the development of robust and effective dosage forms. The work was funded by the Croatian Science Foundation (HRZZ-UIP-2017-05-3949).

Inflammatory bowel disease is a complex disorder characterized by chronic gastrointestinal inflammation. Thus, patients prefer to use herbal dietary supplements containing turmeric, Indian frankincense, green chiretta, and black pepper in an attempt to cope better with their chronic condition. The dietary supplements’ dosage forms and herbal ingredients were assessed in terms of the products’ physicochemical parameters (weight uniformity, friability, disintegration, rupture test, tablet’s breaking force, and powder flowability) in view of the USP-NF requirements. In addition, contaminants such as organic solvents and ethylene oxide were evaluated using gas chromatography. Assessment of gluten via an Enzyme-Linked Immunosorbent Assay was also performed. Most of the products met USP requirements. The high average weight of one multicomponent tablet sample with a high breaking force value can explain the observed negative results of the disintegration test. A total of 26% of samples tested positive for gluten, but the most alarming fact is that the ethylene oxide levels found in two samples were up to 30 times higher than the EU limit. Accordingly, dietary supplement quality control is of fundamental importance.

Inflammatory bowel disease (IBD) is a continuously increasing worldwide disease and affects people of all ages. More than half of these patients use herbal dietary supplements to alleviate the symptoms of IBD [1]. Curcuminoid compounds present in turmeric, boswellic acids as pentacyclic terpenoid molecules from frankincense resin, andrographolides as diterpene lactones isolated from green chiretta, and piperine as a piperidine alkaloid from black pepper fruits are the most abundant biologically active substances in herbal dietary supplements for the treatment of IBD. Since today the use of dietary supplements is widespread and constantly increasing, this study aimed to analyze the dissolution of those mentioned biologically active components from oral herbal preparations in biorelevant media. Samples in tablet dosage form were collected from local pharmacies and purchased from the Internet. All dietary supplements were classified as botanical monopreparations and multipreparations. In vitro dissolution studies were conducted using a USP dissolution (paddle) apparatus 2 (Erweka DT600, Erweka GmbH, Langen, Germany). The herbal tablets were dispersed in 900 mL of USP- recommended dissolution medium (1 % sodium lauryl sulfate), fasted/fed simulated gastric and small intestinal fluids (FaSSGF, FaSSIF/FeSSIF) at 37 °C [2]. During dissolution, 1 mL of sample was collected at each time point up to 120 min and filtered through Minisart RC4, 0.45 μ m filters (Sartorius, Germany), which did not retain any of the analytes. All samples were analyzed in duplicate. The concentration of biologically active components in the filtrate was analyzed using an HPLC-DAD assay. Chromatographic analyses were performed on a Shimadzu Nexera chromatographic system. Based on the obtained results (similarity factor, f2 value < 50 %), the in vitro release profile of the biologically active substance from herbal tablets depends on the composition of the biorelevant medium.

Aloe vera (syn.: Aloe barbadensis Miller) is the most commercialized Aloe species belonging to the Xanthorrhoeaceae family. Aloin is the major anthraquinone of aloe and characterized as the C- glycoside of aloe-emodin. Due to polyphenolic structure, aloin and aloe-emodin are considered as physiologically active compounds responsible for aloe biological effects such as reduction of inflammation. Despite the frequent use of aloe, limited information is available for their bioavailability which is a very common phenomenon for other phytochmicals as well. A study providing deeper mechanistic insightsinto the bioavailability of aloin and aloe-emodin is essential to develop better treatment strategies in the future based on botanical products. Although traditional evaluation of drugs bioavailability and performance in human body require long lasting and expensive clinical studies, a more affordable and faster approach is being developed. Innovative biomimetic chromatographic methods have shown to be good alternative for such studies. The aim of this work was to evaluate parameters such as hydrophobicity, lipophilicity, and plasma protein binding using chromatographic tools, especially growing population of biomimetic columns mimicking cell membranes, as well as ones containing plasma proteins (human serum albumin (HSA) and alpha-1- acid glycoprotein (AGP). As expected, aloin as glycoside showed lower hydrophobicity (RM0=1.85, logk0=1.37) compared to aloe-emodin (RM0=3.06, logk0=3.01). On the other hand, aloin showed superior affinity to phospholipids compared to aloe-emodin (Δlogk0 IAM=1.68). Both compounds showed the higher affinity for HSA (62.7% aloin ; 97.5% aloe-emodin) than AGP (44.1% aloin ; 84.2% aloe-emodin).

Evaluation of parameters such as lipophilicity, solubility and plasma protein binding through conventional approaches is time consuming and expensive, especially in high throughput screening. With the development of advanced prediction tools, in silico approaches enabled fast and inexpensive insight into characteristics of compounds based on their structure. Curcuminoids, andrographolides, boswellic acids and piperine, are part of food supplements used in patients suffering from inflammatory bowel diseases. The aim of this work was to evaluate the accuracy of in silico predicted log P, log S and plasma protein binding (PPB) values with the experimentally observed ones obtained using thin layer chromatography and high precision liquid chromatography with conventional reversed phase columns (C-18) and biomimetic columns with human serum albumin (HSA) and alpha-1-acid glycoprotein (AGP). These parameters are important to understand their pharmacokinetics which plays an important role in the therapy outcome. For comparison of experimentally observed and in silico calculated data for 10 examined compounds (curcumin, demethoxycurcumin, bisdemethoxycurcumin, α-boswellic acid, β- boswellic acid, 11-keto-β-boswellic acid , 3- acetyl-11-keto-β-boswellic acid, andrographolide, neoandrographolide and piperine), principal component analysis (PCA) and cluster analysis (CA) were used. Results of PCA and CA analysis showed that close relation between in silico observed values compared to experimentally observed ones, implying that in silico prediction can be used for fast insight into physicochemical properties of compounds.

Andrographolides are diterpenoids found in the plant of Andrographis paniculata (Burm.f.) Nees (Acanthaceae) and widely used in traditional medicine, exhibiting anti-inflammatory property. In this research, a novel micro-extraction by packed sorbent (MEPS) procedure for sample preparation was examined with the aim of extraction of pharmacological active andrographolide, neoandrographolide and 14- deoxyandrographolide from plasma samples. The analysis of samples was performed using developed and validated high-performance liquid chromatography with diode array detection (HPLC/DAD) method. The validation results showed acceptable linearity of the method (R2>0.99) for all analytes within the calibration range. Acceptable accuracy and precision of the method were indicated by recovery of 91–100% and Relative Standard Deviation (RSD) values below 7.6%.The extraction recovery was found to be between 91.04 and 100.34% with RSD values not higher than 11.5%. The limit of quantification was found to be between 62 and 322 ng/mL while the limit of detection was found between 18 and 97 ng/mL. Short-term (1 - 3 days at room temperature) and long-term stability (3 days at -80 °C) were also tested and showed no significant difference (RSDs were lower than 14.8%). The development of this fast and easy MEPS pretreatment procedure granted agreeable validation results and it represents a bioanalytical improvement (cheaper, greener and less time-consuming) over classical solid-phase extraction methods.

Gluten is naturally found in food, especially in certain grains such as wheat, rye, and barley. The consumption of gluten causes damage to the lining of the small intestine, preventing the absorption of nutrients and causing deficiencies that can lead to severe conditions and diseases. Inflammatory bowel diseases, Crohn's disease and ulcerative colitis, are chronic relapsing diseases affecting millions worldwide and disrupting their daily lives and requiring prescribing of drugs and dietary supplements. In addition to the active substance, orally formulated drugs contain a whole range of ingredients, such as excipients. Excipients can be synthetic or from natural sources originating from plants. One common excipient is starch. Corn, potatoes, and wheat are the most common starch types. Therefore, oral drugs and dietary supplements in the form of tablets and capsules are potential sources of gluten contamination. Plants and dietary supplements are important sources of biologically active components that have a positive effect on human health. In addition, the use of dietary supplements is constantly increasing. On the other hand, health professionals do not recognize the potential sources of gluten in orally formulated drugs. For all the listed reasons, this work aimed to evaluate gluten content in dietary supplements. Forty-five samples were collected from local pharmacies, food health stores and purchased from the Internet (products online-only available in Croatia). All dietary supplements were classified as botanical monopreparations and multipreparation, as they contained one or more of the investigated herbal extracts: Indian frankincense, Green chiretta and Turmeric. Enzyme-linked immunosorbent assay (ELISA) was used to detect and quantitate gluten in investigated samples. All samples were analyzed in duplicate prior to stated expiry date. Based on the obtained results, the gluten content was above the LOQ value (0.15 ng/mL) in 12 samples, while in one dietary supplement was over 20 ppm (28 µg/mL). Therefore, there are currently no labeling requirements for gluten found in orally formulated drugs and dietary supplements, while all foods labeled "gluten-free" must contain less than 20 ppm gluten. To sum up, all analyzed dietary supplements except one sample contained gluten less than the FDA criteria. ACKNOWLEDGEMENTS This work has been supported in part by the Croatian Science Foundation under the project numbers [UIP-2017-05-3949, DOK-2021-02- 7922].

Andrographolides are diterpenoids found in the plant Andrographis paniculata and are widely used in traditional medicine, exhibiting anti- inflammatory properties.1 In this research we developed and validated high-performance liquid chromatography coupled with electrospray ionization triple-quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) method for quantification of andrographolides in human plasma. The chromatographic analysis of pharmacological active andrographolide (AND), neoandrographolide (NAND) and 14-deoxy 11, 12- didehydroandrographolide (DAND) was performed on a 15 cm HSS Cyano column with a gradient mobile phase of ultra-pure water and acetonitrile with the addition of formic acid (0.1%) as a modifier at a flow rate of 0.8 mL/min in 21 min. The validation results showed acceptable linearity of the method (r>0.999) for all analytes within the calibration range 0.7–10 µg/mL. The acceptable accuracy of the method was indicated by recovery of 90–107 %. The limit of quantification was found to be between 2 and 70 ng/mL while the limit of detection was found between 0.6 and 20 ng/mL. Multiple reaction monitoring (MRM) mode was optimized to quantify data under monitoring precursor-product ion transitions of m/z 351.1 → 105.0, 351.5 → 91.0, 351.1 → 79.0 and 351.1 → 77.0 for AND, m/z 481.6  319.0, 481.6  301.2, 481.6  289.2, 481.6  95.1 for NAND and m/z 333.1  314.9, 333.1  297.2, 333.1  257.3, 333.1  91.0 for DAND (Figure 1). This advanced method is simple, selective, accurate and cost-effective. As a result, this assay can be easily transferred in clinical settings for pharmacokinetic studies and therapeutic drug monitoring programs. ACKNOWLEDGEMENTS This work has been supported in part by the Croatian Science Foundation under the project numbers [UIP-2017-05-3949, DOK-2021- 02-7922] and project FarmInova (KK.01.1.1.02.0021) funded by the European Regional Development Fund.

Upalne bolesti crijeva (eng. Inflammatory Bowel Disease, IBD), Crohnova bolest i ulcerozni kolitis, spadaju u kronične i progresivne bolesti. Etiologija im nije u potpunosti razjašnjena, a smatra se da uključuje kombinaciju genskih i okolišnih čimbenika što dovodi do neravnoteže između proupalnih i protuupalnih citokina u gastrointestinalnom traktu. Tijekom posljednjeg desetljeća biološka terapija u potpunosti je izmijenila strategiju liječenja. Ipak, 30 % bolesnika ne odgovori na biološku terapiju, a čak kod 40 % onih koji su inicijalno odgovorili terapija izgubi djelotvornost. Upravo za ove bolesnike postoji potreba za razvojem novih lijekova s drugačijim mehanizmima djelovanja. Modulatori sfingozin-1-fosfat (S1P) receptora predstavljaju novu generaciju lijekova koji čine prekretnicu u liječenju bolesnika s upalnim bolestima crijeva. U 2021. godini Agencija za hranu i lijekove Sjedinjenih Američkih Država i Europska agencija za lijekove su odobrili ozanimod kao prvi selektivni modulator S1P1 i S1P5 izoformi receptora, dok etrasimod predstavlja drugu generaciju modulatora S1P receptora koji trenutno prolazi kroz fazu III. kliničkih ispitivanja. Kako bi ovaj lijek našao svoje mjesto u terapiji upalnih bolesti crijeva potreban je promišljen i sveobuhvatan pristup razvoju učinkovitog i sigurnog farmaceutskog proizvoda s ciljanom dostavom aktivne farmaceutske tvari u područje upale. Za ispitivanje kompatibilnosti etrasimoda i 130 pomoćnih tvari korištena je platforma PharmDE. CACD (eng. Computer Assisted Compatibility Design) pristup pokazao je kako etrasimod nije kompatibilan s pomoćnim tvarima koje u strukturi sadrže hidroksilne skupine.

Trace and heavy metal impurity in dietary supplements (DS) poses a serious potential risk to health. Furthermore, trace metals play a significant role in reactions that lead to the formation of the active chemical plant constituents and are responsible for their curative and toxic properties. For all mentioned above, this study aimed to evaluate the content of metal impurity in DS containing one or more plant species used to alleviate the symptoms of inflammatory bowel disease (IBD): Indian frankincense, Green chiretta and turmeric. Fifty- four collected samples were in different dosage forms: tablet, hard and soft capsule, tea mixture, bulk and tincture. Total Hg content in the studied samples was analyzed using mercury analyzer, while the contents of Ag, As, Ba, Cd, Co, Cr, Cu, Mo, Ni, Pb, Sb, Se and Sn were analyzed by GFAAS after microwave digestion. The obtained results of metal impurities were in a wide range. In conclusion, trace and heavy metal impurity in DS used in treating IBD were borderline or higher than the safety level. Notably, Pb and Hg posed the highest risk.

Tiopurin S-metiltransferaza (TPMT, E.C. 2.1.1.67) citosolni je enzim koji metilira sulfhidrilne skupine kako aromatskih, tako i heterocikličkih spojeva. Primjeri lijekova koje TPMT inaktivira su azatioprin, 6- merkaptopurin (6MP) i 6-tiogvanin koji se koriste kao imunosupresivi za liječenje leukemija, ali i upalnih bolesti crijeva. Cilj ovog istraživanja bio je razviti HPLC metodu za određivanje 6-metilmerkaptopurina (6MMP) kao metabolita 6-merkaptopurina u biološkom uzorku hemolizata kako bi se odredila enzimska kinetika, ali i postavili temelji za ispitivanje eventualnih interakcija biljnih dodataka prehrani sa lijekovima koji se metaboliziraju preko TPMT, a koje bi mogle dovesti do promjena u njihovim terapijskim koncentracijama. Krv korištena za istraživanje izvađena je zdravom dobrovoljcu na antikoagulant EDTA, eritrociti su odvojeni od krvne plazme, lizirani sa suviškom hladne ultračiste vode te skladišteni na -20 °C. Razvijena je HPLC metoda u trajanju od 22 min. Kao nepokretna faza korištena je Cortecs Phenyl kolona (150 × 4, 6 mm, veličina čestica 2, 7 um). Provedena je gradijentna eluacija s dvije sastavnice mobilne faze (0, 1 % mravlje kiseline u vodi, odnosno metanolu). Reakcijske otopine sadržavale su hemolizat, 6MP, S- adenozilmetionin (SAM, kofaktor u reakcijama metilacije), ditiotreitol i HClO4 pomoću koje je reakcija zaustavljana nakon inkubacije. Uzorci su inkubirani 90 minuta na 37 °C. Nakon utvrđivanja selektivnosti kao ključnog validacijskog parametra, ispitani su linearnost (r = 0, 9994), točnost (analitički prinosi između 97, 07 % i 98, 69 %) te preciznost (RSD = 0, 81 %, n = 6 ; analiza varijance pokazala je statistički neznačajnu razliku prinosa za α = 0, 05 kroz tri dana). Nadalje, ispitana je kinetika metilacije 6MP putem TPMT pri čemu kao produkt nastaje 6MMP. Vmax za 6MP iznosio je 41, 94 nmol 6MMP/h/g Hb, a Km 239, 5 µM. Vmax za SAM bio je 40, 93 nmol 6MMP/h/g Hb, a Km 7, 6 µM. Dobiveni rezultati poslužit će daljnjem ispitivanju mogućnosti interakcija biljnih dodataka prehrani i terapije azatioprinom ili 6MP. Ovaj rad je sufinancirala Hrvatska zaklada za znanost projektom [UIP-2017-05-3949].

Crohnova bolest i ulcerozni kolitis, poznatiji kao upalne bolesti crijeva, predstavljaju kronične upalne bolesti gastrointestinalnog trakta. Zacjeljivanje sluznice i postizanje duboke remisije omogućuje se ciljanom terapijom, međutim postoje skupine pacijenata kojima koristi isključivo liječenje protuupalnim lijekovima iz skupine 5-aminosalicilata; mesalazin i njegovi prolijekovi sulfasalazin, balsalazid i olsalazin. Nedostatak folne kiseline primijećen je u više od polovine oboljelih od Crohnove bolesti i više od trećine pacijenata s ulceroznim kolitisom. Njezin nedostatak može dovesti do razvoja megaloblastične anemije, a povezan je i s povećanom prevalencijom karcinoma debelog crijeva, stoga se uz redovitu terapiju često propisuju povišene doze folne kiseline od 1 do 5 mg na dan. Razvojem fiksnih kombinacija lijekova potencijalni rizik od nepridržavanja propisane terapije nastoji se svesti na najmanju moguću mjeru, što je posebno važno kod pacijenata koji boluju od kroničnih bolesti gdje se terapija sastoji od više lijekova iz različitih farmakoloških skupina. Razvoj fiksnih kombinacija lijekova složen je postupak u kojem važnu ulogu ima ispitivanje fizikalno-kemijskih svojstava svakog pojedinog lijeka kao i njihovih smjesa. U ovom radu predložene su analitičke metode kao podrška razvoju fiksnih kombinacija 5-aminosalicilata i folne kiseline pri čemu su korištene različite analitičke tehnike, poput termalnih, spektroskopskih te kromatografskih, s ciljem ispitivanja fizikalno-kemijske kompatibilnosti 5-aminosalicilata i folne kiseline. Provedena istraživanja pokazala su da su ispitivani 5-aminosalicilati; mesalazin, sulfasalazin, balsalazid i olsalazin, fizikalno-kemijski kompatibilni s folnom kiselinom, što otvara mogućnost za daljnja ispitivanja potrebna za razvoj predloženih fiksnih kombinacija. Crohn's disease and ulcerative colitis, better known as inflammatory bowel diseases, are chronic inflammatory diseases of the gastrointestinal tract. Healing of the mucosa and achieving deep remission is achieved by targeted therapy, however there are groups of patients who benefit exclusively from treatment with anti-inflammatory drugs from the group of 5-aminosalicylates; mesalazine and its prodrugs sulfasalazine, balsalazide and olsalazine. Folic acid deficiency has been observed in more than half of Crohn’s disease patients and more than a third of patients with ulcerative colitis. Its deficiency can lead to the development of megaloblastic anemia, and is associated with an increased prevalence of colorectal cancer, so in regular therapy, increased doses of folic acid of 1 to 5 mg per day are often prescribed. With the development of fixed-dose combinations, the potential risk of non-compliance with prescribed therapy is minimized, which is especially important in patients with chronic diseases where therapy consists of several drugs from different pharmacological groups. The development of fixed drug combinations is a complex process in which an important role is played by the examination of the physicochemical properties of each individual drug as well as their mixtures. In this paper, analytical methods are proposed to support the development of fixed combinations of 5-aminosalicylate and folic acid using various analytical techniques, such as thermal, spectroscopic and chromatographic, to test the physicochemical compatibility of 5-aminosalicylate and folic acid. Studies have shown that 5-aminosalicylates; mesalazine, sulfasalazine, balsalazide and olsalazine, are physicochemically compatible with folic acid, which opens the possibility for further testing required for the development of the proposed fixed combinations.

Fourier-transform infrared spectroscopy (FTIR) and X-ray powder diffraction (XRPD) are widely used techniques in the drug compatibility studies. As obtaining results in both techniques are based on the same principle sometimes the interpretation of obtained spectra can be difficult. Comparing obtained spectra of pure substances with the spectra of their blends can lead to ambiguous conclusions due to the overlapping of peaks in the obtained spectra. In this work we introduced a multivariate statistical approach, the principal component analysis (PCA) followed by cluster analysis (CA). Two 5-aminosalicylates (5-ASAs), olsalazine (OSZ) and balsalazide (BSZ) as well as their blends with folic acid (FA), prepared in different ratio (10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10), were measured using FTIR and XRPD and compared to evaluate any changes in the spectra which could indicate the chemical reaction of the components of blends. PCA followed by CA was performed on spectral region from 550 – 1800 cm-1 (using Pearson correlation, Euclidean distance measure and Ward’s agglomerative clustering). A 9 x 625 matrix was created. The number of rows represents the used 5-ASA and FA as well as their blends, whilst spectral data was placed in columns. In the case of BSZ and FA as well as OSZ and FA all blends were positioned in the plot in the increasing PC1 values as the ratio of 5-aminosalicylates reduces and FA share in blends increases, with the 1:1 being placed between measured blends. Placement of pure compounds and their blends in the PCA plot, as well as the formation of clusters in CA dendrogram, are in favour of BSZ/FA and OSZ/FA compatibility. Any deviation from the obtained order in which blends are positioned would indicate incompatibility between compounds, as well as the formation of clusters with random blends. Pearson’s correlation was calculated between obtained diffractogram of blend and theoretical diffractogram (calculated as the average of two spectra of pure compounds). Obtained correlation coefficients (r) for BSZ/FA and OSZ/FA blends are 0.99 and 0.98, respectively. High correlation value between observed and theoretical spectra indicates lack of component interaction. These results indicate that FA can be in the same formulation containing BSZ or OSZ.

Regulation of dietary supplements regarding their quality is still not well developed worldwide [1]. Many such products, mostly those available online, are not subjected to quality control and are either fraudulently underdosed, demonstrating no efficacy, or adulterated with synthetic active substances or alien herbal species, possibly leading to overdosage and toxicity. Furthermore, inadequate dissolution of active substances can lead to decreased bioavailability. The aims of this work were, therefore, to assess the content of curcuminoids and piperine, active substances of turmeric and black pepper (often used in conjunction with turmeric as piperine enhances the absorption of curcuminoids [2]), in widely used marketed turmeric dietary supplements and food samples, as well as to examine the dissolution of tableted and capsulated products according to the United States Pharmacopoeia (USP). A total of 20 dietary supplement and 13 food samples were investigated. High-performance liquid chromatography with spectrophotometric detection was the technique of choice. Firstly, optimization of extraction of active substances from a representative food sample (as the more complex sample type) was performed using Box Behnken response surface methodology. After model optimization, maximum yield for all substances was achieved using 76.9% (V/V) ethanol as extraction solvent, 75 °C as extraction temperature and 30 min as sonication time. Food samples contained 1.7 to 4.7% curcuminoids (of turmeric rhizome mass) save for two purchased online, which showed higher content (9.1 and 10.5%, possibly implying adulteration). The content of piperine ranged from 3.9 to 5.8% of black pepper fruit mass. Only 9 of 20 and 1 of 8 dietary supplement samples conformed to the USP regulations of 90 to 110% of declared curcuminoid and piperine content, respectively. Dissolution studies using USP Apparatus 2 revealed only 3 of 15 formulated products conformed to minimum of 75% active substance release, which could be due to lower expected curcuminoid amounts present in products on the American market. In conclusion, more than half of the investigated samples failed to conform to active substance content and/or dissolution criteria of revelant regulations, which points to an increased need of quality control of herbal preparations and dietary supplements.

Background: Dietary supplements are readily accessible in pharmacies, health food stores and on-line. Although these products are extensively used worldwide, they are inadequately regulated, and patients are misled by the manufacturers. Thus, quality control assays are required to ensure safety of products available to the public. Objective: In this presentation, recently developed, validated and applied methods for determination of active and toxic ingredients in dietary supplements used for treatment of various conditions by chromatographic and spectrometric techniques will be presented. Methods: Direct- injection mass spectrometry was used for identification of biologically active ingredients while liquid chromatography coupled with mass spectrometry was used for their quantitation. On the other hand, gas chromatography coupled with flame ionization detector and atomic absorption spectrometry were used for comprehensive assessment of toxic ingredients. Results: Our investigations revealed high deviations between label content and levels of active compounds found in dietary supplements used for treatment of various conditions. Furthermore, high amounts of ethanol were found in several products especially designed for children. Metal impurities were below the limits established by USP. Conclusion: The only source of information for patients about dietary supplements and their composition is their label. In order to ensure a high level of patients’ protection and facilitate their choice, dietary supplements must be safe and appropriately labeled. Foundings: This research was funded by the Croatian Science Foundation, grant numbers HRZZ- UIP-2017-05-3949 and HRZZ-DOK-2018- 01-9047.

Soybeans, red clover, chastetree, 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. Since polyphenolic compounds are responsible for the potential antioxidant activity and radical scavenging capacity of plant food, this study aimed to evaluate total polyphenol content (TPC) and antioxidant activity (AA) of phytoestrogen- containing food and dietary supplement (DS) formulated products prepared. The HPLC-DPPH method was applied for the analysis of various phytoestrogen-containing samples. Polyphenol content and antioxidant activity in formulated DS (TPC: 17.65±10.28 mg/g ; AA: 103.01±58.42 mM TROLOX/g) was higher than in functional food samples (TPC: 6.18±1.37 mg/g dry matter ; AA: 40.01±15.80 mM TROLOX/g dry matter). Also, the results indicate that multi-botanical products (TPC: 23.70±9.09 mg/g ; AA: 143.49±45.63 mM TROLOX/g) have higher polyphenol content and antioxidant activity than monopreparation (TPC: 15.63±10.11 mg/g ; AA: 89.52±57.07 mM TROLOX/g). Furthermore, the correlation between polyphenol content and the antioxidant activity was strongly statistically significant, so it can be concluded that antioxidant activity is proportional to the content of secondary metabolites. The most eye- catching batch-to-batch deviations were represented by two chasteberry-based products (RSD 12.6% and 41.3%) and one red clover (RSD 57.9%) derived product.

Differential scanning calorimetry (DSC) is the first technique of choice when it comes to compatibility evaluation between components of physical blends in the development of pharmaceuticals. Its main advantage is fast acquisition of information regarding the potential interactions of components in blends which are manifested through changes in characteristic thermograms of blends compared to those of pure compounds. Physicochemical compatibility of folic acid (FA) and two 5-aminosalicylates (5-ASA), balsalazide (BSZ) and olsalazine (OSZ), used in the therapy of inflammatory bowel diseases was investigated using DSC and isothermal stress testing (IST), as a prerequisite to the development of 5-ASA/FA fixed-dose combinations. Thermograms of pure compounds as well as their blends in various ratios (5-ASA : FA 10:1, 5:1, 2:1 and 1:1, m/m) were obtained on a Diamond DSC (Perkin Elmer Inc., Waltham, MA, USA) with a heating rate of 10 °C/min, under the atmosphere of pure nitrogen with the gas flow of 25 mL/min. IST was conducted on pure drug substances and their blends, in a ratio of 10:1, 5:1, 2:1 and 1:1 in favour of both 5-ASAs and FA, by keeping the samples at 50 °C for four weeks. IST was followed by chromatographic analysis of stressed samples using high-performance liquid chromatography. OSZ and FA showed peaks that correspond to their melting, whilst BSZ showed no characteristic melting peak, however, one peak that occurred corresponds to its dehydration since it comes in the form of a dihydrate, which can provide relevant information regarding its physicochemical properties. DSC results implied compatibility between FA and two 5-ASAs (no notable shifts in peak temperatures, < 5°C). Results obtained by chromatographic analysis of samples subjected to IST showed that recoveries of both 5-ASAs and FA in all stressed samples did not markedly differ (97.9 – 103.0%, with the relative standard deviation not exceeding 2.4%). Results obtained by DSC and IST imply that there was no notable change in the physical or chemical nature of 5-ASAs and FA when combined in the same blend. Although DSC measurements can sometimes be misleading due to the exposure of samples to extreme temperatures, it is always recommended to support those results by using other techniques, such as IST.

Active substance content is one of the essential parameters of any preparation, as it often governs both efficacy and safety of the overall product. It is well known herbal products are not subjected to such rigorous control as conventional pharmaceuticals, so their quality can drastically differ. Therefore, the goal of this work was to assess the active substance content of turmeric, green chiretta and Indian frankincense-based products available locally and online. As these products are often comprised of two or more stated herbal constituents, a universal HPLCDAD method for determination of three curcuminoids, piperine (curcuminoid bioavailability enhancer), three andrographolides and six boswellic acids was developed and validated for the first time. Sonication extraction step was optimized using three-factorial Box-Behnken design, while the optimal parameters were extraction solvent of 81.5% (V/V) ethanol/water, sonication time of 30 min and extraction temperature of 60 °C (model desirability 0.927). The method was validated according to ICH guidelines, with selectivity (evaluated using standard addition method) and robustness of the method (evaluated using Plackett-Burman design) being paid special attention to. In total, 54 samples (19 raw material and 35 herbal preparation) were analyzed. Drastic differences in declared content of herbal preparations (1.4 to 176.8%) were observed. Regarding raw material, two online-marketed samples showed suspiciously high amounts of curcuminoids, pointing to possible adulteration. Overall, most online-marketed products showed either very little active substances (39% of Internet-bought preparation samples showed less than 20% declared content, implying no efficacy) or substantially higher amounts which could potentially cause adverse effects (one sample containing andrographolides) or lead to an interaction with concomitantly used drugs (one sample containing piperine). In conclusion, consumers should purchase dietary supplements and similar products from certified manufacturers in local pharmacies and refrain from buying them online.

U ovom preglednom radu opisana je tehnika ekstrakcije čvrstom fazom te je prikazana njena primjena u bioanalitici. Rad obuhvaća pregled bioanalitičkih metoda namijenjenih određivanju terapijske doze lijekova te ispitivanju njihove farmakokinetike. Nadalje, opisane su bioanalitičke metode kojima se određuju biomarkeri različitih bolesti, opijatne droge, sintetički stimulansi i štetne tvari koje se unose prehrambenim i kozmetičkim proizvodima. Navedeni su raznovrsni reaktivni sorbensi od široko komercijalno dostupnih modificiranih silikagelova i polimera do visoko selektivnih inovativnih sorbensa poput nanomaterijala, polimera s molekulskim otiskom, imunosorbensa itd. Osim seruma, plazme i urina, istaknute su i metode kojima su analizirane druge vrste uzoraka poput sline, bronhalnog sekreta, koštane srži, majčinog mlijeka, mekonija i kose. Automatizacija bioanalitičkih metoda je danas imperativ, posebice u kliničkim laboratorijima stoga su obrađene i metode koje koriste formate ekstrakcije čvrstom fazom namijenjene analizi velikog broja uzoraka poput 96-ekstrakcijskih pločica i on-line kolona.

U svrhu što učinkovitijeg praćenja terapije upalnih bolesti crijeva uz analizu koncentracija metabolita tiopurinskih imunosupresiva potrebna je i analiza plazmatskih koncentracija aktivnog oblika folne kiseline, 5-metiltetrahidrofolata (5-MTHF) koji je u ovih pacijenata u potencijalnom nedostatku. Cilj ovog rada bio je razviti jednostavnu i brzu kromatografsku metodu za praćenje obiju skupina analita uz prihvatljivo vrijeme trajanja metode te što manji manualni rad analitičara. Biološki uzorci su složenog sastava, a analiti koji se određuju u vrlo niskim koncentracijama, stoga je potrebno optimizirati pripremu uzorka plazme za analizu 5-MTHF da bi se poboljšala osjetljivost metode te se postigle niže granice određivanja. Također, uzevši u obzir prirodu analita, cilj je pripremiti i stabilan uzorak kako bi se osigurala reprezentativna slika biološkog stanja. RP-HPLC-FLD tehnika korištena je za praćenje koncentracije 5-MTHF u plazmi. U optimizaciji pripreme ispitani su razni omjeri plazme i acetonitrila/metanola (2:1 i 3:1), utjecaj dodane količine antioksidansa askorbinske kiseline (ASK) (od 15 do 90 µg na 200 µL plazme) te ditiotreitola (DTT) (25 µg na 200 µL plazme) te otapala nakon uparivanja uzorka (1 i 3 g/L ASK). U svrhu što boljeg uklanjanja interferencija u uzorku, ispitani su i Phree Phospholipid Removal i Isolute PLD+ sorbensi. Priprema uzorka s 90 µg ASK uz omjer 2:1 MeOH:plazma pokazala je najveću iskoristivost uz najveću površinu pika 2 sata nakon prvog injektiranja, dok je najstabilnija otopina bila ona s dodanih 15 μg ASK i 25 μg DTT, pokazujući najmanje relativno smanjenje površine pika nakon 2 sata. Sorbensi su pokazali manji broj interferirajućih pikova uz nešto manji analitički prinos u odnosu na postupak taloženja proteina. U ovom radu optimizirana je priprema uzorka plazme za analizu 5-MTHF. Daljnjim tijekom istraživanja provest će se i optimizacija pripreme uzorka za analizu tiopurina te će metoda biti validirana. Ovaj rad sufinancirala je Hrvatska zaklada za znanost projektom [UIP-2017-05-3949].

The success of the food and dietary supplement industry in bringing to market safe and high- quality phytoestrogencontaining products is important for the well-being of an increasing number of post-menopausal women consuming these products. On the other hand, a serious safety issue associated with food and herbal supplements is heavy metal contamination. Direct-injection mass spectrometric (DI-MS) technology has evolved as a powerful tool for screening of active ingredients in food, herbal and dietary supplement samples. In this work the developed DI-MS method provided simple and fast multi-target screening of phytoestrogens in food, raw materials and dietary supplement products without prior analyte separation by chromatography. Phytoestrogen marker compounds were identified in two of four herbal raw material samples and eight of twenty dietary supplement products. As for the heavy metal content determination, graphite furnace–atomic absorption spectroscopy (GF-AAS) was utilized. The average daily intake of heavy metals is well below the recommended tolerable daily intakes if the products are used according to the manufacturer’s instructions, with the exception of one sample.

Kontrola kvalitete važan je korak u farmaceutskoj industriji kako bi se osigurala i potvrdila sigurnost farmaceutskih proizvoda. Tekućinska kromatografija visoke djelotvornosti igra važnu ulogu u kontroli kvalitete aktivnih farmaceutskih komponenata i farmaceutskih proizvoda od njezine rane pojave. Razvojem novih analitičkih metoda pomiču se granice te metode postaju sofisticiranije a time se otežava njihov razvoj koji je kompleksan i dugotrajan. Folna kiselina (Vitamin B9) igra važnu ulogu u mnogim funkcijama u ljudskom tijelu. Osigurava normalan rad organizma, dok trudnice moraju uzimati folnu kiselinu kako bi se osigurao normalan razvoj ploda. Kako pacijenti koji boluju od upalnih bolesti crijeva, kronove bolesti (eng. Crohn's disease) ili ulceroznog kolitisa (eng. Ulcerative colitis), dokazano imaju manju koncentraciju folne kiseline u svom tijelu, potrebna je njegova nadoknada. Predložene fiksne kombinacije aminosalicilata i folne kiseline osigurale bi unos folne kiseline prilikom liječenja pacijenata sa upalom bolesti crijeva. Cilj rada bio je razviti jednu HPLC metodu koja bi se koristila u laboratorijima za kontrolu kvalitete za analizu sadržaja svih 4 predloženih kombinacija aminosalicilata (mesalazin, sulfasalazin, olsalazin i balsalazid) i folne kiseline. Velika razlika u koncentraciji komponenata u kombinacijama (do 750 puta) te razlika ali i sličnosti u fizikalno-kemijskim karakteristikama spojeva otežalo je razvoj metode ali ga je učinilo i zanimljivim. Prilikom razvoja metode ispitane su razne kolone, u kombinaciji s raznim mobilnim fazama, protocima, gradijentima, temperaturama. Također, ispitani su i razne metode detekcije (detektor s nizom dioda, fluorescentni detektor i maseni spektrometar). Metoda je razvijena na Agilent 1100 HPLC sustavu koristeći XBridge fenilnu kolonu (150 mm x 4.6 mm, 3.5 µm) sa gradijentnom eluacijom na 25°C pri protoku od 1 mL/min. Kao mobilna faza A koristila se 0.2% otopina mravlje kiseline u vodi te 0.2% otopina mravlje kiseline u metanolu kao mobilna faza B. Za detekciju je odabran detektor s nizom dioda kao najprikladniji a za praćenje apsorbancija uzete su valne duljine od 320 nm za praćenje sulfasalazina, olsalazina i balsalazida, 300 nm za mesalazin i 280 nm za folnu kiselinu. Metoda je validirana na linearnost, točnost, preciznost, granicu detekcije i kvantifikacije, robusnost i selektivnost prema ICH smjernicama. Metoda je ispitana na komercijalno dostupnim aminosalicilatima i folnoj kiselini te su svi dobiveni sadržaji u dozvoljenim granicama. Time je potvrđeno da je metoda primjerena za ciljanu primjenu.

Biomimetička kromatografija danas ima sve važniju ulogu u razvoju lijekova. !mobilizacija raznih bioloških struktura poput humanog serumskog albumina, al-kiselog glikoproteina, fosfolipida te raznih enzima i receptora na nepokretne faze kromatografske kolone dovela je do mogućnosti ispitivanja velikog broja spoj eva u kratkom vremenu. Biomimetičkom kromatografijom mogu se ispitati parametri poput vezanja za proteine plazme, lipofilnosti te volumena raspodjele, kao i afiniteta za određene mete ili receptore. Na taj način moguće je dobiti uvid u farmakokinetička i farmakodinamička svojstva velikog broja spoj eva kandidata za lijek te odabrati najpogodnije za nastavak istraživanja, time ubrzavajući proces razvoja lij ekova.

In the past decade, direct-injection mass spectrometric (DIMS) technology that combines ever-improving mass and time resolution with high sensitivity and robustness has been rapidly developing. Therefore, DIMS represents a powerful analytical tool for the quality assessment of herbal formula in dietary supplements. The aim of our work was to adapt the previously developed method for phytoestrogen profiling of food, medicinal plant raw material and dietary supplement samples. The food samples, soy beans and flax seeds, were crushed to fine powder using Knifetec 1095 sample mill by Foss Tecator AB (Sweden). Afterwards, the samples were placed in a porous cellulose thimble and defatted with petroleum ether using Soxtec 2055 (Foss Tecator AB, Sweden). The content (1 g) of finely ground and homogenized dietary supplement sample was dissolved with 15 mL of ethanol and sonicated for 15 min at room temperature in Elmasonic xtra TT ultrasonic bath (Elma, Germany). The supernatant was obtained by centrifuging (1000 rpm, 5 min, 25 °C) (model Z 326 K, Hermle, Germany). The system consists of an MSD ion trap mass spectrometer (Agilent Technologies, USA) with an electrospray ionization (ESI) probe. ESI was performed in positive ion mode from m/z 100 to 500. Optimized MS conditions were described as follows: source temperature 325 °C, ion spray voltage 3.5 kV, nitrogen flow fate 10 L/min and pressure 20 psi. Structural information for 11 phytoestrogens were collected using standard solutions (0.1 mg/mL). Finally, DIMS method was applied for phytoestrogen profiling in 4 food, 5 herbal materials and 20 dietary supplement products. Multiple reaction monitoring at unit resolution was employed to monitor the following transitions in the investigated samples: 255→227 (daidzein), 271→243 (genistein), 345→327 (secoisolariciresinol), 271→225 (apigenin), 359→341 (matairesinol), 269→254 (formononetin), 375→359 (casticin), 285→270 (biochanin A), 341→285 (8-prenylnaringenin) and 355→299 (xanthohumol and isoxanthohumol). Phytoestrogens were only detected in herbal materials (2 samples) and dietary supplements (5 samples). The DIMS method provided sufficient sensitivity and selectivity for the rapid identification of phytoestrogens in complex samples without prior analyte resolution by chromatography and with a total analysis time of less than 30 min.

Determination of various parameters such as lipophilicity, plasma protein binding etc. through conventional means is time-consuming, therefore the effectiveness of drug discovery is diminished. In silico approaches offer faster insight into several characteristics of the molecule related to pharmacokinetics, which is especially important with two or more compounds in a single formulation. Seeing that folic acid (FA) is malabsorbed in inflammatory bowel disease (IBD) patients, a fixed dose combination of one of the aminosalicylates mesalazine (MSZ), olsalazine (OSZ), sulfasalazine (SSZ) or balsalazide (BSZ) and FA is proposed. The goal of this work was to evaluate the applicability of in silico approach to predict the physico- chemical and ADME parameters of aminosalicylates and FA obtained by two chromatographic techniques. In silico approximations of logP, logD, plasma protein binding and Caco-2 permeability were predicted by 14 on-line based platforms. TLC measurements were done using RP- C18 F254 silicagel plates. HPLC measurements were done using conventional octadecylsilyl column as well as biomimetic chromatographic columns: immobilized artificial membrane, human serum albumin and α-1 acid glycoprotein. Experimental measurements showed increasing lipophilicity in the following order: MSZ (RMwTLC = -0.36, logkwC18 = 0.37) < FA < BSZ ≈ OSZ < SSZ (RMwTLC = 2.98, logkwC18 = 3.71). SSZ and OSZ showed longest retention times on HSA and AGP columns, implying high percentage of plasma protein binding (higher than 90%). Of the 14 in silico approaches, 5 of them have shown significant correlations with at least one of physico- chemical and ADME parameter (R2 0.864 to 0.998): Chemexper, Chemicalize, Molsoft, PreADME and admetSAR. Although further improvements in theoretical approaches should be done, in silico predictions show great potential in high- throughput drug screening due to high processing capability of a large sample pool and acceptable correlations with the experimental data. 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.

In silico prediction of drug metabolism and fate is a growing approach in drug development. Various studies should be performed as early as possible in the drug development process to get a brief insight into molecule characteristics, such as absorption, distribution, metabolism and excretion (ADME). Methods used to examine molecule characteristics such as lipophilicity, solubility and plasma protein binding require active pharmaceutical ingredient to be synthesized and analyzed and that is, in first place, expensive, but also time consuming. In silico approach offers fast and inexpensive way to obtain information about the specific molecule through many on-line prediction platforms. Inflammatory bowel disease is a chronic inflammatory state that can affect the entire gastrointestinal tract, commonly known as Crohn’s disease, or it can take place mainly in the colon, which is known as Ulcerative colitis. Due to damaged sidewalls of intestine folic acid is malabsorbed, and as consequence, leads to development of megaloblastic anemia. Novel fixed dose combination of one aminosalicylate (mesalazine, sulfasalazine, olsalazine and balsalazide) and folic acid could be used in the treatment of inflammatory bowel disease to simplify the therapy and to make sure that folic acid is compensated, especially in the case of sulfasalazine and olsalazine, which additionally inhibit its absorption. To characterize active pharmaceutical ingredients, lipophilicity and ADME parameters should be investigated and therefore correlated with in silico predicted values to investigate their applicability. Furthermore, to control the quality of proposed fixed dose combination, analytical methods should be developed. Experiments were done on HPLC-DAD system using high throughput methods and various columns from conventional octadecylsilyl column to biomimetic chromatographic columns such as: immobilized artificial membrane (IAM), human serum albumin (HSA) and α-1 acid glycoprotein (AGP). Experimental results were correlated with predicted parameters such as lipophilicity, plasma protein binding and Caco-2 permeability from 14 on-line platforms. Six of them showed good correlation with experimentally obtained values (R from 0.89 to 0.96), implying that in silico prediction has great potential in screening of similar properties in drug discovery. Second part of the research was to develop a single RP-HPLC-DAD method for determination of assay in proposed fixed dose combination of each representative of aminosalicylate group and folic acid. The method was developed on Waters XBridge Phenyl (150×4.6 mm, 3.5 µm) column with gradient elution using 0.2% (v/v) HCOOH in water as mobile phase A and 0.2% (v/v) HCOOH in methanol as mobile phase B. The method was validated according to ICH Q2(R1) guideline where all obtained validation parameters were within the acceptable ranges. Therefore, according to obtained results, it can be confirmed that the method is suitable for future research.

Kardiovaskularne bolesti imaju najveću prevalenciju i komorbiditete te najveći mortalitet u općoj populaciji. Dobro poznavanje patofizioloških mehanizama nastanka u kombinaciji s razumijevanjem farmakoloških učinaka dostupnih lijekova temelj je uspješnog liječenja. Beta- blokatori su nezamjenjivi lijekovi u indikacijama poput hipertenzije, angine pektoris, akutnog infarkta miokarda, sekundarne prevencije infarkta miokarda, srčane aritmije i zatajenja srca. Iako svi beta- blokatori dijele zajednički osnovni mehanizam djelovanja, među njima postoje određene razlike, kako u farmakokinetičkim i farmakodinamičkim svojstvima, tako i u dodatnim mehanizmima djelovanja. Upravo te razlike mogu izdvojiti jedan ili više lijekova kao lijekove izbora u odnosu na ostale pripadnike skupine, pogotovo u osoba s komorbiditetima. Cilj ovog rada bio je ispitati antioksidativni učinak beta-blokatora (atenolola, bisoprolola, karvedilola, nebivolola i propranolola) mjereći njihovu sposobnost hvatanja slobodnog 1, 1-difenil-2-pikrilhidrazil radikala (DPPH•). Korištena DPPH metoda temelji se na sposobnosti hvatanja slobodnog radikala potencijalnim antioksidansom (beta-blokatorom) pri čemu dolazi do smanjenja apsorbancije DPPH•. Analize su provedene tekućinskom kromatografijom visoke djelotvornosti (engl. high performance liquid chromatography, HPLC). Korištena je Xbridge C18 kolona (dimenzije 4, 6 mm × 150 mm, veličina čestica 3, 5 μm) uz izokratnu eluaciju korištenjem mobilne faze sastava metanol : ultra-čista voda (80:20, V/V). Brzina protoka mobilne faze bila je 1, 0 mL/min, temperatura kolone kontrolirana na 25 °C, a volumen injektiranja uzoraka iznosioje 20 μL. Kromatogrami su snimani detektorom s nizom dioda (engl. diode array detector, DAD) na valnoj duljini od 517 nm. Vrijeme zadržavanja DPPH• na koloni iznosilo je 3, 83 min, dok je ukupno trajanje analize iznosilo 4, 5 min. Svi uzorci pripremljeni su kao 1 mM otopine i analizirani u triplikatu, a rezultati izraženi kao antioksidativni kapacitet ekvivalentan standardu TROLOX-a. Na temelju dobivenih rezultata mjerljivu antioksidativnu aktivnost pokazali su atenolol i karvedilol. HPLC-DPPH metoda pokazala se brzom i učinkovitom za određivanje antioksidativne aktivnosti beta-blokatora s ciljem ispitivanja dodatnih mehanizama djelovanja pojedinih predstavnika ove široko primjenjivane skupine lijekova u kliničkoj praksi.