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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.

Tijekom predavanja predstavljeni su najvažniji rezultati istraživanja provedeni u sklopu projekta Hrvatske zaklade za znanost - Razvoj naprednih analitičkih metoda za lijekove i biološki aktivne tvari u liječenju upalnih bolesti crijeva (HRZZ-UIP- 2017-05-3949) voditeljice Ane Mornar Turk

U predavanju se prezentirani rezultati ispitivanja kvalitete dodataka prehrani koji se koriste u ublažavanju simptoma upalne bolesti crijeva.

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).

Na predavanju su prezentirana istraživanja na području dodataka prehrani.

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.

Inflammatory bowel disease is a general term that includes ulcerative colitis and Crohn's disease, disorders of unknown causes resulting in inflammation of the colon or small intestine. In addition to various medications, dietary supplements containing herbal extracts are used in the treatment of inflammation. One of them is andrographolide, an active compound from the plant Andrographis paniculate for the treatment of several inflammatory diseases [1, 2]. In this research, different techniques for sample preparation were examined with the aim of analysis of biological samples and determination of chemical components andrographolide (AND), neoandrographolide (NAND) and 14- deoxyandrographolide (DAND) in human plasma by high-performance liquid chromatography/ultraviolet spectroscopy (HPLC – UV). Xanthohumol was selected as the internal standard (IS). For plasma sample preparation and clean-up, solid phase extraction (SPE) and solid phase microextraction (SPME) techniques were used. Simple to use and short sample processing time made them as a good choice for many analytical applications [2]. ISOLUTE® PLD+ Protein and Phospholipid Removal columns and Phenomenex Phree Phospholipid Removal cartridges gave Extraction recovery ≥ 90 % with relative standard deviation not higher than 5.2 % while HybridSPE® Phospholipid removal tips gave Extraction recovery ≥ 80 % with relative standard deviation not higher than 6.8 %.

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].

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.