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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.

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 %.

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.

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.

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.

Mercury is a toxic heavy metal with no known biologic function in humans. Organic form of this metal is more toxic than inorganic form since it is more readily absorbed through ingestion. Beside this the toxicity of mercury is in part due to the fact that it accumulates in biological tissues (bioaccumulation) and high exposure to this dangerous contaminant of environment may cause neurological disorders including seizures and even death. Little is known about mercury contamination in herbal row material and dietary supplement. For all of the above reasons, the aim of this work was to evaluate total mercury content in phytoestrogen-containing food, herbal row material and dietary supplement samples and to compare to maximum allowed value. Two food samples of soybeans and flax were purchased from a local health food store, 5 samples of dietary supplement row material were kindly donated and 20 dietary supplements containing phytoestrogens were supplied from a community pharmacy. The dietary supplements analyzed in this work were in multiple dosage forms including liquid extracts, tablets and capsules. Liquid products contained only ethanolic herbal extract while other products contained additional ingredients, such as vitamins and minerals. Also, two different batches of each product were analyzed. The content of Hg in the investigated phytoestrogen-containing samples was analyzed by using advanced mercury analyzer AMA-254 with HS cuvette (Leco, Czech Republic) under the following conditions: wavelength 253.6 nm, drying time 60 s, decomposition time 150 s, cuvette clear time 45 s and typical sample mass 100 mg. The determined total mercury content of all phytoestrogen-containing samples varied between 0.16 and 15.81 µg/kg ; mean value 4.01 µg/kg. Furthermore, a statistically significant difference was not found between the content of Hg in food (5.88 µg/kg), herbal row material (3.95 µg/kg) and dietary supplement (3.67 µg/kg) samples. It was interesting to note that the batch-to- batch quality consistency of selected products was evaluated using two different batches of each dietary supplement and variable batch-to- batch uniformity was found among various brands. RSD values were between 0.29 – 67.05 % ; mean value: 18.0 %. To sum up, the obtained data provide evidence that the mercury content in the investigated phytoestrogen-containing food, herbal row material and dietary supplement samples was well below the internationally established maximum allowed value (100 µg/kg).