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Breast cancer is the most common cancer worldwide while the HR+/HER2- subtype is the most prevalent [1]. Locally advanced or metastatic HR+/HER2- breast cancers are treated with the combination of CDK4/6 inhibitors and drugs with anti-estrogen effects. One of the common combinations used in clinical practice is ribociclib with letrozole [2]. Many breast cancer patients suffer from various comorbidities and side effects. Thus, different drug classes are regularly used in clinical practice, painkiller ibuprofen being one of the most common. Because of that, we developed a capillary electrophoresis (CE) method for the simultaneous determination of ribociclib, letrozole, and ibuprofen since drugs metabolized by the same enzymes can have interactions. During method development, different buffer types (borate and phosphate with and without SDS) and additives and surfactants (SBE-β-CD and i-PrOH) were tested. Separation was achieved with 75 mM phosphate, 8.5 mM sulfobutylether-β-cyclodextrin, and 5% i-PrOH. The optimal conditions were 15 kV, 25 °C and 5 s hydrodynamic injection under 50 mbar. The developed method was validated according to the ICH guidelines and was shown to be linear in the range of 1 to 75 µg mL-1 (r>0.9957). Intra-day and inter-day repeatabilities were evaluated for method precision with acceptable RSD values. The robustness of the method was tested by changing applied voltage (± 0.5 kV), cyclodextrin concentration (± 0.5 mM), and temperature (± 2 ºC) one variable at a time, and the changes in peak areas and migration times were shown to be in the range of 0.34 to 7.27 %. The validated method is the basis for the further development of an even more sensitive method capable of detecting low plasma concentrations of these drugs using innovative sample preparation techniques and coupling to the mass detector which could then be applied to standard clinical practice.

Breast cancer is one of the most common cancers in women. Approximately two-thirds of breast cancers are HR+/HER2- tumors [1]. Advances in the treatment of this cancer phenotype have been made with abemaciclib (ABE), ribociclib (RIB), and palbociclib (PAL), inhibitors of cyclin D dependent kinases 4 and 6 (CDK4/6) in combination with aromatase inhibitors letrozole or anastrozole, and selective estrogen receptor degrader fulvestrant [2]. Based on a literature search, there is an unmet need for fast and simple methods for their determination in pharmaceutical dosage forms. Thus, we developed a first capillary electrophoresis method for fast simultaneous analysis of three CDK4/6 inhibitors. Separation was achieved by capillary zone electrophoresis in an acidic medium, in accordance with the structures of analytes and their pKa values. The optimal pH of the running buffer was found to be 2.9. The optimal method conditions were 27.5 kV separation voltage, the temperature of 30 °C, 5 s injection under 50 mbar pressure, and 50 mM phosphate buffer. The developed method was validated according to the ICH guidelines and was shown to be linear in the range of 10 to 100 µg mL-1 with correlation coefficients being 0.9981, 0.9984, and 0.9991 for ABE, RIB, and PAL, respectively. Selectivity of the method was tested by calculating the peak purities and was shown to be acceptable. The robustness of the method was tested by changing applied voltage, temperature, and pH one variable at a time, and the changes in peak areas and migration times were shown to be in the range of 0.22 to 1.63 %. Intra-day and inter- day repeatabilities were acceptable with RSD values in the ranges of 0.22 to 1.08 % and 0.64 to 3.95 %, respectively. The validated method was successfully applied to pharmaceutical dosage forms of all CDK4/6 inhibitors.

Breast cancer is the most common cancer in the world with 2.3 million new cases diagnosed in 2020 [1]. This type of cancer is of great public health interest and reducing its burden on the healthcare systems worldwide is of high importance. Over 70 % of breast cancers are hormone receptor-positive (HR+) and human epidermal growth factor receptor 2-negative (HER2-) [2]. Today, the standard therapy for locally advanced or metastatic HR+/ HER2- breast cancer is a combination of cyclin-D dependent kinase (CDK) 4 and 6 inhibitors with endocrine therapy because of better overall survival and progression-free survival. However, despite therapeutic benefits, these drugs exhibit serious side effects and toxicities. This problem can be tackled with therapeutic drug monitoring (TDM) which has been proven to improve clinical outcomes for some anticancer drugs. In order to enable precise determination of plasma concentrations of these drugs, advanced analytical methods have to be developed. One of the most common techniques utilized for this purpose is liquid chromatography coupled with a mass spectrometer. As a greener alternative, capillary electrophoresis with a mass spectrometer can be employed. Often for plasma drug analysis additional concentration and cleanup of a sample are required in which case various on-line or off-line concentrations like dispersive liquid- liquid microextraction (DLLME) or solidphase extraction (SPE) can be implemented. Finally, these methods applied to breast cancer patients' plasma samples can result in a determination of an individual's own pharmacokinetic parameters with subsequent optimal drug dosage, better clinical outcomes and less side effects. This research was funded by Croatian Science Foundation, grant numbers HRZZ-UIP-2019- 04-8461 and DOK-2021-02-4595.

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.

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.

Introduction: Palbociclib and ribociclib are newly registered anticancer agents often used in combination with anastrozole, letrozole or fulvestrant for the treatment of breast cancer (1). Novel anticancer medicines are potential candidates for therapeutic drug monitoring in plasma in order to avoid drug-related toxicities and enhance therapeutic outcomes (2). Therefore, it is desirable to employ a simultaneous high performance liquid chromatographic (HPLC) method for the selective determination of these analytes in a complex biological sample. Materials and Methods: Several chromatographic columns were tested, the influence of mobile phase composition and pH was investigated, gradient elution steps, flow rate and column temperature were optimised. Detection was carried out using a diode-array detector (DAD), a fluorescence detector (FLD), and a mass spectrometer (MS). Results: The optimal conditions were as follows: biphenyl column 4.6x150 mm, 2.6 μm, gradient elution with acetonitrile and water, both containing 0.1 % formic acid, as mobile phase, 25 °C column temperature and 0.5 mL/min flow. All peaks eluted within 12 min and adequate separation was accomplished, as presented in the table: Name Retention Time USP Resolution DAD absorption maximum (nm) Main ion ESI+ (m/z) Limit of detection (ng/mL) Ribociclib 4.163 270 435.45 52.2 Palbociclib 4.911 15.0 366 448.41 39.98 Anastrozole 6.904 37.9 210 294.27 19.8 Letrozole 7.114 3.8 240 217.17 16.6 Fulvestrant 8.748 28.2 210 607.67 1.98 Conclusions: A selective and sensitive new HPLC- DAD-FLD-MS method was developed for the simultaneous determination of five anticancer drugs in human plasma.

HRZZ projekt naziva „Nova bioanalitička rješenja za personalizaciju terapije raka dojke“ u svom fokusu ima razvoj bioanalitičkih metoda za terapijsko praćenje novih lijekova koji se koriste u terapiji raka dojke. Radi se o inhibitorima o ciklinu D ovisnih kinaza (CDK) 4 i 6, palbociklibu, ribociklibu i abemaciklibu, te o antiestrogenim lijekovima koji se s njima koriste u sinergističkim terapijskim kombinacijama – anastrozolu, letrozolu i fulvestrantu. U terapiji citostaticima, lijekovima uske terapijske širine i visokog rizika nuspojava, ali i opasnosti napretka bolesti u slučaju poddoziranja, terapijsko praćenje u plazmi pacijenta može uvelike poboljšati ishode liječenja. Stoga je cilj našeg istraživanja razviti osjetljive, selektivne i pouzdane analitičke metode sposobne detektirati niske koncentracije analita u složenom biološkom matriksu. U tu svrhu koriste se sofisticirane tehnike tekućinske kromatografije (ultra)visoke djelotvornosti (engl. (ultra)high performance liquid chromatography, (U)HPLC) i kapilarne elektroforeze (engl. capillary electrophoresis, CE). Za detekciju analita koriste se detektor niza dioda (engl. diode array detector), detektor fluorescencije (engl. fluorescence detector, FLD) i spektrometar masa (engl. mass spectrometer, MS). Tijekom prve godine provedbe projekta optimizirano je nekoliko HPLC-DAD-FLD metoda za istovremenu analizu svih 6 analita u plazmi korištenjem različitih kromatografskih kolona i mobilnih faza. Kroz suradnje s Plivom i Institutom za antropologiju optimizirani su parametri detekcije single quadrupole (SQD) i quadrupole-time-of- flight (QTOF) MS detektora, kako bi se mogli analizirati realni uzorci vrlo niskih koncentracija analita. Također je posebna pažnja posvećena pripremi uzoraka te je optimizirana metoda disperzivne tekućinske mikroekstrakcije. Ispitivani su parametri poput odabira prikladnog ekstrakcijskog i disperznog sredstva, optimalnog pH, dodatka soli i soniciranja kako bi se, uz dobro pročišćavanje uzoraka, postigli i što bolji ekstrakcijski prinosi. Pored kromatografskih metoda, proučeno je i kapilarno-elektroforetsko ponašanje analita. Optimizirana je metoda za simultanu analizu inhibitora CDK i inhibitora aromataze uz dodatak negativno nabijenih ciklodekstrina u pozadinski pufer, čime je omogućeno selektivno razdvajanje i nabijenih i nenabijenih analita. U sklopu istraživanja izrađeno je i 5 studentskih radova. Rad je financiran sredstvima projekta UIP-2019-04- 8461 Hrvatske zaklade za znanost.

Abemaciclib, palbociclib and ribociclib are novel anticancer agents, inhibitors of the cyclin dependent kinase (CDK) 4 and 6. They are used in combination with aromatase inhibitors anastrozole and letrozole in breast cancer therapy. Since these therapeutic combinations are relatively new and prone to CYP-mediated interactions, as well as showing a risk of toxicity in higher doses, it is advisable to employ therapeutic drug monitoring (TDM) for therapy individualisation [1]. A viable alternative to conventional techniques used in TDM is capillary electrophoresis, as it provides advantageous efficacy, high throughput, cost-effectiveness, automation, low sample and solvent consumption, and simplified sample preparation [2]. All measurements were performed on a bare-fused silica capillary, diameter 50 μm, total length 40 cm and effective length 8.3 cm, as injection was carried out at the outlet end. The analytes were detected using a diode-array detector with total run times of 10 min. Acidic conditions, in which all analytes are either neutral or protonated, proved superior to alkaline in the preliminary studies. pH was varied in a range of 2.5 to 5, using 20 - 150 mM phosphate, acetate and citrate buffers, with pH values around 3.1 showing best resolutions (Rs>1.5) between the positively charged CDK inhibitors. At the same time, the neutral anastrozole and letrozole required a charged buffer additive in order to achieve separation. Different additive concentrations, 15 - 50 mM sodium dodecylsulphate (SDS), 0.5 - 10 mM cetyltrimethylammonium bromide (CTAB) and 2 - 30 mM sulfobutylether-β-cyclodextrin (SBE-β-CD), were tested alone or in combination with organic modifiers (5 - 15 % v/v isopropanol, methanol or acetonitrile). Other conditions such as sample diluent (diluted background electrolyte, organic solvents or water), voltage (5 - 30 kV) and temperature (17.5 - 35 °C) were also optimized to achieve shorter run times and better peak shapes. The proposed method provides a basis for the quantification of these compounds in biological samples using capillary electrophoresis.

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.

Palbociclib, ribociclib and abemaciclib are novel anticancer agents used in combination with anastrozole, letrozole or fulvestrant in breast cancer treatment. They significantly improve therapeutic outcomes, but still exhibit an array of side-effects and a potential for inter- individual variabilities, so therapeutic drug monitoring (TDM) could prove beneficial [1]. A suitable bioanalytical method therefore needs to be developed, encompassing both the sample preparation procedure and the analytical system parameters. Optimal sample preparation ensures maximal analyte extraction recoveries, while minimizing matrix effect and prolonging the instrument lifetime [2]. Dispersive liquid-liquid microextraction is a technique in which a mixture of an organic extractant and disperser solvents is quickly injected into an aqueous solution, forming an emulsion that facilitates fast and efficient extraction. In biological sample preparation, protein precipitation (PPT) usually precedes the extraction. Since acetonitrile is commonly used as both a PPT agent and a disperser solvent, the supernatant after PPT can immediately be mixed with an extractant solvent and injected into an aqueous solution [3]. In this work, various conditions affecting analytes’ extraction recoveries were investigated: type and volume of the extractant and disperser solvent, aqueous phase pH, addition of salt, and ultrasonication. In optimal conditions, for 50 µL of plasma sample, 200 µL of acetonitrile is added for PPT. 200 µL of the supernatant is withdrawn, mixed with 400 µL of chloroform and injected into 100 µL of 5 % w/V aqueous sodium citrate solution. The organic layer is evaporated to dryness, reconstituted in 40 µL 65 % V/V methanol and analysed by liquid chromatography. The proposed method is fast, simple, with low sample and solvent consumption, and offers very high extraction yields (87.2 to 97.8 %) with significantly less matrix interferences compared to PPT alone.