Advances and Challenges in the Analysis of Boswellic Acids by Separation Methods.

Boswellia resin is an exudate from the cut bark of Boswellia trees. The main constituents of pharmacological interest are boswellic acids (pentacyclic triterpenoids), namely α-boswellic acid, ß-boswellic acid, 3-O-acetyl-α-boswellic acid, 3-O-acetyl-ß-boswellic acid, 11-keto-ß-boswellic acid, and 3-O-acetyl-11-keto-ß-boswellic acid. Nowadays, dietary supplements with Boswellia serrata extract are used in the treatment of inflammatory joint diseases. Additionally, the constituents of Boswellia resin have shown potential for the treatment of other chronic inflammatory diseases and various types of cancer. Separation methods including ultra/high-performance liquid chromatography, gas chromatography, thin layer chromatography, supercritical fluid chromatography, and capillary electrochromatography coupled with UV or MS detection have been used for the determination of boswellic acids in various matrices (mostly plant material and biological samples). This review aims to provide a comprehensive summary of these separation methods, offering a critical discussion of their strengths and limitations in the analysis of boswellic acids. The knowledge of various separation methods plays a pivotal role in the quality control of herbal dietary supplements and the monitoring of the metabolism and pharmacokinetics of their constituents. The approaches based on metabolomics and network pharmacology represent new ways of fingerprinting secondary metabolites in Boswellia resin increasing the comprehensiveness of the output of these methods resulting in safer dietary supplements.

Analysis of vitamin D and its metabolites in biological samples - Part I: Optimization and comparison of UHPSFC-MS/MS and UHPLC-MS/MS methods.

Fat-soluble vitamin D is an essential bioactive compound important for human health. Insufficient vitamin D levels can result not only in bone disease but also in other disorders, such as cancer, metabolic disorders, and diseases related to poor immune function. The current methods commonly used for vitamin D analysis are often applied to determine the levels of the most abundant metabolite in plasma, i.e., 25-OH-D2/D3. These methods do not consider the presence of other hydroxylated and esterified metabolites, including isomers and epimers, which are typically found in low concentrations. In this study, we developed a fast and selective ultra-high performance supercritical fluid chromatography (UHPSFC) method using a 150 mm long 1-amino anthracene (1-AA) column and a mobile phase consisting of carbon dioxide and methanol/isopropanol (1/1, v/v) mixed with 8 % water. After thorough optimization of column temperature and back pressure, the separation of four vitamin D3 esters, vitamin D3 and D2, and eight mono- and di-hydroxylated metabolites, including three groups of isomers, was achieved in 10 min. Two ion sources, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization optimized within this study, were compared in tandem mass spectrometry (MS/MS) detection. No significant sensitivity differences were observed. Subsequently, the same 1-AA column chemistry was examined in ultra-high performance liquid chromatography (UHPLC) as the stationary phase that could hypothetically bring different selectivity in the separation of vitamin D and its metabolites. However, this hypothesis was rejected, and C18 was used as a stationary phase in the final optimized UHPLC-MS/MS method. Despite detailed optimization, the final 15 min UHPLC method was not able to separate di-hydroxylated isomers of vitamin D3, while it enabled better resolution of esterified forms compared to UHPSFC. Optimized methods provided similar repeatability of retention times and peak areas, with RSD < 2 % and 10 %, respectively. The lowest limits of quantification were in the range of 1.2 - 4.9 ng/mL for UHPSFC-APCI-MS/MS, while for UHPLC-APCI-MS/MS, they were typically in the range of 2.6 - 9.6 ng/mL. Based on the obtained results, the UHPSFC-APCI-MS/MS method was the most promising approach for fast, selective, and sensitive analysis that could be applied in the analysis of biological samples with emphasis on the separation of both hydroxylated and esterified metabolites, including isomeric forms.

Analysis of vitamin D and its metabolites in biological samples - Part II: Optimization of a sample preparation method for liver tissue.

Extraction of vitamin D, including its hydroxylated and esterified metabolites, from soft tissues such as the liver is challenging due to the lipophilic character of matrix and analytes that are expected in very low concentration levels. In this study, we aimed at the optimization of two-step extraction using solid-liquid extraction as the first step, followed by solid-phase extraction. Various solvents, including ethanol, acetonitrile, methanol, acetone, heptane, and heptane with isopropanol, were investigated to isolate vitamin D compounds from liver tissue in the first step. Acetone was finally selected as the most suitable solvent for the solid-liquid extraction, with the highest recovery in the range of 67 - 98% for polar hydroxylated forms and 3 - 28% for lipophilic vitamin D and esters. Two solid phase extraction (SPE) based on the (i) "bind and elute strategy" and (ii) "removal strategy" using hydrophilic-lipophilic balanced SPE sorbent were optimized as a proceeding step for acetone extracts to increase the method selectivity. Finally, two optimized methods, combining solid-liquid extraction and individual SPE strategy, were examined in terms of sensitivity, recovery, matrix effect, accuracy, and precision. The limits of quantification were in the range of 1 - 10 ng/mL and 3 - 20 ng/mL analyzed by ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography hyphenated a with tandem mass spectrometer, respectively. The absolute recovery determined for the "bind and elute strategy" protocol was in the range of 3 - 24 %. Nevertheless, this method was free of matrix effects, which were determined to be in the 73 - 120 % range. On the contrary, the "removal strategy" approach provided higher recovery values for all compounds (47 - 123 %), but the results for nonpolar vitamin D and esters were strongly affected by signal suppression (matrix effects 3 - 51 %). Both methods fulfilled the criteria for accuracy and precision requested by the European Medicine Agency Guideline on Bioanalysis. "Removal strategy" SPE with decreased manual intervention and lower solvent consumption was finally applied to mouse liver tissue to determine vitamin D and its hydroxylated and esterified metabolites for the first time. The results, i.e., vitamin D esters detected in liver tissue, supported the notion that esters of vitamin D can be stored in lipophilic tissues to release vitamin D.

Microbiota modulates the steroid response to acute immune stress in male mice.

Microbiota plays a role in shaping the HPA-axis response to psychological stressors. To examine the role of microbiota in response to acute immune stressor, we stimulated the adaptive immune system by anti-CD3 antibody injection and investigated the expression of adrenal steroidogenic enzymes and profiling of plasma corticosteroids and their metabolites in specific pathogen-free (SPF) and germ-free (GF) mice. Using UHPLC-MS/MS, we showed that 4 hours after immune challenge the plasma levels of pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone (CORT), 11-dehydroCORT and their 3α/ß-, 5α-, and 20α-reduced metabolites were increased in SPF mice, but in their GF counterparts, only CORT was increased. Neither immune stress nor microbiota changed the mRNA and protein levels of enzymes of adrenal steroidogenesis. In contrast, immune stress resulted in downregulated expression of steroidogenic genes (Star, Cyp11a1, Hsd3b1, Hsd3b6) and upregulated expression of genes of the 3α-hydroxysteroid oxidoreductase pathway (Akr1c21, Dhrs9) in the testes of SPF mice. In the liver, immune stress downregulated the expression of genes encoding enzymes with 3ß-hydroxysteroid dehydrogenase (HSD) (Hsd3b2, Hsd3b3, Hsd3b4, Hsd3b5), 3α-HSD (Akr1c14), 20α-HSD (Akr1c6, Hsd17b1, Hsd17b2) and 5α-reductase (Srd5a1) activities, except for Dhrs9, which was upregulated. In the colon, microbiota downregulated Cyp11a1 and modulated the response of Hsd11b1 and Hsd11b2 expression to immune stress. These data underline the role of microbiota in shaping the response to immune stressor. Microbiota modulates the stress-induced increase in C21 steroids, including those that are neuroactive that could play a role in alteration of HPA axis response to stress in GF animals.

Ultrahigh-Performance Supercritical Fluid Chromatography-Multimodal Ionization-Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts.

Complex analysis of plant extracts usually requires a combination of several analytical approaches. Therefore, in this study, we developed a holistic two-injection approach for plant extract analysis, which is carried out within one instrument without the need for any manual intervention during the analysis. Ultrahigh-performance supercritical fluid chromatography (UHPSFC) was employed for the analysis of 17 volatile terpenes on a porous graphitic carbon column within 7.5 min, followed by analysis on short diol column where flavonoids, phenolic acids, and terpenoic acids were analyzed within 15.5 min. A multimodal ionization source combining electrospray and atmospheric pressure chemical ionization (ESCi) was selected for mass spectrometry detection as a simultaneous ionization of both lipophilic and polar compounds was required. The quantitative aspects of the final UHPSFC-ESI/ESCi-MS/MS two-injection approach were determined, and it was applied to the analysis of Eucalyptus sp. extracts prepared by supercritical fluid extraction. Current methods reported in the literature typically require a labor-intensive combination of liquid and gas chromatography for the complex analysis of plant extracts. We present for the first time a new UHPSFC approach requiring only a single instrument that provides an alternative approach to the analysis of complex plant extracts.

Novel approach to supercritical fluid chromatography-mass spectrometry analysis of metal ions using EDTA complexation.

BACKGROUND: Reliable methods enabling detection of metal ions, and especially heavy metals, in different matrices are necessary in various fields such as ecology, pharmaceuticals and toxicology. As some of the currently used methods suffer from spectral and chemical interferences, this study investigates the applicability of SFC-MS/MS for the determination of metal ions. RESULTS: Effective novel approaches for metal ion analysis using CO2-based mobile phase were developed using three ligands forming metal complexes. As metal-EDTA complexes are prepared by simple addition of EDTA to the solution containing metal ions, this approach to metal ion analysis does not require laborious synthesis and isolation of solid metal-complexes. Besides, two other approaches using diethyldithiocarbamate and acetylacetonate as ligands were compared. Metal complexes of Cu, Co, Cr, Fe, Al, Mn, and Zn with all 3 ligands were synthesized and their identity was confirmed by high-resolution mass spectrometry (HRMS). The suitability of the three developed UHPSFC-MS/MS methods was examined using the determination of calibration range and repeatability of injections. Moreover, the universality of the developed UHPSFC-MS/MS method for the determination of metal-EDTA complexes was proved by analyzing Ni, Bi and Pb as additional metal ions. SIGNIFICANCE AND NOVELTY: This study demonstrates the extended range of applicability for SFC based separations. For the first time, the possibility to analyze metal complexes with EDTA using a fast and reliable ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) method is reported. The three developed UHPSFC-MS/MS methods are able to separate DDC, acac, and EDTA complexes of various metals very efficiently (total cycle times of 5, 2, and 3 min, respectively). They offer a fast and green alternative to chromatographic methods commonly used for metal ion analysis.

Columns in analytical-scale supercritical fluid chromatography: From traditional to unconventional chemistries.

Within this review, we thoroughly explored supercritical fluid chromatography (SFC) columns used across > 3000 papers published from the first study carried out under SFC conditions in 1962 to the end of 2022. We focused on the open tubular capillary, packed capillary, and packed columns, their chemistries, dimensions, and trends in used stationary phases with correlation to their specific interactions, advantages, drawbacks, used instrumentation, and application field. Since the 1990s, packed columns with liquid chromatography and SFC-dedicated stationary phases for chiral and achiral separation are predominantly used. These stationary phases are based on silica support modified with a wide range of chemical moieties. Moreover, numerous unconventional stationary phases were evaluated, including porous graphitic carbon, titania, zirconia, alumina, liquid crystals, and ionic liquids. The applications of unconventional stationary phases are described in detail as they bring essential findings required for further development of the supercritical fluid chromatography technique.

Design of semisynthetic derivatives of the Amaryllidaceae alkaloid ambelline and exploration of their in vitro cytotoxic activities.

Ambelline, an alkaloid from the Amaryllidaceae family with a crinane-type skeleton, has not yet demonstrated any outstanding biological activity. However, its analogues prepared by derivatization of the C-11 hydroxyl group show different interesting effects. Continuing our earlier work, twelve novel aromatic esters were developed (10, 14, 16, 17, 22-25, 30-33) and studied, together with previously synthesized derivatives (2-9, 11-13, 15, 18-21, 26-29) in terms of their cytotoxic activity. The cytotoxic potential was determined on a panel of nine human cancer cell lines and one noncancerous cell line to characterize their biological activity spectrum. To describe and foresee the structure-activity relationship for further research, substances synthesized and described in our previous work were also included in this cytotoxicity study. The most significant activity was associated with analogues having methyl (10), methoxy (14-17), or ethoxy (18) substitution on the phenyl condensed to ambelline. However, the 4-chloro-3-nitrobenzoyl derivative (32) showed the most promising IC50 values, ranging from 0.6 ± 0.1 µM to 9.9 ± 0.2 µM. In vitro cytotoxicity studies indicated the most potent antiproliferative activity of 32 in a dose-dependent and time-dependent manner. Besides, 32 was found to be effective in decreasing viability and triggering apoptosis of MOLT-4 T-lymphoblastic leukemia cells.

What is the role of current mass spectrometry in pharmaceutical analysis?

The role of mass spectrometry (MS) has become more important in most application domains in recent years. Pharmaceutical analysis is specific due to its stringent regulation procedures, the need for good laboratory/manufacturing practices, and a large number of routine quality control analyses to be carried out. The role of MS is, therefore, very different throughout the whole drug development cycle. While it dominates within the drug discovery and development phase, in routine quality control, the role of MS is minor and indispensable only for selected applications. Moreover, its role is very different in the case of analysis of small molecule pharmaceuticals and biopharmaceuticals. Our review explains the role of current MS in the analysis of both small-molecule chemical drugs and biopharmaceuticals. Important features of MS-based technologies being implemented, method requirements, and related challenges are discussed. The differences in analytical procedures for small molecule pharmaceuticals and biopharmaceuticals are pointed out. While a single method or a small set of methods is usually sufficient for quality control in the case of small molecule pharmaceuticals and MS is often not indispensable, a large panel of methods including extensive use of MS must be used for quality control of biopharmaceuticals. Finally, expected development and future trends are outlined.

The intriguing molecular dynamics of Cer[EOS] in rigid skin barrier lipid layers requires improvement of the model.

Omega-O-acyl ceramides such as 32-linoleoyloxydotriacontanoyl sphingosine (Cer[EOS]) are essential components of the lipid skin barrier, which protects our body from excessive water loss and the penetration of unwanted substances. These ceramides drive the lipid assembly to epidermal-specific long periodicity phase (LPP), structurally much different than conventional lipid bilayers. Here, we synthesized Cer[EOS] with selectively deuterated segments of the ultralong N-acyl chain or deuterated or 13C-labeled linoleic acid and studied their molecular behavior in a skin lipid model. Solid-state 2H NMR data revealed surprising molecular dynamics for the ultralong N-acyl chain of Cer[EOS] with increased isotropic motion toward the isotropic ester-bound linoleate. The sphingosine moiety of Cer[EOS] is also highly mobile at skin temperature, in stark contrast to the other LPP components, N-lignoceroyl sphingosine acyl, lignoceric acid, and cholesterol, which are predominantly rigid. The dynamics of the linoleic chain is quantitatively described by distributions of correlation times and using dynamic detector analysis. These NMR results along with neutron diffraction data suggest an LPP structure with alternating fluid (sphingosine chain-rich), rigid (acyl chain-rich), isotropic (linoleate-rich), rigid (acyl-chain rich), and fluid layers (sphingosine chain-rich). Such an arrangement of the skin barrier lipids with rigid layers separated with two different dynamic "fillings" i) agrees well with ultrastructural data, ii) satisfies the need for simultaneous rigidity (to ensure low permeability) and fluidity (to ensure elasticity, accommodate enzymes, or antimicrobial peptides), and iii) offers a straightforward way to remodel the lamellar body lipids into the final lipid barrier.

Lab-in-syringe automated protein precipitation and salting-out homogenous liquid-liquid extraction coupled online to UHPLC-MS/MS for the determination of beta-blockers in serum.

A sample preparation method involving tandem implementation of protein precipitation and salting-out homogenous liquid-liquid extraction was developed for the determination of beta-blockers in serum. The entire procedure was automated using a computer-controlled syringe pump following the Lab-In-Syringe approach. It is based on the denaturation of serum proteins with acetonitrile followed by salt-induced phase separation upon which the proteins accumulate as a compact layer at the interphase of the solutions. The extract is then separated and diluted in-syringe before being submitted to online coupled UHPLC-MS/MS. A 1 mL glass syringe containing a small stir bar for solution mixing at up to 3000 rpm, was used to deal with sample volumes as small as 100 µL. A sample throughput of 7 h-1 was achieved by performing the chromatographic run and sample preparation procedure in parallel. Linear working ranges were obtained for all analytes between 5 and 100 ng mL-1, with LOD values ranging from 0.4 to 1.5 ng mL-1. Accuracy values in the range of 88.2-106% and high precision of <11% RSD suggest applicability for routine analysis that can be further improved using deuterated standards.

Highly selective butyrylcholinesterase inhibitors related to Amaryllidaceae alkaloids - Design, synthesis, and biological evaluation.

Butyrylcholinesterase (BChE) is one of the most frequently implicated enzymes in the advanced stage of Alzheimer's disease (AD). As part of our endeavors to develop new drug candidates for AD, we have focused on natural template structures, namely the Amaryllidaceae alkaloids carltonine A and B endowed with high BChE selectivity. Herein, we report the design, synthesis, and in vitro evaluation of 57 novel highly selective human BChE (hBChE) inhibitors. Most synthesized compounds showed hBChE inhibition potency ranging from micromolar to low nanomolar scale. Compounds that revealed BChE inhibition below 100 nM were selected for detailed biological investigation. The CNS-targeted profile of the presented compounds was confirmed theoretically by calculating the BBB score algorithm, these data were corroborated by determining the permeability in vitro using PAMPA-assay for the most active derivatives. The study highlighted compounds 87 (hBChE IC50 = 3.8 ± 0.2 nM) and 88 (hBChE IC50 = 5.7 ± 1.5 nM) as the top-ranked BChE inhibitors. Compounds revealed negligible cytotoxicity for the human neuroblastoma (SH-SY5Y) and hepatocellular carcinoma (HepG2) cell lines compared to BChE inhibitory potential. A crystallographic study was performed to inspect the binding mode of compound 87, revealing essential interactions between 87 and hBChE active site. In addition, multidimensional QSAR analyses were applied to determine the relationship between chemical structures and biological activity in a dataset of designed agents. Compound 87 is a promising lead compound with potential implications for treating the late stages of AD.

Advanced Tool for Chiral Separations of Anionic and Zwitterionic (Metalla)carboranes: Supercritical Fluid Chromatography.

The continuous expansion of research in the field of stable carboranes and their wide potential in the drug design require carrying out fundamental studies regarding their chiral separations. Although supercritical fluid chromatography (SFC) is a viable technique for fast enantioseparations, no investigation concerning boron cluster compounds has been done yet. We aimed at the development of a straightforward method enabling chiral separations of racemic mixtures of anionic cluster carboranes and metallacarboranes that represent an analytical challenge. The fast gradient screening testing nine polysaccharide-based columns was used. The key parameters affecting the selectivity were the type of chiral selector, the type of alcohol, and the base in cosolvent. Moreover, the addition of acetonitrile or water to the cosolvent was identified as an effective tool for decreasing the analysis time while preserving the resolution. After the optimization, the chiral separations of 19 out of 20 selected compounds were achieved in less than 10 min. These results demonstrate the clear advantage of SFC over chiral separations using HPLC in terms of both analysis time and structural variety of successfully separated compounds.

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial.

The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

Supercritical fluids in analysis of cannabinoids in various Cannabis products.

We developed a fast, selective, and sensitive method for the determination of various neutral and acidic phytocannabinoids with an emphasis on the separation of structurally related compounds. Optimized ultra-high performance supercritical fluid chromatography (UHPSFC) allowed the separation of 2 groups of structural isomers, including isomers of m/z 357: cannabidiolic and Δ9-tetrahydrocannabinolic acid, and isomers of m/z 315: cannabichromene, Δ9-tetrahydrocannabinol, Δ8-tetrahydrocannabinol, cannabicyclol, and cannabidiol only in mere 3.5 min followed by 1.5 min equlibration. The 2-ethylpyridine functionalized stationary phase and gradient elution using mobile phase comprising carbon dioxide and methanol: acetonitrile (25:75) + 5% water mixture were selected after the optimization. Tandem mass spectrometry (MS/MS) with electrospray ionization in positive and negative modes with methanol + 5% water as a make-up solvent provided adequate selectivity and sensitivity needed for analysis of phytocannabinoids in complex matrices. The limits of quantification were in the range 0.01-0.5 ng/mL for most of the monitored cannabinoids. The optimized UHPSFC-MS/MS method was then used for the determination of cannabinoids in various products, such as dietary supplements, nutraceuticals, and cosmetics. Solvent extraction methods were optimized for the cosmetic and nutraceutical products with the accuracy in the range 80.4-120.6% and precision 0.5-18.9%. To extract cannabinoids from the herbal infusion matrix, supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) methods were developed using environmentally friendly solvents water, ethanol, and carbon dioxide. The detailed optimization of extraction solvent composition, temperature, and pressure was carried out with the emphasis on avoiding the thermal degradation of cannabinoids. Optimized SFE and PLE methods were compared and applied to different herbal infusions to confirm declared cannabinoids content.

Winter conditions, not resource availability alone, may drive reversible seasonal skull size changes in moles.

Seasonal changes in the environment can lead to astonishing adaptations. A few small mammals with exceptionally high metabolisms have evolved a particularly extreme strategy: they shrink before winter and regrow in spring, including changes of greater than 20% in skull and brain size. Whether this process is an adaptation to seasonal climates, resource availability or both remains unclear. We show that European moles (Talpa europaea) also decrease skull size in winter. As resources for closely related Iberian moles (Talpa occidentalis) are lowest in summer, we predicted they should shift the timing of size changes. Instead, they do not change size at all. We conclude that in moles, seasonal decrease and regrowth of skull size is an adaptation to winter climate and not to a changing resource landscape alone. We not only describe this phenomenon in yet another taxon, but take an important step towards a better understanding of this enigmatic cycle.

HER-3 molecular classification, expression of PD-L1 and clinical importance in breast cancer.

Receptors of the large HER family play an important role in breast cancer, which is undergoing a gradual development in connection with biological development, both in the field of diagnostics and therapy. Dimerization of HER-2 with other HER members, such as HER-3, is the biggest driver of tumor cell growth and survival. Numerous studies show that HER-3 gene overexpression correlates with poor prognosis. However, other studies have shown HER-3 overexpression to be a positive prognostic factor. HER-3 may confer resistance to certain EGFR or HER-2 receptor therapeutics. An interesting fact, however, is that HER-3 expression can serve as a marker in immunotherapy for triple-negative breast cancer (TNBC). It is thought to be involved not only in cell survival and proliferation, but also in the regulation of PD-L1 expression. In breast cancer, PD-L1 expression is heterogeneous and is generally associated with the presence of tumor­infiltrating lymphocytes and a number of factors with poor prognosis such as young age, hormone receptor negativity, and high HER-2 expression and proliferation index. Our results showed amplification of HER-3 (CERB3) in 2 out of a sample of 20 patients with TNBC, and 13 of 20 HER-2­positive patients. PD-L1 expression was demonstrated in 3 out of 13 HER-3­positive patients and 2 out of 2 HER-3­positive TNBC patients. There was a strong correlation between positive HER-3 and PD-L1 TNBC expression (p = Keywords: breast cancer, HER family, overexpression, HER-3, HER-2, PD-L1, TNBC.

Carbon dioxide expanded liquid: an effective solvent for the extraction of quercetin from South African medicinal plants.

BACKGROUND: Quercetin is one of the most important bioflavonoids having positive effects on the biological processes and human health. Typically, it is extracted from plant matrices using conventional methods such as maceration, sonication, infusion, and Soxhlet extraction with high solvent consumption. Our study aimed to optimize the environmentally friendly carbon dioxide-based method for the extraction of quercetin from quince fruit with an emphasis on extraction yield, repeatability, and short extraction time. RESULTS: A two-step design of experiments was used for the optimization of the key parameters affecting physicochemical properties, including CO2/co-solvent ratio, co-solvent type, temperature, and pressure. Finally, gas expanded liquid combining CO2/ethanol/H2O in a ratio of 10/81/9 (v/v/v) provided the best extraction yield. Extraction temperature 66 °C and pressure 22.3 MPa were the most suitable conditions after careful optimization, although both parameters did not significantly affect the process. It was confirmed by experiments in various pressure and temperature conditions and statistical comparison of obtained data. The optimized extraction procedure at a flow rate of 3 mL/min took 30 min. The repeatability of the extraction method exhibited an RSD of 20.8%. CONCLUSIONS: The optimized procedure enabled very fast extraction in 30 min using environmentally friendly solvents and it was successfully applied to 16 different plant samples, including 14 bulbs and 2 fruits from South Africa. The quercetin content in extracts was quantified using ultra-high performance liquid chromatography (UHPLC) with tandem mass spectrometry. UHPLC hyphenated with high-resolution mass spectrometry was used to confirm chemical identity of quercetin in the analyzed samples. We quantified quercetin in 11 samples of all 16 tested plants. The quercetin was found in Agapanthus praecox from the Amaryllidaceae family and its presence in this specie was reported for the first time.

Vitamin D: sources, physiological role, biokinetics, deficiency, therapeutic use, toxicity, and overview of analytical methods for detection of vitamin D and its metabolites.

Vitamin D has a well-known role in the calcium homeostasis associated with the maintenance of healthy bones. It increases the efficiency of the intestinal absorption of dietary calcium, reduces calcium losses in urine, and mobilizes calcium stored in the skeleton. However, vitamin D receptors are present ubiquitously in the human body and indeed, vitamin D has a plethora of non-calcemic functions. In contrast to most vitamins, sufficient vitamin D can be synthesized in human skin. However, its production can be markedly decreased due to factors such as clothing, sunscreens, intentional avoidance of the direct sunlight, or the high latitude of the residence. Indeed, more than one billion people worldwide are vitamin D deficient, and the deficiency is frequently undiagnosed. The chronic deficiency is not only associated with rickets/osteomalacia/osteoporosis but it is also linked to a higher risk of hypertension, type 1 diabetes, multiple sclerosis, or cancer. Supplementation of vitamin D may be hence beneficial, but the intake of vitamin D should be under the supervision of health professionals because overdosing leads to intoxication with severe health consequences. For monitoring vitamin D, several analytical methods are employed, and their advantages and disadvantages are discussed in detail in this review.

Fast Optimization of Supercritical Fluid Chromatography-Mass Spectrometry Interfacing Using Prediction Equations.

The effect of makeup solvent composition in ultrahigh-performance supercritical fluid chromatography-triple quadrupole mass spectrometry using electrospray ionization was studied using a set of 91 compounds, 3 stationary phases, and 2 organic modifiers of the mobile phase. The 24 tested makeup solvents included pure alcohols and methanol in combination with commonly used additives such as water, formic and acetic acid, ammonia, and ammonia salts with varying molarity. The behavioral trends for different makeup solvent additives were established in the first step. Subsequently, the correlations between physicochemical properties and the MS responses were calculated using the Pearson correlation test and matrix plots. The regression analysis was performed using five descriptors: molecular weight, pKa, log P, number of hydrogen donors/acceptors, and the MS responses obtained with methanol as the makeup solvent. The resulting regression equations had a high prediction rate calculated as R2-predicted coefficient, especially when 10 mmol/L ammonium in methanol was used as an organic modifier of the mobile phase in positive mode. The trueness of these equations was tested via the comparison between experimental and predicted responses expressed as R2. Values of R2 > 0.8 were found for 88% of the proposed equations. Thus, the MS response could be measured using only one makeup solvent and the responses of other makeup solvents could be easily estimated. The suitability and applicability of determined regression equations was confirmed by the analysis of 13 blind probes, i.e., compounds not included in the original set of analytes. Moreover, the predicted and experimental responses followed the same increasing/decreasing trend enabling one to predict makeup solvent compositions leading to the highest sensitivity.