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Background: Reliable quantification of multiple steroid classes in biological fluids within a single method remains an analytical challenge despite many previously published methods. Crosstalk of positional isomers, overlap of stereoisomer fragmentation patterns, differing proton affinities, in-source fragmentation, varying stability of protonated ions in the gas phase across steroid classes, and non-existence of steroid-free matrix are the main challenges limiting the number of simultaneously profiled steroids., Results: In this study, we focused on the development of a derivatization-free, achiral, high-throughput, and cost-effective UHPLC-MS/MS approach that allows simultaneous profiling of a spectrum of 38 steroids covering progestogens, androgens, corticosteroids, and estrogens, while properly addressing the hurdles of steroid analysis. Within a 20-min method, 16 stereoisomers and 15 positional isomers were fully resolved within a single run while separated from 7 additional non-interfering steroids and matrix interferences in rodent plasma. Protein precipitation (PP) and supported liquid extraction (SLE) methods using only 40 μL of sample were developed to achieve the lowest possible limits of quantification. Nevertheless, 5α-dihydroprogesterone and 3α,5α-THDOC could be only qualitatively assessed when using PP. In contrast, DHEA-S could not be quantified or identified when using SLE. A novel surrogate matrix-background subtraction approach, using rat plasma after the animal's adrenalectomy, has been implemented into the optimized PP-UHPLC-MS/MS workflow, successfully validated according to the unified ICH/EMA M10 guidelines, and compared to the traditional quantification strategies. Moreover, the validity of the newly adopted approach has been verified by the targeted profiling of multiple biologically active endogenous steroids in more than 500 samples of mouse plasma in total., Significance: Underestimation of hurdles associated with steroid analysis often compromises the accurate steroid quantification. Our comprehensive, fully validated UHPLC-MS/MS method targeting a wide spectrum of endogenous steroids, mitigating steroid crosstalk and using a minimal sample volume together with a novel surrogate matrix-background subtraction approach significantly advances steroid analysis for research and clinical applications covering multiple biological scopes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The approaches to matrix effects determination and reduction in ultra-high performance supercritical fluid chromatography with mass spectrometry detection have been evaluated in this study using different sample preparation methods and investigation of different calibration models. Five sample preparation methods, including protein precipitation, liquid-liquid extraction, supported liquid extraction, and solid phase extraction based on both "bind and elute" and "interferent removal" modes, were optimized with an emphasis on the matrix effects and recovery of 8 forms of vitamin E, including α-, β-, γ-, and δ-tocopherols and tocotrienols, from plasma. The matrix effect evaluation included the use and comparison of external and internal calibration using three models, i.e., least square with no transformation and no weighting (1/x 0 ), with 1/x 2 weighting, and with logarithmic transformation. The calibration model with logarithmic transformation provided the lowest %-errors and the best fits. Moreover, the type of the calibration model significantly affected not only the fit of the data but also the matrix effects when evaluating them based on the comparison of calibration curve slopes. Indeed, based on the used calibration model, the matrix effects calculated from calibration slopes ranged from +92% to - 72% for α-tocopherol and from -77% to +19% in the case of δ-tocotrienol. Thus, it was crucial to calculate the matrix effect by Matuszewski's post-extraction approach at six concentration levels. Indeed, a strong concentration dependence was observed for all optimized sample preparation methods, even if the stable isotopically labelled internal standards (SIL-IS) were used for compensation. The significant differences between individual concentration levels and compounds were observed, even when the tested calibration range covered only one order of magnitude. In methods with wider calibration ranges, the inappropriate use of calibration slope comparison instead of the post-extraction addition approach could result in false negative results of matrix effects. In the selected example of vitamin E, solid-phase extraction was the least affected by matrix effects when used in interferent removal mode, but supported liquid extraction resulted in the highest recoveries. We showed that the calibration model, the use of a SIL-IS, and the analyte concentration level played a crucial role in the matrix effects. Moreover, the matrix effects can significantly differ for compounds with similar physicochemical properties and close retention times. Thus, in all bioanalytical applications, where different analytes are typically determined in one analytical run, it is necessary to carefully select the data processing in addition to the method for the sample preparation, SIL-IS, and chromatography., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Flavonoids are considered as health-protecting food constituents. The testing of their biological effects is however hampered by their low oral absorption and complex metabolism. In order to investigate the direct effect(s) of unmetabolized flavonoid, a preparation in a biologically friendly solvent for intravenous administration is needed. Isorhamnetin, a natural flavonoid and a human metabolite of the most frequently tested flavonoid quercetin, has very low water solubility (<3.5 μg/mL). The aim of this study was to improve its solubility to enable intravenous administration and to test its pharmacokinetics in an animal model. By using polyvinylpyrrolidone (PVP10) and benzalkonium chloride, we were able to improve the solubility approximately 600 times to 2.1 mg/mL. This solution was then administered intravenously at a dose of 0.5 mg/kg of isorhamnetin to rats and its pharmacokinetics was analyzed. The pharmacokinetics of isorhamnetin corresponded to two compartmental model with a rapid initial distribution phase (t 1/2α : 5.7 ± 4.3 min) and a slower elimination phase (t 1/2β : 61 ± 47.5 min). Two sulfate metabolites were also identified. PVP10 and benzalkonium did not modify the properties of isorhamnetin (iron chelation and reduction, and cell penetration) substantially. In conclusion, the novel preparation reported in this study is suitable for future testing of isorhamnetin effects under in vivo conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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-D 2 /D 3 . 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 D 3 esters, vitamin D 3 and D 2 , 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 C 18 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 D 3 , 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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 CO 2 -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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 13 C-labeled linoleic acid and studied their molecular behavior in a skin lipid model. Solid-state 2 H 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 Δ 9 -tetrahydrocannabinolic acid, and isomers of m/z 315: cannabichromene, Δ 9 -tetrahydrocannabinol, Δ 8 -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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

One undescribed indole alkaloid together with twenty-two known compounds have been isolated from aerial parts of Vinca minor L. (Apocynaceae). The chemical structures of the isolated alkaloids were determined by a combination of MS, HRMS, 1D, and 2D NMR techniques, and by comparison with literature data. The NMR data of several alkaloids have been revised, corrected, and missing data have been supplemented. Alkaloids isolated in sufficient quantity were screened for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7) and butyrylcholinesterase (BuChE; E.C. 3.1.1.8) inhibitory activity. Selected compounds were also evaluated for prolyl oligopeptidase (POP; E.C. 3.4.21.26), and glycogen synthase 3β-kinase (GSK-3β; E.C. 2.7.11.26) inhibition potential. Significant hBuChE inhibition activity has been shown by (-)-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1H-indole with an IC 50 value of 0.65 ± 0.16 μM. This compound was further studied by enzyme kinetics, along with in silico techniques, to reveal the mode of inhibition. This compound is also predicted to cross the blood-brain barrier (BBB) through passive diffusion., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Three dimensions of chromatographic separation, using split-flow two-dimensional liquid chromatography (SF-2D-LC) with two parallel second dimensions, LC × 2LC, combined with quadruple parallel mass spectrometry (LC3MS4) is demonstrated for analysis of NIST SRM 1849a adult/infant formula. The first dimension, 1 D, was a conventional non-aqueous reversed-phase (NARP) HPLC separation using two C18 columns in series, followed by detection using an ultraviolet (UV) detector, a fluorescence detector (FLD), with flow then split to a corona charged aerosol detector (CAD), and then dual parallel mass spectrometry (MS), conducted in atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) modes. The first second dimension, 2 D(1), UHPLC was conducted on a 50.0 mm C30 column using a NARP-UHPLC parallel gradient for separation of short-chain triacylglycerols (TAGs) from long-chain TAGs, with detection by UV and ESI-MS. The second dimension, 2 D(2), UHPLC was conducted using a 100.0 mm C30 column with a NARP-UHPLC parallel gradient for improved separation of TAG isomers, with detection by UV, an evaporative light scattering detector, and high-resolution, accurate-mass (HRAM) ESI-MS. Transferred eluent dilution was used to refocus peaks and keep them sharp during elution in both 2 Ds. The separation space in the 2 D(2) was optimized using multi-cycle (aka, "constructive wraparound") elution, which employed flow rate programming. In the 1 D, calibration lines for quantification of fat-soluble vitamins were constructed. A lipidomics approach to TAG identification and quantification by HRAM-ESI-MS was applied to the 2 D(2). These experiments can be represented: LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4, or three-dimensional liquid chromatography with quadruple parallel mass spectrometry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

In the second part of this study, a systematic comparison was made between two ion fragmentation acquisition modes, namely data-independent acquisition (DIA) and DIA with ion mobility spectrometry (IMS) technology. These two approaches were applied to the analysis of 192 doping agents in urine. Group I included 102 compounds such as stimulants, diuretics, narcotics, and β2-agonists, while Group II contained 90 compounds included steroids, glucocorticoids, and hormone and metabolic modulators. Important method parameters were examined and compared, including the fragmentation, sensitivity, and assignment capability with the minimum occurrence of false positive hits. The results differed between Group I and II in number of detected fragments when exploring the MS/MS spectra. In Group I only 13%, while in the Group II 64% of the substances had a higher number of fragments in DIA-IMS mode vs. DIA. In terms of sensitivity, the performance of the two modes with and without activated IMS dimension was identical for about 50% of the doping agents. The sensitivity was higher without IMS, i.e. in simple DIA mode, for 20-40% of remaining doping agents. Despite this sensitivity reduction with IMS, 82% of compounds from both Groups met the minimum required performance level (MRPL) criteria of the World Anti-Doping Agency (WADA) when the DIA-IMS mode was applied. Automated data processing is important in routine doping analysis. Therefore, processing methods were optimized and evaluated for the prevalence of false peak assignments by analysing the target substances at different concentrations in urine samples. Overall, a significantly higher number of misidentified compounds was observed in Group II, with an almost 2-fold higher number of misidentifications in DIA compared to DIA-IMS. This result highlights the benefit of the IMS dimension to reduce the rate of false positive in screening analysis. The optimized UHPLC-IM-HRMS method was finally applied to the analysis of urine samples from administration studies including nine doping agents from both Groups. However, to limit the number of interferences from the biological matrix, an emphasis is needed on the adequate settings of the data processing method., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Long-term stability of retention times of a wide range of analytes has been evaluated using eight different stationary phases. These were from a single manufacturer to minimize the differences in silanol activity caused by the manufacturing process. The tested stationary phases included bridge ethylene hybrid, 2-ethylpyridine bridge ethylene hybrid with direct modification of silica particles, bidentate crosslinked charged surface hybrid fluorophenyl, bidentate crosslinked high strength silica C18, and propanediol linked phases including diol (pure propanediol linker), and three phases based on diol further modified with 2-picolylamine, diethylamine, and 1-aminoanthracene group. Retention times were monitored at the first injection, after three, nine, twelve months, and after the column regeneration via washing with pure water. The analyses were carried out using three different mobile phases, including methanol, methanol with 10 mmol/L ammonium formate, and methanol with 0.1% ammonium hydroxide. No overall decreasing or increasing trends were observed after evaluating individual contributing parameters such as analyte, stationary phase, and organic modifier. Our results suggest that the silyl-ether formation is not the only factor contributing to changes in the stationary phase pore surface. Indeed, the adsorption of mobile phase additives is probably another significant factor. That was also confirmed by the regeneration procedure using water, which is likely to reverse the silyl-ether formation to achieve the original retention. However, the retention times returned to the original values for all analytes only on three columns. Retention times on other columns remained shifted within ± 15 % RSD depending on the analyte properties and the nature of organic modifier. The retention time variations observed for each analyte group, i.e., acids, bases, and neutrals, were interpreted for each stationary phase. We concluded that the sterically protected surfaces exhibited significantly smaller changes in the retention times. Although the regeneration procedure effect depended on the column type, the results suggested beneficial effect of water. However, as the adsorption of additives on the column surface is an additional factor leading to retention time variations, the recommendation of using only one additive and/or organic modifier in each column will clearly improve the long-term repeatability of the retention times., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

In this series of two papers, 192 doping agents belonging to the classes of stimulants, narcotics, cannabinoids, diuretics, β2-agonists, β-blockers, anabolic agents, and hormone and metabolic modulators were investigated, with the aim to assess the benefits and limitations of ion mobility spectrometry (IMS) in combination with ultra-high performance liquid chromatography (UHPLC) and high resolution mass spectrometry (HRMS) in anti-doping analysis. In this first part, a generic UHPLC-IM-HRMS method was successfully developed to analyze these 192 doping agents in standard solutions and urine samples, and an exhaustive database including retention times, TW CCS N2 values, and m/z ratios was constructed. Urine samples were analyzed using either a simple "dilute and shoot" procedure or a supported liquid-liquid extraction (SLE) procedure, depending on the physicochemical properties of the compounds and sensitivity criteria established by the World Anti-Doping Agency (WADA) as the minimum required performance levels (MRPL). Then, the precision of the generic UHPLC-IM-HRMS method was assessed as intraday, interday as well as interweek variation of UHPLC retention times and TW CCS N2 values, for which RSD the values were always lower than 2% in urine samples. The possibility to filter MS data using IMS dimension was also investigated, and in average, the application of IMS filtration provided low energy MS spectra with 86% less interfering peaks in both standard and urine samples. Therefore, the filtered MS spectra allowed for an easier interpretation and a lower risk of false positive result interpretations. Finally, IMS also offers additional selectivity to the UHPLC-HRMS enabling to separate isobaric and isomeric substances. Among the selected set of 192 doping agents, there were 30 pairs of isobaric or isomeric compounds, and only two pairs could not be resolved under the developed conditions. This illustrates the potential of adding ion mobility to UHPLC-HRMS in anti-doping analyses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Twenty known Amaryllidaceae alkaloids of various structural types, and one undescribed alkaloid of narcikachnine-type, named narcieliine (3), have been isolated from fresh bulbs of Zephyranthes citrina. The chemical structures of the isolated alkaloids were elucidated by a combination of MS, HRMS, 1D and 2D NMR, and CD spectroscopic techniques, and by comparison with literature data. The absolute configuration of narcieliine (3) has also been determined. Compounds isolated in a sufficient quantity were evaluated for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7), butyrylcholinesterase (BuChE; E.C. 3.1.1.8), and prolyl oligopeptidase (POP; E.C. 3.4.21.26) inhibition activities. Significant human AChE/BuChE (hAChE/hBuChE) inhibitory activity was demonstrated by the newly described alkaloid narcieliine (3), with IC 50 values of 18.7 ± 2.3 µM and 1.34 ± 0.31 µM, respectively. This compound is also predicted to cross the blood-brain barrier (BBB) through passive diffusion. The in vitro data were further supported by in silico studies of 3 in the active site of hAChE/hBuChE., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A novel series of aromatic esters (1a-1m) related to the Amaryllidaceae alkaloid (AA) haemanthamine were designed, synthesized and tested in vitro with particular emphasis on the treatment of neurodegenerative diseases. Some of the synthesized compounds revealed promising acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory profile. Significant human AChE (hAChE) inhibition was demonstrated by 11-O-(3-nitrobenzoyl)haemanthamine (1j) with IC 50 value of 4.0 ± 0.3 µM. The strongest human BuChE (hBuChE) inhibition generated 1-O-(2-methoxybenzoyl)haemanthamine (1g) with IC 50 value 3.3 ± 0.4 µM. Moreover, 11-O-(2-chlorbenzoyl)haemanthamine (1m) was able to inhibit both enzymes in dose-dependent manner. The mode of hAChE and hBuChE inhibition was minutely inspected using enzyme kinetic analysis in tandem with in silico experiments, the latter elucidating crucial interaction in 1j-, 1m-hAChE and 1g-, 1m-hBuChE complexes. The blood-brain barrier (BBB) permeability was investigated applying the parallel artificial membrane permeation assay (PAMPA) to predict the CNS availability of the compounds., Competing Interests: Declaration of Competing Interest The authors declare no competing interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In our previous study we proposed a screening approach using ultra-high performance supercritical fluid chromatography for the determination of 10 pharmaceutical quality control mixtures. Most of resulting methods offered baseline separation of all analytes. However, some of these methods had to be further optimized to ensure their successful validation and applicability to impurity control in drug substance and drug products. Several challenges occurred during the optimization including: (i) the necessity of the resolution of active pharmaceutical ingredient and following impurity equaling at least 3, which was especially difficult to achieve for mixtures of structurally close compounds, (ii) unrepeatable elution of compounds eluting close to the dead volume or at the end of the gradient elution, and (iii) shifts in retention times due to the column aging and effects of additive. The most frequent optimization adjustments involved changes in gradient program. Other adjustments such as the substitution of Viridis UPC 2 HSS C18 SB column with a slightly different Acquity UPLC HSS C18 SB column, the addition of acetonitrile in the modifier, and the column coupling also led to beneficial changes in selectivity. Subsequently, validation of all 10 methods was carried out to prove the applicability of ultra-high performance supercritical fluid chromatography methods for the impurity control in pharmaceuticals. Parameters recommended by ICH guidelines Q2 and Q3 including specificity, linearity, range, lower and upper limit of quantification, limit of detection, accuracy, and precision were examined. In addition, intermediate precision and the accuracy profiles were determined for selected methods. Overall, only two impurities did not meet the validation criteria due to low resolution and low sensitivity, respectively. Only identification threshold and not reporting threshold was met for this impurity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Twenty-one known Amaryllidaceae alkaloids of various structural types and one undescribed alkaloid, named narcimatuline, have been isolated from fresh bulbs of Narcissus pseudonarcissus L. cv. Dutch Master. The chemical structures were elucidated by combination of MS, HRMS, 1D and 2D NMR spectroscopic techniques, and by comparison with literature data. Narcimatuline amalgamates two basic scaffolds of Amaryllidaceae alkaloids in its core, namely galanthamine and galanthindole. All isolated compounds were evaluated for their in vitro acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), prolyl oligopeptidase (POP), and glycogen synthase kinase-3β (GSK-3β) inhibitory activities. The most interesting biological profile was demonstrated by newly isolated alkaloid narcimatuline., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, 63 compounds measured as 10 individual mixtures containing in each case an active pharmaceutical ingredient and its relevant impurities, and additional set of 7 basic beta blockers were analyzed using ultra-high performance supercritical fluid chromatography with UV and mass spectrometry detection. The separations were accomplished using 8 different stationary phases (diol, diethylamine, 2-picolylamine, 1-aminoanthracene, BEH 2-ethylpyridine, BEH, CSH pentafluorophenyl, and HSS C18 SB), 6 modifiers (methanol, ethanol, isopropanol, methanol/acetonitrile, methanol/ethanol, ethanol/acetonitrile) and 5 additives in methanol (0.1% formic acid, 10 mmol/L ammonium formate, 10 mmol/L ammonium acetate, 0.4% ammonium hydroxide, and 2% water). The resulting chromatograms were evaluated using 6 selected parameters: number of eluted peaks, number of separated peaks, resolution between active pharmaceutical ingredient and following impurity, peak symmetry, peak width at 50% of the peak height, and selectivity. Volatile additives such as ammonium formate, ammonium acetate, and especially ammonium hydroxide provided overall generic approach with very good chromatographic performance. Combined modifier methanol/acetonitrile (1:1) was important for separations of some critical pairs in case of neutral compounds. Diol stationary phase proved its suitability for wide range of applications when the separations of all mixtures were satisfactory on this column, except for vardenafil and beta blockers mixture. HSS C18 SB stationary phase offered unique complementary selectivity especially for analysis of structurally close compounds and/or isomers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fast, selective, and sensitive ultra-high performance liquid chromatography method with tandem mass spectrometry detection for the determination of quercetin and its metabolites with various physico-chemical properties such as molecular weight, lipophilicity, and acid-base properties has been developed. These compounds included small hydrophilic phenolic acids and more lipophilic metabolites with preserved flavonoid structure in small amount of rat plasma. The developed method enables selective separation of phenolic acids and a pair of isomers tamarixetin and isorhamnetin with satisfactory peak shapes and a high sensitivity using mass spectrometry detection. In addition, two sample preparation procedures including protein precipitation and microextraction in packed sorbent (MEPS) were optimized. The sample acidification included in protein precipitation as well as optimizing of MEPS sorbents and elution solvents improved isolation of quercetin and related compounds from rat plasma. Finally, both methods developed for sample preparation were fully validated to demonstrate sufficient accuracy and precision and acceptable matrix effects. Both sample preparation approaches combined with mass spectrometry-based quantification allowed the simultaneous determination of quercetin and its metabolites from a small amount of biological samples of only 50 μL. Due to the fast and non-selective parallel sample preparation, the protein precipitation was eventually applied to plasma samples derived from pharmacokinetic studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The new microextraction technique named parallel artificial liquid membrane extraction (PALME) was introduced as an alternative approach to liquid-liquid extraction of charged analytes from aqueous samples. The concept is based on extraction of analytes across a supported liquid membrane sustained in the pores of a thin polymeric membrane, a well-known extraction principle also used in hollow fiber liquid-phase microextraction (HF-LPME). However, the new PALME technique offers a more user-friendly setup in which the supported liquid membrane is incorporated in a 96 well plate system. Thus, high-throughput is achievable, in addition to the green chemistry offered by using PALME. The consumption of organic solvent is minimized to 3-5μL per sample. With a sample volume of 250μL and acceptor solution volume of 50μL, a maximal enrichment factor of five is achievable. Based on these parameters, a new method for extraction of polar basic drugs was developed in the present work. The basic drugs hydralazine, ephedrine, metaraminol, salbutamol, and cimetidine were used as model analytes, and were extracted from alkalized human plasma into an aqueous solution via the supported liquid membrane. The extraction was promoted by a carrier dissolved in the membrane, creating a temporary ion-pair complex between the hydrophilic drug and the carrier. As the model analytes were extracted directly into an aqueous solution, there was no need for evaporation of the extract before injection into LC-MS. Hence, the sample preparation is performed in one step. With optimized conditions, the extraction recoveries were in the range 50-89% from human plasma after 45min extraction. The data from the method evaluation were satisfactory and in line with current guidelines, and revealed an extraction method with substantial potential for high throughput bioanalysis of polar basic drugs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

High throughput general chiral screening method using supercritical fluid chromatography was developed. This method takes an advantage of very fast gradient screening (3 min + 1 min isocratic hold) and generic enantioselectivity of the combined additive formed by 0.1% trifluoroacetic (TFA) acid and 0.1% diethylamine (DEA). The TFA/DEA combined additive was systematically added to organic modifiers methanol and isopropanol. Among five tested polysaccharide-based chiral stationary phases, amylose tris(3,5-dimethylphenylcarbamate) and cellulose tris(3,5-dimethylphenylcarbamate) provided the best enantioseparation success rate. Therefore, the proposed initial first-line screening includes four experiments using these two stationary phases and the above mentioned two combinations: CO 2 /methanol and CO 2 /isopropanol + the combined additive. If these stationary phases fail in the screening step, cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(3,5-dichlorophenylcarbamate) can be proposed for the screening in the second line. For further optimization in case of insufficient resolution obtained in the screening phase fine tuning of temperature, BPR pressure and gradient slope was tested with unsuccessful results. An improvement of enantioselectivity was obtained only when gradient elution was replaced by isocratic elution with substantially lower amount of organic modifier, when changing the concentration of the additive or when using combined organic modifier, such as methanol/acetonitrile (1:1). Finally, to enable the MS compatibility, also volatile additives including ammonium formate and ammonium acetate were tested. The results were more encouraging than expected. Volatile buffers thus make an interesting option in chiral SFC screening methods, however, at the cost of somewhat lower enantioselectivity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The goal of this study was to develop an effective supercritical fluid chromatography method using single quadrupole MS for analysis of all isomeric forms of vitamin E. Finally, two fast and effective methods, the high resolution one and the high speed one, for the determination of 8 vitamin E isomers in human serum were developed. Rapid high-throughput liquid-liquid extraction was selected as a sample preparation step. Sample pretreatment of 100 μL human serum was consisted of protein precipitation with 200 μL ethanol and liquid-liquid extraction by 400 μL hexane/dichloromethane (80/20, v/v). The separation was performed on BEH 2-EP (3.0 × 100 mm, 1.7 μm) stationary phase, using isocratic elution with carbon dioxide and 10 mM ammonium formate in methanol in the ratio 98:2 for high resolution method with run time 4.5 min and in the ratio 95:5 for high speed method, where the run time was 2.5 min. The method development included optimization of key parameters: the choice of the suitable stationary phase and the composition of mobile phase, where an influence of various modifiers, their ratio and additives were tested, and optimization of fine tunning parameters including BPR pressure, flow-rate and column temperature. Quantification of all isomeric forms was performed using SIM (single ion monitoring) experiments in ESI positive ion mode. Both high speed and high resolution chromatographic methods were validated in terms of precision, accuracy, range, linearity, LOD, LOQ and matrix effects using the same LLE procedure. The high resolution method provided more sensitive results (LOD: 0.017-0.083 μg mL(-1)) and better linearity (r(2) > 0.9930) than the high speed one (LOD: 0.083-0.25 μg mL(-1), r(2) > 0.9877) at the cost of double time of analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This work describes the development of two methods involving supported liquid extraction (SLE) sample treatment followed by ultra-high performance liquid chromatography or ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS and UHPSFC-MS/MS) for the screening of 43 anabolic agents in human urine. After evaluating different stationary phases, a polar-embedded C18 and a diol columns were selected for UHPLC-MS/MS and UHPSFC-MS/MS, respectively. Sample preparation, mobile phases and MS conditions were also finely tuned to achieve highest selectivity, chromatographic resolution and sensitivity. Then, the performance of these two methods was compared to the reference routine procedure for steroid analyses in anti-doping laboratories, which combines liquid-liquid extraction (LLE) followed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). For this purpose, urine samples spiked with the compounds of interest at five different concentrations were analyzed using the three analytical platforms. The retention and selectivity of the three techniques were very different, ensuring a good complementarity. However, the two new methods displayed numerous advantages. The overall procedure was much faster thanks to high throughput SLE sample treatment using 48-well plates and faster chromatographic analysis. Moreover, the highest sensitivity was attained using UHPLC-MS/MS with 98% of the doping agents detected at the lowest concentration level (0.1ng/mL), against 76% for UHPSFC-MS/MS and only 14% for GC-MS/MS. Finally, the weakest matrix effects were obtained with UHPSFC-MS/MS with 76% of the analytes displaying relative matrix effect between -20 and 20%, while the GC-MS/MS reference method displayed very strong matrix effects (over 100%) for all of the anabolic agents., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study shows the possibility offered by modern ultra-high performance supercritical fluid chromatography combined with tandem mass spectrometry in doping control analysis. A high throughput screening method was developed for 100 substances belonging to the challenging classes of anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids, which should be detected at low concentrations in urine. To selectively extract these doping agents from urine, a supported liquid extraction procedure was implemented in a 48-well plate format. At the tested concentration levels ranging from 0.5 to 5 ng/mL, the recoveries were better than 70% for 48-68% of the compounds and higher than 50% for 83-87% of the tested substances. Due to the numerous interferences related to isomers of steroids and ions produced by the loss of water in the electrospray source, the choice of SFC separation conditions was very challenging. After careful optimization, a Diol stationary phase was employed. The total analysis time for the screening assay was only 8 min, and interferences as well as susceptibility to matrix effect (ME) were minimized. With the developed method, about 70% of the compounds had relative ME within the range ±20%, at a concentration of 1 and 5 ng/mL. Finally, limits of detection achieved with the above-described strategy including 5-fold preconcentration were below 0.1 ng/mL for the majority of the tested compounds. Therefore, LODs were systematically better than the minimum required performance levels established by the World anti-doping agency, except for very few metabolites., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of the work was to synthesize a molecularly imprinted material for the selective solid-phase extraction (SPE) of β-N-methylamino-L-alanine (L-2-amino-3-methylpropionic acid; BMAA) from cyanobacterial extracts. BMAA and its structural analogs that can be used as template are small, polar and hydrophilic molecules. These molecules are poorly soluble in organic solvents that are commonly used for the synthesis of acrylic-based polymers. Therefore, a sol gel approach was chosen to carry out the synthesis and the resulting sorbents were evaluated with different extraction procedures in order to determine their ability to selectively retain BMAA. The presence of imprinted cavities in the sorbent was demonstrated by comparing elution profiles obtained by using molecularly imprinted silica (MIS) and non-imprinted silica (NIS) as a control. The molecularly imprinted solid-phase extraction (MISPE) procedure was first developed in a pure medium (acetonitrile) and further optimized for the treatment of cyanobacterial samples. It was characterized by high elution recoveries (89% and 77% respectively in pure and in real media).The repeatability of the extraction procedure in pure medium, in real medium and the reproducibility of MIS synthesis all expressed as RSD values of extraction recovery of BMAA were equal to 3%, 12% and 5%, respectively. A MIS capacity of 0.34 µmol/g was measured. The matrix effects, which affected the quantification of BMAA when employing a mixed mode sorbent, were completely removed by adding a clean-up step of the mixed-mode sorbent extract on the MIS., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A novel simple, fast and efficient ultra-high performance supercritical fluid chromatography (UHPSFC) method was developed and validated for the separation and quantitative determination of eleven illegal dyes in chili-containing spices. The method involved a simple ultrasound-assisted liquid extraction of illegal compounds with tetrahydrofuran. The separation was performed using a supercritical fluid chromatography system and CSH Fluoro-Phenyl stationary phase at 70°C. The mobile phase was carbon dioxide and the mixture of methanol:acetonitrile (1:1, v/v) with 2.5% formic acid as an additive at the flow rate 2.0 mL min(-1). The UV-vis detection was accomplished at 500 nm for seven compounds and at 420 nm for Sudan Orange G, Butter Yellow, Fast Garnet GBC and Methyl Red due to their maximum of absorbance. All eleven compounds were separated in less than 5 min. The method was successfully validated and applied using three commercial samples of chili-containing spices - Chili sauce (Indonesia), Feferony sauce (Slovakia) and Mojo sauce (Spain). The linearity range of proposed method was 0.50-9.09 mg kg(-1) (r ≥ 0.995). The detection limits were determined as signal to noise ratio of 3 and were ranged from 0.15 mg kg(-1) to 0.60 mg kg(-1) (1.80 mg kg(-1) for Fast Garnet) for standard solution and from 0.25 mg kg(-1) to 1.00 mg kg(-1) (2.50 mg kg(-1) for Fast Garnet, 1.50 mg kg(-1) for Sudan Red 7B) for chili-containing samples. The recovery values were in the range of 73.5-107.2% and relative standard deviation ranging from 0.1% to 8.2% for within-day precision and from 0.5% to 8.8% for between-day precision. The method showed potential for being used to monitor forbidden dyes in food constituents. The developed UHPSFC method was compared to the UHPLC-UV method. The orthogonality of Sudan dyes separation by these two methods was demonstrated. Benefits and drawbacks were discussed showing the reliability of both methods for monitoring of studied illegal dyes in real food constituents., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The conditions for the analysis of selected doping substances by UHPSFC-MS/MS were optimized to ensure suitable peak shapes and maximized MS responses. A representative mixture of 31 acidic and basic doping agents was analyzed, in both ESI+ and ESI- modes. The best compromise for all compounds in terms of MS sensitivity and chromatographic performance was obtained when adding 2% water and 10mM ammonium formate in the CO2/MeOH mobile phase. Beside mobile phase, the nature of the make-up solvent added for interfacing UHPSFC with MS was also evaluated. Ethanol was found to be the best candidate as it was able to compensate for the negative effect of 2% water addition in ESI- mode and provided a suitable MS response for all doping agents. Sensitivity of the optimized UHPSFC-MS/MS method was finally assessed and compared to the results obtained in conventional UHPLC-MS/MS. Sensitivity was improved by 5-100-fold in UHPSFC-MS/MS vs. UHPLC-MS/MS for 56% of compounds, while only one compound (bumetanide) offered a significantly higher MS response (4-fold) under UHPLC-MS/MS conditions. In the second paper of this series, the optimal conditions for UHPSFC-MS/MS analysis will be employed to screen >100 doping agents in urine matrix and results will be compared to those obtained by conventional UHPLC-MS/MS., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7 min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC-MS/MS and for 32% in UHPSFC-MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this study, direct analysis in real time-mass spectrometry (DART-MS) was assessed for the analysis of various pharmaceutical formulations with intention to summarize possible applications for the routine pharmaceutical development. As DART is an ambient ionization technique, it allows direct analysis of pharmaceutical samples in solid or liquid form without complex sample preparation, which is often the most time-consuming part of the analytical method. This makes the technique suitable for many application fields, including pharmaceutical drug development. DART mass spectra of more than twenty selected tablets and other common pharmaceutical formulations, i.e. injection solutions, ointments and suppositories developed in the pharmaceutical industry during several recent years are presented. Moreover, as thin-layer chromatography (TLC) is still very popular for the monitoring of the reactions in the synthetic chemistry, several substances were analyzed directly from the TLC plates to demonstrate the simplicity of the technique. Pure substance solutions were spotted onto a TLC plate and then analyzed with DART without separation. This was the first DART-MS study of pharmaceutical dosage forms using DART-Orbitrap combination. The duration of sample analysis by the DART-MS technique lasted several seconds, allowing enough time to collect sufficient number of data points for compound identification. The experimental setup provided excellent mass accuracy and high resolution of the mass spectra which allowed unambiguous identification of the compounds of interest. Finally, DART mass spectrometry was also used for the monitoring of the selected impurity distribution in the atorvastatin tablets. These measurements demonstrated DART to be robust ionization technique, which provided easy-to-interpret mass spectra for the broad range of compounds. DART has high-throughput potential for various types of pharmaceutical analyses and therefore eliminates the time for sample cleanup and chromatographic separation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This tutorial provides an overview of the possibilities, limitations and analytical conditions of modern analytical supercritical fluid chromatography (SFC) using columns packed with sub-2 μm particles. In particular, it gives a detailed overview of commercially available modern SFC instrumentation and the detectors that can be employed (UV, MS, ELSD, FID, etc.). Some advice on the choice of the stationary phase dimensions and chemistries, the nature of the mobile phase (choice of organic modifier and additives) and its flow rate as well as the backpressure and temperature are also provided. Finally, several groups of potentially problematic compounds, including lipophilic compounds, hydrophilic substances and basic drugs, are discussed in detail. All these families of analytes can be resolved with SFC but require specific analytical conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Estrogen steroids, represented by estradiol and its related substances, include both structurally very close and simultaneously different analogs. Their separation still remains an analytical challenge. Subcritical fluid chromatography (SbFC) on sub-2-micron particles was found to be an appropriate tool to obtain fast and efficient separation of nine target analytes. Among the four tested stationary phases charged hybrid modified with PFP (pentafluorophenyl) moiety was found to be the most convenient providing the fastest separation within 1.6 min using quick gradient elution with carbon dioxide and methanol as an organic modifier. However, complete separation was obtained also on other tested phases including bare hybrid stationary phase, hybrid stationary phase modified with 2-EP (2-ethylpyridine) and also C18, which is less typical in SbFC. The baseline separation on the latter columns was achieved by means of a temperature increase, a change in organic modifier type and gradient time increase respectively. Quantitative performance was evaluated at optimized conditions and method validation was accomplished. Excellent repeatability of both retention times (RSD<0.15%) and peak areas (RSD<1%) was observed. The method was linear in the range of 1.0-1000.0 μg/ml for all steroids with the lowest calibration point being an LOQ, except for Δ-derivatives, that provided better sensitivity and thus LOQ of 0.5 μg/ml. The sensitivity was sufficient for the analysis of real samples although it was still five times lower compared to UHPLC-UV experiments., (Copyright © 2014 Elsevier B.V. All rights reserved.)

For the first time, polymer monolithic capillary columns have been employed at ultra-high-pressure liquid chromatographic conditions (UHPLC) to investigate their potential for high-speed separations of peptides and intact proteins. In comparison to conventional flow rates and gradient conditions, a substantial decrease in analysis time (>factor 4) can be achieved when operating monolithic columns such as ultra-high-pressure conditions while scaling the gradient volume. The effects of flow rate and column length on the peak capacity and the gradient performance limits were assessed for the separation of peptide and protein mixtures applying the maximum system pressure (80MPa) and a fixed gradient steepness. The potential for ultra-fast gradient separations of large biomolecules was further demonstrated for very steep gradients (gradient times≪1min). A tryptic digest of cytochrome c was separated using a gradient time of only 1min. Finally, the run-to-run repeatability and column robustness were assessed at ultra-high pressure conditions (after>800 runs) with consecutive steep 1min separations of peptides, yielding RSD values below 0.12% in retention time., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The development of bioanalytical methods has become more and more challenging over the past years due to very demanding requirements in terms of method reliability, sensitivity, speed of analysis and sample throughput. LC-MS/MS has established itself as a method of choice for routine analysis of biological materials. A development of such method consists of several steps including sample preparation and clean-up step, efficient chromatographic separation, sensitive and selective detection of analytes in complex matrices, a choice of convenient data processing and calibration approach and finally method validation. Each of these steps has its own constraints and challenges, which are discussed in detail in this review. Novel and modern approaches in sample preparation, chromatography and detection are especially emphasized. Attention is paid to proper calibration approach and matrix effects that can seriously affect method accuracy and precision., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this paper two fast and highly sensitive ultra-high performance liquid chromatography (UHPLC) methods for the determination of tetracycline antibiotics (oxytetracycline, tetracycline, doxycycline, demeclocycline, chlortetracycline, minocycline and degradation product epitetracycline) in surface waters have been developed using fluorescence (FL) and mass spectrometry (MS) detection. ACQUITY UPLC BEH C8 and ACQUITY CSH C18 columns were employed for FL and MS detection, respectively, both packed with 1.7μm particles. Mixed-mode separation mechanism of CSH (charged surface technology) sorbent was found particularly useful in analysis of TCs, which possess problematic amphoteric structures. The FL methodology was based on chelation of tetracyclines with calcium ions to perform on-column derivatisation. The developed methods were compared in the terms of validation parameters including linearity, sensitivity, precision and accuracy. The linearity range for FL detection was within 7ngmL(-1) to 50μgmL(-1) with method limit of detection (MLOD) as low as 0.2ngmL(-1) for most of the analytes. MS detection showed even higher sensitivity reaching MLOD of 0.003ngmL(-1), which is the highest sensitivity reported so far in analysis of TCs. Matrix matched calibration curves in the range of 0.01-50ngmL(-1) were used for quantification to compensate for matrix effects with the correlation coefficients demonstrating good linearity (0.9940-0.9999). The extraction of the antibiotics from surface waters was performed using solid phase extraction with Oasis HLB cartridges. Accuracy was expressed as recovery with values ranging from 96.52% to 127.30% and from 91.66% to 123.70% for FL and MS detection, respectively., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Entecavir is a deoxyguanosine nucleotide antiviral agent with the activity against hepatitis B virus (HBV). The agent possesses a polar structure, which is predetermined for hydrophilic interaction chromatography (HILIC). Novel, fast and sensitive HILIC-UHPLC method developed in this study included separation from matrix component on BEH Amide stationary phase by isocratic elution using binary mobile phase composed of acetonitrile/5mM ammonium acetate pH 4.0 (75:25) at flow-rate 0.3 ml/min. Analysis under RP-UHPLC conditions was also possible on BEH C18 stationary phase with mostly aqueous binary mobile phase composed of (4:96) acetonitrile/0.01% formic acid. The comparison of sensitivity of the two UHPLC-MS/MS methods both using selected reaction monitoring (SRM) for quantitation revealed only slightly higher sensitivity for HILIC determination, however much better method linearity, repeatability and accuracy. HILIC separation mode provided also more convenient conditions for straightforward coupling with solid phase extraction (SPE). Entecavir was extracted on Oasis HLB cartridge (1 ml, 30 mg) and eluted by 75% acetonitrile in water, which is actually the HILIC mobile phase used in this study. Therefore the evaporation/reconstitution step was omitted, which substantially accelerated the sample preparation step. The method was validated using stable isotopically labeled internal standard entecavir-C(2)(13) N(15), which is the most appropriate internal standard. Validation results demonstrated good method accuracy (with < 5% error, and 26% at LOQ), recovery (87-114%), precision (<4% RSD), selectivity and sensitivity (LOQ=100 pg/ml). The matrix effects determined by both post-column infusion method as well as post-extraction addition method were negligible (<15%)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this study, the selectivity, retention properties, peak shape and loading capacity for bases were practically evaluated using two UHPLC mixed-mode hybrid CSH stationary phases modified by C18 or Phenyl group. The data were compared with the data obtained on other UHPLC hybrid stationary phases (BEH C18, BEH C8, BEH Phenyl and BEH Shield RP18) at both basic and acidic conditions using conventional HPLC buffers (50mM ammonium formate/acetate) as well as low ionic-strength additives such as, e.g. 0.1-0.01% formic/acetic acid and 1mM solution of ammonium formate/acetate, which are widely used in LC-MS applications. Ten pharmaceutically important compounds encompassing acids, bases and neutral were included into the study. Due to properties of CSH sorbent (which possess positively charged surface besides RP group), much improved peak shapes and weaker retention was obtained for bases even at very low concentration of acidic additives. Such conditions are ideally suited for LC-MS analysis of bases, where typical RP chromatographic separation (retention and good selectivity at basic pH) and LS-MS conditions (efficient ionization at acidic pH) are not in agreement. On the other hand, acids were more strongly retained and for some compounds the peak shape was influenced negatively due to ion-exchange mechanism. Further, the behavior of acidic, basic and neutral solutes is discussed using various additives at both basic and acidic pH for all above stated columns. The robustness of retention times after pH change from basic to acidic was also evaluated. The new CSH stationary phases represent an interesting selectivity tool preferably for separation of basic compounds., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A simple and reproducible method for the determination of pravastatin and pravastatin lactone in rat plasma and urine by means of ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) using deuterium labeled internal standards for quantification is reported. Separation of analytes was performed on BEH C(18) analytical column (50 mm × 2.1mm, 1.7 μm), using gradient elution by mobile phase consisting of acetonitrile and 1mM ammonium acetate at pH 4.0. Run time was 2 min. Quantification of analytes was performed using the SRM (selected reaction monitoring) experiment in ESI negative ion mode for pravastatin and in ESI positive ion mode for pravastatin lactone. Sample treatment consisted of a protein precipitation by ACN and microextraction by packed sorbent (MEPS) for rat plasma. Simple MEPS procedure was sufficient for rat urine. MEPS was implemented using the C8 sorbent inserted into a microvolume syringe, eVol hand-held automated analytical syringe and a small volume of sample (50 μl). The analytes were eluted by 100 μl of the mixture of acetonitrile: 0.01 M ammonium acetate pH 4.5 (90:10, v:v). The method was validated and demonstrated good linearity in range 5-500 nmol/l (r(2)>0.9990) for plasma and urine samples. Method recovery was ranged within 97-109% for plasma samples and 92-101% for the urine samples. Intra-day precision expressed as the % of RSD was lower than 8% for the plasma samples and lower than 7% for the urine samples. The method was validated with sensitivity reaching LOD 1.5 nmol/l and LOQ 5 nmol/l in plasma and urine samples. The method was applied for the measurement of pharmacokinetic plots of pravastatin and pravastatin lactone in rat plasma and urine samples., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In theory, with identical stationary phase chemistry, the transfer of an HPLC method to UHPLC conditions is straightforward and necessitates the calculation of new conditions based on column and instrument geometries. Occasionally, undesirable changes in selectivity, retention or efficiency have been reported and have been attributed to a frictional heating phenomenon that is due to the elevated generated pressure drop. In the present study, the frictional heating in a UHPLC system was evaluated experimentally under gradient elution conditions (acetonitrile/buffer at pH 3 and 9) with generated pressure drops in the range of 100-1000 bar on both 1.0mm and 2.1mm I.D. columns using a mixture of 10 representative basic, acidic and neutral pharmaceutical compounds. Under adiabatic conditions (i.e., still-air oven), the longitudinal temperature gradient was estimated at +4 °C, +8 °C and +16 °C at 300, 600 and 1000 bar, respectively, on a 2.1mm I.D. column using an empirical measurement procedure. With the 1.0mm I.D. column, these values were reduced to +3 °C, +6 °C and +12 °C, respectively. Finally, various approaches to eliminate or at least to reduce the effect of frictional heating are briefly discussed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Residues of steroid hormones have become a cause for concern because they can affect the biological activity of non-target organisms. Steroid hormones are a potential risk for wildlife and humans through the consumption of contaminated food or water. Their determination requires extraction and clean-up steps, prior to detection, to reach low concentration levels. In recent years, a great effort has been made to develop new analytical methodologies, such as microextraction techniques, that reduce environmental pollution. Researchers have modified old methods to incorporate procedures that use less-hazardous chemicals or that use smaller amounts of them. They are able to do direct analysis using miniaturised equipment and reduced amounts of solvents and wastes. These accomplishments are the main objectives of green analytical chemistry. In this overview, we focus on microextraction techniques for the determination of steroid hormones in biological (e.g., human urine, human serum, fish, shrimp and prawn tissue and milk) and environmental (e.g., wastewaters, surface waters, tap waters, river waters, sewage sludges, marine sediments and river sediments) samples. We comment on the most recent applications in sorptive-microextraction modes, such as solid phase microextraction (SPME) with molecularly imprinted polymers (MIPs), in-tube solid-phase microextraction (IT-SPME), stir-bar sorptive extraction (SBSE) and microextraction in packed sorbent (MEPS). We also describe liquid-phase microextraction (LPME) approaches reported in the literature that are applied to the determination of steroid hormones., (Copyright © 2011 Elsevier B.V. All rights reserved.)

UHPLC-MS/MS method using BEH C18 analytical column was developed for the separation and quantitation of 12 phenolic compounds of Chamomile (Matricaria recutita L.). The separation was accomplished using gradient elution with mobile phase consisting of methanol and formic acid 0.1%. ESI in both positive and negative ion mode was optimized with the aim to reach high sensitivity and selectivity for quantitation using SRM experiment. ESI in negative ion mode was found to be more convenient for quantitative analysis of all phenolics except of chlorogenic acid and kaempherol, which demonstrated better results of linearity, accuracy and precision in ESI positive ion mode. The results of method validation confirmed, that developed UHPLC-MS/MS method was convenient and reliable for the determination of phenolic compounds in Chamomile extracts with linearity >0.9982, accuracy within 76.7-126.7% and precision within 2.2-12.7% at three spiked concentration levels. Method sensitivity expressed as LOQ was typically 5-20 nmol/l. Extracts of Chamomile flowers and Chamomile tea were subjected to UHPLC-MS/MS analysis. The most abundant phenolic compounds in both Chamomile flowers and Chamomile tea extracts were chlorogenic acid, umbelliferone, apigenin and apigenin-7-glucoside. In Chamomile tea extracts there was greater abundance of flavonoid glycosides such as rutin or quercitrin, while the aglycone apigenin and its glycoside were present in lower amount., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Twenty nine phenolic compounds comprising nine phenolic acids, sixteen flavonoids (including eight tea catechins, glycosides and aglycones), four coumarins plus caffeine were analysed within 20 min using ultra high performance liquid chromatography (UHPLC) with PDA detection. UHPLC system was equipped with C18 analytical column (100 mm x 2.1mm, 1.7 microm), utilising 0.1% formic acid and methanol mobile phase in the gradient elution mode. The developed method was tested for the system suitability: resolution, asymmetry factor, peak capacity, retention time repeatability and peak area repeatability. The method was fully validated in the terms of linearity (r(2)>0.9990 for all 30 compounds), range (typically 1-100 mg L(-1)), LOD, LOQ, inter/intra-day precision (<3% and <9% respectively) and inter/intra-day accuracy (typically 100+/-10%). Subsequently the method was applied to the identification (spectral information and peak purity calculations were profited) and quantification of phenolic compounds and caffeine present in tea infusions and extracts., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Any bio-analytical method includes several steps, all of them being important in order to achieve reliable results. The first step is taking aliquots of samples for the analysis, followed by the extraction procedure and sample clean-up, chromatographic analysis and detection. Chromatographic methods, particularly liquid chromatography, are the methods of choice in bio-analytical laboratories. Current trends in fast liquid chromatographic separations involve monolith technology, fused core columns, high temperature liquid chromatography and ultra-high performance liquid chromatography (UHPLC). UHPLC has recently become a wide-spread analytical technique in many laboratories which focus on fast and sensitive bio-analytical assays. The key advantages of UHPLC are the increased speed of analysis, higher separation efficiency and resolution, higher sensitivity and much lower solvent consumption as compared to other analytical approaches. This is all enabled by specially designed instruments and sub-2-microne particle packed analytical columns. There is a great contrast between ultra-fast chromatographic analysis and conventional sample preparation, which remains highly labor-intensive and time-consuming. Conventional sample preparation techniques including SPE, solid phase extraction; LLE, liquid-liquid extraction; PP, protein precipitation and many modern approaches (RAM, restricted access material; MIP, molecularly imprinted polymers; SPME, solid phase microextraction; LLME, liquid-liquid microextraction; MEPS, microextraction by packed sorbent and many others) have also been featured as fundamental and critical step of bio-analytical methods.

The widespread occurrence of antibiotics as contaminants in the aquatic environment has increased attention in the last years. The concern over the release of antibiotics into the environment is related primarily to the potential for the development of antimicrobial resistance among microorganisms. This article presents an overview of analytical methodologies for the determination of quinolone (Qs) and fluoroquinolone (FQs), macrolide (MLs), tetracycline (TCs), sulfonamide (SAs) antibiotics and trimethoprim (TMP) in different environmental waters. The analysis of these antibiotics has usually been carried out by high-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) and to a lesser extent by ultraviolet (UV) or fluorescence detection (FD). A very important step before LC analysis is sample preparation and extraction leading to elimination of interferences and prevention of matrix effect and preconcentration of target analytes.

Simvastatin and atorvastatin belong to the group of hypolipidemic drugs, more exactly to the second generation of inhibitors of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. They induce a significant reduction in total cholesterol, low-density lipoprotein cholesterol and plasma triglycerides, therefore they are widely used in the treatment of hypercholesterolemia even of its severe form-familiar hypercholesterolemia. Simvastatin and atorvastatin as the most widely used statins in clinical treatment and their hydroxy-acid/lactone forms were determined by means of UPLC in connection with triple quadrupole mass spectrometer. Deuterium labeled reference standard compounds were used as internal standards for the quantitation. Separation was performed on Acquity BEH C18 (100 mm x 2.1 mm, 1.7 microm) using gradient elution by mobile phase containing acetonitrile and ammonium acetate pH 4.0, which is convenient in order to prevent interconversion of analytes. ESI in positive mode was used for the ionization of all compounds. Two SRM (selected reaction monitoring) transitions were carefully optimized for each analyte in order to get high sensitivity and selectivity. SPE on Discovery DSC-18 was used as a sample preparation step. Intra-day precision was generally within 10% RSD, while inter-day precision within 15% RSD. Method accuracy expressed as recovery ranged from 75 to 100%. The method was validated with the sensitivity reaching LOQ 0.08-5.46 nmol/l and LOD 0.01-1.80 nmol/l in biological samples. Atorvastatin, simvastatin, its metabolites and hydroxy-acid/lactone forms were monitored in human serum and in lipoprotein fractions (LDL, HDL and VLDL) at patients with end stage renal diseases.