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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-, ß-, 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/x0), with 1/x2 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.
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The retention behavior in supercritical fluid chromatography and its stability over time are still unsatisfactorily explained phenomena despite many important contributions in recent years, especially focusing on linear solvation energy relationship modeling. We studied polar stationary phases with predominant -OH functionalities, i.e., silica, hybrid silica, and diol columns, and their retention behavior over time. We correlated molecular descriptors of analytes with their retention using three organic modifiers of the CO2-based mobile phase. The differences in retention behavior caused by using additives, namely, 10 mmol/L NH3 and 2% H2O in methanol, were described in correlation to analyte properties and compared with the CO2/methanol mobile phase. The structure of >100 molecules included in this study was optimized by semiempirical AM1 quantum mechanical calculations and subsequently described by 226 molecular descriptors including topological, constitutional, hybrid, electronic, and geometric descriptors. An artificial neural networks simulator with deep learning toolbox was trained on this extensive set of experimental data and subsequently used to determine key molecular descriptors affecting the retention by the highest extent. After comprehensive statistical analysis of the experimental data collected during one year of column use, the retention on different stationary phases was fundamentally described. The changes in the retention behavior during one year of column use were described and their explanation with a proposed interpretation of changes on the stationary phase surface was suggested. The effect of the regeneration procedure on the retention was also evaluated. This fundamental understanding of interactions responsible for retention in SFC can be used for the evidence-based selection of stationary phases suitable for the separation of particular analytes based on their specific physicochemical properties.
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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.
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Determination of the various forms of vitamin K, which are involved in coagulation and other physiological processes in humans, is challenging and no standardized method is yet available. Therefore, a reliable and practical method was developed to quantify vitamin K levels in serum and additionally in lipoprotein fractions to clarify its distribution. The LC-MS/MS method for the determination of vitamin K1 and the three main isoforms of vitamin K2 (MK-4, MK-7, MK-9) was combined with a gradient ultracentrifugation technique to allow the separation of lipoprotein fractions. The chromatographic separation was carried out on a Kinetex™ C18 column using a mobile phase consisting mainly of methanol. The target analytes were detected by electrospray ionization mass spectrometry. The separation of all four substances was achieved after a simple sample preparation technique based on miniaturized liquid-liquid extraction. Our method of only 8.5 min revealed the levels of the major forms of vitamin K in 59 human and 12 rat sera and confirmed our hypothesis that vitamin K is primarily (about 50 %) found in the high-density lipoprotein fraction. The median concentrations of vitamin K1, MK-4, MK-7, and MK-9 were found to be 1.19, 2.98, 0.43, and < 0.71 nmol/L in human serum and 1.74, 6.75, less than 0.2, and less than 0.5 nmol/L in rat serum, respectively.
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Espectrometría de Masas en Tándem , Vitamina K 1 , Humanos , Ratas , Animales , Vitamina K 1/química , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida/métodos , Cromatografía Líquida de Alta Presión/métodos , Vitamina K , Vitamina K 2/química , LipoproteínasRESUMEN
The extraction efficiencies of thirty types of fibers produced by meltblown, alternating current electrospinning, and meltblown-co-electrospinning technologies were tested as advanced sorbents for on-line solid-phase extraction in a high-performance liquid chromatography system have been tested and compared with a commercial C18 sorbent. The properties of each fiber, which were often depended on the production process, and their applicability were demonstrated with the extraction of the model analytes nitrophenols and chlorophenols from various matrices including river water and to purify complex matrix human serum and bovine serum albumin from macromolecular ballast. Polycaprolactone fibers outperformed other polymers and were selected for subsequent modifications including (i) incorporation of hybrid carbon nanoparticles, i.e., graphene, activated carbon, and carbon black into the polymer prior to fiber fabrication, and (ii) surface modification by dip coating with polyhydroxy modifiers including graphene oxide, tannin, dopamine, hesperidin, and heparin. These novel fibrous sorbents were comparable to commercial C18 sorbent and provided excellent analyte recoveries of 70-112% even from the protein-containing matrices.
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Contaminantes Ambientales , Nanofibras , Humanos , Nanofibras/química , Ríos/química , Agua Dulce , Extracción en Fase Sólida/métodos , Polímeros/química , AguaRESUMEN
The development of new drug delivery platforms including the use of nanotechnology has been found of great interest in recent years. Two different loading approaches of the model antimycotic drug clotrimazole into the nanofibrous polycaprolactone and polydioxanone structures including electrospinning of a drug-polymer blend and impregnation of nanofibers with drug have been tested. The final amount of clotrimazole in the nanofibrous materials was determined by HPLC analysis and Raman spectroscopy. The electrospinning of blend approach allowed the adsorption of clotrimazole in a quantity of up to 30 % using mixtures with polymer/clotrimazole ratios from 2:1 to 8:1 (w/w). Ethanolic clotrimazole solutions with concentrations from 2.5 to 3.5 mg L-1 were used for adsorbing clotrimazole in blank nanofibers for 1-3 h with final clotrimazole content ranging from 3.0 to 5.7 %. Furthermore, a comparative liberation study including comparison with commercially available creams was carried out in low pressure flow system. The results obtained confirmed well controlled release of clotrimazole from both types of nanofibers. Compared to commercial pharmaceutical formulations containing 1 % clotrimazole where first-order release kinetics was observed, nanofibrous materials provided linear controlled release (zero-order kinetics) in the tested 3 h period.
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Clotrimazol , Nanofibras , Clotrimazol/química , Liberación de Fármacos , Preparaciones de Acción Retardada , Nanofibras/química , Polímeros/químicaRESUMEN
Oxime reactivators of acetylcholinesterase (AChE) are used as causal antidotes for intended and unintended poisoning by organophosphate nerve agents and pesticides. Despite all efforts to develop new AChE reactivators, none of these drug candidates replaced conventional clinically used oximes. In addition to the therapeutic efficacy, determining the safety profile is crucial in preclinical drug evaluation. The exact mechanism of oxime toxicity and the structure-toxicity relationship are subjects of ongoing research, with oxidative stress proposed as a possible mechanism. In the present study, we investigated four promising bispyridinium oxime AChE reactivators, K048, K074, K075, and K203, and their ability to induce oxidative stress in vitro. Cultured human hepatoma cells were exposed to oximes at concentrations corresponding to their IC50 values determined by the MTT assay after 24 h. Their potency to generate reactive oxygen species, interfere with the thiol antioxidant system, and induce lipid peroxidation was evaluated at 1, 4, and 24 h of exposure. Reactivators without a double bond in the four-carbon linker, K048 and K074, showed a greater potential to induce oxidative stress compared with K075 and K203, which contain a double bond. Unlike oximes with a three-carbon-long linker, the number of aldoxime groups attached to the pyridinium moieties does not determine the oxidative stress induction for K048, K074, K075, and K203 oximes. In conclusion, our results emphasize that the structure of oximes plays a critical role in inducing oxidative stress, and this relationship does not correlate with their cytotoxicity expressed as the IC50 value. However, it is important to note that oxidative stress cannot be disregarded as a potential contributor to the side effects associated with oximes.
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Reactivadores de la Colinesterasa , Humanos , Reactivadores de la Colinesterasa/farmacología , Reactivadores de la Colinesterasa/química , Acetilcolinesterasa/metabolismo , Células Hep G2 , Inhibidores de la Colinesterasa/toxicidad , Oximas/farmacología , Oximas/química , Antídotos/farmacología , Organofosfatos/toxicidad , Estrés Oxidativo , Carbono , Compuestos de Piridinio/farmacología , Compuestos de Piridinio/químicaRESUMEN
Sample preparation involving the cleavage of proteins into peptides is the first critical step for successful bottom-up proteomics and protein analyses. Time- and labor-intensiveness are among the bottlenecks of the commonly used methods for protein sample preparation. Here, we report a fast online method for postinjection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7-8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.
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Ricina , Cromatografía Liquida/métodos , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Proteínas/análisis , PéptidosRESUMEN
Challenges and pitfalls in the application of diethyldithiocarbamate derivatization for LC analysis of cisplatin and oxaliplatin, as well as the suitability of this method for different biological matrices with implications for use in routine practice have been identified. The LC of platinum drugs presents a significant challenge. They are polar compounds with poor retention on reverse phase packings. Cisplatin also exhibits poor absorption in UV and ionization in mass spectrometry. Therefore, we developed and optimized a derivatization approach for the LC analysis of total platinum in plasma, plasma ultrafiltrate, peritoneal fluid, and urine. Derivatization in urine proved to be difficult due to the complexity of the matrix, and extended testing was required. Our results highlight the important issues affecting the efficiency, reliability, and suitability of platinum drug derivatization. Although precolumn derivatization is less selective than its postcolumn counterpart, the application of precolumn derivatization is a simple, rapid, and universal approach for the determination of platinum drugs by HPLC. One of its major advantages is that it allows a more affordable analysis using UV detection without the need for additional high-end instrumentation such as a MS detector.
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Cisplatino , Platino (Metal) , Cromatografía Líquida de Alta Presión/métodos , Ditiocarba , Reproducibilidad de los ResultadosRESUMEN
A novel method for the extraction of river water contaminants as model analytes of ranging polarities, including bisphenols A, C, S, Z, fenoxycarb, kadethrin, and deltamethrin, using small compact fibrous disks has been developed and validated. Polymer nanofibers and microfibers prepared from poly(3-hydroxybutyrate), polypropylene, polyurethane, polyacrylonitrile, poly(lactic acid), and polycaprolactone doped with graphene were evaluated in terms of extraction efficiency, selectivity, and stability in organic solutions. Our novel extraction procedure comprised preconcentration of analytes from 150 mL river water to 1 mL of eluent using a compact nanofibrous disk freely vortexed in the sample. Small nanofibrous disks with a diameter of 10 mm were cut from a compact and mechanically stable 1-2 mm thick micro/nanofibrous sheet. After 60 min extraction in a magnetically stirred sample located in a beaker, the disk was removed from the liquid and washed with water. Then, the disk was inserted into a 1.5 mL HPLC vial and extracted with 1.0 ml methanol upon short intensive shaking. Our approach avoided the undesired problems related to the manual handling typical of "classical" SPE procedure since the extraction was carried out directly in the HPLC vial. No sample evaporation, reconstitution, or pipetting was required. The nanofibrous disk is affordable, needs no support or holder, and its use avoids creation of plastic waste originating from disposable materials. Recovery of compounds from the disks was 47.2-141.4% depending on the type of polymer used and the relative standard deviations calculated from 5 extractions ranged from 6.1 to 11.8% for poly(3-hydroxybutyrate), 6.3-14.8% for polyurethane, and 1.7-16.2% for polycaprolactone doped with graphene. A small enrichment factor was obtained for polar bisphenol S using all sorbents. A higher preconcentration reaching up to 40-fold was achieved for lipophilic compounds such as deltamethrin when using poly(3-hydroxybutyrate) and graphene-doped polycaprolactone.
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A novel ultra-high performance chromatography method with multichannel detection that allows fast, sensitive, and robust analysis of an antifungal drug terbinafine and its three main impurities ß-terbinafine, (Z)-terbinafine, and 4-methylterbinafine in just 5.0 min has been developed. Analysis of terbinafine is important in pharmaceutical analysis since it enables the detection of its impurities at very low concentrations. In this study, we focused on the development, optimization, and validation of the UHPLC method as well as its subsequent application in the evaluation of terbinafine and its three main impurities in the dissolution medium to reveal the incorporation of terbinafine in two poly(lactic-co-glycolic acid) (PLGA) carriers and testing of the drug release at pH 5.5. PLGA based drug delivery systems such as solid dispersions, thin films, microparticles, and nanoparticles are new favorable ways of terbinafine administration. PLGA features excellent tissue compatibility, biodegradation, and adjustable drug release profile. Our pre-formulation study indicates that poly(acrylic acid) branched PLGA polyester has more suitable properties than tripentaerythritol branched PLGA polyester. Therefore, the former is likely to enable design of a new drug delivery system for topically applied terbinafine that could facilitate its administration and increase patient compliance.
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Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Humanos , Terbinafina , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Portadores de Fármacos/química , Cromatografía Líquida de Alta PresiónRESUMEN
An efficient sample preparation based on pipette tip microextraction that can be used for the analysis of retinol in human serum has been developed. Altogether, nine commercial pipette tips were compared based on recovery, sample volume, use of organic solvent, handling difficulty, duration of the preparation process, price, and greenness of the method. Retinol acetate was used as the internal standard. The extraction efficiency for both compounds was evaluated to optimize and select the best pipette tip for sample preparation, which was the WAX-S XTR pipette tip containing an ion exchanger and salt. This tip combined solid phase extraction and salting-out assisted liquidâliquid extraction. Satisfying recoveries of 100 and 80% for retinol and retinol acetate, respectively, and good repeatability were demonstrated. The action of this pipette tip was based on the clean-up workflow in which the interferences were retained on the sorbent. The presence of residual interferences in the extracted samples did not affect the HPLC separation of compounds of interest. The simplicity of the clean-up workflow reduced the time of the sample preparation compared to the bind-wash-elute counterpart workflow. The advantages of our technique are its environmental friendliness and cost effectiveness. The selected pipette tip with an excellent microextraction efficiency enables sample preparation in both clinical research and practice.
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Diterpenos , Vitamina A , Humanos , Extracción en Fase Sólida/métodos , Ésteres de Retinilo , Cloruro de SodioRESUMEN
New extraction protocols, gas-expanded liquid extraction (GXLE), and ultrasound extraction (UE) have been optimized with an emphasis on using green solvents and maximizing the extraction of 14 selected phenolic compounds, including flavonoid-based compounds and phenolic acids from dried apples. The design of the experiments' approach was applied to optimize the main extraction parameters. Fine tuning included optimization of the flow rate in GXLE and the extraction time for GXLE and UE. Optimized GXLE was carried out with CO2-ethanol-water (34/53.8/12.2; v/v/v) at a flow rate of 3 mL/min at a temperature of 75 °C and pressure of 120 bar for 30 min. UE with ethanol-water 26/74 (v/v) lasted for 10 min at 70 °C. Both methods differed in solvent consumption and sample throughput, while providing a comparable total phenolic content of 2442 µg/g with an RSD < 10% and 2226 µg/g with RSD < 6%, for GXLE and UE, respectively. Both methods were used in determining the phenolic compounds in five apple cultivars, 'Angold', 'Artiga', 'Golden Delicious', 'Meteor', and 'Topaz'. Phenolic profiles were plotted with chlorogenic acid, catechin, epicatechin, hirsutrin, phloridzin, and guaiaverin as the main components. Statistical evaluation, including pair t-test, Bland-Altman test, and linear regression did not reveal any differences between UE and GXLE results.
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Advanced solid phase extraction (SPE) fibrous sorbents including polyethylene, polypropylene poly (hydroxybutyrate), and polyamide 6 nanofibers, polycaprolactone microfibers/nanofibers, polycaprolactone microfibers/polyvinylidene difluoride nanofibers, and poly (hydroxybutyrate) microfibers/polypropylene microfibers composites, as well as commercial molecularly imprinted polymers and restricted access media sorbent were compared in terms of bisphenols extraction from milk and their clean-up efficiency. Three on-line SPE-HPLC methods were completely validated for the extraction and detection of bisphenols A, AF, C, A diglycidyl ether, and F diglycidyl ether in bovine milk. Polycaprolactone composite nanofibers compared favorably to restricted access media, enabled excellent clean-up of bisphenols from the proteinaceous matrix, and yielded recoveries 98.0-124.5% and 93.0-115.0%, respectively, with RSD less than 10%. Total analysis time including on-line SPE step lasted only 12 min, which represents a significant reduction in time compared with previously reported as well as official European Union and AOAC methods defined for the determination of bisphenols in various matrices.
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Impresión Molecular , Nanofibras , Animales , Polímeros Impresos Molecularmente , Adsorción , Nanofibras/química , Leche , Polipropilenos , Extracción en Fase Sólida/métodos , Cromatografía Líquida de Alta Presión/métodos , Hidroxibutiratos , Éteres , Impresión Molecular/métodosRESUMEN
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.
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Cromatografía con Fluido Supercrítico , Cromatografía con Fluido Supercrítico/métodos , Estereoisomerismo , Cromatografía Líquida de Alta Presión/métodos , Polisacáridos/química , EtanolRESUMEN
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.
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Cromatografía de Fase Inversa , Proteómica , Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Espectrometría de Masas/métodos , Péptidos/análisis , Proteómica/métodosRESUMEN
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.
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Cannabidiol , Cannabinoides , Cannabis , Cromatografía con Fluido Supercrítico , Espectrometría de Masas en Tándem/métodos , Cannabis/química , Dronabinol/análisis , Cannabidiol/análisis , Metanol/química , Dióxido de Carbono/química , Cannabinoides/análisis , Cromatografía con Fluido Supercrítico/métodos , Solventes/análisis , Agua , Etanol , Acetonitrilos , Cromatografía Líquida de Alta Presión/métodosRESUMEN
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.
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Dynamics of pesticides decomposition in sweet cherry fruits at different technologies of long-term storage, ultra-low oxygen and modified atmosphere packing, and after post-harvesting application of 1-methylcyclopropen and ozone has been studied. We assumed that type of pesticide and fruit storage conditions may have a profound effect on pesticide residues content. Therefore, levels of residues after applying combinations of active ingredients including acetamiprid, boscalid, cyprodinil, fenhexamid, fenpyrazamine, fludioxonil, fluopyram, pyraclostrobin, pirimicarb, tebuconazole, thiacloprid, and trifloxystrobin were monitored. We found these contents below tolerated maximum residue limits when applied at recommended times and depended on period prior to withdrawal. Low contents of acetamiprid, boscalid, fenpyrazamine, thiacloprid, pirimicarb, and fludioxonil were found when fruit were stored in modified atmosphere packages. Ozone affected degradation of tebuconazole, pyraclostrobin, and cyprodinil. However, differences between storage regimens were not statistically significant (p ≥ 0.05). Kinetic of degradation was studied with fruits stored after treatment with 1-methylcyclopropen and ozone.
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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.