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Unsymmetrically disubstituted metallocene derivatives, characterized as the first sandwich structure, have found interest in asymmetrical synthesis and in medicinal chemistry as well. Besides, they present a particular case of chirality. Twenty original and six commercially available molecules presenting either i) a planar chirality or ii) an asymmetrical carbon containing group or iii) being symmetrically substituted were analyzed in supercritical fluid chromatography on eleven polysaccharide-based chiral stationary phases with carbon dioxide containing 30% of methanol or 2-propanol as a co-solvent mobile phase. A basic additive, either diethylamine, triethylamine or n-butylamine was also required at 1% to the co-solvent for elution. While some of the tested chiral stationary phases provided enantioseparation for the racemates, chlorinated cellulosic phases proved to be both highly retentive and highly enantioselective towards these particular species with the highest rate of success compared to their non-chlorinated counterparts. For instance, the resolution value was equal to 14.1 for one ferrocene derivative in one-hour analysis time on cellulose tris(3,5-dichlorophenylcarbamate) column with 30% 2-propanol/1% n-butylamine while a single peak was observed under the same conditions on cellulose tris(3,5-dimethylphenylcarbamate) column. Experimental parameters were arbitrarily set at 150 bar outlet pressure, 40 °C temperature and 3 mL/min flow-rate.

Fluindapyr is a pyrazolamide chiral fungicide of succinate dehydrogenase inhibitor (SDHIs) with two enantiomers. Pesticide enantiomers often exhibit different biological activities, toxicity due to their different enantioselectivity. Therefore, it is important to separate fluindapyr enantiomers and assess each enantiomer. In this study, fluindapyr enantiomers were baseline separated by supercritical fluid chromatography-mass spectrometry in 2 min. The limit of quantification (LOQ) of this method was 5 μg/kg. The developed method was applied to monitor the fluindapyr enantiomers in cucumber and tomato, the data showed that R-(-)-fluindapyr was preferentially degraded in tomato leaves, S-(+)-fluindapyr was preferentially degraded in cucumber leaves, and fluindapyr enantiomers had no enantioselective degradation behavior in two fruits. It is proved again that enantiomers have different enantioselective degradation behavior with the different plant species and even to different parts of the same plant. The enantioselectivity is likely to be caused by different biodegradation enzyme systems.

A supercritical fluid chromatography with electrochemical detection system was developed for the simultaneous determination of tocopherol and tocotrienol isomers. The supercritical fluid chromatography with electrochemical detection system was connected with an additional pump to create a flow path to add a supporting electrolyte solution. The supporting electrolyte solution was mixed with a mobile phase in a post-column fashion, enabling the independent control of the separation and detection. After optimization of the measurement conditions, vitamin E isomers and an internal standard substance (2,2,5,7,8-pentamethyl-6-hydroxychroman) were separated within 30 min using a mixture of supercritical carbon dioxide and methanol (99:1, v/v) as a mobile phase and a cyanopropyl column (4.6 mm inner diameter × 250 mm length, 5 μm). For the electrochemical detection, methanol containing 1.0 mol/L ammonium acetate was used as a supporting electrolyte solution, and the applied potential was set at +0.8 V. This analytical method showed good linearity (5-100 μg/mL) and repeatability (less than 2.5% relative standard deviation, n = 6) and was applicable to the determination of tocopherol and tocotrienol isomers in nutrition supplements.

Background Origanum vulgare L. essential oil (OEO) is widely known for its good biological activity, but different extraction methods with significant implications on the yield of OEO and the content of the thymol and carvacrol. As an efficient method for extracting essential oils (EO), the supercritical carbon dioxide extraction (SC-CO2) can improve the yield of EOs while protecting their main active components from loss. Objective In this study, the process optimization of SC-CO2 of OEO was carried out. The effects of extraction pressure, temperature, time, and modifier concentration on the composite score of OEO extraction process were investigated. Method Response surface analysis was performed using a Box-Behnken design with three levels and four independent variables. Steam distillation (SD) and lipophilic solvents (n-hexane) extraction (LSE) were compared with SC-CO2 for OEO yields. OEOs extracted by the three methods were qualitatively and semi-quantitatively analyzed by gas chromatography quadrupole-time-of-flight mass spectrometry and gas chromatography-flame ionization detector. Results The results showed that extraction pressure was the most significant factor affecting the OEO yield, thymol, and carvacrol content. In the optimal conditions (pressure: 217 bar, temperature: 54°C, time: 2 h, modifier concentration: 14%), the yield of OEO reached up to 1.136%, and the contents of thymol and carvacrol reached 53.172 and 41.785 mg/g, respectively. Conclusions SC-CO2 was the best extraction method compared to the other two methods. Under the optimal conditions, yield and the content of main components can be effectively improved. It can provide a theoretical basis for the industrial extraction of OEO. Highlights Taking the comprehensive score as the index, the interaction between the four independent variables in the supercritical fluid extraction process was evaluated by the response surface method. The effects of extraction parameters on the yield of EOs and the contents of thymol and carvacrol were comprehensively investigated.

Gynura procumbens is a medicinal herb that contains bioactive compounds that can relieve coughs and prevent liver cancer. Supercritical fluid extraction (SFE) was suggested as one of the techniques that can be used to extract the valuable compounds from the G. procumbens. SFE was widely applied in extracting medicinal ingredients from herbs. However, most of them were performed only at the laboratory scale. Moreover, study to increase the yield performance, economic studies and safety assessments of the SFE process were also performed; however, these tests were conducted individually. Moreover, to date, there is no integration study between all the factors stated for determining the overall performance of SFE with herbs specifically G. procumbens. The integration between all the factors is beneficial because the data on the overall performance can assist in developing the SFE process with G. procumbens at the pilot or industrial scale. Therefore, this study incorporated a multifactor approach to measure the overall performance of the SFE process towards G. procumbens by using a rating and index approach. A summary of factors, such as the solubility of G. procumbens in CO2, operational cost and safety assessment elements, were taken into consideration as the main influences that determine the overall performance index of this study. Iperformance or overall performance of SFE from G. procumbens was successfully assessed and compared with response surface methodology (RSM). Overall, the results from Iperformance exhibit satisfactory solubility values when compared to the optimized value from RSM when considering the lowest operational costs in the safest SFE environment.

Acephate is widely used in crops as racemate. However, the enantioselective dissipation of acephate enantiomers has not been investigated in pakchoi. A sensitive and effective approach was established for determining residues of acephate and its highly toxic metabolite methamidophos enantiomers by supercritical fluid chromatography tandem mass spectrometry. Baseline separations for their enantiomers were achieved by using a Chiralcel OD-H column. The optimal chromatographic conditions were obtained as follows: CO

Powerful chromatographic techniques are required for lipidomic analyses due to the extreme complexity of natural lipidomes. In the past few years, ultrahigh-performance supercritical fluid chromatography (UHPSFC) has proven to be a good alternative to conventional LC methods for comprehensive lipidomic analysis. The goal of this work was to study UHPSFC intraclass separation of lipids according to the fatty acyl composition. The effects of column chemistry, mobile phase composition and gradient, flow rate, back pressure, temperature, and column coupling on intraclass separation of lipids were widely investigated and discussed. In general, UHPSFC exhibited interclass selectivity together with intraclass separation of lipids according to their total number of double bonds and acyl chain lengths. Moreover, separations of diacylglycerol and lysophosphatidylcholines regioisomers were achieved in some cases. The nature of the stationary phase showed the most prominent effect on UHPSFC intraclass selectivity, while other chromatographic conditions were used for partial improvement in resolution of lipid species. An octadecyl-based stationary phase showed excellent separation of nonpolar lipid species, including triacylglycerol isobars; however, it provided poor peak shapes and limited retention time reproducibility for polar lipids. Diol- and 1-aminoanthracene-based columns provided the best inter- and intraclass resolution of most lipids. The main benefit for UHPSFC separation of complex lipid samples is the combination of the acyl chain/double bond intraclass separation of lipids with excellent lipid class selectivity, which can facilitate mass spectrometry detection and quantitation of trace species without ion suppression effects.

Supercritical carbon dioxide extraction was used to extract carotenoids from dry paprika

Since the quality and safety of food highly depend on its preservation and protection, the use of food packaging materials increases the risk of chemical contamination of the packaged food by migration. Herein, we focused on antioxidants, photoinitiators, UV absorbers and plasticizers which are extensive additives used in food packaging materials. In the present study, a rapid, simple, green and reliable method was developed and validated for the determination of twelve chemical additives in edible vegetable oils using SFC together with a modified QuEChERS procedure. Under the optimum conditions, twelve additives were separated within 10 min, and the consumption of the organic solvent was significantly reduced, which improved the environmentally friendliness. The performance of the developed method was evaluated. Good linearity (r > 0.999) was obtained in the range of 0.20–20.0 µg/mL and 0.50–20.0 µg/mL, respectively. The limits of detection and limits of quantification of the twelve additives in vegetable oils were 0.05–0.15 µg/mL and 0.15–0.50 µg/mL, respectively. Recoveries of all the chemical additives for the spiked samples were between 60.9% and 106.4%, with relative standard deviations (RSD) lower than 9.9%. The results demonstrated that the proposed method was efficient, reliable and robust for the routine analysis of additives in edible vegetable oils and can be an alternative to the multi-residue analysis of chemical additives for other packaged foods.

The goals of this study were to determine the phytochemical profile of

By-products of

Triacylglycerol (TAG) regioisomers containing palmitic acid (16:0) was identified using ultra-performance supercritical fluid chromatography and quadrupole time-of-flight mass spectrometry (UPSFC-Q-TOF-MS) and quantified using calibration curve method and calculation equation method. There were negative linear correlation between [R

In this study, the impact of introducing water in the sample solvent upon the injection in SFC is investigated. Adsorption of water on the stationary phase was indicated. Using a set of ten neutral test compounds and four ionizable test compounds, spread all along the co-solvent gradient, several parameters were scrutinized (i.e. water content in the sample diluent, nature of the sample diluent, nature of the co-solvent) in regards to peak broadening. From this systematic investigation, the competition for adsorption on the stationary phase between the analytes and the water molecules contained in the diluent was highlighted. The chromatographic peaks of neutral molecules eluting before water molecules were compressed and the ones eluting after were broadened. While the extent of this phenomenon was related to the peak position for neutral molecules, it was not observed on acidic molecules.

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.

Triterpenoids are among the bioactive components of Chaga, the sterile conk of the medicinal fungus

Fungal infections of cultivated food crops result in extensive losses of crops at the global level, while resistance to antifungal agents continues to grow. Supercritical fluid extraction using CO

Japanese Spanish mackerel (JSM) (Scomberomorus niphonius) is a marine fish species containing health-beneficial polyunsaturated fatty acids (PUFAs). In the present study, the quality of JSM by-products oils extracted by supercritical CO2 (SC-CO2) and organic solvent extraction was compared in terms of physico-chemical properties of the oils. Eicosapentaenoic acid (EPA) is one of the important polyunsaturated fatty acids present in SC-CO2-extracted skin and muscle oil 5.81 ± 0.69% and 4.93 ± 0.06%, respectively. The amount of docosahexaenoic acid (DHA) in SC-CO2-extracted skin and muscle oil was 12.56 ± 0.38% and 15.01 ± 0.28%, respectively. EPA and DHA are considered as important PUFAs for the development of brain function and the prevention of coronary heart diseases. Extracted oils showed considerable antioxidant activity. In the obtained oils, atherogenic index (AI) values varied from 0.72 to 0.93 and thrombogenic index (TI) ranged from 0.75 to 0.92, which is considered an acceptable level. Fatty acid composition, bio potentiality, thermogravimetric, and vitamin D analysis showed that oils extracted from JSM by-products can be a good source of oil for application in food, pharmaceutical and cosmetic industries. Therefore, the present research revealed the potentiality of green valorisation of S. niphonius by-products as a possible sustainable approach for targeting the era of zero waste.

A widely disseminated native species from Australia

In this study, quality evaluations of Hedyotis diffusa (H. diffusa) batches by rapid fingerprint analysis based on supercritical fluid chromatography (SFC) were accomplished. Abundant chemical components of H. diffusa were effectively extracted by optimal supercritical fluid extraction conditions (20 % MeOH as modifier, 45 °C, 300 bar and 60 min). Then, the extract was separated by SFC on a Torus 1-AA column (100 × 3.0 mm i.d., 1.7 µm) within 10 min by gradient elution increasing from 5 % to 45 % modifier (MeOH containing 0.05 % TFA) at 1.2 mL/min, 30 °C and 2000 psi. The SFC approach exhibited short analysis time, while maintaining good peak shape and resolution. Seven major compounds were further identified by SFC coupled with tandem mass spectrometer to be phenylpropanoid, iridoids and anthraquinones. Finally, fingerprint analysis of 10 batches of H. diffusa by the developed SFC method was accomplished. The similarity values were between 0.894 and 0.968, indicating quality differences of H. diffusa from depending on origin and harvest year exist. The result demonstrates the feasibility of the SFC in batch quality evaluation of H. diffusa.

The applicability of ultrahigh-performance supercritical fluid chromatography coupled with mass spectrometry (UHPSFC/MS) for the qualitative analysis of metabolites with a wide polarity range (log P: -3.89-18.95) was evaluated using a representative set of 78 standards belonging to nucleosides, biogenic amines, carbohydrates, amino acids, and lipids. The effects of the gradient shape and the percentage of water (1, 2, and 5%) were investigated on the Viridis BEH column. The screening of eight stationary phases was performed for columns with different interaction sites, such as hydrogen bonding, hydrophobic, π-π, or anionic exchange type interactions. The highest number of compounds (67) of the set studied was detected on the Torus Diol column, which provided a resolution parameter of 39. The DEA column had the second best performance with 58 detected standards and the resolution parameter of 54. The overall performance of other parameters, such as selectivity, peak height, peak area, retention time stability, asymmetry factor, and mass accuracy, led to the selection of the Diol column for the final method. The comparison of additives showed that ammonium acetate gave a superior sensitivity over ammonium formate. Moreover, the influence of the ion source on the ionization efficiency was studied by employing atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The results proved the complementarity of both ionization techniques, but also the superior ionization capacity of the ESI source in the negative ion mode, for which 53% of the analytes were detected compared to only 7% for the APCI source. Finally, optimized analytical conditions were applied to the analysis of a pooled human plasma sample. 44 compounds from the preselected set were detected in human plasma using ESI-UHPSFC/MS in MS

Cinnarizine is a weak base, which can produce supersaturation and precipitation during gastrointestinal transit, affecting its absorption in vivo. Therefore, it is necessary to investigate whether the oral bioavailability of cinnarizine can be improved after co-administration with precipitation inhibitors or not. In order to evaluate the pharmacokinetic behavior of cinnarizine in rats, a simple, rapid, sensitive, and environmentally friendly supercritical fluid chromatography-tandem mass spectrometric method was established and validated. In this method, flunarizine, a structural analogue of cinnarizine, was selected as the internal standard, and cinnarizine was extracted from rat plasma using evaporation-free liquid-liquid extraction method. The analytes were separated on a Torus 1-AA column (3.0 mm × 100 mm, 1.7 μm) within 2.0 min, using a gradient elution procedure. The transitions of cinnarizine and flunarizine were m/z 369.1 → 167.1 and m/z 405.1 → 203.1, respectively. Cinnarizine showed good linear correlation in the range of 1-500 ng/ml with a lower limit of quantification of 1 ng/ml. The intra- and interday precision and accuracy of all quality control samples were within ±15%. This high-throughput, accurate, sensitive, and reproducible method has been successfully applied to study the effects of the precipitation inhibitor cinnarizine on the pharmacokinetics in rats.

Humans often respond to sensory impulses provided by aromas, and current trends have generated interest in natural sources of fragrances rather than the commonly used synthetic additives. For the first time, the resulting aroma of a selected culture of

A comparative study of chiral separation of pantoprazole and rabeprazole is carried out using supercritical fluid chromatography and high-performance liquid chromatography. The columns used were Chiralpak IA and Chiralpak IE. The best mobile phase in supercritical fluid chromatography was carbon dioxide-0.2% triethylamine in methanol (60:40) and 0.1% triethylamine in n-hexane-ethanol (50:50) in high-performance liquid chromatography. For supercritical fluid chromatography, values of the retention factor of pantoprazole enantiomers were 3.97 and 4.88. These values for rabeprazole enantiomers were 6.10 and 7.52. The values of separation and resolution factor for pantoprazole and rabeprazole were 1.23 and 1.23 and 2.20 and 3.36, respectively. Similarly, for high-performance liquid chromatography, the values of retention factor for enantiomers of pantoprazole were 4.02 and 7.32. These values for rabeprazole enantiomers were 5.32 and 7.88, respectively. The values of separation and resolution factor for pantoprazole and rabeprazole were 1.82 and 1.48 and 9.22 and 6.58, respectively. A comparison was carried out, which confirmed supercritical fluid chromatography as the best method due to its fastness, eco-friendly, and inexpensiveness. The reported methods are effective, efficient, and reproducible and may be used to separate and identify pantoprazole and rabeprazole in any unknown samples.

Improving efficiency while maintaining high quality separations is a central theme for specialized analytical/purification groups supporting discovery chemistry programs. Supercritical fluid chromatography (SFC) has become the prevalent technique for chiral separation and a complementary technique to reverse phase high-pressure liquid chromatography (RP-HPLC). In this manuscript we demonstrate the successful micro-isolation of chiral racemates, small molecules, and peptides using a sub-minute method on an analytical SFC system. The addition of a custom gas liquid separator (GLS) and alterations to the fluidic pathways allow the fractionation of desired products on a micro-scale SFC platform, providing analytical method development, purifications, and purity confirmation on a single SFC system. This enables micro-purification of pharmaceuticals including chiral racemates at high speed and reduced cost of materials. The resulting small-quantity, high-purity products enable follow-up enantioselective isolations from racemic products of parallel synthesis libraries. The processes established here will be beneficial for the isolations of other desired products in complex crude mixtures.

Liquid chromatography-mass spectrometry methods were required to afford the rapid separation and detection of purines and small organic acids. These compounds are found in sweat and sebum and are potential biomarkers for the early detection of pressures sores. Two ultrahigh-performance supercritical fluid chromatography-mass spectrometry assays have been successfully developed for both classes of compounds. Separation for purines was achieved using a gradient of supercritical carbon dioxide and methanol with a 1-aminoanthracene sub 2 µm particle size column followed by positive ion electrospray ionization. Separation for organic acids was achieved using a gradient of supercritical carbon dioxide and methanol (50 mM ammonium acetate 2% water) with a Diol sub 2 µm particle size column followed by negative ion electrospray ionization. Calibration curves were created in the absence of internal standards and R2 values > 0.96 were achieved using single ion monitoring methods for the protonated purines and the deprotonated acids. The two new assays afford rapid analytical methods for the separation and detection of potential biomarkers in human sweat leading to the early detection and prevention of pressure sores.

Phenolic compounds from mango (M. indica) seed kernels (MSK) var. Sugar were obtained using supercritical CO2 and EtOH as an extraction solvent. For this purpose, a central composite design was carried out to evaluate the effect of extraction pressure (11–21 MPa), temperature (40–60 °C), and co-solvent contribution (5–15% w/w EtOH) on (i) extraction yield, (ii) oxidative stability (OS) of sunflower edible oil (SEO) with added extract using the Rancimat method, (iii) total phenolics content, (iv) total flavonoids content, and (v) DPPH radical assay. The most influential variable of the supercritical fluid extraction (SFE) process was the concentration of the co-solvent. The best OS of SEO was reached with the extract obtained at 21.0 MPa, 60 °C and 15% EtOH. Under these conditions, the extract increased the OS of SEO by up to 6.1 ± 0.2 h (OS of SEO without antioxidant, Control, was 3.5 h). The composition of the extract influenced the oxidative stability of the sunflower edible oil. By SFE it was possible to obtain extracts from mango seed kernels (MSK) var. Sugar that transfer OS to the SEO. These promissory extracts could be applied to foods and other products.

Phenolic compounds present in common beans (Phaseolus vulgaris L.) have been reported to possess antimicrobial, anti-inflammatory and ultraviolet radiation (UVR) protective properties. UVR from sunlight, which consists of UV-B and UV-A radiations, induces reactive oxygen species (ROS) and free radical formation, consequently activating proteinases and enzymes such as elastase and tyrosinase, leading to premature skin aging. The objective of this work was to extract, characterize and evaluate the antioxidant and antiaging potential of polyphenols from a black bean endemic variety. The polyphenolic extract was obtained from black beans by supercritical fluid extraction (SFE) using CO2 with a mixture of water–ethanol as a cosolvent and conventional leaching with a mixture of water–ethanol as solvent. The polyphenolic extracts were purified and characterized, and antioxidant potential, tyrosinase and elastase inhibitory potentials were measured. The extract obtained using the SFE method using CO2 and H2O–Ethanol (50:50 v/v) as a cosolvent showed the highest total phenolic compounds yield, with 66.60 ± 7.41 mg GAE/g coat (p &gt, 0.05) and 7.30 ± 0.64 mg C3GE/g coat (p &lt, 0.05) of anthocyanins compared to conventional leaching. Nineteen tentative phenolic compounds were identified in leaching crude extract using ESI-QTOF. Quercetin-3-D-galactoside was identified in crude and purified extracts. The purified SFC extract showed IC50 0.05 ± 0.002 and IC50 0.21 ± 0.008 mg/mL for DPPH and ABTS, respectively. The lowest IC50 value of tyrosinase inhibition was 0.143 ± 0.02 mg/mL and 0.005 ± 0.003 mg/mL of elastase inhibition for leaching purified extract. Phenolic compounds presented theoretical free energy values ranging from −5.3 to −7.8 kcal/mol for tyrosinase and −2.5 to −6.8 kcal/mol for elastase in molecular docking (in silico) studies. The results suggest that the purified extracts obtained by SFE or conventional leaching extraction could act as antioxidant and antiaging ingredients for cosmeceutical applications.

Early detection of cancer is one of the unmet needs in clinical medicine. Peripheral blood analysis is a preferred method for efficient population screening, because blood collection is well embedded in clinical practice and minimally invasive for patients. Lipids are important biomolecules, and variations in lipid concentrations can reflect pathological disorders. Lipidomic profiling of human plasma by the coupling of ultrahigh-performance supercritical fluid chromatography and mass spectrometry is investigated with the aim to distinguish patients with breast, kidney, and prostate cancers from healthy controls. The mean sensitivity, specificity, and accuracy of the lipid profiling approach were 85%, 95%, and 92% for kidney cancer; 91%, 97%, and 94% for breast cancer; and 87%, 95%, and 92% for prostate cancer. No association of statistical models with tumor stage is observed. The statistically most significant lipid species for the differentiation of cancer types studied are CE 16:0, Cer 42:1, LPC 18:2, PC 36:2, PC 36:3, SM 32:1, and SM 41:1 These seven lipids represent a potential biomarker panel for kidney, breast, and prostate cancer screening, but a further verification step in a prospective study has to be performed to verify clinical utility. Včasná detekce rakoviny je jednou z nenaplněných potřeb klinické medicíny. Periferní analýza krve je preferovanou metodou pro efektivní populační screening, protože odběr krve je dobře zakotven v klinické praxi a je minimálně invazivní pro pacienty. Lipidy jsou důležité biomolekuly a odchylky v koncentracích lipidů mohou odrážet patologické poruchy. Lipidomická profilace lidské plazmy spojením ultra-vysokoúčinné superkritické kapalinové chromatografie a hmotnostní spektrometrie je zkoumána s cílem odlišit pacienty s karcinomem prsu, ledvin a prostaty od zdravých kontrol. Průměrná citlivost, specifičnost a přesnost při profilování lipidů byla 85 %, 95 % a 92 % u rakoviny ledvin; 91 %, 97 %, a 94 % pro rakovinu prsu; a 87 %, 95 % a 92 % pro rakovinu prostaty. Není pozorována žádná souvislost statistických modelů se stádiem nádoru. Statisticky nejvýznamnějšími druhy lipidů pro diferenciaci zkoumaných typů nádorů jsou CE 16:0, Cer 42:1, LPC 18:2, PC 36:2, PC 36:3, SM 32:1 a SM 41:1. Těchto sedm lipidů představuje potenciální biomarkerový panel pro screening rakoviny ledvin, prsu a prostaty, ale k ověření klinické užitečnosti musí být proveden další ověřovací krok v prospektivní studii.

The separation of structural analogues in natural products has always been one of the challenges in separation science, where supercritical fluid chromatography (SFC) with chiral stationary phases (CSPs) is an unconventional but potential solution. In this study, a preparative two-dimensional chiral SFC (2D cSFC) method that was established with two kinds of CSPs was applied in the isolation of the aliphatic acid derivatives in Piper kadsura (P. kadsura). The RPLC unseparated peaks of two samples A and B of P. kadsura were evenly scattered on the CSP-1 column while they clustered into two groups on the CSP-2 column by SFC. There was impressively complementary selectivity between CSP-1 and CSP-2, which were used for construction of 2D cSFC. The first dimension (

Supercritical fluid chromatography (SFC) has unique separative characteristics distinguished from those of HPLC and gas chromatography. At present, SFC is widely used and there are many applications in various biological, medical, and pharmaceutical fields. In this review, we focus on recently developed novel techniques related to SFC separation including: new column stationary phases, microfluidics, two-dimensional separation, and gas-liquid separation. In addition, we discuss the application of SFC using a water-containing modifier to biological molecules such as amino acids, peptides, and small proteins that had been challenging analytes.

The extraction of clary sage (Salvia sclarea L.) using supercritical carbon dioxide (SC-CO2) was systematically studied by using thin layer chromatography-direct bioautography (TLC-DB) and response surface methodology (RSM). The three parameters temperature, pressure, and cosolvent ratio were optimized for the maximum antibacterial activity of clary sage extracts against Pseudomonas aeruginosa (P. aeruginosa) and methicillin-resistant Staphylococcus aureus (MRSA). The highest inhibition zone was 7.51 mm for P. aeruginosa and 7.57 mm for MRSA. According to RSM analysis, the predicted optimum extraction parameters are 18.6 MPa pressure, 40 °C temperature, and 2% ethanol (EtOH) ratio. The combination of this analytical and statistical method allows saving time, money, and instrument runtime in the optimization of essential oil composition, which is tailored to a specific task and could be useful on any kind of herbs in a wide range of use from perfume manufacturing to the food industry.

The prediction of chromatographic retention under supercritical-fluid chromatography (SFC) conditions was studied, using established and novel theoretical models over ranges of modifier content, pressure and temperature. Whereas retention models used for liquid chromatography often only consider the modifier fraction, retention in SFC depends much more strongly on pressure and temperature. The viability of combining several retention models into surfaces that describe the effects of both modifier fraction and pressure was investigated. The ability of commonly used retention models to describe retention as a function of modifier fraction, expressed either as mass or volume fraction, pressure and density was assessed. Using the multivariate surfaces, retention-time prediction for isocratic separations at constant temperature improved significantly compared to univariate modelling when both pressure and modifier fractions were changed. The “mixed-mode” model with an additional exponential pressure or density parameter was able to predict retention times within 5%, with the majority of the predictions within 2%. The use of mass fraction and density further improves retention modelling compared to volume fraction and pressure. These variables however, do require extra computations.

Plant-derived natural dyes are used in a variety of formulated products, from food to cosmetics and pharmaceutics. In addition to their color, they also provide some bioactivity. While they are mostly analyzed with high-performance liquid chromatography, supercritical fluid chromatography was also employed for several dye families, mostly for carotenoids and chlorophylls, and more recently for anthraquinones and flavonoids. These supercritical fluid chromatography methods are described in this review. Because the dyes have different structures and structural variations (polarity, isomers, etc.), the best chromatographic system to achieve their separation is not always the same. Hydrophobic stationary phases are preferred for the most hydrophobic dyes (chlorophylls and carotenoids) while polar stationary phases are preferred for the polar dyes (anthraquinones and flavonoids). Regarding the mobile phase composition, chlorophylls and carotenoids are best eluted with moderate proportions of co-solvent in CO2 (about 40%), while the most polar glycosylated flavonoids require higher proportions of co-solvent and acidic additives. Because dyes are colorful, ultraviolet-visible detection is often sufficient, while mass spectrometry offers additional structural information. Furthermore, fundamental information can also be gained through chromatographic analysis of dyes: either solubility in supercritical fluids, in view of their extraction, or retention behavior providing an understanding of stationary phase properties.

Supercritical-fluid chromatography (SFC) is regaining popularity in various fields of analytical chemistry owning to significant advances in instrumentation made in the past decade. However, due to the CO2 based mobile phase and the high flow rates often employed, detection of trace amounts of analytes and coupling with certain detectors or other chromatography techniques are still difficult under many circumstances. In this study we propose a post-column re-focusing approach for SFC analysis to achieve not only signal enhancement in UV-Vis detection, but also actual concentration enhancement of the analyte. By heart-cutting and transporting a selected fraction from the SFC flow into a trapping column with a flushing solvent, re-focusing of the collected analytes can be achieved by re-mobilization with another solvent once the depressurized CO2 is eliminated. By carefully selecting the trapping stationary phase and the two solvents, signal-enhancement ratios between 2.2 and 6.4 were realized for four representative compounds eluting with very different percentages of SFC modifier (methanol). The actual concentration enhancement was lower (ratios between 1.7 to 2.9), because the UV response of the analytes was found to differ significantly under SFC and LC conditions.

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) has a great potential for the high-throughput lipidomic quantitation of biological samples; therefore, the full optimization and method validation of UHPSFC/MS is compared here with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) in hydrophilic interaction liquid chromatography (HILIC) mode as the second powerful technique for the lipid class separation. First, the performance of six common extraction protocols is investigated, where the Folch procedure yields the best results with regard to recovery rate, matrix effect, and precision. Then, the full optimization and analytical validation for eight lipid classes using UHPSFC/MS and HILIC-UHPLC/MS methods are performed for the same sample set and applied for the lipidomic characterization of pooled samples of human plasma, human serum, and NIST SRM 1950 human plasma. The choice of appropriate internal standards (IS) for individual lipid classes has a key importance for reliable quantitative workflows illustrated by the selectivity while validation and the calculation of the quantitation error using multiple internal standards per lipid class. Validation results confirm the applicability of both methods, but UHPSFC/MS provides some distinct advantages, such as the successful separation of both non-polar and polar lipid classes unlike to HILIC-UHPLC/MS, shorter total run times (8 vs. 10.5 min), and slightly higher robustness. Various types of correlations between methods (UHPSFC/MS and HILIC-UHPLC/MS), biological material (plasma and serum), IS (laboratory and commercially mixtures), and literature data on the standard reference material show the intra- and inter-laboratory comparison in the quantitation of lipid species from eight lipid classes, the concentration differences in serum and plasma as well as the applicability of non-commercially available internal standard mixtures for lipid quantitation.

This report presents the first ultra high performance supercritical fluid chromatography diode array detector based assay for simultaneous determination of iridoid glucosides, flavonoid glucuronides, and phenylpropanoid glycosides in Verbena officinalis (Verbenaceae) extracts. Separation of the key metabolites was achieved in less than 7 min on an Acquity UPC2 Torus Diol column using a mobile phase gradient comprising subcritical carbon dioxide and methanol with 0.15% phosphoric acid. Method validation for seven selected marker compounds (hastatoside, verbenalin, apigenin‐7‐O‐glucuronide, luteolin‐7‐O‐glucuronide, apigenin‐7‐O‐diglucuronide, verbascoside, and luteolin‐7‐O‐diglucuronide) confirmed the assay to be sensitive, linear, precise, and accurate. Head‐to‐head comparison to an ultra high performance liquid chromatography comparator assay did prove the high orthogonality of the methods. Quantitative result equivalence was evaluated by Passing‐Bablok‐correlation and Bland‐Altman‐plot analysis. This cross‐validation revealed, that one of the investigated marker compound peaks was contaminated in the ultra high performance liquid chromatography assay by a structurally related congener. Taken together, it was proven that the ultra high performance supercritical fluid chromatography instrument setup with its orthogonal selectivity is a true alternative to conventional reversed phase liquid chromatography in quantitative secondary metabolite analysis. For regulatory purposes, assay cross‐validation with highly orthogonal methods seems a viable approach to avoid analyte overestimation due to coeluting, analytically indistinguishable contaminants.

A novel online two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography-triple-quadrupole mass spectrometry (2D SFC/RPLC-QQQ MS) method based on a vacuum solvent evaporation interface was developed for lipid profiling in human plasma, in which lipid classes were separated by the first-dimension SFC and different lipid molecular species were further separated by the second-dimension RPLC. All separation condition parameters were carefully optimized, and their influence on the chromatographic behavior of lipids is discussed. Finally, the recoveries of 11 lipid standards were all more than 88% for the interface. Besides, the limit of detection for these lipid standards was on the order of nanograms per milliliter, and the relative standard deviations of the peak area and retention time ranged from 1.54% to 19.85% and from 0.00% to 0.10%, respectively. The final 2D SFC/RPLC-QQQ MS method allowed the identification of 370 endogenous lipid species from ten lipid classes, including diacylglycerol, triacylglycerol, ceramide, glucosylceramide, galactosylceramide, lactosylceramide, sphingomyelin, acylcarnitine, phosphatidylcholine, and lysophosphatidylethanolamine, in human plasma within 38 min, which was used for screening potential lipid biomarkers in breast cancer. The 2D SFC/RPLC-QQQ MS method is a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery. Graphical Abstract.

A novel method to quantitate vitamin D and its main metabolites (vitamin D3, vitamin D2, and their 25-hydroxy metabolites) in breast milk by supercritical fluid chromatography has been developed and fully validated. A small volume of sample (1 mL) is subjected to ethanolic protein precipitation and liquid-liquid extraction. Final extracts are derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione and vitamin D derivatives analyzed by supercritical fluid chromatography hyphenated to tandem mass spectrometry with atmospheric pressure chemical ionization. Multiple reaction monitoring is used for quantitation. Separation conditions were optimized using a gradient of methanol-water-ammonium formate into carbon dioxide. Make-up solvent was methanol containing ammonium formate. The quantitation limit reached levels as low as 50 pmol/L, with intra- and inter-day relative standard deviations lower than 15% and 20% for all analytes. Accuracy was evaluated by spiking experiments and was well within acceptability ratios (± 15%). The method was then applied to a subset of commercially available human milk samples. The newly developed method provides opportunities to determine the nutritional status of mother-infant dyads from a non-invasive measure, or for interventional or observational studies building knowledge on the composition of human milk. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-019-02248-5) contains supplementary material, which is available to authorized users.

A lyophilization-supercritical fluid extraction coupled with supercritical fluid chromatography-quadrupole tandem mass spectrometry online method was developed for the determination of lipid mediators in breast cancer cells. Supercritical fluid extraction was applied to the cell samples for the first time due to the use of lyophilization. The conditions of supercritical fluid extraction and supercritical fluid chromatography-quadrupole tandem mass spectrometry were investigated systematically. Under the optimized conditions, all the calibration curves for the lipid mediators showed good linearity (correlation coefficient > 0.99). The limits of detection and the limits of quantification were in the range of 0.190-5.36 pg and 0.560-16.2 pg, respectively. The recoveries were in the range of 70.3-125%. The relative standard deviations of the precision ranged from 1.49-18.7% and the accuracies were higher than 84%. Compared with liquid-liquid extraction coupled with liquid chromatography and tandem mass spectrometry method, the present approach reduced the manual labor and obtained higher sensitivity as well as higher extraction recoveries for all 15 lipid mediators. Finally, the online method was applied to the quantification of lipid mediators in breast cancer cells and normal mammary epithelial cells. On the basis of the results, this lyophilization-supercritical fluid extraction online coupled with supercritical fluid chromatography-quadrupole tandem mass spectrometry method showed great promise in the analysis of lipid mediators in complex biological samples.

To circumvent the detrimental effects of large‐volume injection with fixed‐loop injector in modern supercritical fluid chromatography, the feasibility of performing multiple injection was investigated. By accumulating analytes from a certain number of continual small‐volume injections, compounds can be concentrated on the column head, and this leads to signal enhancement compared with a single injection. The signal to noise enhancement of different compounds appeared to be associated with their retention on different stationary phases and with type of sample diluent. The diethylamine column gave the best signal to noise enhancement when acetonitrile was used as sample diluent and the 2‐picolylamine column showed the best overall performance with water as the sample diluent. The advantage of multiple injection over one‐time large‐volume injection was proven with sulfanilamide, with both acetonitrile and water as sample diluents. The multiple injection approach exhibited comparable within‐ and between‐day precision of retention time and peak area with those of single injections. The potential of the multiple injection approach was demonstrated in the analysis of sulfanilamide‐spiked honey extract and diclofenac‐spiked ground water sample. The limitations of this approach were also discussed.

Different parts of Prunus persica as fruits, flowers, leaves and kernels have been consumed with dietary and therapeutic purposes traditionally. During fruit production, remarkable amount of leaves which can hold important bioactive groups as phenolics, have been left unutilized. the aim of this study was to investigate cytotoxic, antimicrobial and nitric oxide inhibitory activities of supercritical carbondioxide extracts of Prunus persica leaves. Among studied cell lines, supercritical carbon dioxide extract which was processed at 150 bar, 60 degrees C, and 6% co-solvent ethanol, exhibited remarkable cytotoxic activity against HeLa, MPanc-96 and MCF-7 cell lines with IC50 values of 12.22 mu g/ml, 28.17 mu g/ml and 35.51 mu g/ml respectively, whereas IC50 value of conventional solvent extract was above 50 mu g/ml. Minimum inhibitory concentration values determined for antibacterial and antifungal activities against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium and Candida albicans were found as 62.50 mu g/ml. Strong nitric oxide inhibition was achieved with IC50 of 9.30 mu g/ml. the promising results revealed that Prunus persica leaves may have remarkable potential as supplement both for drug and food industries. This study is the first report revealing cytotoxic, antimicrobial and nitric oxide inhibitory activity of supercritical carbon dioxide extract of Prunus persica leaves.

Background Benign prostatic hyperplasia (BPH) is a pathological condition affecting older men. BPH complications often lead to deterioration in the quality of life. Serenoa repens (Saw Palmetto) is used for treating lower urinary tract infections in traditional medicine. Methods This study was performed to compare the efficacy of β-sitosterol enriched saw palmetto oil (VISPO) and conventional saw palmetto oil (SPO) extracted using supercritical fluid extraction, in alleviating the BPH complications using testosterone-induced BPH model rats. The animals received testosterone (5 mg/kg s.c.) with or without SPO and VISPO (200 and 400 mg/kg b.w.) or Finasteride (1 mg/kg b.w.) p.o. for 28 days. At the end of the experiment, overnight fasted animals were euthanized, blood samples collected for serum analysis of testosterone. Prostate tissue histomorphology was examined by hematoxylin and eosin (H&E) staining. Western blot analysis was performed using prostate tissue homogenates. Results VISPO exhibited superior efficacy compared to SPO as evident from the significant decrease in prostate weight to body weight ratio, serum testosterone level and increase in growth inhibition of prostate tissue compared to BPH group (p Conclusion Our data provide experimental evidence that β-sitosterol enriched saw palmetto oil could be higher efficacious in treating the BPH complications compared to the conventional saw palmetto oil preparations.

Supercritical fluid chromatography (SFC) holds the potential to become an orthogonal method to HPLC/UHPLC in xenobiotic metabolism studies, due to its outstanding capacity to simultaneously separate highly similar (as HPLC) and physicochemically different analytes (problematic using HPLC). Paucity of guideline-conform validation, however, has been a major obstacle to clinical application of SFC, even in cases where biotransformation yields chemically dissimilar metabolites that require more than one HPLC method for comprehensive analysis. Here, a method based on supercritical fluid chromatography coupled to single quadrupole MS detection was developed to simultaneously quantify the divisive analgesic flupirtine and its acidic and basic metabolites, represented by 4-fluorohippuric acid (4-FHA) and the active metabolite D-13223 respectively, using custom-made synthetic internal standards. Experimental data on the fundamental retention mechanisms under supercritical conditions, indicating the importance of halogen and π-π-bonding for specific retention on polysaccharide-based stationary-phases, is discussed. Compared to previous HPLC methods, the novel method offers higher versatility in terms of the target metabolite range (addressing both acidic and basic metabolites within a singular method), faster analysis (7.5 min), and compliance with green chemistry principles. Validation was performed according to EMA criteria on bioanalytical method validation, demonstrating selectivity, carry-over, calibration curve parameters (LLOQ, range, and linearity), within- and between-run accuracy and precision, dilution integrity, matrix effect and stability. For proof-of-concept, the SFC method was applied to clinical samples of human urine obtained after single intravenous (100 mg), single oral (100 mg), and repeated oral administration (400 mg). Flupirtine, D-13223, and 4-FHA could be quantified, shedding light on the extent of oxidative flupirtine metabolism in humans in the context of the unresolved biotoxification that has led to the withdrawal of specific neuronal KV7 openers.