Evaluation of greenness and analytical performances of separative methods for chiral separation of novel lactam-based P2RX7-antagonists.

In this work, a preparative supercritical fluid chromatography (SFC) method was first developed to separate a series of chiral compounds evaluated as lactam-based P2RX7 antagonists. Subsequently, high-performance liquid chromatography, SFC, and capillary electrophoresis (CE) were comparatively investigated as QC tools to determine the enantiomeric purity of the separated isomers, including analytical performance and greenness. The screening of the best conditions was carried out in liquid and SFC on the nine derivatives and the amylose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase was found to be highly efficient. The same screening was carried out in CE and very different conditions, either in acidic or basic background electrolyte and different cyclodextrins used as chiral selectors, allowed the separation of six of the nine derivatives. 1-((3,4-Dichlorophenyl)carbamoyl)-5-oxopyrrolidine-2-carboxylic acid (compound 1) was chosen as a probe, and its semi-preparative separation by SFC and enantiomeric verification using the three techniques are presented. Its limit of detection and limit of quantification are calculated for each method. Finally, the greenness of each quality control method was evaluated.

Mass spectrometry coupling of chip-based supercritical fluid chromatography enabled by make-up flow-assisted backpressure regulation.

This work introduces a novel microfluidic backpressure pressure control developed for chip-based supercritical fluid chromatography (chipSFC). The presented on-chip pressure control mechanism involves the post-column addition of a viscous make-up stream, which enables pressure regulation within the range of 73 to 130 bar range. In contrast to approaches using mechanical backpressure regulators, this chip-based make-up-assisted pressure regulation offers a wear-free alternative that functions entirely through fluidic means and contributes minimally to extra column volume. It prevents phase separation of the supercritical mobile phase and, therefore, expands the analytical scope of chipSFC to detection systems with an ambient pressure interface. This was demonstrated by a proof-of-principle experiment, where a model mixture was separated within 30 s and detected using atmospheric pressure ionisation mass spectrometry.

Exploring the application limits of different hold-up time markers in supercritical fluid chromatography.

The study focuses on the application range of nitrous oxide as a hold-up time marker in supercritical fluid chromatography (SFC). This compound has been suggested a decade ago to be used as unretained marker, something that the field of SFC was missing for a long time, since its beneficial properties make it an ideal candidate as hold-up time marker. Determination of the hold-up volume and actual volumetric flow rates have always been problematic in SFC due to the compressibility of carbon dioxide and one part of this is the difficulty of hold-up time measurements. Depending on the mobile phase, different methods have been used to measure the hold-up time with varying results. Nitrous oxide and other molecules have been compared in different conditions, mobile phases and stationary phases. In all cases, nitrous oxide gave the lowest elution times. However, detection was difficult in mobile phases containing 10% or more of organic modifier, because most solvents mask the signal of nitrous oxide. Interestingly, the choice of stationary phase also had a slight effect on detection, while different pressure and temperature settings affected each compound in a different manner.

Investigation into the performance and stability of immobilized and coated polysaccharide columns in supercritical fluid chromatography.

In this study, the performance of the widely used "golden four" coated chiral stationary phases (Chiralpak AD-3, Chiralcel OD-3, Chiralpak AS-3, and Chiralcel OJ-3) was compared with their corresponding immobilized versions (Chiralpak IA-3, Chiralpak IB-3, Chiralpak IB N-3, Chiralpak IH-3, and Chiralpak IJ-3) under supercritical fluid chromatography (SFC) conditions with a set of 30 racemic compounds. Using the traditional modifiers, methanol and isopropanol, the immobilized columns (Chiralpak IB N-3 and Chiralpak IH-3) showed an improved general ability to successfully resolve the enantiomers of the target analytes relative to their coated versions (Chiralcel OD-3 and Chiralpak AS-3), while the coated columns (Chiralpak AD-3, Chiralcel OD-3, and Chiralcel OJ-3) performed better than their immobilized versions (Chiralpak IA-3, Chiralpak IB-3, and Chiralpak IJ-3). An investigation of the non-traditional modifiers, dichloromethane, ethyl acetate, and tetrahydrofuran with immobilized columns, revealed a generally decreased ability to successfully resolve the enantiomers of the target analytes, relative to the use of the traditional modifiers, methanol and isopropanol. The stability of the coated columns (Chiralpak AD-H and Chiralcel OD-H) was evaluated by injecting "forbidden" solvents, including dichloromethane, dimethyl sulfoxide, and tetrahydrofuran. After 200 injections of these solvents on coated columns, the retention factors and resolutions slightly decreased, and a significant increase in column backpressure was observed, indicating some degree of stationary phase degradation.

Analytical and preparative chiral supercritical fluid chromatography resolutions using crown ether-derived column.

In this study, crown ether-derived column Crownpak® CR-I (+) was evaluated under SFC conditions using 12 primary amines, and the chromatographic results were compared against eight immobilized polysaccharide-based columns. Crownpak® CR-I (+) achieved a significantly higher success rate. It was found that the addition of 5% water to the modifier dramatically improved the peak shape for chiral separation of primary amines on Crownpak® CR-I (+). The first reported preparative SFC separations on Crownpak® CR-I (+) are shown, offering a new approach for the preparative resolution of primary amines. The case studies demonstrate that Crownpak® CR-I (+) is a very useful column in the chiral separation of challenging compounds that contain a primary amine group in the pharmaceutical industry.

Evaluation of supercritical fluid chromatography coupled to tandem mass spectrometry for the analysis of pesticide residues in grain.

A supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) technique was developed for the rapid and simultaneous detection of nine pesticides (carbendazim, isoprocarb, paclobutrazol, isoprothiolane, flusilazole, quinalphos, piperonylbutoxide, propargite, and bioresmethrin) in rice, wheat, and maize. The cereal samples were extracted with a solution of 0.5% acetic acid in acetonitrile and purified using quick, easy, cheap, effective, rugged, and safe method. The samples were characterized using multi-reaction monitoring and quantified with the external standard method. Excellent linearities (R2  > 0.9991) and limits of quantification (0.4-40.0 µg/kg) were established for all nine pesticides. Satisfactory pesticide recovery rates (62.2%-107.4%) were obtained at three standard concentrations (50, 100, and 200 µg/kg), with relative standard deviations in the range of 2.1%-14.3%. The results confirmed that the proposed method was suitable for the routine detection of these pesticides in grain samples. Compared with high-performance liquid chromatography-MS/MS, the overall test run time and the amount of solvent required were reduced by 66% and 90%, respectively, when SFC-MS/MS was applied. Therefore, the use of SFC-MS/MS permits a shorter run time and affords greater analytical efficiency, such that it is both economical and environmentally sustainable.

Chiral analysis and semi-preparative separation of metconazole stereoisomers by supercritical fluid chromatography and cytotoxicity assessment in vitro.

Metconazole is one of the widely-used chiral triazole fungicides in controlling wheat leaf rust, powdery mildew, Fusarium head blight with high efficacy, and so forth. In the current work, the effects of chiral stationary phases, alcoholic modifiers, and column temperature on the chiral separation of metconazole were discussed in detail. Amylose tris(3,5-dimethylphenylcarbamate)-coated chiral stationary phase exhibited much stronger chiral recognition ability toward metconazole stereoisomers in the CO2 /ethanol mixture as compared to the others. Then, a two-step semi-preparative separation of metconazole was performed through supercritical fluid chromatography and high-performance liquid chromatography, and the enantiomeric excess values of four stereoisomers were achieved over 98%. Moreover, the enantioselective cytotoxicity of cis-metconazole against HepG2 cells has been investigated, and the order of the cell proliferation toxicity against HepG2 cells was (1R, 5S)-metconazole > (1S, 5R)-metconazole > the mixture. Briefly, this study would provide valuable information in the preparative separation of optically pure metconazole products through chromatographic techniques and their environmental risk assessment.

Simultaneously characterization of multiple constituents in Valeriana jatamansi Jones using an online supercritical fluid extraction-high-performance liquid chromatography/supercritical fluid chromatography-mass spectrometry system.

Valeriana jatamansi Jones is a commonly used traditional Chinese medicine, boasting rich effective compositions with versatile chemical structures and wide polarity, including iridoids, chlorogenic acid, and flavonoids. Previous reports indicate that conventional high-performance liquid chromatography (HPLC) analytical methods have proven inefficient performance in comprehensively characterizing components in Valeriana jatamansi. In the present study, a hybrid online analytical platform combining supercritical fluid extraction with both conventional HPLC separation (reverse phase) and supercritical fluid chromatography (normal phase) has been established and validated. This system can provide online extraction with two different chromatographic separation modes to increase separation ability and has been connected to a mass spectrometer to acquire high-resolution mass spectrometry data. Then, the online platform was applied to screening components in Valeriana jatamansi. A total of 117 compounds were identified, including five lignans, 18 organic acids, six flavonoids, and 88 iridoids. Thirty-three compounds were reported from Valeriana jatamansi for the first time. These results enrich our understanding of the components of Valeriana jatamansi and prove that the developed online platform in this study is a robust approach for accelerating working efficiency in comprehensively analyzing complicated samples.

The Impact of Water as an Additive on the Elution of Some Basic Organic Compounds in Supercritical Fluid Chromatography.

In this study, water was used as an additive in the methanol-modified carbon dioxide-based eluent for the elution of some basic organic compounds from a hybrid silica column via supercritical fluid chromatography (SFC). The experiments were applied to sulfonamides, propranolol, and other organic nitrogen compounds involving aromatic rings from different classes of amine, pyrimidine, and purine with different pKa values (the pKa values for the studied analytes range from 4.6 to 10.4). The results revealed different responses to the different percentages of water addition. Adding 1~2% of water to the modifier (methanol) led to a positive effect manifested by more symmetrical peak shapes and reduced retention times for most compounds. The key factor for this improvement in the properties of chromatographic peaks is due to the adsorption of water on the silanol groups of the stationary phase, consequently resembling the phenomena observed in hydrophilic interaction liquid chromatography (HILIC). Moreover, the availability of hydrogen bond acceptor and donor sites in the analyte structure is an important factor to be considered when adding water as an additive to the modifier for improving the chromatographic peaks. However, introducing water in an amount higher than 3% resulted in perturbed chromatographic signals. It was also found that water as an additive alone could not successfully elute propranolol from the hybrid silica column with an acceptable peak shape; thus, the addition of a strong base such as amine salts was also necessary. The proposed use of a particular amount of water in the mobile phase could have a positive effect compared to the same mobile phase without water, improving the chromatographic peak properties of the elution of some basic organic compounds from the hybrid silica column.

Ternary eluent compositions in supercritical fluid chromatography improved fingerprinting of therapeutic peptides.

Currently, little information has been published on the application of ternary eluent compositions in supercritical fluid chromatography for separating peptides. This work investigates the benefits of adding acetonitrile to methanol as the modifier. Three cyclic antibiotic peptides (bacitracin, colistin, and daptomycin) ranging between 1000 and 2000 Da were chosen as model substances. The ternary mixture of carbon dioxide, methanol, and acetonitrile is optimized to increase the resolution of the peptide's fingerprint. In addition, varying compositions of methanol and acetonitrile were found to change the elution order of the analytes, which is a valuable tool during method development. An individual gradient method using two Torus 2-PIC columns (each 100 × 3.0 mm, 1.7 µm), carbon dioxide, and a modifier consisting of acetonitrile/methanol/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v) was optimized for each of the peptides. Subsequently, a generic method development protocol applicable to polypeptides is proposed.

Separation of diarylethene-based photoswitchable isomeric compounds by high-performance liquid chromatography and supercritical fluid chromatography.

Diarylethene-based photoswitches have become very popular over the last few decades for potential applications in chemistry, materials science, and biotechnology due to their unique physical and chemical properties. We report the isomeric separation of a diarylethene-based photoswitchable compound using high-performance liquid chromatography. The separated isomers were characterized by ultraviolet-visible spectroscopy and mass spectrometry confirmed the isomeric nature of the compounds. The isomers were purified by preparative high-performance liquid chromatography, providing fractionated samples to study the isomers individually. A total amount of 13 mg of an isomer of interest was fractionated from a solution of 0.4 mg/ml of the isomeric mixture. Because the preparative high-performance liquid chromatographic method required large quantities of solvent, we explored the use of supercritical fluid chromatography as an alternative separation mode which, to the best of our knowledge, is the first time this technique is used to separate diarylethene-based photoswitchable compounds. Supercritical fluid chromatography provided faster analysis times while maintaining sufficient baseline resolution for the separated compounds and consuming less organic solvent in the mobile phase compared to high-performance liquid chromatography. It is proposed that the supercritical fluid chromatographic method be upscaled and used in future fractionation of the diarylethene isomeric compounds, becoming a more environmentally benign approach for compound purification.

Ultra-high-performance supercritical fluid chromatography-mass spectrometry for the analysis of organic contaminants in sediments.

A nontarget screening method was developed based on D-optimal designs for ultra-high performance supercritical fluid chromatography with positive and negative electrospray ionization mode mass spectrometry. A mixture of organic contaminants such as pesticides, steroids, surfactants, phenolic and fatty acids, and polycyclic aromatic hydrocarbon derivatives, was used for the optimization. An aprotic mixture of dichloromethane and acetone [3:1] performed overall best as the injection solvent. The highest peak capacities (n) were accomplished at the shallowest gradient (1%B/min), ammonium formate (n = 378 in negative ionization mode), or ammonium acetate (n = 327 in positive ionization mode) in methanol as the modifier. Capillary voltage, make-up solvent flow rate, water, and additive concentration were the most significant factors for improving peak intensity: higher peak intensities were obtained at lower additive concentrations (5mM ammonium formate), and with 5% water in positive ionization mode. Conversely, water had detrimental effects in negative ionization mode. The optimized method was used to quantify organic contaminants in 17 freshwater sediment samples from Copenhagen, Denmark. Out of 50 monitored contaminants, 35 were detected in at least one sample. Further, the method has a potential for target and nontarget screening analysis of organic contaminants in solid matrices.

An alternative strategy based on ultra-high-performance supercritical fluid chromatography for full monosaccharide compositional analysis of polysaccharides in Schisandra chinensis fruits.

Due to green and environment-friendly characteristics, ultra-high-performance supercritical fluid chromatography has been widely used in analytical fields in recent years, but until now few reports are available for monosaccharide compositional analysis of macromolecule polysaccharides. In this study, an ultra-high-performance supercritical fluid chromatography technology with an unusual binary modifier is used to determine the monosaccharide compositions of natural polysaccharides. Each carbohydrate herein is simultaneously labeled as 1-pheny-3-methyl-5-pyrazolone and acetyl-derivative via pre-column derivatizations aiming to increase UV absorption sensitivity and decrease water solubility. Ten common monosaccharides are fully separated and detected on ultra-high-performance supercritical fluid chromatography combined with a photo-diode array detector by systematic optimization of multiple relevant parameters, for example, column stationary phases, organic modifiers, additives, flow rates, and so on. Compared with carbon dioxide as a mobile phase, the addition of a binary modifier increases the resolution of analytes. Additionally, this method has the advantages of small consumption of organic solvent, safety, and being environmental-friendly. It has been successfully applied for full monosaccharide compositional analysis of heteropolysaccharides from Schisandra chinensis fruits. To sum up, a new alternative approach is provided for monosaccharide compositional analysis of natural polysaccharides.

Separation and determination of triadimefon and its metabolites triadimenol enantiomers in fruit puree by supercritical fluid chromatography.

A method was established for the separation and determination of triadimefon and its metabolite triadimenol enantiomer residues in major complementary fruit puree for infants and young children (banana puree, pineapple puree, and grape puree) by supercritical fluid chromatography. After the samples were extracted with acetonitrile and purified with a solid phase extraction cartridge, Acquity Trefoil CEL2 chiral chromatographic column was adopted for separation, and gradient elution was conducted at the flow rate of 1.0 ml/min under the mobile phase of supercritical carbon dioxide - 0.5% ammonia methanol, the detection wavelength was 220 nm and quantification was conducted with the external standard method. The limits of quantitation of triadimefon and triadimenol enantiomers were both 0.05 mg/kg, the linear ranges were 0.5-50 mg/L, and the linear correlation coefficients were greater than 0.9993. The recoveries in the spiked samples at 0.05, 0.2, and 3.0 mg/kg were from 80.1 to 106%, and the relative standard deviation reached 3.3-7.6%. The method is efficient, rapid, reproducible, and environmentally friendly, enabling accurate analysis of pesticide enantiomers, which can detect the enantiomer residues of triadimefon and its metabolite triadimenol in major complementary fruit puree for infants and young children.

Controlling the positive ion electrospray ionization of poly(ethylene glycols) when using ultra-high-performance supercritical fluid chromatography-mass spectrometry.

Poly(ethylene glycols) are complex polymers often added to pharmaceutical formulations to improve drug solubility and delivery. One of the main challenges when using chromatographic techniques coupled to mass spectrometry is the unselective ionization of poly(ethylene glycols) oligomers. Additionally, when the chain length is large enough, multiple charged species are formed, further complicating the mass spectra and processing. This study uses the advanced oligomer separation provided by supercritical fluid chromatography with a mass spectrometry approach that selectively ionizes poly(ethylene glycols) as ammoniated molecules to simplify data analysis and facilitate batch-to-batch comparisons. Several visual representations of the response of the ionization events based on the polymer molecular weight and the repeating unit were used to elucidate trends in ionization. Evaluation of the influence of the oligomer length and end-group on the electrospray ionization of the polymer allowed the development of a process to enable selective ionization for these complex polymers.

Use of N-(4-aminophenyl)piperidine derivatization to improve organic acid detection with supercritical fluid chromatography-mass spectrometry.

The analysis of organic acids in complex mixtures by LC-MS can often prove challenging, especially due to the poor sensitivity of negative ionization mode required for detection of these compounds in their native (i.e., underivatized or untagged) form. These compounds have also been difficult to measure using supercritical fluid chromatography (SFC)-MS, a technique of growing importance for metabolomic analysis, with similar limitations based on negative ionization. In this report, the use of a high proton affinity N-(4-aminophenyl)piperidine derivatization tag is explored for the improvement of organic acid detection by SFC-MS. Four organic acids (lactic, succinic, malic, and citric acids) with varying numbers of carboxylate groups were derivatized with N-(4-aminophenyl)piperidine to achieve detection limits down to 0.5 ppb, with overall improvements in detection limit ranging from 25-to-2100-fold. The effect of the derivatization group on sensitivity, which increased by at least 200-fold for compounds that were detectable in their native form, and mass spectrometric detection are also described. Preliminary investigations into the separation of these derivatized compounds identified multiple stationary phases that could be used for complete separation of all four compounds by SFC. This derivatization technique provides an improved approach for the analysis of organic acids by SFC-MS, especially for those that are undetectable in their native form.

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

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

Extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance using supercritical fluid extraction followed by supercritical fluid chromatography.

In this paper, an off-line combination method of supercritical fluid extraction and supercritical fluid chromatography was developed for the selective extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance. The enrichment of target components was successfully achieved using supercritical fluid extraction with the following conditions (8% ethanol as co-solvent at 45°C and 30 MPa for 30 min). Taking full advantage of the complementarity of supercritical fluid chromatography stationary phases, a two-step preparative supercritical fluid chromatography strategy was constructed. The extract was firstly divided into seven fractions on a Diol column (250 × 20 mm internal diameter, 10 µm) within 8 min by gradient elution increasing from 5% to 20% modifier (methanol) at 55 ml/min and 15 MPa. Then the seven fractions were separated by using a 1-AA or a DEA column (250 × 19 mm internal diameter, 5 µm) at 50 ml/min and 13.5 MPa. This two-step strategy showed superior separation ability for structural analogs. As a result, seven compounds, including four diphenylheptanes and three flavonoids with high purity, were successfully obtained. The developed method is also helpful for the extraction and isolation of other structural analogs of traditional Chinese medicines.

Comparison of supercritical fluid chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry for the stereoselective analysis of chlorfenvinphos and dimethylvinphos in tobacco.

This study used reversed-phase liquid chromatography-tandem mass spectrometry and supercritical fluid chromatography-tandem mass spectrometry for determination of the stereoisomers of chlorfenvinphos and dimethylvinphos in tobacco. Tobacco samples were extracted and purified with a modified quick, easy, cheap, effective, rugged, and safe technique using spherical carbon. The performance of both methodologies was comprehensively compared in terms of methods validation parameters (separation efficiency, linearity, selectivity, recovery, repeatability, sensitivity, matrix effect, etc.). Under optimized conditions, the calibration curves of the stereoisomers of chlorfenvinphos and dimethylvinphos in the range of 10-500 ng/mL showed excellent linearity with R2 ≥ 0.997 in both methods. The adequate recoveries of analytes from three different spiked tobaccos were obtained using reversed-phase liquid chromatography-tandem mass spectrometry (86.1-95.7%) as well as supercritical fluid chromatography-tandem mass spectrometry (86.5-94.0%). The relative standard deviations for spiked samples were all below 7.0%. Compared with supercritical fluid chromatography-tandem mass spectrometry, lower matrix effects and LODs can be obtained in reversed-phase liquid chromatography-tandem mass spectrometry.

A highly sensitive method (supercritical fluid chromatography coupled with ion mobility mass spectrometry) for determination of multiple compounds in radix curcumae.

A highly sensitive method was developed for simultaneously separating and identifying multiple compounds in radix curcumae. The determination of these compounds was achieved by combining supercritical fluid chromatography with drift tube ion mobility quadrupole time-of-flight MS. Related parameters were optimized: the RX-SIL column was used as the stationary phase, methanol was selected as the organic modifier, back pressure was 120 bar, back temperature was 60°C, the mobile phase flow rate was 1.75 mL/min, the makeup solvent was 0.2% formic acid/methanol with a flow rate of 0.7 mL/min. Under optimal conditions, multipolar compounds were separated. Furthermore, these compounds were identified by the values of collision sectional areas. The established method was verified by related parameters and exhibited good linearity, sensitivity, precision and accuracy. It could be extended to analyze other curcuminoids and sesquiterpenoids in natural products.