Supercritical Fluid Chromatography Method Research Articles

Automated multifactorial design of experiment and Bayesian optimisation algorithm approaches to method development for the green analysis by supercritical fluid chromatography of a pharmaceutical ingredient

During drug development, chromatography is frequently used for purity and stability testing of both drug substance and drug product. Reversed phase liquid chromatography (RPLC) is one of the most widely used methodologies due to its wide scope of application. In the later stages of drug development, the specified impurities and degradation products that define the critical quality attribute of the final API, also known as Key Predictive Sample Set (KPSS), are usually well defined and controlled. At this point, a method review enables selecting the most appropriate technique which should be the one providing optimal robustness (ICH-Q14[1]), with the support of Quality by Design (QbD) approaches. Supercritical Fluid Chromatography (SFC) is a preferred technique for its proven diversity in selectivity. The adoption of a technique which presents the most favourable environmental impact, such as, but not limited to, SFC, is also becoming increasingly important as laboratories strive to reduce carbon footprint. Re-developing a method requires high resource-demands in terms of staff, materials, and time. Any step of the process that can be automated can facilitate this approach, speeding up the delivery of the method whilst preserving robustness.In this article we describe how an SFC method was developed for the purity profiling of a late-stage oncology candidate, taking advantage of the superior selectivity of SFC towards structurally similar analytes, owed to the high orthogonality with R2 as low as 0.014 towards the KPSS. We describe two approaches to automate the method development. Firstly, a multifactorial design of experiments (DoE) and secondly, an optimization via a Bayesian algorithm, which was completed in one night, highlighting the potential and limitations, with an insight into the robustness. Both methods achieved baseline separation with varying levels of automation embedded into the process and a large reduction of the resource demands when compared to traditional optimisation methods. Finally, we describe the beneficial environmental impact that implementing SFC methods can yield, with a calculated green score reduced to a value between 17 and 30 % compared to RPLC, depending on the number of runs per sequence.

Large-scale purification of a deprotected macrocyclic peptide by supercritical fluid chromatography (SFC) integrated with liquid chromatography in discovery chemistry

A macrocyclic peptide A was successfully purified in large quantities (∼30 g) in >95 % purity by an integrated two-step orthogonal purification process combining supercritical fluid chromatography (SFC) with medium-pressure reverse-phase liquid chromatography (MP-RPLC). MP-RPLC was used to fractionate the crude peptide A, remove unwanted trifluoroacetic acid (TFA) originating from the peptide A cleavage off the resin, and convert the peptide A into ammonium acetate salt form, prior to the final purification by SFC. A co-solvent of methanol/acetonitrile containing ammonium acetate and water in CO2 was developed on a Waters BEH 2-Ethylpyridine column. The developed SFC method was readily scaled up onto a 5 cm diameter column to process multi-gram quantities of the MP-RPLC fraction to reach > 95 % purity with a throughput/productivity of 0.96 g/h. The incorporation of SFC with MP-RPLC has been demonstrated to have a broader application in other large-scale polypeptide purifications.

Hyphenation of supercritical fluid chromatography and trapped ion mobility-mass spectrometry for quantitative lipidomics

BackgroundLipidomics studies require rapid separations with accurate and reliable quantification results to further elucidate the role of lipids in biological processes and their biological functions. Supercritical fluid chromatography (SFC), in particular, can provide this rapid and high-resolution separation. The combination with trapped ion mobility spectrometry (TIMS) has not yet been applied, although the post-ionization separation method in combination with liquid chromatography or imaging techniques has already proven itself in resolving isomeric and isobaric lipids and preventing false identifications. However, a multidimensional separation method should not only allow confident identification but also provide quantitative results to substantiate studies with absolute concentrations. ResultsA SFC method was developed and the hyphenation of SFC and TIMS was further explored towards the separation of different isobaric overlaps. Furthermore, lipid identification was performed using mass spectrometry (MS) and parallel accumulation serial fragmentation (PASEF) MS/MS experiments in addition to retention time and collision cross section (CCS). Quantification was further investigated with short TIMS ramps and performed based on the ion mobility signal of lipids, since TIMS increases the sensitivity by noise filtering. The final method was, as an exemplary study, applied to investigate the function of different ceramide synthases (CerS) in the nematode and model organism Caenorhabditis elegans (C. elegans). Loss of three known CerS hyl-1, hyl-2 and lagr-1 demonstrated different influences on and alterations in the sphingolipidome. SignificanceThis method describes for the first time the combination of SFC and TIMS-MS/MS, which enables a fast and sensitive quantification of lipids. The results of the application to C. elegans samples prove the functionality of the method and support research on the metabolism of sphingolipids in nematodes.

Analytical Method Lifecycle of SFC Methods from Development Use to Routine QC Implementation; Supporting Small Molecule R&D and Commercialization

Selection of the correct analytical technique from the outset of method development is key in securing the optimal and most robust method to be used throughout a product’s development journey from R&D to commercialization. In this article, we review some key considerations for chromatographic technique selection and method development across the full drug process—from early-stage API synthesis to routine commercial release activities. We describe the implementation of supercritical fluid chromatography (SFC) in the pharmaceutical industry, especially for chiral, water sensitive analytes, and low to high LogP and LogD hydrophobic compounds, while demonstrating high levels of method robustness and support of “green” analytics.

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

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

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.

Differentiating Between Three Licorice Species Using SFC-TOF/MS Analysis With Principal Component Analysis

Objective Although the discrimination of crude drugs by metabolomics analysis using liquid chromatography–mass spectrometry (LC–MS) has been widely reported, there are few reports on natural products analysis using supercritical fluid chromatography (SFC)–MS. This study aimed to compare the results of metabolomic analysis of licorice obtained using SFC–MS and LC–MS. Methods We analyzed 14 CHCl3 extracts of licorice roots ( Glycyrrhiza glabra, G. uralensis, and G. inflata) using SFC-time-of-flight (TOF)/MS and LC-TOF/MS in conjunction with principal component analysis (PCA). The three species were assessed for discrimination based on the PCA results. The characteristic constituents of each variety were purified, and their chemical structures were elucidated using various spectroscopic analyses. Results The PCA score plots of the results of both SFC and LC classified the licorice samples into three groups. Seven compounds (six known compounds and one new phenolic compound, glyglabone) were isolated from G. glabra, five known compounds were isolated from G. uralensis, and four known compounds were isolated from G. inflata. Conclusions The 16 isolated compounds may be characteristic compounds in the three licorice species. The SFC and LC methods showed comparable results in distinguishing the three species. SFC was highly effective for directly analyzing CHCl3 extracts, reducing analysis time and organic solvent consumption by over 50% compared to the LC analysis. Thus, SFC-TOF/MS in combination with PCA can be used to differentiate between G. glabra, G. uralensis, and G. inflata.

Development of an ultra-high-performance supercritical fluid chromatography method for the analysis of phenols in the pyrolysis aqueous fraction.

The pyrolysis process consists of the thermal decomposition of biomass in an inert atmosphere, which produces a liquid (bio-oil) composed of a complex mixture of organic compounds, including an oil and water phase. The aqueous fraction can reach up to 45% w/w, and understanding its composition is of utmost importance in determining its intended destination, whether for the reuse of compounds in industrial applications or for treating the effluent for disposal. In this study, a fast, direct, and efficient method using ultra-high-performance supercritical fluid chromatography (UHPSFC) was developed and optimized for monitoring phenols in aqueous samples obtained from the pyrolysis processing of six different biomass sources. The following parameters were evaluated for method optimization: stationary phase type, mobile phase flow, organic modifier, sample diluent, temperature, pressure, and modifier gradient time. With a total analysis time of 26min, out of the fourteen (14) investigated phenolic compounds, eleven (11) were successfully separated after method optimization, and among them, five (5) were quantified in all six aqueous fractions. The aqueous fractions of residue from cowpea pod (1.89mg.mL-1), sugar apple (3.09mg.mL-1), and acerola (4.79mg.mL-1) presented lower concentrations compared to grape (8.16mg.mL-1), pine nuts (6.68mg.mL-1), and guava (6.05mg.mL-1) fractions. However, even at lower concentrations, all biomasses showed promising results regarding the phenolic compound content, analytes that have high added value for the chemical industry.

Facilitated on-line supercritical fluid extraction - supercritical fluid chromatography for nonpolar and polar compounds from milk thistle seeds

Plant seeds, as those from milk thistle (Silybum marianum), are a valuable source of nonpolar and polar compounds with potentially interesting biological activity. The main nonpolar compounds are triglycerides, which are also the main components of all vegetable oils. In addition, specific polar compounds – flavonolignans, called silymarin, have been found in large amounts in milk thistle seeds extract. These flavonoids derivatives have different biological activity, for instance hepatoprotective effects. In order to extract and analyze both nonpolar (triglycerides) and polar compounds (flavonolignans) from milk thistle seeds through a sequential methodology, an on-line supercritical fluid extraction - supercritical fluid chromatography (SFE-SFC) method was developed. Different ways of transferring the extracts from SFE to SFC (i.e. direct on-column transfer and loop transfer) were compared, and particularly for their effect on chromatographic quality. In this respect, nonpolar and polar compounds caused different issues, especially as polar compounds required a significant portion of co-solvent in the extraction step, favoring early elution in the chromatographic column. First, on-line SFE-SFC was used for triglycerides analysis and allowed the comparison of transfer modes. Then, on-line kinetics were performed to measure defatting time before polar molecules extraction. Finally, the eventual benefit of loop transfer was also investigated for the analysis of flavonolignans, polar molecules whose analysis can be difficult by on-line SFE-SFC. The aim of this paper is to discuss the versatility of on-line SFE-SFC and how challenging the coupling can be, especially when both non-polar and polar molecules must be analyzed independently in a single sample.

Supercritical fluid chromatography based on reversed-phase/ ion chromatography mixed-mode stationary phase for separation of spirooxindole alkaloids

The present paper illustrates the versatility of the supercritical fluid chromatography (SFC) since, for the first time, four spirooxindole alkaloids (SOAs) including two pairs of isomers were separated by using two types of reversed-phase/ ion chromatography (RP/IC) mixed-mode stationary phases. Two mixed-mode stationary phases (C8SAX and C8SCX) was simultaneously provided dispersive and electrostatic interactions, which were suitable for the separation of such alkaloids. This study tried to provide an in-depth understanding of the SFC separation mechanism of the mixed-mode stationary phase through investigation of the impact of changes in mobile phase composition on alkaloids’ retention behavior. On C8SAX, due to the strong electrostatic repulsion, there was a very narrow elution window of the alkaloids, of which behaviors were hardly affected by adding diethylamine in mobile phase. When adding formic acid or acidic ammonium formate, the prolonged retention time of alkaloids was presented because of the shielded effect of formate anions on the electrostatic repulsion. In particular, better peak shape and improved resolution were obtained by using acidic ammonium formate due to the deactivation of silanol groups by ammonium cations. On the other hand, both formic acid and acidic ammonium formate can strengthen the electrostatic attraction of C8SCX, causing difficult elution of the alkaloids. Ammonium cations from either the protonated diethylamine or the ionized ammonium formate, were considered as counter ions to effectively mask the electrostatic attraction of C8SCX, to significantly reduce the retention of alkaloids, but improve the resolution. Finally, utilizing two developed SFC methods, i.e., C8SAX with EtOH+ 10 mM acidic ammonium formate in CO2, or C8SCX with EtOH+0.1% diethylamine in CO2, the baseline separation of corynoxeine and isocorynoxeine, rhynchophylline and isorhynchophylline was achieved within 5 min.

Rapid enantiomeric analysis of zopiclone in serum by supercritical fluid chromatography-tandem mass spectrometry.

Zopiclone (ZOP) is a hypnotic drug prescribed to treat insomnia. Due to the chiral nature of ZOP, the psychologically active S-form and inactive R-form need to be determined enantiomerically in a forensic drug analysis. In the present study, a supercritical fluid chromatography (SFC) method was designed with a faster analysis ability than that of previously reported techniques. The SFC-tandem mass spectrometry (SFC-MS/MS) method was optimized using a column with a chiral polysaccharide stationary phase (Trefoil CEL2). ZOP was extracted from pooled human serum using solid-phase extraction (Oasis HLB) and analyzed. The developed SFC-MS/MS method achieved the baseline separation of S-ZOP and R-ZOP within 2 min. The fit-for-purpose method validation indicated that the optimized solid-phase extraction achieved near complete recovery and approximately 70% of the matrix effect. Both the retention time and peak area showed sufficient precision. The lower and upper limits of quantification (LOQ) were 5.7 × 10-2 ng/mL and 25 ng/mL for R-ZOP, and 5.2 × 10-2 ng/mL and 25 ng/mL for S-ZOP. The calibration line was linear in the range from lower LOQ to upper LOQ. The stability test indicated that ZOP in serum stored in a refrigerator (4°C) degraded and about 55% remained in 31 days. The quick analysis of the SFC-MS/MS method makes it a valid option for the enantiomeric analysis of ZOP.

Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of four target flavonoids components in citrus samples

AbstractA new method for the analysis of four target flavonoids in two kinds of citrus samples by ultra-high performance supercritical fluid chromatography (UHPSFC) method was developed. Main variables affecting the UHPSFC separation were optimized, and under the optimized conditions the four target compounds (tangeretin, nobiletin, hesperetin and naringenin) can be separated within 10 min. The UHPSFC method allowed the determination of the four target compounds in the diluted stock solutions with limit of detection (LOD) ranging from 1.08 to 2.28 μg mL−1, and limit of quantification (LOQ) ranging from 1.45 to 4.52 μg mL−1, respectively. The coefficients of determination (R2) of the calibration curves were higher than 0.9950. The recoveries of the four target compounds at three different concentrations were in the range of 82.4–117.6%. The validation results demonstrated that the proposed method is simple, accurate, time-saving and environment friendly, and it is applicable to a variety of complex samples such as medicine-food dual purpose herbs and functional foods.

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 13mg of an isomer of interest was fractionated from a solution of 0.4mg/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.

Comparison of supercritical fluid chromatographic methods to predict the skin permeability of pharmaceutical and cosmetic compounds

Supercritical fluid chromatography (SFC) was explored as an alternative for liquid chromatography to predict the skin permeability of pharmaceutical and cosmetic compounds. Nine dissimilar stationary phases were applied to screen a test set of 58 compounds. The experimental retention factors (log k), in addition to two sets of theoretical molecular descriptors, were applied to model the skin permeability coefficient. Different modelling approaches, i.e. multiple linear regression (MLR) and partial least squares (PLS) regression, were used. In general, the MLR models performed better than the PLS models for a given descriptor set. The results obtained on a cyanopropyl (CN) column provided the best correlation with the skin permeability data. The retention factors obtained on this column were included in a simple MLR model, together with the octanol-water partition coefficient and the number of atoms (r² = 0.81, RMSEC = 0.537 or 20.5% and RMSECV = 0.580 or 22.1%). The overall best MLR model included the chromatographic descriptor from a phenyl column and 18 descriptors (r² = 0.98, RMSEC = 0.167 or 6.2% and RMSECV = 0.238 or 8.9%). This model showed a good fit, on top of very good predictive features. However, stepwise MLR models with a reduced complexity could also be determined, with the best performance parameters obtained with the CN-column based retention and eight descriptors (r² = 0.95, RMSEC = 0.282 or 10.7% and RMSECV = 0.353 or 13.4%). SFC thus provides a suitable alternative to the liquid chromatographic techniques previously applied to model the skin permeability.

Chiral separation of α-tocopherol and α-tocopheryl acetate by supercritical fluid chromatography for accurate vitamin E nutrition labeling

Separations of respective stereoisomers of α-tocopherol and α-tocopheryl acetate by supercritical fluid chromatography (SFC) using polysaccharide-based chiral stationary phases (CSP) were studied. Two separate methods have been developed for α-tocopherol and α-tocopheryl acetate with total runtime of 35 min and 15 min, respectively. Under the optimized conditions, all-rac-α-tocopherol is separated into four peaks (peak area ratio 5:1:1:1) on an amylose tris(3,5-dimethylphenylcarbamate) CSP, and all-rac-α-tocopheryl acetate is separated into 2 peaks with equal peak areas on a cellulose tris(3,5-dimethylphenylcarbamate) CSP. The characteristic peak profiles of these racemic mixtures are distinctive enough to differentiate synthetic vitamin E (racemate) from the naturally occurring vitamin E (RRR-stereoisomer only). The α-tocopherol method has been demonstrated free of interference from other natural tocopherols, such as β-, γ-, and δ-tocopherol. Evaluation of the α-tocopherol method with commercially available vitamin E dietary supplements and an infant formula showed acceptable results in the qualitative analysis of α-tocopherol. These SFC methods provide fast, simple, and “green” solutions to verify or determine the type of vitamin E for dietary supplement and food manufacturers to ensure accurate vitamin E nutrition label claims.

An accurate and reliable analytical strategy for simultaneous determination of target furanocoumarins and flavonoids in cosmetic and pharmaceutical samples by ultra-high performance supercritical fluid chromatography

Furanocoumarins and flavonoids have various important biological activities and wide application. In the present study, a rapid and reliable supercritical fluid chromatography method was proposed for the separation of 10 target components including 8 furanocoumarins and 2 flavonoids. After detailed condition optimization, the 10 target compounds can be baseline separated on a Trefoil CEL1 (3.0 mm × 150 mm, 2.5 µm) column using gradient elution. A 0.07% (v/v) trifluoroacetic acid in ethanol was determined to be the most proper mobile phase for the separation of target compounds. The column temperature, back pressure, flow rate were set at 36 ℃, 2000 psi, 1.0 mL min−1 to 1.4 mL min−1, respectively. The ten target compounds were analyzed within 24 min using the optimized conditions. Under the optimized conditions, all the target compounds showed good linearity with linear correlation coefficients higher than 0.995, and satisfactory recovery in the range of 83.52–112.92%. All these results showed that the developed ultra-high performance supercritical fluid chromatography method was reliable and effective. Finally, the application of the developed method to cosmetic, Psoraleae fructus and Angelicae dahuricae radix samples were presented. The results highlight the applicability of the ultra-high performance supercritical fluid chromatography method to the analysis of interested compounds in pharmaceutical and cosmetic samples.

Development of a Preparative Method for Obtaining an Enantiomerically Pure Substance of Salbutamol by Supercritical Fluid Chromatography

Introduction. An increasing number of studies conducted in various countries in the field of biopharmacy convincingly show the differences in the physiological effects on the human body of stereoisomers of pharmaceutical substances. As a rule, one of the enantiomers has the necessary pharmacological effect, the other enantiomer is either inert or has a negative side effect. Currently, it is important to obtain enantiomerically pure pharmacological substances from their racemic mixtures.Aim. Obtaining the R-isomer of salbutamol from a racemic mixture of salbutamol and developing a preparative procedure for chiral separation by supercritical fluid chromatography (SFC) with a yield of the target product sufficient for the production process.Materials and Methods. Supercritical fluid chromatography (SFC) is widely used for analytical and preparative separations of enantiomers of pharmaceutical substances. The relevance of the use of supercritical fluid chromatography SFC is largely due to the fact that it uses sub- or supercritical carbon dioxide (SC-CO2) as the main component of the mobile phase (MP). Studies were carried out on semi-preparative Investigator SFC (Waters Corporation, USA) and preparative Prep 200 qSFC (Waters Corporation, USA) supercritical fluid chromatographs with PDA detectors. Samples were weighed to the nearest 0.0001 g on a XPE206DR balance (Mettler Toledo, USA).Results and discussion. The process of chiral separation of salbutamol sulfate by the SFC method on Prep 200 qSFC (Waters Corporation, USA) was studied. It was revealed that in the chromatographic system under supercritical conditions, the salt is separated into acidic and basic residues, which significantly reduces productivity and shortens the duration of continuous operation of the chromatograph. Conditions for the preparative chiral separation of the racemic mixture of salbutamol base into R- and S-isomers with high enantioselectivity and productivity have been developed. The resulting R-isomer of salbutamol base can be converted into the pharmaceutical substance in the form of sulfate or other salt without loss of enantiomeric purity, the S-isomer can be subjected to racemization and subsequent use.Conclusion. A preparative method has been developed for the chiral separation of a racemic mixture of salbutamol by supercritical fluid chromatography (SFC) with a yield of the target product (R-isomer) of 5.5 g per shift (8 hours).

Development of a Chiral Supercritical Fluid Chromatography-Tandem Mass Spectrometry and Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry Platform for the Quantitative Metabolic Profiling of Octadecanoid Oxylipins.

Octadecanoids are broadly defined as oxylipins (i.e., lipid mediators) derived from 18-carbon fatty acids. In contrast to the well-studied eicosanoids, there is a lack of analytical methods for octadecanoids, hampering further investigations in the field. We developed an integrated workflow combining chiral separation by supercritical fluid chromatography (SFC) and reversed-phase liquid chromatography (LC) coupled to tandem mass spectrometry detection for quantification of a broad panel of octadecanoids. The platform includes 70 custom-synthesized analytical and internal standards to extend the coverage of the octadecanoid synthetic pathways. A total of 103 octadecanoids could be separated by chiral SFC and complex enantioseparations could be performed in <13 min, while the achiral LC method separated 67 octadecanoids in 13.5 min. The LC method provided a robust complementary approach with greater sensitivity relative to the SFC method. Both methods were validated in solvent and surrogate matrix in terms of linearity, lower limits of quantification (LLOQ), recovery, accuracy, precision, and matrix effects. Instrumental linearity was good for both methods (R2 > 0.995) and LLOQ ranged from 0.03 to 6.00 ng/mL for SFC and 0.01 to 1.25 ng/mL for LC. The average accuracy in the solvent and surrogate matrix ranged from 89 to 109% in SFC and from 106 to 220% in LC, whereas coefficients of variation (CV) were <14% (at medium and high concentrations) and 26% (at low concentrations). Validation in the surrogate matrix showed negligible matrix effects (<16% for all analytes), and average recoveries ranged from 71 to 83%. The combined methods provide a platform to investigate the biological activity of octadecanoids and expand our understanding of these little-studied compounds.

Chiral separation of conazole pesticides using supercritical fluid chromatography

ABSTRACT Supercritical fluid chromatographic methods for enantiomer separation of six conazole-type antifungal pesticides (bromuconazole, cyproconazole, prothioconazole, tebuconazole, ipconazole, triticonazole) are presented in this work. Screening tests on six different chiral analytical columns (Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralpak AS, Chiracel OJ and Kromasil Cellucoat) were carried out using methanol as organic modifier. Cellulose-based stationary phases were found to be better for separation of each tested conazole, than the amylose-based columns. The influence of the most commonly used co-solvents (methanol, ethanol and 2-propanol) on the separation was also studied on selected columns. Suitable analytical methods were found for enantiomer separations for all studied conazoles; moreover, the methods are good starting points to semi-preparative separation. The importance of sample solvent selection in the semi-preparative separation of triticonazole isomers was proved. Acetonitrile as sample solvent resulted in a narrow, nice peaks compared to those obtained with methanol or dichloromethane. Twenty milligrams of each enantiomer of triticonazole was collected for ecotoxicity testing using the analytical size column.

Chiral separation and characterization of methoxphenidine – A popular street drug

Methoxphenidine (MXP) belongs among dissociative anesthetics from the group of new psychoactive substances. According to the unofficial user experiences, MXP is less active than common dissociative anesthetics like ketamine, yet, several intoxications were associated with this substance up to this date. Within our previous work, we discovered that at least some MXP samples on the drug market may contain zincate complex which we believe may be responsible for the unusual properties of the purported HCl salts. MXP comprise a stereogenic center in its molecular structure, which is disregarded by the manufacturers and, therefore, MXP is available on the drug scene as a racemic mixture only. However, the differences between the effects and activity of the enantiomers remain to be discovered. Hereby we present a chiral resolution by crystallization, which we used to obtain both the enantiomers of MXP. To control their optical purity, we have developed a new supercritical fluid chromatography (SFC) method. Finally, a combination of electron circular dichroism (ECD) and vibrational circular dichroism (VCD) spectra supported by density functional theory (DFT) calculation has been used to assign the absolute configuration of both enantiomers. Racemic Methoxphenidine hydrochloride was prepared in-house. Then individual enantiomers were obtained by process of crystallization using chiral (−) or (+)-2,3-dibenzoyl-tartaric acid monohydrate. An Acquity UltraPerformance Convergence ChromatographyTM (UPC2) from Waters (Milford, MA, USA) was used for HPLC and SFC enantioseparation. For control of the purity of the obtained enantiomers after crystallization, a HPLC method has been used. To improve efficiency of the existing chiral HPLC method, an SFC enantioseparation method was develop. Four polysaccharide-based CSPs (Alcyon SFC CSP Amylose-SA, Cellulose-SB and Cellulose-SC and CHIRALPAK® IE-3) and CO2-containing mobile phases were employed. The ECD and UV absorption spectra were obtained on a Jasco J-815 spectrometer (Jasco, Japan) in a quartz cuvette (Hellma, Germany) with an optical pathlength of 1 mm. The VCD and IR absorption spectra were measured on Fourier transform infrared spectrometer IFS 66/S equipped with a PMA 37 VCD/IRRAS module (both Bruker, Germany), BaF2 polarizer, and MCT detector (InfraRed Associates, USA). Measurements were carried out in a BioCell cuvette with CaF2 windows (Biotools, Inc, USA) and an optical pathlength of 27.3 μm. DFT simulations were performed to find the stable structures of MXP in solution and to determine the absolute configuration of enantiomers. It was important to develop a fast and robust method, which would enable high-throughput screening of samples, e.g. in toxicology laboratories. Therefore, one of the goals of this study was to develop a novel SFC enantioseparation method for the chiral resolution of MXP enantiomers using a polysaccharide chiral stationary phase. In comparison, the SFC assay provides significant decreasing of retention time with satisfactory resolution. Repeatability of the method was proven by an intra- and an interday validation with respect to retention time and resolution factor. Using DFT calculation, we found two stable conformers in solution, calculated their relative abundances based on Boltzmann distribution and simulated their Boltzmann-weighted spectra. Their comparison with experimental spectra allowed us to determine the absolute configuration of enantiomers. We present an enantioseparation method for methoxphenidine using SFC and offer a comparison of four commercial chiral stationary phases. Using this method, we show that crystallization with a chiral acid provides pure enantiomers of MXP, which was confirmed by chiroptic methods supported with DFT calculations.