Secondary phosphane—borane derivatives and phosphane oxide (VI) react with α-amido enol phosphate... more Secondary phosphane—borane derivatives and phosphane oxide (VI) react with α-amido enol phosphate reagents to afford new chiral or achiral tertiary α-enamido phosphane derivatives.
The characterization of chromatographic columns is of interest to column manufacturers willing to... more The characterization of chromatographic columns is of interest to column manufacturers willing to define the properties of the material they have designed, and to chromatographers looking for adequate columns to suit their separation needs. Nonchromatographic methods provide limited information, while chromatographic methods provide information on column stability, efficiency, retention, and selectivity. In the case of chromatographic methods, probe analytes must be selected. They may be limited to a small number for simple tests, or large numbers then requiring the use of chemometric tools to extract meaningful information (principal component analysis, quantitative structure–retention relationships, etc.).
In the first part of this study, a unified chromatography (UC) analysis method, which is similar ... more In the first part of this study, a unified chromatography (UC) analysis method, which is similar to supercritical fluid chromatography (SFC) but with wide mobile phase gradients of pressurized CO2 and solvent, was developed to analyse short-chain peptides, with UV and mass spectrometry (MS) detection. In this second part, the method is compared to a reference reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) method, based on the analysis of 43 peptides, including 10 linear peptides and 33 cyclic ones. First, the orthogonality between the two methods was examined, based on the retention patterns. As the UC method was developed on a polar stationary phase (Ascentis Express OH5), the elution orders and selectivities were expected to be significantly different from RPLC on a non-polar stationary phase (ACQUITY CSH C18). Secondly, the success rate of the methods was examined, based on successful retention / elution of the peptides and the absence of observed co-elutions between the main peak and impurities. A successful analysis was obtained for 81% of the peptides in UC and 67% in RPLC. Thirdly, the performance of the methods for the intended application of impurity profiling of peptide drug candidates was assessed, based on the comparison of peak purities, the number of impurities detected and the thorough examination of impurity profiles. Excellent complementarity of the two methods for the specific task of impurity profiling, and for the separation of isomeric species was observed, with only one isomeric pair in this set remaining unresolved. The method sensitivity was however better with RPLC than UC. Finally, the operational costs in terms of solvent cost per analysis were the same between the two methods.
A method to analyse short-chain bioactive peptides (MW < 800 Da) and their impurities was deve... more A method to analyse short-chain bioactive peptides (MW < 800 Da) and their impurities was developed with a unified chromatography (UC) analysis, including a wide mobile phase gradient ranging from supercritical fluid to near-liquid conditions, with UV and electrospray ionization mass spectrometry detection (ESI-MS). Four stationary phases and three mobile phase compositions were examined. Ten model peptides were first selected to identify the best operating conditions, including five linear tripeptides and five cyclic pentapeptides, with log P values ranging from -5.9 to 3.6, and including isomeric species. Derringer desirability functions were designed to identify optimal operating conditions based on 7 criteria, namely the number of peaks detected (including all impurities resolved), the proportion of the chromatogram occupied by target peaks, the least favourable resolution observed between the main peptide and impurities, peak shape features (asymmetry and peak width at half height), and finally the signal-to-noise ratio observed both with UV (210 nm) and ESI-MS in positive ionization mode. The optimum conditions were obtained on Ascentis Express OH5 stationary phase, with a mobile phase composed of carbon dioxide and methanol, comprising 2% water and 20 mM ammonium hydroxide. The final gradient program ranged from 5 to 80% co-solvent in CO2, with a reversed flow rate gradient ranging from 3.0 to 1.5 mL/min. Back-pressure was set at 120 bar and the column oven temperature at 60°C. Optimal conditions were applied to a large set of 76 peptides (34 linear tripeptides and 42 cyclic pentapeptides) and provided adequate scattering of the peaks in the retention space, together with some separation of isomeric species, particularly for the cyclic peptides.
Selective extraction is a great concern in the field of natural products. The interest is to appl... more Selective extraction is a great concern in the field of natural products. The interest is to apply specific conditions favouring the solubility of targeted secondary metabolites and avoiding the simultaneous extraction of unwanted ones. Different ways exist to reach selective extractions with suited conditions. These conditions can be determined from experimental studies through experimental design, but a full experimental design takes time, energy, and uses plant samples. Prediction from varied solubility models can also be applied allowing a better understanding of the final selected conditions and eventually less experiments. The aim of this work was to develop and use a chromatographic model to determine optimal extraction conditions without the need for numerous extraction experiments. This model would be applied on the selective extraction of the desired antioxidant compounds in rosemary leaves (rosmarinic and carnosic acids) vs chlorophyll pigments to limit the green colour in extracts. This model was achieved with Supercritical Fluid Chromatography (SFC) and then applied to Supercritical Fluid Extraction (SFE) and Pressurised Liquid Extraction (PLE) assays. SFC models predicted low solubility of chlorophylls for low (5%) and high (100%) percentage of solvent in carbon dioxide. Also, low solubility was predicted with acetonitrile solvent compared to methanol or ethanol. This was confirmed with different extractions performed using SFE with different percentages of solvent (5, 30, and 70%) and with the three solvents used in the SFC models (acetonitrile, methanol and ethanol). Also extractions using PLE were carried out using the same neat solvents in order to confirm the SFC models obtained for 100% of solvent. Globally, extractions validated the SFC models. Only some differences were observed between ethanol and methanol showing the complexity of plant extraction due to matrix effect. For all these extracts, the content of carnosic acid and rosmarinic acid was also monitored and selective extraction conditions of bioactive compounds could be determined.
This paper presents a review of the recent literature covering separations of enantiomers achieve... more This paper presents a review of the recent literature covering separations of enantiomers achieved with supercritical fluid chromatography (SFC) or subcritical fluid chromatography. First of all, the recent progress in SFC technology are explained in relation to chiral separations. Mobile phases and operating conditions mostly encountered in current practise, instruments and stationary phases are discussed. Some elements related to fundamental studies are also given. While the pharmaceutical industry was long the principal user of chiral SFC for preparative purposes, it is now more and more in use for analytical purposes and especially pharmacokinetics. Other application fields are now emerging like the determination of different degradation rates in chiral agrochemicals, forensics, natural products and cosmetics. This review does not aim at being comprehensive but rather provides a global view of current practise of chiral resolution with SFC.
This study is an investigation of numerical and graphical tools for the comparison of the chromat... more This study is an investigation of numerical and graphical tools for the comparison of the chromatographic stationary phases previously characterized with the solvation parameter model [C. West, E. Lesellier, J. Chromatogr. A, in press]. Numerous methods are presented and discussed: the coefficient ratio comparison, principal component analysis (PCA) and calculation of the distance. The coefficient ratio comparison allows to estimate the weight of a particular type of interaction relative to dispersive interactions, but is not practical when a lot of chromatographic systems need to be compared. The principal component analysis is mainly used to reduce the set of correlated variables, but is not useful for non correlated variables such as the solvation parameters. The distance calculation is an interesting tool to measure the differences between two systems but suffers from a confusion between two notions: the difference in the type of interactions involved in the chromatographic system and the intensity of these interactions. Finally, we chose to associate each stationary phase to a vector in a five-dimensional space, according to a method proposed by Ishihama and Asakawa [Y. Ishihama, N. Asakawa, J. Pharm. Sci., 88(12) (1999) 1305] for the comparison of lipophilicity scales. Thus, the angles between the different vectors are used to compare the selectivities of the stationary phases, and the lengths of the vectors are used to compare the relative intensities of the interactions. This method was applied to different alkyl phases characterized in subcritical fluid chromatography (SubFC). The angle values are well suited to the description of the differences in chromatographic behaviour. As expected, a small angle value is obtained between C8, C12 and C18, when a greater one is noticed between C4 and the longer chain length alkyl bonded phases, showing the different acidity and basicity of the C4 phase. Moreover, a satisfactory correlation is obtained between the length of the vectors and the carbon number of the alkyl chain. The differences between classical alkyl-bonded silica phases and polar-embedded alkyl-bonded phases or fluorinated phases are also conveniently evidenced. Finally, graphical tools are investigated and a new type of representation, based in part on a radar plot, is proposed. This plot allows the comparison of stationary phases without reducing the number of studied variables. The comparisons based on this method are consistent with the observed chromatographic behaviour of the phases compared.
This study is an investigation of numerical and graphical tools for the comparison of the chromat... more This study is an investigation of numerical and graphical tools for the comparison of the chromatographic stationary phases previously characterized with the solvation parameter model [C. West, E. Lesellier, J. Chromatogr. A, in press]. Numerous methods are presented and discussed: the coefficient ratio comparison, principal component analysis (PCA) and calculation of the distance. The coefficient ratio comparison allows to estimate the weight of a particular type of interaction relative to dispersive interactions, but is not practical when a lot of chromatographic systems need to be compared. The principal component analysis is mainly used to reduce the set of correlated variables, but is not useful for non correlated variables such as the solvation parameters. The distance calculation is an interesting tool to measure the differences between two systems but suffers from a confusion between two notions: the difference in the type of interactions involved in the chromatographic system and the intensity of these interactions. Finally, we chose to associate each stationary phase to a vector in a five-dimensional space, according to a method proposed by Ishihama and Asakawa [Y. Ishihama, N. Asakawa, J. Pharm. Sci., 88(12) (1999) 1305] for the comparison of lipophilicity scales. Thus, the angles between the different vectors are used to compare the selectivities of the stationary phases, and the lengths of the vectors are used to compare the relative intensities of the interactions. This method was applied to different alkyl phases characterized in subcritical fluid chromatography (SubFC). The angle values are well suited to the description of the differences in chromatographic behaviour. As expected, a small angle value is obtained between C8, C12 and C18, when a greater one is noticed between C4 and the longer chain length alkyl bonded phases, showing the different acidity and basicity of the C4 phase. Moreover, a satisfactory correlation is obtained between the length of the vectors and the carbon number of the alkyl chain. The differences between classical alkyl-bonded silica phases and polar-embedded alkyl-bonded phases or fluorinated phases are also conveniently evidenced. Finally, graphical tools are investigated and a new type of representation, based in part on a radar plot, is proposed. This plot allows the comparison of stationary phases without reducing the number of studied variables. The comparisons based on this method are consistent with the observed chromatographic behaviour of the phases compared.
Insights into chiral recognition mechanisms in supercritical fluid chromatography. II. Factors co... more Insights into chiral recognition mechanisms in supercritical fluid chromatography. II. Factors contributing to enantiomer separation on tris-(3,5-dimethylphenylcarbamate) of amylose and cellulose stationary phases
In the present study, we characterize a famous Pirkle-type enantioselective stationary phase ((R,... more In the present study, we characterize a famous Pirkle-type enantioselective stationary phase ((R,R)-Whelk-O1 from Regis Technologies) and an equivalent enantiomeric phase (ReproSil Chiral-NR from Dr. Maisch) in supercritical fluid chromatography (SFC) with carbon dioxide-methanol (90:10 v/v) mobile phase. First, the interactions contributing to retention are evaluated with a modified version of the solvation parameter model, comprising five Abraham descriptors (E, S, A, B, V), two additional descriptors to take account of molecular shape (flexibility F and globularity G), and two additional descriptors to take account of interactions with ionizable species (D − and D +). Linear solvation energy relationships (LSER) are established based on the retention of 212 achiral analytes. As expected, ππ interactions are the most significant to explain retention, while dipole-dipole, hydrogen bonding and ionic interactions with cationic species are of secondary importance. Secondly, the contributions of interactions to enantioseparations are discussed, based on the analysis of 79 racemates. Discriminant analyses (DA) were computed to gain some insight on retention mechanisms. The set of racemates is first divided between racemates eluted earlier than expected based on the LSER models, and those eluted later than expected. Small spherical molecules are more retained than expected, as they may better fit inside the cleft of the chiral selector. They are also most frequently resolved, probably for the same reason. Among the molecules that are less retained than expected, which are rather large and/or non-spherical, other features are favourable to enantiorecognition: π-electrons, dipoles and electron-donating properties. Contrary to the observations on other sorts of chiral selectors, flexibility was found to have no contribution on the enantiorecognition process.
2-Ethylpyridine-bonded silica is one of the most famous stationary phases employed in supercritic... more 2-Ethylpyridine-bonded silica is one of the most famous stationary phases employed in supercritical fluid chromatography, especially for the analysis of basic compounds and even without an additive in the mobile phase. In the present paper, we present the synthesis and characterization of three original stationary phases based on poly(vinylpyridine) polymers supported on silica. The position of nitrogen atom relative to the polymer chain was varied to be in the 2, 3, or 4 position. All these phases were prototypes, while the poly(4-vinylpyridine) phase was subsequently commercialized (DCPak P4VP from Daicel Corporation). The stationary phases obtained are characterized in supercritical fluid chromatography with carbon dioxide-methanol mobile phase, with a modified version of the solvation parameter model, to take account of ionic interactions. The three phases are also compared to a 2-ethylpyridine-bonded silica phase and a 2-picolylamine-bonded silica phase. It appears that the polymer-based pyridine phases are significantly more retentive than brush-type pyridine phases and adequately shield residual silanol groups to prevent unwanted interactions with basic compounds. The different selectivities and chromatographic performances are also evidenced with sample applications on pharmaceutical compounds, notably with a selection of 140 drug candidates.
In reversed-phase liquid chromatography (RPLC), the comparison of experimental results obtained f... more In reversed-phase liquid chromatography (RPLC), the comparison of experimental results obtained from different columns is a complex problem. A correspondence factor analysis (CFA) and a linear solvation energy relationship (LSER) were applied on retention data to characterize second-order intermolecular interactions responsible for retention on a set of RPLC columns. Seven octadecyl-C 18 columns with different packing materials are obtained from different manufacturers and one octyl-C 8 column. The retention data were determined under isocratic conditions using a methanol-water (65:35, v/v) mobile phase. The chromatographic retention indices based on alkan-2-ones and alkyl aryl ketones retention index scales are calculated using a multiparametric least-squares regressions iterative method. The CFA and LSER results permitted to highlight that the retention indices were appropriate for studying the second-order retention mechanisms on the eight chromatographic systems investigated and exhibited the best reproducibility. Although many earlier studies have reported the use of chemometric methods to characterize chemical factors affecting retention in RPLC using retention factors as retention parameters, this is the first study based on retention indices.
To achieve the most complete impurity profiling of synthetic drugs with a single chromatographic ... more To achieve the most complete impurity profiling of synthetic drugs with a single chromatographic technique, high resolution is required, which may be gained with a combination of high efficiency and versatile selectivity, allowing to separate most similar analytes. Compared to a single-column chromatographic method, coupling complementary stationary phases promises both an increase in efficiency and an increase in selectivity possibilities. With supercritical fluid chromatography (SFC), the use of long columns is facilitated by the low viscosity of the mobile phase. In this paper, we investigate the interest of coupling two achiral stationary phases (Acquity UPC 2 HSS C18 SB and Nucleoshell HILIC) that were previously observed to have excellent complementarity in SFC to carry out impurity profiling on 25 individual drug substances containing varied numbers and amounts of impurities. The single-column gradient methods are compared to tandem-column gradient methods with the two possible ordering of columns (C18 phase in first or second position) based on selectivity, peak capacity, sensitivity, UV-estimated purity of the active pharmaceutical ingredient and number of impurities detected with UV-estimated concentration >0.04 %. It appears that it could be more beneficial to have two
The use of anabolic substances to promote growth in livestock is prohibited within the European U... more The use of anabolic substances to promote growth in livestock is prohibited within the European Union as laid down in Directive 96/22/EC. Nowadays, efficient methods such as steroid profiling or isotopic deviation measurements allow to control natural steroid hormones abuse. In both cases, urine is often selected as the most relevant matrix and, due to its relatively high content of potential interferents, its preparation before analysis is considered as a key step. In this context, the use of a selective sorbent such as molecularly imprinted polymer (MIP) was investigated. A MIP was synthesized based on 17-estradiol, methacrylic acid and acetonitrile as template, monomer and porogen, respectively. Two approaches were then tested for non-conjugated (aglycons and glucuronides deconjugated) steroid purification: (i) molecularly imprinted solid phase extraction (MISPE) and (ii) semi-preparative supercritical fluid chromatography with a commercial MIP as stationary phase (SFC-MIP). Parameters for both approaches were optimized based on the main bovine metabolites of testosterone, estradiol, nandrolone and boldenone. The MISPE protocol developed for screening purposes allowed satisfactory recoveries (upper 65% for the 12 target steroids) with sufficient purification for gas chromatography-mass spectrometry (GC-MS) analysis. For confirmatory purposes, the use of isotopic ratio mass spectrometry (IRMS) requires a higher degree of purity of the target compounds, which can be reached by the SFC-MIP protocol with three steps less compared to the official and current method. Purity, concentration and absence of isotopic fractionation of target steroids extracted from urine of treated cattle (treated with testosterone, estradiol, androstenedione, and boldenone) allowed the measurement of 13 C/ 12 C isotopic ratios of corresponding metabolites and endogenous reference compounds (ERC) and proved the relevance of the strategy.
Impurity profiling of organic products that are synthesized as possible drug candidates requires ... more Impurity profiling of organic products that are synthesized as possible drug candidates requires complementary analytical methods to ensure that all impurities are identified. Supercritical fluid chromatography (SFC) is a very useful tool to achieve this objective, as an adequate selection of stationary phases can provide orthogonal separations so as to maximize the chances to see all impurities. In this series of papers, we have developed a method for achiral SFC-MS profiling of drug candidates, based on a selection of 160 analytes issued from Servier Research Laboratories. In the first part of this study, focusing on mobile phase selection, a gradient elution with carbon dioxide and methanol comprising 2% water and 20mM ammonium acetate proved to be the best in terms of chromatographic performance, while also providing good MS response [1]. The objective of this second part was the selection of an orthogonal set of ultra-high performance stationary phases, that was carried out in two steps. Firstly, a reduced set of analytes (20) was used to screen 23 columns. The columns selected were all 1.7-2.5μm fully porous or 2.6-2.7μm superficially porous particles, with a variety of stationary phase chemistries. Derringer desirability functions were used to rank the columns according to retention window, column efficiency evaluated with peak width of selected analytes, and the proportion of analytes successfully eluted with good peak shapes. The columns providing the worst performances were thus eliminated and a shorter selection of columns (11) was obtained. Secondly, based on 160 tested analytes, the 11 columns were ranked again. The retention data obtained on these columns were then compared to define a reduced set of the best columns providing the greatest orthogonality, to maximize the chances to see all impurities within a limited number of runs. Two high-performance columns were thus selected: ACQUITY UPC(2) HSS C18 SB and Nucleoshell HILIC.
Secondary phosphane—borane derivatives and phosphane oxide (VI) react with α-amido enol phosphate... more Secondary phosphane—borane derivatives and phosphane oxide (VI) react with α-amido enol phosphate reagents to afford new chiral or achiral tertiary α-enamido phosphane derivatives.
The characterization of chromatographic columns is of interest to column manufacturers willing to... more The characterization of chromatographic columns is of interest to column manufacturers willing to define the properties of the material they have designed, and to chromatographers looking for adequate columns to suit their separation needs. Nonchromatographic methods provide limited information, while chromatographic methods provide information on column stability, efficiency, retention, and selectivity. In the case of chromatographic methods, probe analytes must be selected. They may be limited to a small number for simple tests, or large numbers then requiring the use of chemometric tools to extract meaningful information (principal component analysis, quantitative structure–retention relationships, etc.).
In the first part of this study, a unified chromatography (UC) analysis method, which is similar ... more In the first part of this study, a unified chromatography (UC) analysis method, which is similar to supercritical fluid chromatography (SFC) but with wide mobile phase gradients of pressurized CO2 and solvent, was developed to analyse short-chain peptides, with UV and mass spectrometry (MS) detection. In this second part, the method is compared to a reference reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) method, based on the analysis of 43 peptides, including 10 linear peptides and 33 cyclic ones. First, the orthogonality between the two methods was examined, based on the retention patterns. As the UC method was developed on a polar stationary phase (Ascentis Express OH5), the elution orders and selectivities were expected to be significantly different from RPLC on a non-polar stationary phase (ACQUITY CSH C18). Secondly, the success rate of the methods was examined, based on successful retention / elution of the peptides and the absence of observed co-elutions between the main peak and impurities. A successful analysis was obtained for 81% of the peptides in UC and 67% in RPLC. Thirdly, the performance of the methods for the intended application of impurity profiling of peptide drug candidates was assessed, based on the comparison of peak purities, the number of impurities detected and the thorough examination of impurity profiles. Excellent complementarity of the two methods for the specific task of impurity profiling, and for the separation of isomeric species was observed, with only one isomeric pair in this set remaining unresolved. The method sensitivity was however better with RPLC than UC. Finally, the operational costs in terms of solvent cost per analysis were the same between the two methods.
A method to analyse short-chain bioactive peptides (MW < 800 Da) and their impurities was deve... more A method to analyse short-chain bioactive peptides (MW < 800 Da) and their impurities was developed with a unified chromatography (UC) analysis, including a wide mobile phase gradient ranging from supercritical fluid to near-liquid conditions, with UV and electrospray ionization mass spectrometry detection (ESI-MS). Four stationary phases and three mobile phase compositions were examined. Ten model peptides were first selected to identify the best operating conditions, including five linear tripeptides and five cyclic pentapeptides, with log P values ranging from -5.9 to 3.6, and including isomeric species. Derringer desirability functions were designed to identify optimal operating conditions based on 7 criteria, namely the number of peaks detected (including all impurities resolved), the proportion of the chromatogram occupied by target peaks, the least favourable resolution observed between the main peptide and impurities, peak shape features (asymmetry and peak width at half height), and finally the signal-to-noise ratio observed both with UV (210 nm) and ESI-MS in positive ionization mode. The optimum conditions were obtained on Ascentis Express OH5 stationary phase, with a mobile phase composed of carbon dioxide and methanol, comprising 2% water and 20 mM ammonium hydroxide. The final gradient program ranged from 5 to 80% co-solvent in CO2, with a reversed flow rate gradient ranging from 3.0 to 1.5 mL/min. Back-pressure was set at 120 bar and the column oven temperature at 60°C. Optimal conditions were applied to a large set of 76 peptides (34 linear tripeptides and 42 cyclic pentapeptides) and provided adequate scattering of the peaks in the retention space, together with some separation of isomeric species, particularly for the cyclic peptides.
Selective extraction is a great concern in the field of natural products. The interest is to appl... more Selective extraction is a great concern in the field of natural products. The interest is to apply specific conditions favouring the solubility of targeted secondary metabolites and avoiding the simultaneous extraction of unwanted ones. Different ways exist to reach selective extractions with suited conditions. These conditions can be determined from experimental studies through experimental design, but a full experimental design takes time, energy, and uses plant samples. Prediction from varied solubility models can also be applied allowing a better understanding of the final selected conditions and eventually less experiments. The aim of this work was to develop and use a chromatographic model to determine optimal extraction conditions without the need for numerous extraction experiments. This model would be applied on the selective extraction of the desired antioxidant compounds in rosemary leaves (rosmarinic and carnosic acids) vs chlorophyll pigments to limit the green colour in extracts. This model was achieved with Supercritical Fluid Chromatography (SFC) and then applied to Supercritical Fluid Extraction (SFE) and Pressurised Liquid Extraction (PLE) assays. SFC models predicted low solubility of chlorophylls for low (5%) and high (100%) percentage of solvent in carbon dioxide. Also, low solubility was predicted with acetonitrile solvent compared to methanol or ethanol. This was confirmed with different extractions performed using SFE with different percentages of solvent (5, 30, and 70%) and with the three solvents used in the SFC models (acetonitrile, methanol and ethanol). Also extractions using PLE were carried out using the same neat solvents in order to confirm the SFC models obtained for 100% of solvent. Globally, extractions validated the SFC models. Only some differences were observed between ethanol and methanol showing the complexity of plant extraction due to matrix effect. For all these extracts, the content of carnosic acid and rosmarinic acid was also monitored and selective extraction conditions of bioactive compounds could be determined.
This paper presents a review of the recent literature covering separations of enantiomers achieve... more This paper presents a review of the recent literature covering separations of enantiomers achieved with supercritical fluid chromatography (SFC) or subcritical fluid chromatography. First of all, the recent progress in SFC technology are explained in relation to chiral separations. Mobile phases and operating conditions mostly encountered in current practise, instruments and stationary phases are discussed. Some elements related to fundamental studies are also given. While the pharmaceutical industry was long the principal user of chiral SFC for preparative purposes, it is now more and more in use for analytical purposes and especially pharmacokinetics. Other application fields are now emerging like the determination of different degradation rates in chiral agrochemicals, forensics, natural products and cosmetics. This review does not aim at being comprehensive but rather provides a global view of current practise of chiral resolution with SFC.
This study is an investigation of numerical and graphical tools for the comparison of the chromat... more This study is an investigation of numerical and graphical tools for the comparison of the chromatographic stationary phases previously characterized with the solvation parameter model [C. West, E. Lesellier, J. Chromatogr. A, in press]. Numerous methods are presented and discussed: the coefficient ratio comparison, principal component analysis (PCA) and calculation of the distance. The coefficient ratio comparison allows to estimate the weight of a particular type of interaction relative to dispersive interactions, but is not practical when a lot of chromatographic systems need to be compared. The principal component analysis is mainly used to reduce the set of correlated variables, but is not useful for non correlated variables such as the solvation parameters. The distance calculation is an interesting tool to measure the differences between two systems but suffers from a confusion between two notions: the difference in the type of interactions involved in the chromatographic system and the intensity of these interactions. Finally, we chose to associate each stationary phase to a vector in a five-dimensional space, according to a method proposed by Ishihama and Asakawa [Y. Ishihama, N. Asakawa, J. Pharm. Sci., 88(12) (1999) 1305] for the comparison of lipophilicity scales. Thus, the angles between the different vectors are used to compare the selectivities of the stationary phases, and the lengths of the vectors are used to compare the relative intensities of the interactions. This method was applied to different alkyl phases characterized in subcritical fluid chromatography (SubFC). The angle values are well suited to the description of the differences in chromatographic behaviour. As expected, a small angle value is obtained between C8, C12 and C18, when a greater one is noticed between C4 and the longer chain length alkyl bonded phases, showing the different acidity and basicity of the C4 phase. Moreover, a satisfactory correlation is obtained between the length of the vectors and the carbon number of the alkyl chain. The differences between classical alkyl-bonded silica phases and polar-embedded alkyl-bonded phases or fluorinated phases are also conveniently evidenced. Finally, graphical tools are investigated and a new type of representation, based in part on a radar plot, is proposed. This plot allows the comparison of stationary phases without reducing the number of studied variables. The comparisons based on this method are consistent with the observed chromatographic behaviour of the phases compared.
This study is an investigation of numerical and graphical tools for the comparison of the chromat... more This study is an investigation of numerical and graphical tools for the comparison of the chromatographic stationary phases previously characterized with the solvation parameter model [C. West, E. Lesellier, J. Chromatogr. A, in press]. Numerous methods are presented and discussed: the coefficient ratio comparison, principal component analysis (PCA) and calculation of the distance. The coefficient ratio comparison allows to estimate the weight of a particular type of interaction relative to dispersive interactions, but is not practical when a lot of chromatographic systems need to be compared. The principal component analysis is mainly used to reduce the set of correlated variables, but is not useful for non correlated variables such as the solvation parameters. The distance calculation is an interesting tool to measure the differences between two systems but suffers from a confusion between two notions: the difference in the type of interactions involved in the chromatographic system and the intensity of these interactions. Finally, we chose to associate each stationary phase to a vector in a five-dimensional space, according to a method proposed by Ishihama and Asakawa [Y. Ishihama, N. Asakawa, J. Pharm. Sci., 88(12) (1999) 1305] for the comparison of lipophilicity scales. Thus, the angles between the different vectors are used to compare the selectivities of the stationary phases, and the lengths of the vectors are used to compare the relative intensities of the interactions. This method was applied to different alkyl phases characterized in subcritical fluid chromatography (SubFC). The angle values are well suited to the description of the differences in chromatographic behaviour. As expected, a small angle value is obtained between C8, C12 and C18, when a greater one is noticed between C4 and the longer chain length alkyl bonded phases, showing the different acidity and basicity of the C4 phase. Moreover, a satisfactory correlation is obtained between the length of the vectors and the carbon number of the alkyl chain. The differences between classical alkyl-bonded silica phases and polar-embedded alkyl-bonded phases or fluorinated phases are also conveniently evidenced. Finally, graphical tools are investigated and a new type of representation, based in part on a radar plot, is proposed. This plot allows the comparison of stationary phases without reducing the number of studied variables. The comparisons based on this method are consistent with the observed chromatographic behaviour of the phases compared.
Insights into chiral recognition mechanisms in supercritical fluid chromatography. II. Factors co... more Insights into chiral recognition mechanisms in supercritical fluid chromatography. II. Factors contributing to enantiomer separation on tris-(3,5-dimethylphenylcarbamate) of amylose and cellulose stationary phases
In the present study, we characterize a famous Pirkle-type enantioselective stationary phase ((R,... more In the present study, we characterize a famous Pirkle-type enantioselective stationary phase ((R,R)-Whelk-O1 from Regis Technologies) and an equivalent enantiomeric phase (ReproSil Chiral-NR from Dr. Maisch) in supercritical fluid chromatography (SFC) with carbon dioxide-methanol (90:10 v/v) mobile phase. First, the interactions contributing to retention are evaluated with a modified version of the solvation parameter model, comprising five Abraham descriptors (E, S, A, B, V), two additional descriptors to take account of molecular shape (flexibility F and globularity G), and two additional descriptors to take account of interactions with ionizable species (D − and D +). Linear solvation energy relationships (LSER) are established based on the retention of 212 achiral analytes. As expected, ππ interactions are the most significant to explain retention, while dipole-dipole, hydrogen bonding and ionic interactions with cationic species are of secondary importance. Secondly, the contributions of interactions to enantioseparations are discussed, based on the analysis of 79 racemates. Discriminant analyses (DA) were computed to gain some insight on retention mechanisms. The set of racemates is first divided between racemates eluted earlier than expected based on the LSER models, and those eluted later than expected. Small spherical molecules are more retained than expected, as they may better fit inside the cleft of the chiral selector. They are also most frequently resolved, probably for the same reason. Among the molecules that are less retained than expected, which are rather large and/or non-spherical, other features are favourable to enantiorecognition: π-electrons, dipoles and electron-donating properties. Contrary to the observations on other sorts of chiral selectors, flexibility was found to have no contribution on the enantiorecognition process.
2-Ethylpyridine-bonded silica is one of the most famous stationary phases employed in supercritic... more 2-Ethylpyridine-bonded silica is one of the most famous stationary phases employed in supercritical fluid chromatography, especially for the analysis of basic compounds and even without an additive in the mobile phase. In the present paper, we present the synthesis and characterization of three original stationary phases based on poly(vinylpyridine) polymers supported on silica. The position of nitrogen atom relative to the polymer chain was varied to be in the 2, 3, or 4 position. All these phases were prototypes, while the poly(4-vinylpyridine) phase was subsequently commercialized (DCPak P4VP from Daicel Corporation). The stationary phases obtained are characterized in supercritical fluid chromatography with carbon dioxide-methanol mobile phase, with a modified version of the solvation parameter model, to take account of ionic interactions. The three phases are also compared to a 2-ethylpyridine-bonded silica phase and a 2-picolylamine-bonded silica phase. It appears that the polymer-based pyridine phases are significantly more retentive than brush-type pyridine phases and adequately shield residual silanol groups to prevent unwanted interactions with basic compounds. The different selectivities and chromatographic performances are also evidenced with sample applications on pharmaceutical compounds, notably with a selection of 140 drug candidates.
In reversed-phase liquid chromatography (RPLC), the comparison of experimental results obtained f... more In reversed-phase liquid chromatography (RPLC), the comparison of experimental results obtained from different columns is a complex problem. A correspondence factor analysis (CFA) and a linear solvation energy relationship (LSER) were applied on retention data to characterize second-order intermolecular interactions responsible for retention on a set of RPLC columns. Seven octadecyl-C 18 columns with different packing materials are obtained from different manufacturers and one octyl-C 8 column. The retention data were determined under isocratic conditions using a methanol-water (65:35, v/v) mobile phase. The chromatographic retention indices based on alkan-2-ones and alkyl aryl ketones retention index scales are calculated using a multiparametric least-squares regressions iterative method. The CFA and LSER results permitted to highlight that the retention indices were appropriate for studying the second-order retention mechanisms on the eight chromatographic systems investigated and exhibited the best reproducibility. Although many earlier studies have reported the use of chemometric methods to characterize chemical factors affecting retention in RPLC using retention factors as retention parameters, this is the first study based on retention indices.
To achieve the most complete impurity profiling of synthetic drugs with a single chromatographic ... more To achieve the most complete impurity profiling of synthetic drugs with a single chromatographic technique, high resolution is required, which may be gained with a combination of high efficiency and versatile selectivity, allowing to separate most similar analytes. Compared to a single-column chromatographic method, coupling complementary stationary phases promises both an increase in efficiency and an increase in selectivity possibilities. With supercritical fluid chromatography (SFC), the use of long columns is facilitated by the low viscosity of the mobile phase. In this paper, we investigate the interest of coupling two achiral stationary phases (Acquity UPC 2 HSS C18 SB and Nucleoshell HILIC) that were previously observed to have excellent complementarity in SFC to carry out impurity profiling on 25 individual drug substances containing varied numbers and amounts of impurities. The single-column gradient methods are compared to tandem-column gradient methods with the two possible ordering of columns (C18 phase in first or second position) based on selectivity, peak capacity, sensitivity, UV-estimated purity of the active pharmaceutical ingredient and number of impurities detected with UV-estimated concentration >0.04 %. It appears that it could be more beneficial to have two
The use of anabolic substances to promote growth in livestock is prohibited within the European U... more The use of anabolic substances to promote growth in livestock is prohibited within the European Union as laid down in Directive 96/22/EC. Nowadays, efficient methods such as steroid profiling or isotopic deviation measurements allow to control natural steroid hormones abuse. In both cases, urine is often selected as the most relevant matrix and, due to its relatively high content of potential interferents, its preparation before analysis is considered as a key step. In this context, the use of a selective sorbent such as molecularly imprinted polymer (MIP) was investigated. A MIP was synthesized based on 17-estradiol, methacrylic acid and acetonitrile as template, monomer and porogen, respectively. Two approaches were then tested for non-conjugated (aglycons and glucuronides deconjugated) steroid purification: (i) molecularly imprinted solid phase extraction (MISPE) and (ii) semi-preparative supercritical fluid chromatography with a commercial MIP as stationary phase (SFC-MIP). Parameters for both approaches were optimized based on the main bovine metabolites of testosterone, estradiol, nandrolone and boldenone. The MISPE protocol developed for screening purposes allowed satisfactory recoveries (upper 65% for the 12 target steroids) with sufficient purification for gas chromatography-mass spectrometry (GC-MS) analysis. For confirmatory purposes, the use of isotopic ratio mass spectrometry (IRMS) requires a higher degree of purity of the target compounds, which can be reached by the SFC-MIP protocol with three steps less compared to the official and current method. Purity, concentration and absence of isotopic fractionation of target steroids extracted from urine of treated cattle (treated with testosterone, estradiol, androstenedione, and boldenone) allowed the measurement of 13 C/ 12 C isotopic ratios of corresponding metabolites and endogenous reference compounds (ERC) and proved the relevance of the strategy.
Impurity profiling of organic products that are synthesized as possible drug candidates requires ... more Impurity profiling of organic products that are synthesized as possible drug candidates requires complementary analytical methods to ensure that all impurities are identified. Supercritical fluid chromatography (SFC) is a very useful tool to achieve this objective, as an adequate selection of stationary phases can provide orthogonal separations so as to maximize the chances to see all impurities. In this series of papers, we have developed a method for achiral SFC-MS profiling of drug candidates, based on a selection of 160 analytes issued from Servier Research Laboratories. In the first part of this study, focusing on mobile phase selection, a gradient elution with carbon dioxide and methanol comprising 2% water and 20mM ammonium acetate proved to be the best in terms of chromatographic performance, while also providing good MS response [1]. The objective of this second part was the selection of an orthogonal set of ultra-high performance stationary phases, that was carried out in two steps. Firstly, a reduced set of analytes (20) was used to screen 23 columns. The columns selected were all 1.7-2.5μm fully porous or 2.6-2.7μm superficially porous particles, with a variety of stationary phase chemistries. Derringer desirability functions were used to rank the columns according to retention window, column efficiency evaluated with peak width of selected analytes, and the proportion of analytes successfully eluted with good peak shapes. The columns providing the worst performances were thus eliminated and a shorter selection of columns (11) was obtained. Secondly, based on 160 tested analytes, the 11 columns were ranked again. The retention data obtained on these columns were then compared to define a reduced set of the best columns providing the greatest orthogonality, to maximize the chances to see all impurities within a limited number of runs. Two high-performance columns were thus selected: ACQUITY UPC(2) HSS C18 SB and Nucleoshell HILIC.
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Papers by Caroline West