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This review article summarizes the results obtained from the combined efforts of a joint academic and industrial initiative to solve the real-life challenge of determining low levels of peptide-related impurities (typically 0.05–1% of the drug substance) in the presence of the related biologically active peptide at a high concentration. A rational screening strategy for pharmaceutically important peptides has been developed that uses combinations of reversed‑phase ultrahigh-pressure liquid chromatography (UHPLC) columns and mobile phases that exhibit complementary reversed-phase chromatographic selectivity using either UV- or mass spectrometry (MS)-compatible conditions. Numerous stationary and mobile phases were categorized using the chemometric tool of principal component analysis (PCA), employing a novel characterization protocol utilizing specifically designed peptide probes. This was successfully applied to the development of a strategy for the detection of impurities (especially isomers) in peptide drug substances using two-dimensional liquid chromatography coupled with MS detection (2D-LC–MS).

This study describes the first reported development of a rapid, generic gradient Ultra-High Performance Liquid Chromatography (UHPLC) methodology with targeted triple quadrupole MS/MS using electrospray positive ionisation to detect and unambiguously confirm the identity of 33 substituted 1, 2-diarylethamine (or diphenidine) derivatives in solid drug samples. The in-house synthesised library included a range of derivatives possessing either electron donating/withdrawing substituents, commonly included in combinatorial libraries, of varying size and lipophilicity on the phenyl ring. These test probes were used to investigate if their order of elution and that of their regioisomers were dependent on the position and type of the substituent on the phenyl ring. In addition, investigations into the retention mechanism of the diphenidines under reverse-phase UHPLC conditions were undertaken. Common adulterants found within seized bulk samples were assessed to prove that the methodology was specific, and the developed UHPLC-MS/MS (t G = 10 min) protocol was applied to confirm the identity of the psychoactive components within four seized bulk samples provided by law enforcement.

A simple, rapid and robust protocol for the characterisation of strong cation exchange columns for the analysis of small molecular weight bases is described. A range of ten different phases were characterised, and the resultant selectivity and retention factors analysed using Principal Component Analysis. The score plots for the first and second principal components described 83% of the variability within the dataset. Score plots highlighted the large chromatographic differences observed between the phases, the validity of which was established using a larger range of bases. All the strong cation exchange materials demonstrated a synergistic mixed mode (i.e. ion exchange and hydrophobic) retention mechanism. Principal Component Analysis also highlighted the potential difficulty in locating suitable strong cation exchange “back-up” columns for the analysis of small molecular weight bases in that the characterised columns all displayed very different selectivities. The robustness of the protocol was confirmed by a factorial design experiment.

The paper describes a simple and rapid reversed-phase UHPLC method development screening strategy for the purity determination of peptide-based pharmaceuticals. The protocol utilises five disparate column and six volatile or non-volatile mobile phases (i.e., 30 combinations). The method development strategy has been demonstrated to be highly effective in identifying conditions which generate complementary selectivity and good peak shape. Columns with varying degrees of charge (positive and negative), in addition to their differing hydrophobic character, were used in combination with mobile phases within the pH range of 2.3 to 5.1. The novel ion-pair / chaotropic reagent ammonium hexafluorophosphate at pH 2.3 was shown to be an extremely useful mobile phase additive in that it produced excellent complementary separation and good peak shape. Methanesulfonic acid was demonstrated to be a good alternative to the ubiquitously employed trifluoroacetic acid which failed to generate optimum separation for the peptides investigated highlighting the importance of screening disparate mobile phase additives. Both ammonium hexafluorophosphate and methanesulfonic acid were shown not to adversely affect the stability of C18 columns or demonstrated any irreversible adsorption / memory effects. No pH hysteresis effects were demonstrated with any of the stationary phases on mobile phase pH cycling. No major problems have been observed with the novel mobile phase additives ammonium hexafluorophosphate and methanesulfonic acid, however, it is recommended that they be used with caution until long-term routine use has been established.

Supercritical Fluid Chromatography (SFC-UV) employing a carbon dioxide (CO 2) and 10 mM ammonium acetate in MeOH-water (95:5 v/v) gradient provides a rapid analysis (t G

A protocol was defined which utilised peptides as probes for the characterisation of reversed phase chromatography peptide separation systems. These peptide probes successfully distinguished between differing stationary phases through the probe's hydrophobic, electrostatic, hydrogen bonding and aromatic interactions with the stationary phase, in addition, to more subtle interactions such as the phase's ability to separate racemic or isomeric probes. The dominating forces responsible for the chromatographic selectivity of peptides appear to be hydrophobic as well as electrostatic and polar in nature. This highlights the need for other types of stationary phase ligands with possibly mixed mode functionalities / electrostatic / polar interactions for peptide separations rather than the hydrophobic ligands which dominate small molecule separations. Selectivity differences are observed between phases, but it appears that it is the accessibility differences between these phases which play a crucial role in peptide separations i.e. accessibility to silanols, the hydrophobic acetonitrile / ligand layer or a thin adsorbed water layer on the silica surface.

The robustness of the Peptide Reversed Phase Chromatography (RPC) Column Characterisation Protocol was evaluated using reduced factorial design, to ascertain the degree of control required for parameters including temperature, flow rate, dwell volume, a systematic shift in the gradient, amount of formic acid in the aqueous and organic, pH of the ammonium formate and amount of acetonitrile (%MeCN) in the strong solvent, where a loss of MeCN resulted in an unacceptable variation. Mitigations have been introduced to ensure the integrity of the data to allow RPC columns to be characterised using peptides as probes, with the definitive protocol described. In addition, the instrument and column batch to batch variability were assessed with good reproducibility.

The differentiation of mobile phase compositions between sub-classes which exhibit distinct chromatographic selectivity (i.e. termed characterisation) towards a range of peptide probes with diverse functionality and hence the possibility for multi-modal retention mechanisms has been undertaken. Due to the complexity of peptide retention mechanisms in given mobile phase conditions, no attempt has been made to explain these, instead mobile phases have simply been classified into distinct groups with an aim of identifying those yielding differing selectivities for use in strategic method development roadmaps for the analysis of peptide mixtures. The selectivity differences between nine synthetic peptides (fragments of [Ile27]-Bovine GLP-2) were used to assess how fifty-one RPC mobile phase compositions of differing pH (range 1.8 – 7.8), salt types, ionic strengths, ion-pair reagents and chaotropic / kosmotropic additives affected chromatographic selectivity on a new generation C18 stationary phase (Ascentis Express C18). The mobile phase compositions consisted of commonly used and novel UV or MS compatible additives. The chemometric tool of Principal Component Analysis (PCA) was used to visualise the differences in selectivity generated between the various mobile phases evaluated. The results highlight the importance of screening numerous mobile phases of differing pH, ion-pair reagents and ionic strength in order to maximise the probability of achieving separation of all the peptides of interest within a complex mixture. PCA permitted a ranking of the relative importance of the various mobile phase parameters evaluated. The concept of using this approach was proven in the analysis of a sample of Bovine GLP-2 (1-15) containing synthesis related impurities. Mobile phases with high ionic strength were demonstrated to be crucial for the generation of symmetrical peaks. The observations made on the C18 phase were compared on three additional stationary phases (i.e. alkyl amide, fluorophenyl and biphenyl), which had previously been shown to possess large selectivity differences towards these peptides, on a limited sub-set of mobile phases. With the exception of the ion-pair reagent, similar trends were obtained for the C18, fluorophenyl and biphenyl phases intimating the applicability of these findings to the vast majority of RPC columns (i.e. neutral or weakly polar in character) which are suitable for the analysis of peptides. The conclusions were not relevant for columns with a more disparate nature (i.e. containing a high degree of positive charge).

The Peptide RPC Column Characterisation Protocol was applied to 38 stationary phases, varying in ligand chemistry, base silica, end capping and pore size, which are suitable for the analysis of peptides. The protocol at low and intermediate pH is based on measuring retention time differences between peptides of different functionality to calculate selectivity delta values. The characterisation was designed to explore increases / decreases in positive or negative charge (deamidation), steric effect (i.e. racemisation / switch in amino acid order), oxidation and addition / removal of aromatic moieties. The necessity of developing a characterisation protocol specifically for peptide analysis was highlighted by the fact that the small molecule databases (Snyder's Hydrophobic Subtraction Model and the extended Tanaka protocol) failed to correlate with the Peptide RPC Column Characterisation Protocol. Principal Component Analysis was used to demonstrate that the protocol could be used to identify columns with similar or dissimilar chromatographic selectivity for the purpose of selectivity back-up or method development columns respectively. This was validated using peptide fragments derived from the tryptic digest of bovine insulin and carbonic anhydrase. It was also demonstrated that the presence of positively charged functional groups on the stationary phase was advantageous as it yielded very different chromatographic selectivity and improved peak shape.

© 2018 Elsevier B.V. A detailed investigation into the chromatographic retention behaviour and separation of the three regioisomers of the Novel Psychoactive Substance (NPS) methoxphenidine (i.e. 2-, 3- and 4-MXP isomers) has revealed the ionization state of the analyte and stationary phase, to be the controlling factor in dictating which retention mechanism is in operation. At low pH, poor separation and retention was observed. In contrast, at intermediate pH, enhanced retention and separation of the three MXP isomers was obtained; it appeared that there was a synergistic effect between the electrostatic and hydrophobic mechanisms. At high pH, the MXP isomers were retained by hydrophobic retention. Accurate retention time predictions ( < 0.5%) were achievable using non-linear retention models (3 × 3). This allowed the optimization of the gradient separation of the MXP isomers using a two-dimensional gradient and temperature design space. Prediction errors for peak width and resolution were, in most cases, lower than 5%. The use of linear models (2 × 2) still afforded retention time and resolution accuracies of < 2.3 and 11% respectively. A rapid and highly sensitive LC–MS friendly method (i.e. R smin > 5 within 4 min) was predicted and verified. The developed methodology should be highly suitable for the rapid, specific and sensitive detection and control of MXP regioisomers.

In order to assess the effect of silica gel structure on retention in hydrophilic interaction chromatography, a test system was developed which used quaternary ammonium ions as probes with tetramethylammonium acetate (TMAA) as the counter-ion competing against the interaction of the test probes with ionised silanols in the stationary phase. Four silica gel columns and a silica hydride column were examined. Retention times were obtained for the test probes at 20, 40, 60, 80 and 90 % acetonitrile (ACN) with all the mobile phase mixtures containing 10-mM TMAA buffer at pH 6.0. All phases gave “U”-shaped plots for log k against percentage of ACN with the steepest rise in retention occurring between 80 and 90 % ACN. Benzyltrimethylammonium, the smallest quaternary ammonium ion, was the most strongly retained probe at 90 % ACN and was most retained on a high surface area 60 A Kromasil column and least retained on a 300 A ACE silica gel column. The ionic strength of the mobile phase was varied at 80 and 90 % ACN and plots of log k against the inverse of buffer strength followed by fitting of second-order polynomial curves allowed an assessment of the contribution from HILIC to the mixed HILIC/ion-exchange retention mechanism. Toluene and pentylbenzene were used to assess the decrease in accessible pore volume due to water absorption in HILIC mode.

This paper reports the influence of a diverse range of stationary phases and differing mobile phase modifiers on pressure-induced retention changes in reversed-phase liquid chromatography (RP-LC). The practical implications of these effects in the Tanaka column characterization using ultra high-performance liquid chromatography (UHPLC) conditions, and implications for HPLC to UHPLC translations in order to increase productivity and resolution are investigated. The stationary and mobile phase combinations responded to a similar degree to elevated pressure; hence, the authors believe that reliable column characterization parameters should be obtainable when UHPLC format columns are evaluated using the Tanaka approach. Analytes exhibited differing pressure-induced retention changes even for only modest increases in pressure (i.e. ΔP(total) 85 bar as shown when one transfers from a 3- to 2-μm particle). The degree of pressure-induced retention changes correlated with the analyte's molar volume and refractivity. The hydrophobicity of the analytes, as measured by logD, only exhibited a weak correlation. Hence, translating a RP-LC methodology from large to smaller particle size material of the same type may result in an increased or decreased selectivity and hence resolution between two analytes depending on their differing response to the pressure-induced retention changes. This potentially has a major impact on LC method development/optimization strategies and LC method translations.

Mobile phase pH and temperature are major factors in determining retention, selectivity and chromatographic performance of ionizable compounds. This imposes a requirement that stationary phases must ideally be stable in both acidic and basic conditions coupled with good thermal stability, in order to be able to chromatograph these compounds in either their ionized or ion-suppressed modes. The development of a range of new high and/or low pH stable silica based RPLC stationary phases (including sub-2 μm fully porous and sub-3 μm fused core-shell materials), which are specially designed for the analysis of ionizable compounds and their chemical and thermal stability is reviewed. The ability to utilize both pH and temperature as selectivity variables allows the chromatographer to exploit a much wider method development design space including previously prohibited alkaline conditions. This greatly increases the probability of satisfying the desired chromatographic selectivity and performance criteria.

The retention behaviour of a series of 28 monosubstituted benzenes, representing a diverse range of functional groups and substituent shape, were investigated using porous graphitic carbon (PGC) and octadecyl-bonded silica (ODS) stationary phases. For the majority of analytes retention on PGC was greater than on ODS, and in most cases this effect occurred at both pH 2.5 and 7.0. The main trends observed on PGC (in comparison with ODS) were: (i) similar or reduced retention of low polarity molecules such as the hydrocarbon and halogenated analytes; (ii) increased retention of conjugated analytes with extended planarity; (iii) increased retention of polar and charged species; and (iv) substantial increases in retention for selected polar and negatively charged analytes, including some ionised and unionised acid analytes. Poor retention of positively charged analytes was observed on both stationary phases. Molecular modelling studies have explored the geometry of π – π stacking interactions in retention on PGC and have highlighted the strong retention of large conjugated analytes, with extended planar conformations, which can interact with the graphite surface with cofacial geometry. Quantitative structure–retention relationships showed the importance of hydrophobic ( π ) and electronic factors (e.g. mean polarisability and LUMO energy) in retention on PGC, whilst retention on ODS was correlated to hydrophobicity (log P and π ).

Stationary-phase evaluation in reversed-phase liquid chromatography (RP-LC) is not a straightforward process. A number of tests to characterize and classify stationary phases have been suggested. The results of these various tests, however, do not always describe the real properties of the stationary phase. This study critically compares several tests for RP-LC stationary phases, including the Engelhardt, Tanaka, and SRM 870 tests, as well as an in-house test, with emphasis on the stationary-phase descriptors of hydrophobicity and silanol activity. The stationary phases were prepared by thermal immobilization of poly(methyloctylsiloxane) onto silica. Hydrophobicity data from the tests were generally good and interchangeable between the several tests. In contrast, the silanol activity results of the various tests differ significantly. As a consequence, stationary phase classification with respect to silanol activity depends considerably on the test method applied. A new classification method for silanol activity is proposed.

Cyano (CN), butyl (C4), phenyl and octadecyl (C18) phases prepared from the same base silica gel were chromatographically characterized in order to assess the relative importance of lipophilic, π–π and dipole–dipole interactions in governing retention on these differing phases. Dipole interactions of analytes (possessing dipole moments and low lipophilicity) with CN phases were primarily responsible for the elution order. However, as the analytes’ lipophilicity increased, the lipophilic interaction predominated over the dipole interaction. In comparison, retention on the phenyl phase appeared to be complex, being controlled by a mixture of lipophilic, π–π and dipole–dipole interactions. Retention on the C4 and C18 phases was dictated by the analyte’s lipophilicity and its accessibility into the phase.

Eight commercially available sub-2 microm octadecyl silane columns (C18 columns) have been characterised by the Tanaka protocol. The columns can be grouped into two groups that display large differences in selectivity and peak shape due to differences in hydrophobicity, degree of surface coverage and silanol activity. Measurements of particle size distributions were made using automated microscopy and electrical sensing zone measurements. Only a weak correlation could be found between efficiency and particle size. Large differences in column backpressure were observed. These differences are not related to particle size distribution. A more likely explanation is differences in packing density. In order to take full advantage of 100-150 mm columns packed with sub-2 microm particles, it is often necessary to employ not only an elevated pressure but also an elevated temperature. A comparison between columns packed with sub-2, 3 and 5 microm versions of the same packing indicates potential method transferability problems for several of the columns due to selectivity differences. Currently, the best alternative for fast high-resolution LC is the use of sub-2 microm particles in combination with elevated pressure and temperature. However, as shown in this study additional efforts are needed to improve transferability as well as column performance.

Twenty-one commercially available phenyl type RPLC packing materials have been characterized in terms of their surface coverage, hydrophobic selectivity, shape selectivity, hydrogen bonding capacity, ion exchange capacity at pH 2.7 and 7.6 and aromatic selectivity (i.e. pi-pi interaction). The phases have been compared to their corresponding C-alkyl phases, three pentafluorophenyl phases and a series of experimental phenyl phases of known bonding chemistry. Principal component analysis has been used to provide a graphical comparison of the differences and similarities between the phases. The phase's aromatic selectivity was found to be dependent on the length of the alkyl spacer between the silicon atom and the phenyl ring.

The retention and overload of bases were studied on two new mixed-mode, silica based phases possessing ionic carboxylate functionalities of different acidity embedded within a hydrophobic ligand (SiELC Primesep). At low pH, good peak shapes were obtained for small solute mass, suggesting that the mere presence of a mixed-mode hydrophobic/ionic retention mechanism is not responsible for the poor peak shape that can occur on conventional reversed-phases with ionised silanols. Somewhat inferior, but still acceptable peak shape for bases was obtained on a column containing a mixture of discrete ion exchange and reversed-phase particles (Hypersil Duet). In both types of column, the ionic sites favourably increased the capacity for ionised bases, reducing considerably the deterioration of peak shape with load observed with conventional RP columns. The combined ionic and reversed-phase interaction can give strong retention of bases under certain conditions, necessitating careful choice of stationary and mobile phase.

The retention behaviour of acidic, basic and quaternary ammonium salts and polar neutral analytes has been evaluated on acidic, basic and neutral hydrophilic interaction chromatography (HILIC) stationary phases as a function of HILIC operating parameters such as MeCN content, buffer concentration, pH and temperature. Numerous empirical HILIC retention models (existing and newly developed ones) have been assessed for their ability to describe retention as a function of the HILIC operating parameters investigated. Retention models have been incorporated into a commercially available retention modelling programme (i.e. ACD/LC simulator) and their accuracy of retention prediction assessed. The applicability of HILIC modelling using these equations has been demonstrated in the two-dimensional isocratic (i.e. buffer concentration versus MeCN content modelling) and one-dimensional gradient separations for a range of analytes of differing physico-chemical properties on the three stationary phases. The accuracy of retention and peak width prediction was observed to be comparable to that reported in reversed-phase chromatography (RPC) retention modelling. Intriguingly, our results have confirmed that the use of gradient modelling to predict HILIC isocratic conditions and vice versa is not reliable. A relative ranking of the importance of the retention and selectivity of HILIC operating parameters has been determined using statistical approaches. For retention, the order of importance was observed to be organic content > stationary phase > temperature ≈ mobile phase pH (i.e. pH 3-6 which mainly effects the ionization of the analyte) ≈ buffer concentration. For selectivity, the nature of the stationary phase > mobile phase pH > buffer concentration > temperature > organic content.

The retention and overloading properties for eight basic solutes and two quaternary ammonium compounds were studied over the pH range 2.7–10.0 using phosphate and carbonate buffers. At low pH, a hybrid inorganic–organic silica-ODS phase (XTerra RP-18, 15 cm × 0.46 cm) showed substantial loss in efficiency when sample masses exceeded about 0.5 μg; these results were similar to those obtained previously on pure silica ODS and wholly polymeric phases, suggesting a common overloading mechanism. At pH 7–8.5, substantial improvements in loading capacity were obtained on XTerra due apparently to the unexpectedly strong influence of small decreases in solute ionisation. Data from the quaternary compounds suggested that silanol ionisation on this phase was still small even at intermediate pH. For many bases, loading capacity continued to improve as the pH was raised to 10, in line with the decrease in the proportion of ionised solute. However, for the highest p K a solutes, peak shape worsened at high pH, possibly due to the negative influence of increasing column silanol ionisation.

The idea that liquid water is not a uniform and random arrangement of molecules has been taken very seriously by the scientific community. Many experimental and computational investigations show that clathrate- or ice-like structures probably exist at a short time scale in solution. We have designed a new program to simulate water structure around solutes. Our model is based on the geometrical constraints of hydrogen bonding in order to be capable of producing clathrate-like structures. Simulations with small molecules and bio-molecules, using the new software, produce networks of water with specific patterns made of small water rings. The water structures built are consistent with the classification of molecules in terms of structure breaking and making. This approach may give insight into, and a more accurate description of, drug-receptor interactions. The results also suggest that water structure may impart sufficient energy to modify the conformational space of organic molecules through hydrogen bonding.

Polar embedded phases have become increasingly popular in liquid chromatography (LC) analysis. These phases can produce diverse chromatographic selectivities as a result of their differing base silica, the type of polar embedded group (i.e. amide, urea, carbamate, ether or sulphonamide moieties) and the length of the alkyl ligand. Four column characterization protocols, using differing test probes, have been used to characterize 18 of these phases together with 17 alkyl phases (some of which contained novel polar endcapping, i.e. amino), which have been evaluated using principal component analysis (PCA). PCA provided graphical comparisons of the differences/similarities between these phases and between their corresponding C-alkyl, amino endcapped and enhanced polar selectivity phases.

In this study, a porous mixed-mode n-alkyl methacrylate-based monolith has been used in the separation of therapeutic peptides. While the sulfonic acid (SCX) moiety derived from 2-acrylamido-2-methyl-1-propanesulfonic acid supports the generation of a stable electroosmotic flow (EOF) at both acidic and basic pH values, the butyl ligands provide the nonpolar sites for chromatographic resolution. The performance of the monolith was evaluated regarding the influence of pH on chromatographic resolution of peptides. The suitability of the butylmethacrylate/SCX monolith for the analysis of therapeutic peptides containing basic centres, for example arginine, at moderately high pH 9.5 and the stability to repeat injections of a mixture of peptides was demonstrated. Separations with efficiencies as high as 5.0 x 10(5) plates/m were obtained and the migration behaviour of the peptides at both low (2.8) and high (9.5) pH values could be rationalised based on their charge, molecular mass/shape and relative hydrophobicities.

The pKa of eight organic bases was determined in aqueous and aqueous methanol solutions of 0-70% (v/v) methanol using capillary electrophoresis. The bases investigated include compounds commonly used to test the activity of RP columns in HPLC. The variation of pKa with temperature in aqueous methanol solutions was also investigated and found to closely resemble temperature coefficients reported for bases in purely aqueous solutions. pKa values determined by CE were compared to those reported using NMR spectroscopy. The good agreement of the results is evidence that either technique is suitable to perform pKa measurements.

A range of ten perfluorophenyl and perfluoroalkyl stationary phases has been evalua- ted using standard chromatographic tests and probes. Principal Component Analysis of the data has indicated that the phases can be divided into distinct groupings. Extending the dataset to include standard alkyl and phenyl phases provided further data interpretation to support the orthogonal selectivity claims made for perfluorinated phases. The analysis of a range of basic analytes showed an unusual extended retention of hydrophilic basic analytes with perfluorophases. Furthermore, a non- linear relationship between the amount of organic modifier and the logarithm of the retention factor was observed, for the hydrophilic bases, which could not be modelled with LC prediction softwares. This was in sharp contrast to the alkyl and phenyl pha- ses examined. Basic analyte retention on perfluoroalkyl phases could be modelled adequately for the lipophilic bases. Exploration of the retention mechanism of these perfluoro phases indicated that silanol interactions were important in retention and selectivity. Using a rapid, isocratic, high organic modifier methodology, it was possible to analyse a mixture containing a lipophilic steroid, hydrophilic base and an internal standard in a 4 minutes with a perfluorophenyl phase. This had previously only been achievable with an alkyl phase under gradient elution conditions.

A range of C18 bonded silica stationary phases, two specifically designed for capillary electrochromatography (CEC) use, have been evaluated for their performance in the CEC analysis of basic analytes of differing physico-chemical properties. The performance of the phases towards basic analytes will be discussed in terms of various CEC parameters and by Principal Component Analysis (PCA) of five LC chromatographic parameters – hydrophobic and steric selectivity, hydrogen bonding and ion-exchange capacities at pH 7.6 and 2.7. The CEC phases, which the PCA highlighted as possessing high non-acidic silanol activity, were shown to give substantially enhanced EOF at pH 7.8 and did not exhibit the phenomenon of “drying-out” at pH values below 6.1 which was pronounced with hydrophobic LC phases. The Hypersil CEC Basic C18 phase was found to provide superior performance in analysing the basic analytes compared to the Hypersil CEC C18 phase, this was attributed by PCA to its non-acidic base silica. Through the use of experimentally derived electrophoretic mobility values, LC retention factor, and electrosmotic flow values in CEC, it was possible to predict the CEC retention behaviour of ten basic analytes to an accuracy of ± 6%.

The chromatographic isolation and characterisation of the four compounds present in the quaternary phenanthridine veterinary trypanocidal agent, isometamidium chloride hydrochloride (ISM), is reported. The isolated compounds were unambiguously characterised using spectroscopic (NMR, UV, IR and MS) methods as 3-amino-8-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium (1a) and related isomers, 8-amino-3-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium, 3,-8-diamino-7-[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium and 3,-8-bis[3-(3-carbamimidoyl-phenyl)-triazenyl]-5-ethyl-6-phenylethidium. During the course of this study, it was realised that the nature of the solvent used in the NMR study was critical as in DMSO-d6 the quaternary group in the compounds was reduced to dihydro forms (e.g. 2a).

Silica stationary phases of varying purity have been compared and evaluated for the LC separation of pharmaceutically relevant basic compounds of differing molecular weight, log P, and pK a using aqueous/organic mobile phases. The least pure silica gave the best separations. All phases showed a linear relationship between In k and volume fraction of ACN (30-70%) or MeOH (15-35%). The investigations confirmed that all the unbonded phases examined possessed a hydrophobic/adsorption and ion-exchange character. The use of silica with MS friendly mobile phases, and high volume fractions of ACN, has been shown to be applicable to LC/MS analysis of basic analytes.

In this work, the use of three Hypersil unbonded silica phases of different purity, with simple aqueous-organic mobile phase eluents has achieved excellent separations of a basic analyte mixture. LC determination of the ion-exchange capacity of the three silicas highlighted a significant difference in their acidic silanol activity i.e. Hypersil silica >Hypersil BDS silica >Hypurity silica. The total silanol activity for the three phases was very similar. Isocratic CEC analysis on the acidic Hypersil unbonded silica using RP-CEC mobile phase conditions (pH 7.8) generated excellent peak shape, good efficiencies (148–508,000 plates/metre) and baseline separation within 14 min for a mixture of six pharmaceutically relevant basic analytes. The less acidic unbonded silicas gave better peak shapes with reduced analysis times than the Hypersil silica, however baseline separation of the basic analytes was not achieved. It was postulated that the separation mechanism with unbonded silica is a balance between the analyte's electromobility and its interaction (i. e. ion-exchange and reversed-phase/adsorption mechanisms) with the silica surface. The magnitude of the interaction is dependent upon the proportion and distribution of acidic silanol groups present on the surface and the pH of the mobile phase.

The capillary electrochromatographic (CEC) separation of a range of pharmaceutical bases was investigated on a commercially available silica stationary phase using aqueous mobile phases. The effects of mobile phase composition, buffer pH, applied voltage, and buffer anion on the retention behaviour of these bases were studied. Promising chromatography was obtained at pH 7.8 but was later found to be irreproducible. However, successful and reproducible chromatography of the bases was achieved at pH 2.3.

The capillary electrochromatographic separation of six substituted barbiturates in the ion-suppressed mode was investigated on commercially available C18, C8, and phenyl-bonded packing materials. The effect on the retention behavior of mobile phase composition, buffer pH, buffer ionic strength, and temperature was studied. Successful chromatography of the barbiturates was achieved with all three packing materials over a pH range from 2.3 to 7.8. It was notable that a significant electroosmotic flow (EOF) was generated with a pH 2.3 mobile phase for all the packing materials, suggesting that capillary electrochromatography (CEC) in the ion suppression mode can be applied to a wide range of weakly acidic compounds of pharmaceutical interest. The C18, C8, and phenyl-bonded packing materials gave similar selectivities and retention times, and approximately linear relationships between percentage organic modifier in the mobile phase or temperature versus ln k were observed. Increasing the buffer ionic strength reduced the EOF but did not result in changes in selectivity. The use of methanol compared with acetonitrile had no effect on selectivity but resulted in a large increase in retention of the analytes. The experimental investigations have shown that the CEC separation of a series of weakly acidic compounds of pharmaceutical relevance can be achieved readily using a range of commercially available packed capillaries.

The analytical benefits of using “short-end injection” rapid analysis in capillary electrochromatography (CEC) have been demonstrated. These include extremely rapid analysis times, the potential for voltage gradients to be exploited, high hydrodynamic loadability and the convenience of using short detachable lengths of packed capillaries. “Short-end injection” rapid CEC has been demonstrated for the analysis of a standard neutral test mixture and three pharmaceutically active steroids. Extremely short analysis times were obtained due to the increased effective field strength experienced by the packed bed and the reduced length to the detector. In conjunction with an appropriate sampling rate, peak efficiencies in excess of 554,000 plates/metre (i.e. 41,500 plates per column) could be obtained. The repeatability of retention time and peak area compared favourably to that obtained using a conventional CEC approach.

Capillary electrochromatography (CEC) has seen a resurgence of interest during the 1990s, despite having origins in the 1970s. The technique combines the desirable features of both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE): the separation process is based on differential interactions between the stationary and mobile phases, whilst the electroosmotic flow transports the mobile phase through the capillary. Thus, it has demonstrated advantages over both HPLC and CE, which are yet to be fully exploited over a wide field of application; already the popularity of CEC is on the increase, as reflected in the number of scientific publications and seminars held. The aim of this tutorial review is to increase awareness and understanding of both theoretical and practical aspects of CEC. Whilst it does not provide an in-depth account of CEC, it does attempt to cover the more important, relevant work available in the open literature: only major advancements associated with CEC applications are highlighted. Material presented in the review was typically obtained by literature searches involving Analytical Abstracts, Chemical Abstracts and ‘BIDS’ (for academic use only).

The ever-increasing number of commercially available reversed phases with which the analyst is confronted can cause problems in column selection. These and the non-standard test procedures used by the column manufacturers and packing companies cause further confusion. In order to independently compare and contrast a range of well established C18 stationary phases, we have performed a modified column characterization approach, based on Tanaka's methodology, on thirty commonly used phases in our laboratory. These results have been evaluated and presented in various formats as Principal Component Analysis, Cluster Analysis, Tabular Format and Radar Plots in order to assist in observing similarities and differences between phases. The results indicate that, while no two phases are exactly the same (with the exception of “me-to” clones), it is possible to characterize phases into different classes based on their chromatographic performance against selected test probes. The paper illustrates the similarities and differences between these phases. The column characterization described in the paper can form the basis of a rational column selection protocol by either careful matching of the appropriate column characteristics to the analyte's physico-chemical properties or by a systematic evaluation of columns from our various categories.

The operational parameters and factors which control the performance of capillary electrochromatography (CEC) using commercially available CEC instrumentation are evaluated and discussed. The CEC of neutral or ion-suppressed acidic analytes shows marked advantages such as increased column efficiency and reduced analysis times compared to conventional high-performance liquid chromatography (HPLC). Development of the CEC method using C-18 stationary phases follows similar guidelines to that of HPLC and, due to the high efficiencies obtained, the time required for method development can be substantially reduced. The technique of CEC is extremely attractive for the analysis of pharmaceuticals due to its excellent quantitative characteristics: good repeatability, reproducibility, wide UV detector linearity, and excellent detection limits. The analysis of acidic and neutral analytes by CEC using reverse-phase material is highly successful. However, in order to analyze highly basic analytes a strong cation exchange stationary phase is needed. This phase can produce staggering and as yet unexplained “apparent” efficiencies of over 40×106 plates per meter; however, to-date these results are highly non-reproducible. The application of CEC to the pharmaceutical industry has been demonstrated in the analysis of a wide range of structurally diverse pharmaceutical compounds using capillaries packed with reverse-phase materials. © 1997 John Wiley & Sons, Inc. J Micro Sep9: 373–387, 1997

The methyl- and ethylsulphoxide diastereoisomers (V and VI) of the corticosteroid tipredane (INN I) have been shown to undergo further stereoselective S-oxidation to yield diastereoisomeric disulphoxides (II). Interactive computer optimisation software was employed to develop semi-preparative chromatography conditions for the isolation of the disulphoxide diastereoisomers (II) and to develop a multiselective gradient HPLC analysis of tipredane (I), the four monosulphoxide diastereoisomeric pairs (V, VI, IX and X), the four disulphoxide diastereoisomers (II), the vinyl methyl and ethyl derivatives (XI and XII) and the methylsulphone of tipredane (VII). The four diastereoisomeric disulphoxides (II) have been isolated by semi-preparative HPLC and their structures unambiguously confirmed by high resolution multinuclear NMR and mass spectrometry. The stereochemical assignment of the four disulphoxide diastereoisomers (II), the ethylsulphoxide diastereoisomeric pair (VI), and the vinyl methyl and ethylsulphoxide diastereoisomeric paris (IX and X) was determined by degradation/synthesis and relation to the S/R-disulphoxide (II) whose stereochemistry was determined by X-ray crystallography. The monosulphoxides (V and VI) showed a high degree of site and stereoselectivity towards further S-oxidation. S-Oxidation on the C-17 β-substituent of tipredane occurred at a rate approximately 50-fold faster than that on the α-substituent. The disulphoxides (II) have been shown to be susceptible to thermolysis yielding the vinyl methylsulphoxide diastereoisomers (IX) preferentially. The loss of the ethylsulphenic acid from the disulphoxide diastereoisomers (II) could be rationalised in terms of the preferred rotamers of the C-17 substituents.

The epimeric α-ethylsulphoxides of tipredane have been shown to undergo sulphoxide epimerisation and elimination of ethylsulphenic acid during reversed-phase (RP) HPLC, prior to the chromatographic separation. This has been shown to be promoted by increased oven temperature, acidic mobile phase, extended analysis time, column chemistry (i.e. silanol population and distribution) and increased metal content (i.e. iron(II) and titanium) of the RP stationary material. Their propensity to undergo such reactions is related to the facile C-17α C-S(O)Et bond breakage. Epimerisation and elimination have been postulated to arise via a common intermediate: an immobilised ethylsulphoxide-metal-geminal silanol complex.

The degree of sulphoxide epimerisation (and concomitant ethylsuphenic acid elimination) of the α-ethylsuphoxide epimers of tipredane showed no direct correlation with measured chromatographic parameters for a wide range of commercially available octadecylsilyl stationary phases. Correlation was however established with the metal content (i.e. iron(II) and titanium) of these phases. Low metal content stationary phases have been shown to cause epimerisation (in addition to poor chromatography for analytes with chelating properties) via contamination of the top of the column with metal ions extracted from the stainless-steel or titanium frits when the column had been shipped in neat acetonitrile or methanol. The total metal content of packed columns could be conveniently assessed using the highly sensitive 2,3- and 2,7-dihydroxynaphthalene efficiency ratio test.

The retention properties of a silica gel column and a type C silica (silicon hydride) column for bases, sugars and polar acids were compared in hydrophilic interaction chromatography (HILIC) mode with formic acid or ammonium acetate as aqueous phase modifiers. The type C silica column was much more retentive for a series of model bases than the silica gel column and, surprisingly, retention of bases increased on the type C silica column when, the higher pH, ammonium acetate containing mobile phase was used. The retention of sugars was greater on the type C silica column than on the silica gel column and also increased on the type C silica column with increased pH suggesting either a silanophilic mechanism of retention or some unknown mechanism. Three type C silica based columns, type C silica, cogent diamond hydride and a β-pinene modified column, which it was hoped might exert some additional stereochemical discrimination, were tested for metabolomic profiling of urine. In general the unmodified type C silica column gave the strongest retention of the many polar metabolites in urine and could provide a useful complement to established HILIC methods for metabolomic profiling.

The validity of the extended Tanaka column characterization procedure against the retention behavior of 101 analytes of widely differing properties chromatographed on five differing stationary phase chemistries has been established using a chemometric technique called principal component analysis (PCA). It was concluded that the simple and conveniently determined column characterization parameters covered the same space in the PCA loading plot as the retention times for the 101 differing analytes. This confirms that the ten column characterization parameters of the extended Tanaka protocol encode the same information as the retention times of the 101 analytes. Significant selectivity differences were observed between stationary phases and the mobile-phase modifiers – MeOH and MeCN. PCA contribution plots served as a convenient way to highlight specific selectivity differences between stationary phases. logD values exhibited a poor correlation with retention indicating that retention in RP-LC is not solely dictated by the analyte's hydrophobicity. The use of MeOH was found to generate greater selectivity differences with the five stationary phases than when MeCN is used.

The C-17 dithioketal moiety of the corticosteroid tipredane (INN, I ) has been shown to undergo site and stereoselective S-oxidations with a range of achiral oxidants. S-Oxidation was favoured on the methylthio substitutent (β-plane) in preference to the ethylthio substituent (α-plane) in a ratio of 1.8–3.5:1. S-Oxidation on both substituents appeared to be stereoselective yielding an approximate ratio of 4:1 and 1:3.5–6 for the S- : R -methyl and ethylsulphoxide diastereoisomers, ( II and VI ) respectively. The preferred sites for S-oxidation have been explained in terms of the preferred rotamers of the C-17 substituents and the steric factors imposed on the attack of the oxidant on the lone pair of electrons at each sulphur atom. Optimized HPLC conditions using a Hypersil ODS column and an acetonitrile—0.025 M NH 4 OAc pH 7.2 gradient at 26°C were developed to prevent degradation of the ethylsulphoxide diastereoisomers ( VI ) occurring during chromatographic analysis, via elimination of ethylsulphenic acid to yield the corresponding C-17 vinylmethylthio derivative ( VII ).

The instability of the rodent metabolites of tipredane towards sodium hydroxide has been investigated. The major degradant has been characterized as a 9-, 11β-epoxide and represents the first reported case of the elimination of hydrogen fluoride from 9α-fluoro, 11β-hydroxy substituted steroids. The 7α-hydroxy moiety in these metabolites has been shown to promote the hydrogen fluoride elimination reaction in the presence of sodium hydroxide. The rate of hydrogen fluoride elimination versus dehydration of the 7α-hydroxy substituent has been shown to be influenced by the sodium hydroxide concentration and the solvent composition.

A novel stationary phase prepared by the thermal immobilization of poly(dimethylsiloxane) onto the surface of silica (PDMS-SiO(2)) has been described, evaluated and compared with 229 commercially available RP-LC stationary phases using the Tanaka column classification protocol. The phase exhibited many unique chromatographic properties and, based on the phases in the database, was most similar to the fluoroalkylated phases (aside from the obvious lack of fluoro selectivity imposed by the C-F dipole). The phase exhibited classic reversed-phase behaviour in acid mobile phase conditions and mixed-mode reversed-phase/cation-exchange retention behaviour in neutral mobile phase conditions. The phase exhibited acceptable stability at both low and intermediate pH, conditions which should impart optimum chromatographic selectivity to the phase. Retention of basic analytes was shown to occur by a "three site model" as proposed by Neue. This new PDMS-SiO(2) stationary phase is extremely interesting in that the dominancy of its hydrophobic and ion-exchange interactions can be controlled by the influence of mobile phase pH, buffer type and concentration. The PDMS-SiO(2) stationary phase may provide a complementary tool to reversed-phase and HILIC stationary phases. The present results highlight the fact that the type of buffer, its concentration and pH can not only affect peak shape but also retention, selectivity and hence chromatographic resolution. Therefore, in method development and optimization strategies it is suggested that more emphasis should be given to the evaluation of these mobile phase operating parameters especially when basic solutes are involved.

The practical implications of performing column characterization protocols (i.e. Tanaka) and their resultant chromatographic selectivity parameters using small dimension columns (i.e. 50 × 2.1 mm I.D.) at high pressures have been critically compared to those obtained using conventional LC methodology. Retention factors should be corrected for the system extra column volume even when determined on ultra high performance liquid chromatographic (UHPLC) systems with low system volumes. An increase in pressure resulted in a general increase in the retention factor for most analytes, the degree being dependent on the physico/chemical properties of each analyte and the chromatographic conditions employed. However, analytes chromatographed at pH values close to their pK a values exhibited a substantial decrease in retention factor. Performing the Tanaka and extended column characterization procedures at pressures that would be encountered during the characterization of small particle sizes packed into 50 × 2.1 mm I.D. column formats at a constant linear velocity according to standard protocols, resulted in comparable chromatographic selectivity parameters to those determined using standard HPLC systems and column formats. However, due to the wide structural diversity of analytes employed in other popular column characterization protocols, it is imperative to demonstrate comparable results when small columns packed with small particle sizes are chromatographed at increased pressure and compared to standard column formats – otherwise erroneous comparisons and conclusions may be made.

The retention behaviour of a series of 15 n-alkylbenzenes and pentylbenzene structural isomers and benzene were investigated using porous graphitic carbon (PGC) and octadecyl-bonded silica (ODS) stationary phases. Shorter chain n-alkylbenzenes and benzene (n = 0-6), and all the pentylbenzene isomers were more strongly retained on ODS, although the selectivity was greater with PGC. For the pentylbenzene analytes the degree of branching in the alkyl chain at the position adjacent to the aromatic ring affects retention on PGC, with higher retention in less branched molecules. Molecular modelling studies have provided new insights into the geometry of aromatic pi-pi stacking interactions in retention on PGC. For alkylbenzenes with high branching at the position adjacent to the ring, the preferred geometry of association with the surface is with the branched chain directed away from the surface, a geometry not seen in the other alkylbenzenes. The most energetically favoured orientation for interaction between analytes and the PGC surface was found to be cofacial for toluene and ethylbenzene, whereas for other analytes this interaction was in a face-edge orientation. The alternative geometry of association observed with both toluene and ethylbenzene may explain the enhanced retention of these two analytes on PGC compared with their longer chain analogues. Quantitative structure-retention relationships revealed the importance of compactness in analyte structure during retention on PGC, with decreased compactness (associated with longer chain length and reduced chain branching) improving retention. (c) 2010 Elsevier B.V. All rights reserved.