Structure Driven Prediction of Chromatographic Retention Times: Applications to Pharmaceutical Analysis.

Pharmaceutical drug development relies heavily on the use of Reversed-Phase Liquid Chromatography methods. These methods are used to characterize active pharmaceutical ingredients and drug products by separating the main component from related substances such as process related impurities or main component degradation products. The results presented here indicate that retention models based on Quantitative Structure Retention Relationships can be used for de-risking methods used in pharmaceutical analysis and for the identification of optimal conditions for separation of known sample constituents from postulated/hypothetical components. The prediction of retention times for hypothetical components in established methods is highly valuable as these compounds are not usually readily available for analysis. Here we discuss the development and optimization of retention models, selection of the most relevant structural molecular descriptors, regression model building and validation. We also present a practical example applied to chromatographic method development and discuss the accuracy of these models on selection of optimal separation parameters.

Retention characteristics of some antibiotic and anti-retroviral compounds in hydrophilic interaction chromatography using isocratic elution, and gradient elution with repeatable partial equilibration.

The separation of some zwitterionic, basic and neutral antibiotic and antiretroviral compounds was studied using hydrophilic interaction chromatography (HILIC) on bare silica, bonded amide and urea superficially porous phases. The differences in the selectivity and retentivity of these stationary phases were evaluated for compounds with widely different physicochemical properties (logD -3.43 to 2.41 at wwpH 3.0). The mobile phase was acetonitrile-ammonium formate buffered at low wwpH. Compounds containing quinolone and serine groups showed poor peak shapes on all columns, attributed to metal-oxide interactions with system metals. Peak shapes were improved by addition of citrate buffers. Gradient elution, particularly with regard to column equilibration, was also studied due to the large differences in retention factors observed under isocratic conditions. Full equilibration in HILIC was slow for both ionogenic and neutral solutes, requiring as much as ∼40 column volumes. However, highly repeatable partial equilibration, suitable for gradient elution, was achieved in only a few minutes. Pronounced selectivity differences in the separations were shown dependent on the partial equilibration time.

Integrated Analytical and Statistical Two-Dimensional Spectroscopy Strategy for Metabolite Identification: Application to Dietary Biomarkers.

A major purpose of exploratory metabolic profiling is for the identification of molecular species that are statistically associated with specific biological or medical outcomes; unfortunately, the structure elucidation process of unknowns is often a major bottleneck in this process. We present here new holistic strategies that combine different statistical spectroscopic and analytical techniques to improve and simplify the process of metabolite identification. We exemplify these strategies using study data collected as part of a dietary intervention to improve health and which elicits a relatively subtle suite of changes from complex molecular profiles. We identify three new dietary biomarkers related to the consumption of peas (N-methyl nicotinic acid), apples (rhamnitol), and onions (N-acetyl-S-(1Z)-propenyl-cysteine-sulfoxide) that can be used to enhance dietary assessment and assess adherence to diet. As part of the strategy, we introduce a new probabilistic statistical spectroscopy tool, RED-STORM (Resolution EnhanceD SubseT Optimization by Reference Matching), that uses 2D J-resolved 1H NMR spectra for enhanced information recovery using the Bayesian paradigm to extract a subset of spectra with similar spectral signatures to a reference. RED-STORM provided new information for subsequent experiments (e.g., 2D-NMR spectroscopy, solid-phase extraction, liquid chromatography prefaced mass spectrometry) used to ultimately identify an unknown compound. In summary, we illustrate the benefit of acquiring J-resolved experiments alongside conventional 1D 1H NMR as part of routine metabolic profiling in large data sets and show that application of complementary statistical and analytical techniques for the identification of unknown metabolites can be used to save valuable time and resources.

Some factors that can lead to poor peak shape in hydrophilic interaction chromatography, and possibilities for their remediation.

Some factors which present difficulties for obtaining good peak shape in hydrophilic interaction chromatography (HILIC) were studied. The effect of injection solvent composition and volume was systematically investigated using a selection of weak and stronger basic compounds on a hybrid bare silica phase. Increasing the mismatch between the injection solvent (range 95-0% ACNv/v) and the mobile phase (maintained at 95% ACNv/v) gave increasing deterioration in peak shape. With the 2.1mm ID columns used, injections in the mobile phase of increasing volume (1-20 µL) gave poorer peak shape, but the magnitude of the effect was considerably smaller than that of solvent mismatch over this range. Some solute structural features such as galloyl (trihydroxy benzene), catechol (benzene diol) and phosphate (in nucleotides) gave serious peak tailing, attributed to interactions with metals in the stationary phase or the chromatographic hardware. These undesirable effects can be moderated by including complexing agents in the mobile phase, by changing the stationary phase chemistry, or by altering the mobile phase pH.

Performance of charged aerosol detection with hydrophilic interaction chromatography.

The performance of the charged aerosol detector (CAD) was investigated using a diverse set of 29 solutes, including acids, bases and neutrals, over a range of mobile phase compositions, particularly with regard to its suitability for use in hydrophilic interaction chromatography (HILIC). Flow injection analysis was employed as a rapid method to study detector performance. CAD response was 'quasi-universal', strong signals were observed for compounds that have low volatility at typical operating (room) temperature. For relatively involatile solutes, response was reasonably independent of solute chemistry, giving variation of 12-18% RSD from buffered 95% ACN (HILIC) to 10% ACN (RP). Somewhat higher response was obtained for basic compared with neutral solutes. For cationic basic solutes, use of anionic reagents of increasing size in the mobile phase (formic, trifluoroacetic and heptafluorobutyric acid) produced somewhat increased detector response, suggesting that salt formation with these reagents is contributory. However, the increase was not stoichiometric, pointing to a complex mechanism. In general, CAD response increased as the concentration of acetonitrile in the mobile phase was increased from highly aqueous (10% ACN) to values typical in the HILIC range (80-95% ACN), with signal to noise ratios about four times higher than those for the RP range. The response of the CAD is non-linear. Equations describing aerosol formation cannot entirely explain the shape of the plots. Limits of detection (determined with a column for solutes of low k) under HILIC conditions were of the order of 1-3ng on column, which compares favourably with other universal detectors. CAD response to inorganic anions allows observation of the independent movement through the column of the cationic and anionic constituents of basic drugs, which appear to be accompanied by mobile phase counterions, even at quite high solute concentrations.

Comparison of the kinetic performance and retentivity of sub-2 µm core­shell, hybrid and conventional bare silica phases in hydrophilic interaction.

The separation performance and retention properties of four sub-2µm underivatised silica materials were evaluated in the hydrophilic interaction chromatography (HILIC) mode. These included an organic/inorganic hybrid silica, conventional silica, narrow particle size distribution silica and a core-shell silica. Van Deemter characterisation was performed using conditions to give high retention factors (k=5.5-6.0) with 10cm columns to limit the contribution of extra-column dispersion. The core-shell 1.6µm bare silica (Cortecs) was shown to be kinetically superior to fully porous particle types. Little column-to-column variation in the reduced b-coefficient was observed for the test analytes as corroborated by arrested elution experiments. However, the reduced b-coefficient was shown to be different between analytes, e.g. cytosine versus nortriptyline. It is speculated that the nature of the retention mechanism (hydrophilic versus ionic retention) and solute physiochemical properties perhaps influence the b-coefficient. Maxwell-Effective Medium Theory (EMT) applied to results for a wider range of solutes indicated that the intra-particle diffusion (Dpart) behaviour for individual compounds is broadly similar irrespective of the particle morphology in HILIC. Finally, the impact of varying buffer concentration for a test mix showed that retention and peak shape varied considerably between different silicas. High efficiency separations can be achieved for hydrophilic and basic solutes using a combination of sub-2µm core shell bare silica particles and appropriate buffer concentrations.

Comparison of peak shape in hydrophilic interaction chromatography using acidic salt buffers and simple acid solutions.

The retention and peak shape of neutral, basic and acidic solutes was studied on hydrophilic interaction chromatography (HILIC) stationary phases that showed both strong and weak ionic retention characteristics, using aqueous-acetonitrile mobile phases containing either formic acid (FA), ammonium formate (AF) or phosphoric acid (PA). The effect of organic solvent concentration on the results was also studied. Peak shape was good for neutrals under most mobile phase conditions. However, peak shapes for ionised solutes, particularly for basic compounds, were considerably worse in FA than AF. Even neutral compounds showed deterioration in performance with FA when the mobile phase water concentration was reduced. The poor performance in FA cannot be entirely attributed to the negative impact of ionic retention on ionised silanols on the underlying silica base materials, as results using PA at lower pH (where their ionisation is suppressed) were inferior to those in AF. Besides the moderating influence of the salt cation on ionic retention, it is likely that salt buffers improve peak shape due to the increased ionic strength of the mobile phase and its impact on the formation of the water layer on the column surface.

Practical observations on the performance of bare silica in hydrophilic interaction compared with C18 reversed-phase liquid chromatography.

The kinetic performance of a bare silica and C18 phase prepared from the same sub-2µm and 3.5µm base materials were compared in the HILIC and RP mode using both charged and neutral solutes. The HILIC column was characterised using the neutral solute 5-hydroxymethyluridine, the weak base cytosine, and the strong base nortriptyline, the latter having sufficient retention also in the RP mode to allow comparison of performance. Naphthalene was also used as a simple neutral substance to evaluate the RP column alone. The retention factors of all substances were adjusted to give similar values (k'∼5.5) at their respective optimum linear velocities. Reduced van Deemter b-coefficients (determined by curve fitting and by the peak parking method, using a novel procedure involving switching to a dummy column) were significantly lower in HILIC for all substances compared with those found under RP conditions. Against expectation, c-coefficients were always lower in RP when compared with HILIC using sub-2µm particles. While measurement of these coefficients is complicated by retention shifts caused by the influence of high pressure and by frictional heating effects, broadly similar results were obtained on larger particle (3.5µm) phases. The mechanism of the separations was further investigated by examining the effect of buffer concentration on retention. It was concluded that HILIC can sometimes show somewhat inferior performance to RP for fast analysis at high mobile phase velocity, but clearly shows advantages when high column efficiencies, using longer columns at low flow velocity, are employed. The latter result is attributable to the lower viscosity of the mobile phase in HILIC and the reduced pressure requirement as well as the lower b-coefficients.

Practical investigation of the factors that affect the selectivity in hydrophilic interaction chromatography.

Retention data for a series of 29 compounds comprising acids, bases and neutrals were obtained on six different hydrophilic interaction (HILIC) columns including bare silica, zwitterionic and those bonded with neutral bonded ligands. The principal aim of the work was to evaluate the effect of various experimental variables such as the nature of the stationary phase, buffer pH, buffer concentration, organic solvent and its concentration, and temperature, in order to determine which factors gave the most effect on the selectivity of the separation. The influence of solute properties on the separation, such as logD values, was also considered. In this way, it was hoped to provide a practical guide to aid in the selection of conditions to achieve a separation in HILIC. The nature of the stationary phase was found to have the greatest effect on selectivity.