Development of tandem-column liquid chromatographic methods for pharmaceutical compounds using simulations based on hydrophobic subtraction model parameters.

The liquid chromatography (LC) analysis of small molecule pharmaceutical compounds and related impurities is crucial in the development of new drug substances, but developing these separations is usually challenging due to analyte structural similarities. Tandem-column LC (TC-LC) has emerged as a powerful approach to achieve alternative separation selectivity compared to conventional single column separations. However, one of the bottlenecks associated with use of tandem column approaches is time-consuming column pair screening and selection. Herein, we compared critical resolution (Rc) in single column vs. TC-LC separations for a given set of small molecule pharmaceutical compounds and developed a column selection workflow that uses separation simulations based on parameters from the hydrophobic subtraction model (HSM) of reversed-phase selectivity. In this study, HSM solute parameters were experimentally determined for a small molecule pharmaceutical (Linrodostat) and ten of its related impurities using multiple linear regression of their retentions on 16 selected RPLC columns against in-house determined HSM column parameters. Rc values were calculated based on HSM database column parameters for a pool of about 200 available stationary phases in both single-phase column (2.1 mm i.d. × 100 mm) or tandem column paired (two 2.1 mm i.d. × 50 mm) formats. Four column configurations (two single and two tandem) were predicted to achieve successful separations under isocratic HSM separation conditions, with a fifth tandem pair predicted to have a single co-elution. Of these five potential candidates, one tandem pair yielded compete baseline resolution of the 11-component mixture in an experimental separation. In this specific case, the tandem column pairs outperformed single-phase columns, with better predicted and experimental Rc values for the Linrodostat mixture under the HSM separation conditions. The results reported in this study demonstrated the enormous selectivity potential of TC-LC in pharmaceutical compound separations and are consistent with our previous study that examined the potential of tandem column approaches using purely computational means, though there is room for substantial improvement in the prediction accuracy. The proposed workflow can be used to prioritize a small number of column combinations by computational means before any experiments are conducted. This is highly attractive from the point of view of time and resource savings considering over 200,000 different tandem column pairings are possible using columns for which there are data in the HSM database.

Are two liquid chromatography columns in tandem better than one?: Answers from the hydrophobic subtraction model.

An approach is described for determining if there is an intrinsic advantage, from a selectivity and resolution perspective, of using two different UHPLC/HPLC reversed-phase columns in tandem for a separation of a given sample compared to a single U/HPLC reversed-phase column that provides the same plate number. Retention data for 16 compounds extracted directly from the hydrophobic subtraction model (HSM) database at HPLCColumns.org are used to simulate and then compare the critical resolution of those compounds obtained using HSM conditions (isocratic elution at 35°C using 50% acetonitrile, 50% aqueous phosphate buffer at pH 2.8 or 7) for each of 662 U/HPLC single columns or 218,791 combinations of tandem columns and assuming a modest plate number of 8000. The critical resolution obtained for 16 additional "n-1" samples created by the systematic removal of one of the original 16 compounds was also compared using single- and tandem-column LC, as was the critical resolution obtained for thousands of synthetic samples generated by randomly varying HSM solute descriptors for each synthetic compound. When all possible single-column or tandem-column results were compared, a significant advantage was observed with tandem-column liquid chromatography (TC-LC), with an average increase in critical resolution of 0.63 (pH 2.8) or 0.75 (pH 7) units observed for the synthetic samples with the smallest number of components (m = 5). As the number of components in a sample increased, the average improvement in critical resolution (∆Rs,crit) using TC-LC gradually decreased from about 0.70 for m = 5 to 0.18 for m = 32 components. The average improvement in critical resolution achieved by switching from SC-LC to TC-LC was also lower when a smaller number of columns and column combinations were available to explore, as would be the case for a finite column inventory in a real laboratory. Nevertheless, on average there does appear to be an intrinsic advantage of tandem-column liquid chromatography, however small, which can be amplified by using high efficiency columns.

Simultaneous separation of small interfering RNA and lipids using ion-pair reversed-phase liquid chromatography.

RNA interference offers a novel approach for the development of new therapeutics for targets that are otherwise "undruggable" using traditional modalities. The safety and efficacy of siRNA-based therapy mainly rely on lipid or polymer-based nanocarriers to overcome inherent barriers to a systemic delivery of siRNA. A multicomponent lipid nanoparticle (LNP) system is a promising delivery platform, typically consisting of a cationic lipid, phospholipid, PEG-containing short-chain lipid, and cholesterol. Characterization and chemical analysis of the LNP formulation is important to assure drug product stability, a key consideration for chemistry, manufacturing and control strategy. Here we report an ion-pair reversed phase UHPLC method capable of simultaneously separating both siRNA and functional lipids in LNPs with a minimal retention gap for two classes of biologically essential yet chemically distinct molecules. Key chromatographic parameters critical to the separation are discussed, including the structure of the ion-pair agent, stationary phase chemistry, column temperature and an organic additive. The results showed that the retention time of siRNA is tunable by using various ion-pair reagents. The retention factor of the siRNA exhibited a first order relationship with the number of carbons in the alkyl chain of the ion-pair reagents. In contrast, the type of ion-pair reagent has no significant impact on the separation of phospholipids. Separations using a BEH phenyl column and dibutylammonium acetate as the ion-pair reagent showed satisfactory selectivity for a range of double-stranded siRNAs and phospholipids, key components for lipid nanoparticle formulations. Furthermore, the method was applied to the separation of an experimental LNP formulation, demonstrating good selectivity for siRNA, functional lipids and their potential degradation products.

Organic solvent modifier and temperature effects in non-aqueous size-exclusion chromatography on reversed-phase columns.

Common reversed-phase columns (C18, C4, phenyl, and cyano) offer inert surfaces suitable for the analysis of polymers by size-exclusion chromatography (SEC). The effect of tetrahydrofuran (THF) solvent and the mixtures of THF with a variety of common solvents used in high performance liquid chromatography (acetonitrile, methanol, dimethylformamide, 2-propanol, ethanol, acetone and chloroform) on reversed-phase stationary phase characteristics relevant to size exclusion were studied. The effect of solvent on the elution of polystyrene (PS) and poly(methyl methacrylate) (PMMA) and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e., 10°C-60°C) on the SEC partition coefficients KSEC of PS and PMMA polymers, were also investigated. The bonded phases show remarkable differences in size separations when binary mixtures of THF with other solvents are used as the mobile phase. The solvent impact can be two-fold: (i) change of the polymeric coil size, and possible shape, and (ii) change of the stationary phase pore volume. If the effect of this impact is properly moderated, then the greatest benefit of optimized solute resolution can be achieved. Additionally, this work provides an insight on solvent-stationary phase interactions and their effects on column pore volume. The only effect of temperature observed in our studies was a decreased elution volume of the polymers with increasing temperature. SEC partition coefficients were temperature-independent in the range of 10°C-60°C and therefore, over this temperature range elution of PS and PMMA polymers is by near-ideal SEC on reversed-phase columns. Non-ideal SEC appears to occur for high molar mass PMMA polymers on a cyano column when alcohols are used as mobile phase modifiers.

Aqueous size-exclusion chromatography of polyelectrolytes on reversed-phase and hydrophilic interaction chromatography columns.

The size-exclusion separation of a water-soluble polyelectrolyte polymer, sodium polystyrene sulfonate (NaPSS), was demonstrated on common reversed-phase (C18, C4, phenyl, and cyano) and hydrophilic interaction chromatography (HILIC) columns. The effect of common solvents - acetonitrile (ACN), tetrahydrofuran (THF), and methanol (MeOH), used as mobile phase modifiers - on the elution of NaPSS and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e., 10 °C-60 °C) on the partition coefficient, KSEC, were also investigated. Non-size-exclusion chromatography (non-SEC) effects can be minimized by the addition of an electrolyte and an organic modifier to the mobile phase, and by increasing the column temperature (e.g., to 50 °C or 60 °C). Strong solvents such as THF and ACN are more successful in the reduction of such effects than is the weaker solvent MeOH. The best performance is seen on medium polarity and polar stationary phases, such as cyanopropyl- and diol-modified silica (HILIC), where the elution of the NaPSS polyelectrolyte is by a near-ideal SEC mechanism. Hydrophobic stationary phases, such as C18, C4, and phenyl, require a higher concentration of a strong solvent modifier (THF) in the mobile phase to reduce non-SEC interactions of the solute with the stationary phase.

Peak capacity and peak capacity per unit time in capillary and microchip zone electrophoresis.

The origins of the peak capacity concept are described and the important contributions to the development of that concept in chromatography and electrophoresis are reviewed. Whereas numerous quantitative expressions have been reported for one- and two-dimensional separations, most are focused on chromatographic separations and few, if any, quantitative unbiased expressions have been developed for capillary or microchip zone electrophoresis. Making the common assumption that longitudinal diffusion is the predominant source of zone broadening in capillary electrophoresis, analytical expressions for the peak capacity are derived, first in terms of migration time, diffusion coefficient, migration distance, and desired resolution, and then in terms of the remaining underlying fundamental parameters (electric field, electroosmotic and electrophoretic mobilities) that determine the migration time. The latter expressions clearly illustrate the direct square root dependence of peak capacity on electric field and migration distance and the inverse square root dependence on solute diffusion coefficient. Conditions that result in a high peak capacity will result in a low peak capacity per unit time and vice-versa. For a given symmetrical range of relative electrophoretic mobilities for co- and counter-electroosmotic species (cations and anions), the peak capacity increases with the square root of the electric field even as the temporal window narrows considerably, resulting in a significant reduction in analysis time. Over a broad relative electrophoretic mobility interval [-0.9, 0.9], an approximately two-fold greater amount of peak capacity can be generated for counter-electroosmotic species although it takes about five-fold longer to do so, consistent with the well-known bias in migration time and resolving power for co- and counter-electroosmotic species. The optimum lower bound of the relative electrophoretic mobility interval [µr,Z, µr,A] that provides the maximum peak capacity per unit time is a simple function of the upper bound, but its direct application is limited to samples with analytes whose electrophoretic mobilities can be varied independently of electroosmotic flow. For samples containing both co- and counter-electroosmotic ions whose electrophoretic mobilities cannot be easily manipulated, comparable levels of peak capacity and peak capacity per unit time for all ions can be obtained by adjusting the EOF to devote the same amount of time to the separation of each class of ions; this corresponds to µr,Z=-0.5.

In Silico Screening of Two-Dimensional Separation Selectivity for Ion Chromatography × Capillary Electrophoresis Separation of Low-Molecular-Mass Organic Acids.

A prerequisite for ordered two-dimensional (2D) separations and full utilization of the enhanced 2D peak capacity is selective exploitation of the sample attributes, described as sample dimensionality. In order to take sample dimensionality into account prior to optimization of a 2D separation, a new concept based on construction of 2D separation selectivity maps is proposed and demonstrated for ion chromatography × capillary electrophoresis (IC×CE) separation of low-molecular-mass organic acids as test analytes. For this purpose, 1D separation selectivity maps were constructed based on calculation of pairwise separation factors and identification of critical pairs for four IC stationary phases and 28 levels of background electrolyte pH in CE. The derived IC and CE maps were then superimposed and the effectiveness of the respective 2D separations assessed using an in silico approach, followed by testing examples of one successful and one unsuccessful 2D combination experimentally. The results confirmed the efficacy of the predictions, which require a minimal number of experiments compared to the traditional one-at-a-time approach. Following the same principles, the proposed framework can also be adapted for optimization of separation selectivity in various 2D combinations and for other applications.

Separation of small interfering RNA stereoisomers using reversed-phase ion-pairing chromatography.

Small interfering RNA (siRNA) therapeutics have received significant interest in recent years owing to their ability to elicit sequence-specific gene knockdown and subsequent suppression of protein expression. Chemical modifications can improve the hydrolytic stability and susceptibility of siRNAs to enzymatic degradation. One commonly used modification to improve hydrolytic stability, the replacement of the native phosphodiester linkage with a phosphorothioate moiety, introduces an additional phosphorous stereocenter into the molecule, resulting in the formation of diastereomers. The chromatographic separation of stereoisomeric siRNAs containing such phosphothioates is a challenging problem, especially when multiple phosphothioates are present within a modified siRNA duplex giving rise to multiple stereoisomers. In this study, we report an investigation into the use of an ion-pairing reversed phase UHPLC (or IP-RP UHPLC) method for the baseline separation of closely related diastereomers of an Apo-B gene targeting siRNA duplex under denaturing conditions. The related siRNA species consist of two diastereomers from the sense strand and the two pairs of diastereomers from the antisense strand. Key chromatographic parameters critical to stereoisomer separation are highlighted, including the structure of the ion-pairing agent, chemical composition of the stationary phase, and organic modifier. The method was applied to the separation of an siRNA stressed with iodine and demonstrated satisfactory selectivity for the parent siRNA and the desulfurization product.

Longitudinal On-Column Thermal Modulation for Comprehensive Two-Dimensional Liquid Chromatography.

Longitudinal on-column thermal modulation for comprehensive two-dimensional liquid chromatography is introduced. Modulation optimization involved a systematic investigation of heat transfer, analyte retention, and migration velocity at a range of temperatures. Longitudinal on-column thermal modulation was realized using a set of alkylphenones and compared to a conventional valve-modulator employing sample loops. The thermal modulator showed a reduced modulation-induced pressure impact than valve modulation, resulting in reduced baseline perturbation by a factor of 6; yielding a 6-14-fold improvement in signal-to-noise. A red wine sample was analyzed to demonstrate the potential of the longitudinal on-column thermal modulator for separation of a complex sample. Discrete peaks in the second dimension using the thermal modulator were 30-55% narrower than with the valve modulator. The results shown herein demonstrate the benefits of an active focusing modulator, such as reduced detection limits and increased total peak capacity.

Multidimensional liquid-phase separations combining both chromatography and electrophoresis - A review.

Described as intrinsically powerful building blocks for two-dimensional separations by Giddings [Anal. Chem., 56 (1984) 1258A-1270A], the coupling of chromatography and electrophoresis has been proven to enhance the resolution of a wide array of molecules in complex biological, environmental and food samples. This review provides a comprehensive overview of multidimensional chromato-electrophoretic (LC - E) and electrophero-chromatographic (E - LC) separation systems from inception to the most recent published examples. LC separation modes include reversed phase, ion exchange, and size exclusion. Electromigration separation modes include capillary, microchip or free flow electrophoresis; micellar electrokinetic chromatography; electrochromatography; and isoelectric focusing. The advantages and disadvantages of various non-gel based off-line and on-line hyphenation technologies of LC - E and E - LC are discussed, with conditions and system characteristics also provided.

Dual-opposite injection capillary electrophoresis: Principles and misconceptions.

Dual-opposite injection capillary electrophoresis (DOI-CE) is a separation technique that utilizes both ends of the capillary for sample introduction. The electroosmotic flow (EOF) is suppressed to allow all ions to reach the detector quickly. Depending on the individual electrophoretic mobilities of the analytes of interest and the effective length that each analyte travels to the detection window, the elution order of analytes in a DOI-CE separation can vary widely. This review discusses the principles, applications, and limitations of dual-opposite injection capillary electrophoresis. Common misconceptions regarding DOI-CE are clarified.

Stochastic approach for an unbiased estimation of the probability of a successful separation in conventional chromatography and sequential elution liquid chromatography.

A stochastic approach was utilized to estimate the probability of a successful isocratic or gradient separation in conventional chromatography for numbers of sample components, peak capacities, and saturation factors ranging from 2 to 30, 20-300, and 0.017-1, respectively. The stochastic probabilities were obtained under conditions of (i) constant peak width ("gradient" conditions) and (ii) peak width increasing linearly with time ("isocratic/constant N" conditions). The isocratic and gradient probabilities obtained stochastically were compared with the probabilities predicted by Martin et al. [Anal. Chem., 58 (1986) 2200-2207] and Davis and Stoll [J. Chromatogr. A, (2014) 128-142]; for a given number of components and peak capacity the same trend is always observed: probability obtained with the isocratic stochastic approach

Direct determination of amino acids by hydrophilic interaction liquid chromatography with charged aerosol detection.

A chromatographic analytical method for the direct determination of amino acids by hydrophilic interaction liquid chromatography (HILIC) was developed. A dual gradient simultaneously varying the pH 3.2 ammonium formate buffer concentration and level of acetonitrile (ACN) in the mobile phase was employed. Using a charged aerosol detector (CAD) and a 2(nd) order regression analysis, the fit of the calibration curve showed R(2) values between 0.9997 and 0.9985 from 1.5mg/mL to 50µg/mL (600ng to 20ng on column). Analyte chromatographic parameters such as the sensitivity of retention to the water fraction in the mobile phase values (mHILIC) were determined as part of method development. A degradation product of glutamine (5-pyrrolidone-2-carboxylic acid; pGlu) was observed and resolved chromatographically with no method modifications. The separation was used to quantitate amino acid content in acid hydrolysates of various protein samples.

Size exclusion chromatography of synthetic polymers and biopolymers on common reversed phase and hydrophilic interaction chromatography columns.

This work describes the applicability of common reversed phase and HILIC columns for size exclusion chromatography of synthetic and natural polymers. Depending on the nature of the solute and column stationary phase, a "non-retention" condition must be created with the aid of the mobile phase to achieve a unique size-based separation in isocratic mode. The various bonded phases show remarkable differences in size separations that are controlled by mobile phase conditions. Polymer-mobile phase and column-mobile phase solvation interactions determine polymer hydrodynamic volume (or solute bulkiness) and polymer-column steric interaction. Solvation interactions in turn depend on polymer, mobile phase and stationary phase polarities. Column-mobile phase solvation interactions determine the structural order of the bonded ligands that can vary from ordered (extended, aligned away from the silica substrate) to disordered (folded, pointing toward the silica substrate). Chain order increases with increased solvent penetration into the bonded phase. Increased chain order reduces pore volume, and therefore decreases the size-separation efficiency of a column. Conversely, decreased chain order increases pore volume and therefore increases the size-separation efficiency. The thermodynamic quality of the mobile phase also plays a significant role in the separation of polymers. "Poor" solvents can significantly reduce the hydrodynamic diameter of a solute and thus change their retention behavior. Medium polarity stationary phases, such as fluoro-phenyl and cyano, exhibit a unique retention behavior. With an appropriate polarity mobile phase, polar and non-polar synthetic polymers of the same molecular masses can be eluted at the same retention volumes.

A systematic approach for avoiding co-detection of oppositely charged analytes in dual-opposite-injection capillary electrophoresis.

Dual-opposite-injection capillary electrophoresis (DOI-CE) is a separation technique that utilizes both ends of the capillary for sample introduction. Depending on the individual electrophoretic mobilities of the analytes of interest and the migration distance that each analyte travels to the detection window, the elution order of analytes in a DOI-CE separation can vary widely. One consequence of a variable elution order is that co-detection of oppositely charged analytes becomes possible under certain experimental conditions. While several approaches currently exist to avoid co-detection, they are often time consuming and inefficient. This paper describes a systematic approach to easily avoid co-detection by analyzing the net mobilities of the analytes of interest to aid in choosing appropriate capillary lengths for a particular separation. Simulated electropherograms that highlight various aspects of this new approach are shown. An electropherogram from the literature is replicated and the derived relationship is applied to show the potential advantages of this new approach.

Nonequilibrium capture rates induce protein accumulation and enhanced adsorption to solid-state nanopores.

Single molecule capturing of analytes using an electrically biased nanopore is the fundamental mechanism in which nearly all nanopore experiments are conducted. With pore dimensions being on the order of a single molecule, the spatial zone of sensing only contains approximately a zeptoliter of volume. As a result, nanopores offer high precision sensing within the pore but provide little to no information about the analytes outside the pore. In this study, we use capture frequency and rate balance theory to predict and study the accumulation of proteins at the entrance to the pore. Protein accumulation is found to have positive attributes such as capture rate enhancement over time but can additionally lead to negative effects such as long-term blockages typically attributed to protein adsorption on the surface of the pore. Working with the folded and unfolded states of the protein domain PDZ2 from SAP97, we show that applying short (e.g., 3-25 s in duration) positive voltage pulses, rather than a constant voltage, can prevent long-term current blockades (i.e., adsorption events). By showing that the concentration of proteins around the pore can be controlled in real time using modified voltage protocols, new experiments can be explored which study the role of concentration on single molecular kinetics including protein aggregation, folding, and protein binding.

Investigation of a new core-shell particle column for ion-pair reversed-phase liquid chromatography analysis of oligonucleotides.

A new core-shell particle column showed excellent performance and durability for separation of short (∼21-mer) ribonucleic acid (RNA) oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC). Previously investigated core-shell C18 columns showed excellent peak shapes and separations of closely eluting impurities by IP-RPLC. However, these columns showed only modest long-term stability at the neutral pH and elevated column temperatures of ≥60°C, typically used for IP-RPLC analysis of oligonucleotides. The newly introduced SunShell C18 column provided separations comparable to the previously evaluated core-shell columns, but with significantly improved long-term column stability when operated at neutral pH and elevated column temperature.

Sequential elution liquid chromatography can significantly increase the probability of a successful separation by simultaneously increasing the peak capacity and reducing the separation disorder.

This paper demonstrates that sequential elution liquid chromatography (SE-LC), an approach in which two or more elution modes are employed in series for the separation of two or more groups of compounds, can be used to separate not only weak acids (or weak bases) from neutral compounds, but weak acids and weak bases from neutral compounds (and each other) by the sequential application of either of two types of an extended pH gradient prior to a solvent gradient. It also details a comparison, based on peak capacity and separation disorder, of the probability of success of this approach with the unimodal elution approach taken by conventional column liquid chromatography. For an HPLC peak capacity of 120 and samples of moderate complexity (e.g., 12 components), the probability of success (Rs≥1) increases from 37.9% (HPLC) to 85.8% (SE-LC). Different columns were evaluated for their utility for SE-LC using the following criteria: (1) the prediction of the elution order of the groups based on the degree of ionization of the compounds; and (2) the closeness of the peak shape to the ideal Gaussian distribution. The best columns overall were the Zorbax SB-AQ and Waters XBridge Shield columns, as they provided both between-class and within-class separations of all compounds, as well as the lowest degree of tailing of 4-ethylaniline using the pH 2 to pH 8 gradient.

Factors influencing the separation of oligonucleotides using reversed-phase/ion-exchange mixed-mode high performance liquid chromatography columns.

New mixed-mode columns consisting of reversed-phase and ion-exchange separation modes were evaluated for the analysis of short RNA oligonucleotides (∼20mers). Conventional analysis for these samples typically involves using two complementary methods: strong anion-exchange liquid chromatography (SAX-LC) for separation based on charge, and ion-pair reversed-phase liquid chromatography (IP-RPLC) for separation based on hydrophobicity. Recently introduced mixed-mode high performance liquid chromatography (HPLC) columns combine both reversed-phase and ion-exchange modes, potentially offering a simpler analysis by combining the benefits of both separation modes into a single method. Analysis of a variety of RNA oligonucleotide samples using three different mixed-mode stationary phases showed some distinct benefits for oligonucleotide separation and analysis. When using these mixed-mode columns with typical IP-RPLC mobile phase conditions, such as ammonium acetate or triethylammonium acetate as the primary ion-pair reagent, the separation was mainly based on the IP-RPLC mode. However, when changing the mobile phase conditions to those more typical for SAX-LC, such as salt gradients with NaCl or NaBr, very different separation patterns were observed due to mixed-mode interactions. In addition, the Scherzo SW-C18 and SM-C18 columns with sodium chloride or sodium bromide salt gradients also showed significant improvements in peak shape.

Evaluation of core-shell particle columns for ion-pair reversed-phase liquid chromatography analysis of oligonucleotides.

An investigation into the use of core-shell particle columns for separation of short (∼21 base pairs) RNA oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC) showed improved resolution for a number of test analytes relative to conventional (fully-porous) reversed-phase columns. The best resolutions were obtained using columns packed with smaller sub-2µm core-shell particles.