A simulation-guided approach to the selection of RPLC columns for platform small molecule separations.

Simulations were conducted to evaluate the potential of several hundred reversed-phase columns to separate small molecules. By calculating the retention factor of compounds in randomly generated virtual mixtures via the HSM (hydrophobic subtraction model) and applying basic chromatography theory, the simulation can estimate the retention time and peak width of every virtual compound and calculate the resolution between every adjacent pair of compounds. A preferred column set based on the number of successful separations of randomly generated virtual mixtures was developed. The tandem-column liquid chromatography (TC-LC) approach can separate 53.2 % of the 16-compound samples using 20 tandem-column pairs, while a single-column approach can only separate 42.6 % of the 16-compound samples with 20 single columns. The preferred set of columns obtained from the simulation was almost the same as the empirical set of columns previously obtained. In screening applications, TC-LC can achieve a comparably successful separation factor (selectivity) with a smaller column inventory (nine 50-mm columns) compared to the larger inventory needed by single-column LC (twenty-one 100-mm columns).

Separation of weak acids, neutral compounds, and permanent anions using sequential elution liquid chromatography with tandem columns.

This paper discusses the development of an analytical method by an alternative separation approach, sequential elution liquid chromatography (SE-LC), to separate permanently charged ions (anions), weak acids, and neutral compounds using anion exchange and reversed-phase columns in tandem. SE-LC separates classes of compounds by group by employing two or more elution modes. Advantages to using SE-LC over conventional HPLC are a greater peak capacity and a reduced separation disorder. Importantly, the same HPLC as used for a conventional HPLC separation may be used to afford a successful SE-LC separation. Mobile phase selection and gradient optimization are integral for a successful SE-LC class separation of permanent anions, weak acids, and neutral compounds and will be discussed in detail in this paper. The most successful (best resolution and repeatability) SE-LC separation was achieved by applying isocratic elution at low pH to elute the weak acids, followed by an acetonitrile gradient to elute the neutral compounds, and last a sodium methanesulfonate gradient to elute the anionic compounds using a superficially porous C18 column coupled with a strong anion exchange (SAX) column. Repeatability (RSD) in the retention times and peak areas of the analytes was less than 0.25 % and 1.5 %, respectively.

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.

Chiral high-performance liquid and supercritical fluid chromatographic enantioseparations of limonene-based bicyclic aminoalcohols and aminodiols on polysaccharide-based chiral stationary phases.

Enantioseparation of limonene-based bicyclic 1,3-aminoalcohols and 1,3,5- and 1,3,6-aminodiols was performed by normal-phase high-performance liquid chromatographic and supercritical fluid chromatographic (SFC) methods on polysaccharide-based chiral stationary phases. The effects of the composition of the mobile phase, the column temperature and the structures of the analytes and chiral selectors on retention and selectivity were investigated by normal-phase LC and SFC technique. Thermodynamic parameters derived from selectivity-temperature-dependence studies were found to be dependent on the chromatographic method applied, the nature of the chiral selector and the structural details of the analytes. Enantiorecognition in most cases was enthalpically driven but an unusual temperature behavior was also observed: decreased retention times were accompanied by improved separation factors with increasing temperature, i.e. some entropically driven separations were also observed. The elution sequence was determined in all cases. The separation of the stereoisomers was optimized in both chromatographic modalities.

Development of simultaneous targeted metabolite quantification and untargeted metabolomics strategy using dual-column liquid chromatography coupled with tandem mass spectrometry.

Targeted quantification and untargeted global profiling are the two mainstream approaches, a merging of which could provide enhanced analytical potential in metabolomics research. Here, a simultaneous targeted quantification and untargeted metabolomics (STQUM) strategy was developed for more efficient, accurate and comprehensive metabolomics research by using ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UPLC-HRMS/MS). First, we selected 110 cancer-related metabolites as targets and established a dual LC sequential separation method for simultaneous analysis of strong and weak polar metabolites. In order to achieve efficient acquisition for synchronous qualitative and quantitative analysis, high-resolution, data-dependent parallel reaction monitoring (PRM) method and data-independent all ion fragmentation (AIF) method were established. Their performance in targeted confirmation and quantification, and untargeted analysis were systematically investigated and assessed. In total, 78 metabolites were confidently confirmed in positive ion mode in both PRM and AIF assays, in which 73 metabolites can be accurately quantified. In addition, simultaneously untargeted profiling of 4651 features of high reliability and validity were achieved. Both AIF and PRM methods revealed high confidence, sensitivity and accuracy. In the STQUM approach, another 15 metabolites could be accurately quantified in negative ion mode. The method offers a new perspective for merging the hypothesis-based targeted quantitative validation and untargeted biomarkers discovery in one run for improved analysis efficiency and integrity.

Method development for comprehensive extraction and analysis of marine toxins: Liquid-liquid extraction and tandem liquid chromatography separations coupled to electrospray tandem mass spectrometry.

A variety of toxins are produced by marine and freshwater microorganisms that present a threat to human health. These toxins have diverse chemical properties and specifically, a range of hydrophobicity. Methods for extraction and identification of these toxins are often geared toward specific classes of toxin depending on the sample type. There is a need for a general method of toxin extraction and identification for screening samples where the likely toxin content is not known a priori. We have applied a general method for metabolite extraction to toxin containing samples. This method was coupled with a simple dual liquid chromatography approach for separating a broad range of toxins. This liquid chromatography approach was coupled to triple quadrupole and quadrupole time-of-flight MS/MS platforms. The method was testing on a fish matrix for recovery of palytoxin as well as marine corals for detection of natural mixtures of palytoxin analogues. The recovery of palytoxin was found to produce a linear response (R2 of 0.95) when spiked into the fish matrix with a limit of quantitation of 2.5 ng/µL and recovery efficiency of 73% + /- 9%. The screening of corals revealed varying amount of palytoxin, and in one case, different palytoxin structural analogues. This demonstration illustrates the potential utility of this method for toxin extraction and detection.

Effects of N-methylation and amidination of cyclic ß-amino acids on enantioselectivity and retention characteristics using Cinchona alkaloid- and sulfonic acid-based chiral zwitterionic stationary phases.

In the present work the effects of N-methylation and N-amidination of ampholytic cyclic ß-amino acids on their retention and enantioseparation characteristics on Cinchona alkaloid- and sulfonic acid-based zwitterionic chiral selectors, namely Chiralpak ZWIX(+)™ and ZWIX(-)™ columns are described. In a polar-ionic mobile phase, a double ion-pairing interaction mechanism takes place between the charged sites of the chiral analytes (the selectands) and the chiral selector moieties. As a support to correlate the chromatographic results with the structural details of the analytes, pKa values and van der Waals volumes of the substituted amino groups were calculated. In order to ensure better understanding of the mechanistic details of the chromatographic system the composition of the bulk solvent, the role of acid base additives, the concentration of the counter-ions, temperature and the structures of the ampholytic analytes have been investigated. Applying N-Fmoc protection, the ampholytic character of the analytes diminished, leading to a marked loss of retention. In the temperature range studied (5-40 °C) thermodynamic parameters, such as the difference in the standard enthalpy change Δ(ΔH°), entropy Δ(ΔS) and Gibbs energy Δ(ΔG°) were calculated from linear van't Hoff plots derived from the ln α vs. 1/T curves. The values of the thermodynamic parameters depended on the structures of the chiral selectors applied and the analytes studied.

High-performance liquid chromatographic separation of paclitaxel intermediate phenylisoserine derivatives on macrocyclic glycopeptide and cyclofructan-based chiral stationary phases.

High-performance liquid chromatographic methods were developed for the separation of enantiomers of four unnatural paclitaxel precursor phenylisoserine analogs on chiral stationary phases containing macrocyclic glycopeptides and cyclofructans as chiral selectors. The effects of the mobile phase composition, the nature and concentration of different mobile phase additives (alcohols, amines and acids) in different chromatographic modes, temperature and the structures of the analytes on the separations were investigated. Separations were carried out at constant mobile phase compositions in the temperature range 10-50°C on macrocyclic antibiotic-based and 5-35°C on cyclofructan-based columns and the changes in enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°), were calculated. The elution sequence was determined in most cases; no general rule could be observed.

Investigation of the structure-selectivity relationships and van't Hoff analysis of chromatographic stereoisomer separations of unusual isoxazoline-fused 2-aminocyclopentanecarboxylic acids on Cinchona alkaloid-based chiral stationary phases.

The enantiomers of four unusual, rather rigid isoxazoline-fused 2-aminocyclopentanecarboxylic acids were directly separated on a quinine- or a quinidine-based zwitterionic ion-exchanger as chiral selector. The effects of the mobile phase composition, the structures of the analytes and temperature on the separations were investigated. Experiments were performed at constant mobile phase composition in the temperature range 10-50°C to study the effects of temperature, and thermodynamic parameters were calculated from plots of ln α versus 1/T. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. It was found that the enantiomer separations were in most cases predominantly enthalpy-driven, but entropically-driven separations were also observed. The sequence of elution of the enantiomers was determined in all cases.

Thermodynamics Study of Solvent Adsorption on Octadecyl-Modified Silica.

Elution and solvation processes in liquid chromatography may be controlled by temperature changes. In the case of solvent adsorption, the temperature influences the amount of adsorbed solvent as well as the enthalpy and entropy of the solvation process. In this work, the thermodynamic parameters of organic solvents used as organic modifiers in the reversed-phase high-performance liquid chromatography elution process were determined. The changes of enthalpy and entropy in a series of chemically bonded stationary phases were measured to determine the effects of the temperature and surface coverage density of octadecyl ligands on the thermodynamic parameters of the solvation. For both the enthalpy and entropy a parabolic trend was observed with the minimum for medium surface coverage. The correlation of solvent adsorption values with the enthalpy of solvation was also investigated. The highest influence of the temperature on solvation process was observed for stationary phases with high surface coverage.

An Approach to Transfer Methods from HPLC to UHPLC Techniques in Some Carbapenems.

Stability-indicating LC methods were developed and validated for the quantitative determination of doripenem, meropenem and tebipenem in the presence of their degradation products formed during forced degradation studies. Isocratic HPLC and UHPLC separations were performed with a core-shell Kinetex 1.7, 2.6 and 5 µm, all C18, 100A, 100 × 2.1 mm columns and the mobile phase composed of acetonitrile and 12 mmol L-1 ammonium acetate in different ratios. The flow rates of the mobile phase were: 0.5 mL min-1 for 1.7 µm column, and 1.0 mL min-1 for 2.6 and 5 µm ones. Detection wavelength was 298 nm and temperature was set at 30 °C. All analysed drugs were exposed to stress conditions which caused their hydrolysis and thermal degradation. The methods were validated by evaluation of linearity, accuracy, precision, selectivity and robustness. Proposed methods were successfully applied for the determination of investigated antibiotics during kinetic studies in aqueous solutions and in the solid state. The advantages of chromatographic procedures which are based on the use of C18 stationary phases with different particle sizes in the analysis of selected carbapenems were discussed.

Assay of Diastereoisomers of Cefuroxime Axetil in Amorphous and Crystalline Forms Using UHPLC-DAD.

A sensitive UHPLC-DAD method was developed for determination of diastereoisomers of cefuroxime axetil in bulk substance in amorphous and crystalline forms as well as in pharmaceutical preparations. Chromatographic separation was achieved on Kinetex C-18 (100 mm × 2.1 mm, 1.7 µm) column with mobile phase consisting of 0.1 % formic acid:methanol (88:12, v/v), at the flow rate of 0.7 mL min-1 and total run time of 3 min. The wavelength of the DAD detector was set at 278 nm. Inter-day precision (RSD) was less than 3 % and accuracy level ranged between 98.31 and 104.99 %. Degradation products of cefuroxime axetil in aqueous solutions and in the solid state were identified with a EIS-Q-MS mass spectrometer. The solubility of above-mentioned polymorphic forms of cefuroxime axetil in suitable solvents is a crucial factor during preparation of samples and is essential for chromatographic separation of its diastereoisomers.

Direct high-performance liquid chromatographic enantioseparation of secondary amino acids on Cinchona alkaloid-based chiral zwitterionic stationary phases. Unusual temperature behavior.

Two chiral stationary phases containing a quinine- or a quinidine-based zwitterionic ion-exchanger as chiral selector were applied for the enantioseparation of 27 unusual cyclic secondary α-amino acids. The effects of the nature and concentration of the bulk solvent, the acid and base additives, the structures of the analytes and temperature on the enantioresolution were investigated. To study the effects of temperature and to obtain thermodynamic parameters, experiments were carried out at constant mobile phase compositions in the temperature range -5 to 55 °C. The thermodynamic parameters indicated that in most cases the separations were enthalpy-driven, but some entropy-driven separations were also observed. The sequence of elution of the enantiomers was determined in most cases.

Unusual temperature-induced retention behavior of constrained ß-amino acid enantiomers on the zwitterionic chiral stationary phases ZWIX(+) and ZWIX(-).

The effects of temperature on the chiral recognition of cyclic ß-amino acid enantiomers on zwitterionic [Chiralpak ZWIX(+) and ZWIX(-)] chiral stationary phases were investigated. Experiments were performed at different mobile phase compositions and under 10°C column temperature increments in the temperature range 10-50°C. Apparent thermodynamic parameters and T(iso) values were calculated from plots of ln k and ln α versus 1/T, respectively. Unusual temperature behavior was observed, especially on the ZWIX(-) column, where the application of MeOH/MeCN (50/50 v/v) containing 25 mM triethylamine and 50 mM formic acid as mobile phase led to nonlinear van't Hoff plots and increasing retention time with increasing temperature. On both columns, both enthalpically and entropically driven separations were observed.

A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog.

Nitrosylcobalamin (NO-Cbl), a novel vitamin B12 analog and anti-tumor agent, functions as a biologic 'Trojan horse', utilizing the vitamin B12 transcobalamin II transport protein and cell surface receptor to specifically target cancer cells. a stability-indicating HPLC method was developed for the detection of NO-Cbl during forced degradation studies. This method utilized an ascentis® RP-amide (150 mm × 4.6 mm, 5 µm) column at 35 °C with a mobile phase (1.0 mL min-1) combining a gradient of methanol and an acetate buffer at pH 6.0. Detection wavelengths of 450 and 254 nm were used to detect corrin and non-corrin-based products, respectively. NO-Cbl, synthesized from hydroxocobalamin and pure nitric oxide gas, was subjected to degradative stress conditions including oxidation, hydrolysis and thermal and radiant energy challenge. The method was validated by assessing linearity, accuracy, precision, detection and quantitation limits and robustness. The method was applied successfully for purity assessment of synthesized NO-Cbl and for the determination of NO-Cbl during kinetic studies in aqueous solution and in solid-state degradation assessments. This HPLC method is suitable for the separation of cobalamins in aqueous and methanolic solutions, for routine detection of NO-Cbl and for purity assessment of synthesized NO-Cbl. additionally, this method has potential application in identification and monitoring of diseases involving altered nitric oxide homeostasis where vitamin B12 therapy is utilized to scavenge excess nitric oxide, subsequently resulting in the in vivo production of NO-Cbl.

Vortex-assisted magnetic dispersive solid-phase microextraction for rapid screening and recognition of dicofol residues in tea products.

A simple and rapid vortex-assisted magnetic dispersive solid-phase microextraction (VAMDSME) method coupled with gas chromatography-electronic capture detection was developed for rapid screening and selective recognition of dicofol in tea products. The magnetic molecularly imprinted microspheres (mag-MIMs) synthesised by aqueous suspension polymerisation using dichlorodiphenyltrichloroethane (DDT) as a dummy template showed high selectivity and affinity to dicofol in aqueous solution and were successfully applied as special adsorbents of VAMDSME for rapid isolation of dicofol from complex tea matrix. Good linearity was obtained in a range of 0.2-160 ng g(-1) and the limit of detection based on a signal to noise ratio of 3 was 0.05 ng g(-1). The recoveries at three spiked levels ranged from 83.6% to 94.5% with the related standard deviations (RSD) ⩽ 5.0%. The VAMDSME-GC protocol, which took advantages of the selective adsorption of molecularly imprinted microspheres and rapid magnetic phase separation, as well as the short equilibrium time by vortex-assisted, could avoid the time-consuming procedures related to other traditional extraction methods.

LC-MS/MS Method for the Simultaneous Determination of Free Urinary Steroids.

Cortisol homeostasis is implicated in hypertension and metabolic syndrome. Two enzymes modulate cortisol availability; 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) preferentially converts inactive cortisone to cortisol, whereas 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) converts cortisol to cortisone. In contrast, 5α and 5ß reductases inactivate cortisol by conversion to its tetrahydrometabolites: tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone. A subtle local increase in cortisol can be detected by measuring 24-h urine metabolites, LC-MS/MS being the reference method. The 11ß-HSD2 activity is assessed based on the cortisol/cortisone ratio, and the 11ß-HSD1 activity on the (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ratio. To better understand hypertension and/or metabolic syndrome pathogenesis a method for simultaneous determination of cortisol, cortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone was developed and validated in an LC coupled with the new detector AB Sciex QTrap® 4500 tandem mass spectrometer. The steroids were extracted from 1 mL urine, using cortisol-D4 as internal standard. The quantification range was 0.1-120 ng/mL for cortisol and cortisone, and 1-120 ng/mL for tetrahydrometabolites, with >89 % recovery for all analytes. The coefficient of variation and accuracy was <10 %, and 85-105 %, respectively. Our LC-MS/MS method is accurate and reproducible in accordance with Food and Drug Administration guidelines, showing good sensitivity and recovery. This method allows the assessment of 11ß-HSD2 and 11ß-HSD1 activities in a single analytical run providing an innovative tool to explain etiology of misclassified essential hypertension and/or metabolic syndrome.

Enantiomeric separation of bicyclo[2.2.2]octane-based 2-amino-3-carboxylic acids on macrocyclic glycopeptide chiral stationary phases.

Direct high-performance liquid chromatographic (HPLC) separation of four bicyclo[2.2.2]octane based 2-amino-3-carboxylic acid enantiomers were developed on chiral stationary phases (CSPs) containing different macrocyclic glycopeptide antibiotic selectors. The analyses were performed under reversed-phase, polar organic and polar ionic mode on macrocyclic-glycopeptide-based Chirobiotic T, T2, TAG, and R columns. The effects of the mobile phase composition including the acid and base modifier, the structure of the analytes, and the temperature on the separations were investigated. Experiments were achieved at constant mobile phase compositions on different stationary phases in the temperature range 5-40°C. Thermodynamic parameters were calculated from plots of ln k or ln α versus 1/T. It was recognized that the enantioseparations in reversed-phase and polar organic mode were enthalpically driven, but under polar-ionic conditions entropically driven enantioseparation was observed as well. Baseline separation and determination of elution sequence were achieved in all cases.

Purification of pituitary autoantigen by column liquid chromatography and chromatofocusing.

INTRODUCTION: Pituitary autoantibodies can be determined both in patients with pituitary disease as well as patients with autoimmune endocrine diseases. The purpose of the study was to isolate and purify pituitary autoantigen using sera of patients and the microsomal fraction of the pituitary. MATERIAL AND METHODS: To isolate a pituitary autoantigen, patient sera were used, which showed a strong immune response to pituitary antigens. Pituitary microsomal fractions were prepared from pituitary tissue homogenates. In the study, sera of patients with pituitary disease, Addison and Graves' disease were used. The initial stages were carried out by affinity chromatography on CN -Br sepharose column whereas purification was continued by column liquid chromatography on AcA54 Ultrogel. Chromatofocusing was performed by Polybuffer exchanger PBE 94. RESULTS: (125)I-labeled pituitary antigens after isolation appeared in column chromatography in three peaks. The first peak contained 50-70 kDa proteins, the second peak - 17 to 22 kDa proteins and the third peak contains (125)-iodides. Three fractions obtained from filtration on Ultrogel were separated in a polyacrylamide gel. In the first peak two bands 67 and 55 kDa appeared. The second peak contained low molecular weight substances, and the third peak contained (125)I. The first peak from Ultrogel was isolated by chromatofocusing - the first peak with pH 5.9 and the second one with pH 4.9. CONCLUSIONS: Isolation and purification of pituitary autoantigen with the use of column liquid chromatography and chromatofocusing resulted in obtainment of two antigenic proteins of specific gravity of 67 and 55 kDa.