Unified chromatography in drug development: Exploiting chaotropic/kosmotropic salts for an accelerated method development.

Recent trends in supercritical fluid chromatography (SFC) introduced an innovative gradient profile called Unified Chromatography (UC), which pushes the amount of liquid modifier up to 80-100 % of the total mobile phase composition. These new conditions allow the full transition from a supercritical to a liquid state, unifying the benefits of both SFC and liquid chromatography. However, to facilitate the use of UC for industrial drug development, a stronger effort is needed to streamline and simplify its method development and optimization. In this work, a quick and novel method development procedure for UC is introduced, enabled by the first-time use of novel additives in SFC/UC that exploit chaotropic/kosmotropic properties. A comprehensive view on some fundamental properties, such as the amount of liquid modifier blended with supercritical CO2 (scCO2) and the percentage of water added in the mobile phase is given, to clarify the benefits of using either a chaotropic salt (NaClO4), kosmotropic (HCOONa) or salt with mixed properties (NaOMs - sodium methanesulfonate). With this expanded knowledge, challenging separations of nucleosides, nucleotide, indoles, triazoles and related derivates have been accomplished with UC. Finally, we provide an example of UC delivering a faster and better method for an AbbVie pipeline compound under accelerated stability study. The combined use of scCO2-based chromatography and the novel additive NaClO4 ensures the retention and elution of all degradation species generated at different conditions, where RP-HPLC failed to provide satisfactory performance.

An HPLC-based Method to Quantify Coronatine Production by Bacteria.

Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This protocol describes an optimized procedure to culture bacterial cells for coronatine production and to quantify the amount of coronatine secreted in the culture medium using an HPLC-based method.

Cellular and cell-free studies of catalytic DNA cleavage by ruthenium polypyridyl complexes containing redox-active intercalating ligands.

The ruthenium(ii) polypyridyl complexes (RPCs), [(phen)2Ru(tatpp)]2+ (32+ ) and [(phen)2Ru(tatpp)Ru(phen)2]4+ (44+ ) are shown to cleave DNA in cell-free studies in the presence of a mild reducing agent, i.e. glutathione (GSH), in a manner that is enhanced upon lowering the [O2]. Reactive oxygen species (ROS) are involved in the cleavage process as hydroxy radical scavengers attenuate the cleavage activity. Cleavage experiments in the presence of superoxide dismutase (SOD) and catalase reveal a central role for H2O2 as the immediate precursor for hydroxy radicals. A mechanism is proposed which explains the inverse [O2] dependence and ROS data and involves redox cycling between three DNA-bound redox isomers of 32+ or 44+ . Cultured non-small cell lung cancer cells (H358) are sensitive to 32+ and 44+ with IC50 values of 13 and 15 µM, respectively, and xenograft H358 tumors in nude mice show substantial (∼80%) regression relative to untreated tumors when the mice are treated with enantiopure versions of 32+ and 44+ (Yadav et al. Mol Cancer Res, 2013, 12, 643). Fluorescence microscopy of H358 cells treated with 15 µM 44+ reveals enhanced intracellular ROS production in as little as 2 h post treatment. Detection of phosphorylated ATM via immunofluorescence within 2 h of treatment with 44+ reveals initiation of the DNA damage repair machinery due to the ROS insult and DNA double strand breaks (DSBs) in the nuclei of H358 cells and is confirmed using the γH2AX assay. The cell data for 32+ is less clear but DNA damage occurs. Notably, cells treated with [Ru(diphenylphen)3]2+ (IC50 1.7 µM) show no extra ROS production and no DNA damage by either the pATM or γH2AX even after 22 h. The enhanced DNA cleavage under low [O2] (4 µM) seen in cell-free cleavage assays of 32+ and 44+ is only partially reflected in the cytotoxicity of 32+ and 44+ in H358, HCC2998, HOP-62 and Hs766t under hypoxia (1.1% O2) relative to normoxia (18% O2). Cells treated with RPC 32+ show up to a two-fold enhancement in the IC50 under hypoxia whereas cells treated with RPC 44+ gave the same IC50 whether under hypoxia or normoxia.

Quinine bonded to superficially porous particles for high-efficiency and ultrafast liquid and supercritical fluid chromatography.

Two new anion-exchange columns were prepared by bonding tert-butyl carbamoylated quinine to 2.7 µm superficially porous particle (SPP) silica to create chiral stationary phases for high-efficiency and ultrafast chromatography. Performance and retention parameters of these new columns are compared with an analogous 5 µm fully porous particle (FPP) based Chiralpak QNAX column and a 3-4 fold increase in efficiency was observed. Ultrafast separations ranging from 12 s down to sub-second are shown using 2.7 µm SPPs bonded via hydrosilation to the selector. Potential benefits of 2.7 µm SPP based columns for increased LC-MS compatibility were investigated. A van Deemter plot comparison showed 2.7 µm SPP based columns provided a lower reduced plate height and a higher optimal linear velocity compared to the 5 µm FPP based column. With geometry-independent kinetic plots, 2.7 µm SPP and 5 µm FPP based columns were assessed for their kinetic performance and the maximal number of plates each column can generate in a given analysis time. The 2.7 µm SPP based column showed remarkable performance improvements in speed and efficiency as indicated by the kinetic plots.

Determination of the interconversion energy barrier of three novel pentahelicene derivative enantiomers by dynamic high resolution liquid chromatography.

Dynamic high resolution liquid chromatography (DHPLC) was used to determine the kinetic and thermodynamic activation parameters of interconversion of three novel pentahelicene derivatives {3,5-bis(trifluoromethyl)benzo[i]pentahelicene, naphtho[1,2-i]pentahelicene and 4-methoxybenzo[i]pentahelicene}. DHPLC was performed on a chiral isopropyl - carbamate cyclofructan 6 (LARIHC CF6-P) column under normal phase conditions. Variation of the column temperature and flow rate was used to study the interconversion process. A computer assisted deconvolution method was employed to determine the individual peak areas and the retention times required for the calculation of apparent enantiomerization energy barriers, enthalphy and entropy of the interconvertion of above defined pentahelicene derivative enantiomers. An ab initio quantum chemistry method was used to estimate theoretical kinetic and thermodynamic interconversion parameters and to evaluate experimental data of these three novel pentahelicene derivative enantiomers.

Separation of peptides on superficially porous particle based macrocyclic glycopeptide liquid chromatography stationary phases: consideration of fast separations.

Macrocyclic glycopeptide based liquid chromatography stationary phases are known for their highly selective peptide separations. Fast and ultrafast (t R < 1 min) high-efficiency separations were achieved with superficially porous particle (SPP)-based stationary phases. Separations of pharmaceutically important classes of peptides such as enkephalins and bradykinins have been achieved in less than 5 min in isocratic elution modes. Selectivity for peptides structurally similar to one another was increased with use of teicoplanin-based stationary phases compared with commercial C18 stationary phases. Ultrafast isocratic separations of structurally related peptides were achieved with teicoplanin- and vancomycin-based short SPP columns. Acidic mobile phases produced better separations. Ammonium formate was the optimal mobile phase buffer additive. Use of an appropriate combination of a macrocyclic glycopeptide stationary phase and a mobile phase permits faster and more electrospray ionization mass spectrometry compatible isocratic separations than previous gradient approaches. The tryptic peptide separation characteristics of the teicoplanin stationary phase are demonstrated. Additionally, compared with commercial C18 stationary phases, teicoplanin showed tryptic peptide separations with different selectivities. Graphical Abstract Ultrafast separation of enkephalin peptide epimers.

Coronatine Facilitates Pseudomonas syringae Infection of Arabidopsis Leaves at Night.

In many land plants, the stomatal pore opens during the day and closes during the night. Thus, periods of darkness could be effective in decreasing pathogen penetration into leaves through stomata, the primary sites for infection by many pathogens. Pseudomonas syringae pv. tomato (Pst) DC3000 produces coronatine (COR) and opens stomata, raising an intriguing question as to whether this is a virulence strategy to facilitate bacterial infection at night. In fact, we found that (a) biological concentration of COR is effective in opening dark-closed stomata of Arabidopsis thaliana leaves, (b) the COR defective mutant Pst DC3118 is less effective in infecting Arabidopsis in the dark than under light and this difference in infection is reduced with the wild type bacterium Pst DC3000, and (c) cma, a COR biosynthesis gene, is induced only when the bacterium is in contact with the leaf surface independent of the light conditions. These findings suggest that Pst DC3000 activates virulence factors at the pre-invasive phase of its life cycle to infect plants even when environmental conditions (such as darkness) favor stomatal immunity. This functional attribute of COR may provide epidemiological advantages for COR-producing bacteria on the leaf surface.

Advances in high-throughput and high-efficiency chiral liquid chromatographic separations.

The need for improved liquid chromatographic chiral separations has led to the advancement of chiral screening techniques as well as the development of new, high efficiency chiral separation methods and stationary phases. This review covers these advancements, which primarily occurred over the last 15 years. High throughput techniques include multi-column screening units, multiple injection sequences, and fast gradient SFC screening. New separation methods and column technologies that aim at high efficiency chiral separations include the use of achiral UHPLC (i.e. sub-2µm) columns for separating derivatized chiral analytes or using chiral additives in the run buffer, UHPLC chiral stationary phases, and superficially porous particle based chiral stationary phases. Finally, the enhancement of chiral separations through these new technologies requires that certain instrumental considerations be made. Future directions in continuing to improve chiral separations are also discussed.

Reduced matrix effects for anionic compounds with paired ion electrospray ionization mass spectrometry.

It is well-known that matrix effects in high performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) can seriously compromise quantitative analysis and affect method reproducibility. Paired ion electrospray ionization (PIESI) mass spectrometry is an approach for analyzing ultra-low levels of anions in the positive ion mode. This approach uses a structurally optimized ion pairing reagent to post-column associate with the anionic analyte, subsequently forming positively charged complexes. These newly formed complex ions are often more surface-active as compared to either the native anion or the ion pairing reagent. No studies have examined whether or not the PIESI approach mitigates matrix effects. Consequently, a controlled study was done using five analytes in highly controlled and reproducible synthetic groundwater and urine matrices. In addition, two different mass spectrometers (linear ion trap and triple quadrupole) were used. Compared to the negative ion mode, the PIESI-MS approach was less susceptible to matrix effects when performed on two different MS platforms. Using PIESI-MS, less dilution of the sample is needed to eliminate ionization suppression which, in turn, permits lower limits of detection and quantitation.

High-Performance Liquid Chromatographic Resolution of Neutral and Cationic Hetero[6]Helicenes.

Cationic hetero[6]helicenes 1+, 2+ and 3+ have been recently disclosed. Herein we report on their enantiomeric separation using high-performance liquid chromatography. Separation of the antipodes can be achieved in preparative scale on neutral adducts with Chiralcel OD-I or Chiralpak ID CSP. Selectivity factors of 1.90, 1.67, and 1.96 were obtained for 1-H, 2-H, and 3-H, respectively. Separation can also be performed on the carbenium ions on regular Chiralpak IA CSP using water-containing eluents, thus allowing for enantiomeric purity determinations in aqueous environments. Resolution of neutral and cationic helicenes is also achieved on more recently developed LARIHC columns. The versatility of the cyclofructan phases allows for baseline separations for both cases and their loading capabilities are demonstrated. Finally, the configurational stability of 1+, 2+, and 3+ was measured. For each replacement of an oxygen atom by an amino group, the racemization barrier increases significantly (ΔG‡ = 29.8, 36.3 and >37 kcal mol(-1) for 1+, 2+, and 3+ respectively).

Enantioseparation of citalopram analogues with sulfated ß-cyclodextrin by capillary electrophoresis.

Capillary electrophoresis methods were developed for the enantiomeric separation of 27 citalopram analogues. Sulfated ß-cyclodextrin was the most broadly selective and useful chiral selector. The separations of most of the citalopram analogue compounds reported in this work have not been reported previously. Excellent enantiomeric separations were obtained for 26 out of 27 compounds, and most of the separations were achieved within 10 min. The effects of chemical parameters such as chiral selector types, buffer types, chiral selector and buffer concentrations, buffer pH and organic modifiers on the separation were investigated. The influence of analyte structure on separation also was examined and discussed.

Gram Scale Conversion of R-BINAM to R-NOBIN.

A mild, operationally simple, and single-step transition-metal-free protocol for the synthesis of enantiomerically pure (R)-(+)-2'-amino-1,1'-binaphthalen-2-ol (R-NOBIN) from (R)-(+)-1,1'-binaphthyl-2,2'-diamine (R-BINAM) is reported. The one-pot conversion proceeds with good yield and shows no racemization. The hydroxyl on the R-NOBIN product was shown to have come from water in the reaction medium via an H2(18)O study. The correct value of the specific rotation of R-NOBIN was reported.

Enantiomeric separations of α-aryl ketones with cyclofructan chiral stationary phases via high performance liquid chromatography and supercritical fluid chromatography.

Normal phase chiral HPLC and SFC methods are presented for the enantiomeric separation of 21 α-aryl ketones with a unique class of chiral stationary phases (CSPs) based on cyclofructans (CFs). Separations were achieved for all but 2 analytes, with 17 compounds attaining baseline separation having resolution values up to 4.0. Most separations obtained in HPLC could be transferred to SFC, but the HPLC resolutions were generally better due to greater enantiomeric selectivity values. A structure-separation relationship (SSR) was developed to identify important structural features for separation of this class of compounds using CF-based CSPs. Preliminary studies are also presented that demonstrate the utility of the CF CSPs to investigate the base-induced enantiomerization of α-aryl ketones. It was demonstrated that even small amounts of base (0.01%v/v) in the mobile phase results in rapid, on-column, enantiomerization. Lastly, CSPs composed of superficially porous particles were used to achieve comparable separations of this class of chiral compounds, but at a fraction of the analysis time compared to CSPs composed of fully porous particles.

Mass spectrometric detection of trace anions: The evolution of paired-ion electrospray ionization (PIESI).

The negative-ion mode of electrospray ionization mass spectrometry (ESI-MS) is intrinsically less sensitive than the positive-ion mode. The detection and quantitation of anions can be performed in positive-ion mode by forming specific ion-pairs during the electrospray process. The paired-ion electrospray ionization (PIESI) method uses specially synthesized multifunctional cations to form positively charged adducts with the anions to be analyzed. The adducts are detected in the positive-ion mode and at higher m/z ratios to produce excellent signal-to-noise ratios and limits of detection that often are orders of magnitude better than those obtained with native anions in the negative-ion mode. This review briefly summarizes the different analytical approaches to detect and separate anions. It focuses on the recently introduced PIESI method to present the most effective dicationic, tricationic, and tetracationic reagents for the detection of singly and multiply charged anions and some zwitterions. The mechanism by which specific structural molecular architectures can have profound effects on signal intensities is also addressed.

Screening primary racemic amines for enantioseparation by derivatized polysaccharide and cyclofructan columns.

It is a challenge to separate the enantiomers of native chiral amines prone to deleterious silanol interactions. A set of 39 underivatized chiral primary amines was screened for enantiomeric separation. Seven recently introduced commercial chiral columns were tested. They included six polysaccharide based chiral stationary phases (CSP) with bonded derivatives, ChiralPak® IA, IB, IC, ID, IE and IF columns and a cyclofructan derivatized CSP, Larihc® CF6-P column. Both the normal phase (NP) mode with heptane/alcohol mobile phases and the polar organic (PO) mode with acetonitrile/alcohol were evaluated. It was found that the cyclofructan based CSP demonstrated the highest success rate in separating primary amines in the PO mode with only one chiral amine not resolved. It is shown that, when screening the columns, there is no standard optimal condition; an excellent mobile phase composition for one column may be poorly suited to another one. Although butylamine was a good mobile phase additive for the polysaccharide columns in both PO and NP modes, it was detrimental to the enantio-recognition capability of the cyclofructan column. Triethylamine was the appropriate silanol screening agent for this latter column.

Ultrafast separation of fluorinated and desfluorinated pharmaceuticals using highly efficient and selective chiral selectors bonded to superficially porous particles.

The separation of fluorinated active pharmaceutical ingredients (APIs) from their desfluoro analogs is a challenging analytical task due to their structural similarity. In this work, fluorine containing APIs and their corresponding desfluorinated impurities were separated on five new 2.7µm superficially porous particles (SPPs) functionalized with bonded chiral selectors. The unique shape selectivity of bonded macrocyclic glycopeptides and oligosaccharides was utilized to separate seven pairs of fluoro/desfluoro APIs resulting in some unprecedented selectivity values. For example, SPP bonded isopropyl cyclofructan 6 yielded a selectivity of 2.73 for voriconazole and desfluoro voriconazole. Further, the SPP based columns allowed for rapid separations ranging from 9 to 55s with very high efficiencies ranging from 45,000 to 70,000plates/m (at high flow rates) in both reversed phase and polar organic modes. Chromatographic separation and detection by HPLC-ESI-MS was demonstrated using ezetimibe and voriconazole and their desfluorinated impurities. Among the tested phases, SPP hydroxypropyl-ß-cyclodextrin separated the most fluorinated and desfluorinated analogs with baseline resolution.

High efficiency, narrow particle size distribution, sub-2 µm based macrocyclic glycopeptide chiral stationary phases in HPLC and SFC.

State of the art chiral chromatography still employs 3-5 µm bonded or immobilized chiral selectors in 10-25 cm columns. With the availability of 1.9 µm narrow particle size distribution (NPSD) silica, it is now possible to make ever shorter, high efficiency columns practical for sub-minute chiral separations. Three macrocyclic glycopeptides (teicoplanin, teicoplanin aglycone, and vancomycin) were bonded onto 1.9 µm NPSD particles. Such packed columns had ∼80% lower backpressure as compared to polydisperse (PD) 1.7 µm silica materials when using the same mobile phase. The decreased backpressure allowed for diminution of frictional heating and allowed for the use of the 1.9 µm NPSD particle based columns at high flow rates. The 1.9 µm NPSD particle based columns showed up to 190,000 plates m(-1) for chiral molecules and 210,000 plates m(-1) for achiral probes. Representative enantiomeric separations are shown for wide classes of compounds, including different types of amino acids, ß-blockers, and pharmaceutically important heterocyclic compounds such as oxazolidinones. Applications in three liquid chromatography modes, namely, reversed phase, polar organic mode and normal phase chiral separations were shown with resolution values ranging from 1.5 to 5.7. Additionally, the same columns were used with supercritical fluid chromatography (SFC) for ultrafast separations.

Combined use of cyclofructans and an amino acid ester-based ionic liquid for the enantioseparation of huperzine A and coumarin derivatives in CE.

Cyclofructans (CFs) and their derivatives have recently been proven to be efficient chiral selectors (CSs) for the enantioseparation of several analytes in CE, HPLC, and GC. In this study, the chiral separation ability of a number of native and derivatized CFs was examined in CE. Particularly, six different CFs, with different derivatization groups and cavity sizes [native CF-6 and CF-7, isopropyl cyclofructan-6 (IPCF-6), IPCF-7, sulfated cyclofructan-6 (SCF-6), and SCF-7] were used as CSs for the enantioseparation of huperzine A, warfarin, and coumachlor. Almost all of the examined CFs, except from SCF-6 & -7, demonstrated relatively low and sometimes no chiral separation ability for huperzine A. In an effort to improve both resolution and efficiency, the chiral ionic liquid D-Alanine tert butyl ester lactate (D-AlaC4Lac) was added into the BGE. In most of the cases, the combination of CF with D-AlaC4Lac resulted in an improvement in peak efficiency and/or resolution. When CF-6 was utilized with D-AlaC4Lac, a resolution of 1.4 was obtained, while the use of IPCF-6/D-AlaC4Lac provided a baseline enantioseparation. Although the combination of SCF-7 and 40 mM D-AlaC4Lac did not affect resolution, it dramatically increased peak efficiency from 24,000 to 117,000. In the case of warfarin and coumachlor, IPCF-6 and IPCF-7 proved to be the most effective CSs. It is, therefore, concluded that the size of the cavity and the CF derivatization are the key parameters for the chiral separation capability. It is also clear from this study that D-AlaC4Lac is necessary for improved peak efficiencies and resolutions.

Problems and Pitfalls in the Analysis of Amygdalin and Its Epimer.

α-[(6-O-ß-d-Glucopyranosyl-ß-d-glucopyranosyl)oxy]-(αR)-benzeneacetonitrile, or R-amygdalin, is the most common cyanogenic glycoside found in seeds and kernels of the Rosaceae family and other plant genera such as Passiflora. Many commercially important seeds are analyzed for amygdalin content. In "alternative medicine", amygdalin has been sold as a treatment for cancer for several decades without any rigorous scientific support for its efficacy. We have found that there are some inconsistencies and possible problems in the published analytical chemistry of amygdalin. It is shown that some analytical approaches do not account for the presence of the S-isomer; therefore, a fast reliable method was developed using a chiral stationary phase and HPLC. This approach allows "real-time" monitoring and complete and highly efficient separations. It is found that the S-amygdalin continuously forms in aqueous solutions. A striking result is that the conversion of amygdalin is glassware dependent. "Clean" vials from various vendors can show drastically different reaction rates of the conversion to the isomer (S-amygdalin, also called neo-amygdalin). The epimerization kinetics are dependent on the solvent, temperature, pH, and the nature of the container. For example, epimerization in water was complete in <15 min in a new glass vial taken from the box, whereas it can take >1 h in specially cleaned glassware. Conversely, epimerization can be significantly delayed at high temperature if high-density polyethylene is used as the container. Hence, inert plastic containers are recommended for storage of aqueous amygdalin solutions. Commercial preparations of R-amygdalin actually contain greater quantities of S-amygdalin and ∼ 5% of other degradation products.

Superficially Porous Particle Based Hydroxypropyl-ß-cyclodextrin Stationary Phase for High-Efficiency Enantiomeric Separations.

A superficially porous particle (SPP)-based hydroxypropyl-ß-cyclodextrin (HPBCD) chiral stationary phase (CSP) was produced and its chromatographic performance was compared to both 5 µm and 3 µm fully porous particle (FPP)-based CSPs. The relative surface coverage of the HPBCD chiral selector on each particle was approximately equal, which resulted in equivalent enantiomeric selectivity (α) values on each phase when constant mobile phase conditions were used. Under such conditions, the SPP column resulted in greatly reduced analysis times and three times greater efficiencies compared to the FPP columns. When higher flow rates were used, efficiency gains per analysis times were five times greater for the SPP column compared to the FPP-based columns. When the mobile phases were altered to give similar analysis times on each column, resolution values were doubled for the SPP column. Finally, the novel SPP based HPBCD column proved to be stable for 500 injections under high flow rate (4.5 mL/min) and high pressure (400 bar) conditions used for an ultrafast (~45 sec) enantiomeric separation.