Journal of Chromatography B (v.875, #1)

by Bezhan Chankvetadze; Eric Francotte (1).

Immobilized-type chiral packing materials for HPLC based on polysaccharide derivatives by Tomoyuki Ikai; Chiyo Yamamoto; Masami Kamigaito; Yoshio Okamoto (2-11).
The polysaccharide-based chiral packing materials (CPMs) for high-performance liquid chromatography (HPLC) have been recognized as the most powerful ones for the analyzing and preparative separating of the chiral compounds. These CPMs have been conventionally prepared by coating polysaccharide derivatives on a silica gel support. This means that the solvents, which swell or dissolve the derivatives on the silica gel and reduce the performance of the chiral columns, do not allow to be applied as components of the eluents. Therefore, the polysaccharide-based CPMs can be used with a rather limited number of eluents. In order to enhance the versatility of the eluent selection for more practical and economical chromatographic enantioseparations, the polysaccharide derivatives must be immobilized onto the silica gel. This review summarizes our latest studies on the development of the immobilized-type CPMs via the radical copolymerization and the polycondensation of the polysaccharide derivatives bearing small amounts of vinyl groups and alkoxysilyl groups, respectively.
Keywords: Amylose; Cellulose; Chiral stationary phase; Chiralpak; High-performance liquid chromatography; Immobilization; Resolution;

Chiral stationary phases (CSPs) based on proteins or glycoproteins have been developed for the enantioseparations of various compounds. In 2001, a review article [J. Haginaka, J. Chromatogr. A, 906 (2001) 253] dealing with CSPs based on proteins and glycoproteins was published. After that serum albumin from other species, penicillin G-acylase, antibodies, fatty acid binding protein and streptavidin were newly introduced as the chiral selectors in CSPs. This review article deals with recent progresses in CSPs based on protein or glycoproteins in LC after 2001, focusing on their enantioselective properties and chiral recognition mechanisms.
Keywords: Reviews; LC stationary phases; Chiral stationary phases; Protein; Glycoprotein; Antibody; Chiral separation; Enantioseparation;

The review examines the most recent achievement of immobilized Penicillin G acylase (PGA) as chiral stationary phases (PGA-CSP) for the separation of acidic enantiomers. Particular attention is paid to the influence of structural variations of a large number of analytes on retention and enantioselectivity with a special emphasis on advances in the elucidation of the chiral recognition mechanism. Docking calculations and molecular dynamic simulations are discussed to rationalize the origins of enantioselective behaviour. The review also touches the chiral behaviour of PGA in partial filling CE. The CE results, obtained using PGA in free solution, suggest that the immobilization procedure used in the development of PGA-CSPs did not alter the enantioselective properties of the enzyme.
Keywords: Penicillin G acylase; Enantioselective discrimination; HPLC; CE; Molecular modelling; Review;

Model of CE enantioseparation systems with a mixture of chiral selectors by Pavel Dubský; Jana Svobodová; Bohuslav Gaš (30-34).
Theory of equilibria, migration and dynamics of interconversion of a chiral analyte in electromigration enantioseparation systems involving a mixture of chiral selectors for the chiral recognition (separation) are proposed. The model assumes that each individual analyte–CS interaction is fast, fully independent on other interactions and the analyte can interact with CS in 1:1 ratio and that the analyte is present in the concentration small enough not to considerably change the concentration of free CSs. Under these presumptions, the system behaves as there was only one chiral selector with a certain overall equilibrium constant, overall mobility of analyte–selector complex (associate) and overall rate constant of interconversion in a chiral environment. We give the mathematical equations of the overall parameters. A special interest is devoted to the dynamics of interconversion. Interconversion in systems with mixture of chiral selectors is governed by two apparent rate constants of interconversion in the same way as in case of singe-selector systems. We propose the experimental design that allows to determine rates of interconversion in both chiral and achiral parts of the enantioseparation system separately. The approach is verified experimentally in the second part of the article.
Keywords: Chiral separation; Chiral selector; Dynamic CE; Interconversion; Rate constant; Mixture of cyclodextrins;

Model of CE enantioseparation systems with a mixture of chiral selectors by Pavel Dubský; Jana Svobodová; Eva Tesařová; Bohuslav Gaš (35-41).
The theoretical assumption that a multi-CS enantioseparation system, the model of which was described in Part I of this work, can be treated as a separation system with only one CS is confirmed by a set of experiments. The model assumes that each individual analyte–CS interaction is fast, fully independent on other interactions and the analyte: CS ratio is 1:1 and that the analyte is present in its concentration small enough not to considerably change the concentration of free CSs. An enantioselective environment in affinity capillary electrophoresis is created using a commercially available mixture of highly sulfated β-cyclodextrines as chiral selectors (CSs) and lorazepam as an analyte that undergoes interconversion during the separation process. Dependencies of the electrophoretic mobilities of the two enantiomers on concentration of the CSs mixture are proved to follow the proposed multi-CS model. Global rate constants of interconversion are determined at various temperatures and concentrations of the CSs mixture. In accord with the proposed theory, linear dependencies of the global rate constants on CSs concentration are achieved. Intercepts and slopes of these plots correspond to local rate constants of interconversion in achiral (without the mixture of CSs in background electrolyte (BGE)) and chiral (with the CSs mixture in BGE) environments, respectively. The experimentally obtained electropherograms show an excellent fit with those resulting from the computer simulation based on our model.
Keywords: Chiral separation; Interconversion; Highly sulfated beta cyclodextrins; Lorazepam; Kinetics;

The software program DCXplorer is introduced to directly access interconversion rate constants in dynamic chromatography and electrophoresis. The program utilizes the unified equation of chromatography which can evaluate reaction rate constants of all kinds of first order reactions of processes taking place during a separation process. Evaluations with DCXplorer are facilitated by a graphical user interface which allows zooming into the area of interest of an interconversion profile and calculating reaction rate constants without a time consuming simulation process. DCXplorer was applied to determine the enantiomerization barrier of the diuretic drug chlorthalidone by pressure supported dynamic capillary electrokinetic chromatography (DEKC) under acidic conditions at pH 5.00 and pH 3.75. Activation parameters ΔH and ΔS were obtained from temperature dependent measurements between 15.0 and 35.0 °C in 5 K steps at pH 3.75 and between 30.0 and 50.0 °C in 10 K steps at pH 5.00.
Keywords: Dynamic chromatography; Electrophoresis; Kinetics; Chlorthalidone; Enantiomer separation; Enantiomerization mechanism; Catalysis; Chirality;

Immobilised polysaccharide-based chiral stationary phases (CSPs) are a new generation of chromatographic materials combining the remarkable enantioselective performance of the polysaccharide derivatives and solvent versatility for enantiomeric resolution. Based on extensive experimental work, this article will focus on the approach to efficient method development with these immobilised CSPs (CHIRALPAK IA, CHIRALPAK IB and CHIRALPAK IC) by applying a limited number of mobile phases. The manuscript will review the development of screening strategies, either by liquid and supercritical chromatography (LC and SFC), for the separation of enantiomers. The rational combination of both modes leads to efficient approaches to get enantiomeric resolutions in reasonable time frames and with high success rates.
Keywords: Chemically immobilised polysaccharide-based chiral stationary phases (CSPs); Enantiomer resolution; HPLC; Organic mobile phases; RP; SFC; Method development;

The screening conditions of an existing column and mobile phase selection strategy for chiral drug substances in polar organic solvent liquid chromatography (POSC) were tested for their applicability on two new chlorine-containing polysaccharide-based stationary phases. The selectors of these phases are cellulose tris(3-chloro-4-methylphenylcarbamate) and amylose tris(5-chloro-2-methylphenylcarbamate). The enantioselectivity of these phases is compared to that of the four phases (Chiralpak® AD-RH, Chiralcel® OD-RH, Chiralpak® AS-RH and Chiralcel® OJ-RH) used in the earlier defined strategy. A test set of 62 structurally diverse chiral drug substances is analyzed using the screening conditions of the strategy on the six phases. The results confirm that the acetonitrile-based mobile phase provides a higher success rate and better resolutions than the methanol-based also on the new phases. However, the importance of the methanol-based mobile phase cannot be neglected for complementarity reasons; the two mobile phases insure enantioselectivity for different compounds. A third (ethanol-based) mobile phase, not part of the strategy, was also used to screen the two new phases. The joint results led to different possibilities to upgrade the current screening strategy so that improved success rates are obtained. The chlorine-containing chiral stationary phases demonstrated to have an added value to the screening process since they provided enantioresolution for compounds not resolved by non-chlorine-containing ones.
Keywords: Chlorine-containing polysaccharide stationary phases; Sepapak®-2; Sepapak®-3; Enantioselectivity; Chiral analyses; Polar organic solvent chromatography;

The Abraham model of linear solvation energy relationship (LSER) was utilized to characterize three recently commercialized chiral stationary phases (CSPs), the Chiralpak IA, IB and IC. Normal phase system constants were determined by HPLC for these three CSPs and compared to literature values for the Chirobiotic T and V CSPs. The results indicate that the Chirobiotic T and V CSPs participate in more polar interactions, such as hydrogen bonding and dipolar interactions, than the three immobilized derivatized polysaccharide CSPs. Additionally, differences were noted for the e and b terms of the Abraham model (polarizable interactions and hydrogen bond acidity) for the IA and IB CSPs, which are nominally similar CSPs in their chemical makeup.
Keywords: LSER; Chiral stationary phase; Macrocyclic glycopeptide; Polysaccharide; HPLC; Abraham model;

The enantioselective and chromatographic properties of Chiralpak AD and Chiralpak IA as well as those of Chiralcel OD and Chiralpak IB have been evaluated using a set of 48 compounds that differ in their physical and chemical properties. The impact of the different immobilisation methodologies of the chiral polysaccharide, i.e., coated or immobilized on retention and enantioselectivity was studied. The study on immobilized chiral stationary phases (CSPs) was expanded to also include mobile phases containing mixtures of alkanes and more non-conventional solvents such as ethyl acetate, ethers, acetone and dichloromethane. In this paper we report some of the general trends observed for the 48 racemic compounds with respect to retention, α and R s. Further, the impact of the immobilisation methodology and the choice of the mobile phase on the elution order of the enantiomers is also discussed.
Keywords: Chiralcel OD; Chiralpak IB; Chiralpak AD; Chiralpak IA; Enantioseparation; Chiral stationary phase; Polysaccharide;

Effect of the solute molecular structure on its enantioresolution on cellulose tris(3,5-dimethylphenylcarbamate) by Rahul B. Kasat; Siao Yee Wee; Ji Xian Loh; Nien-Hwa Linda Wang; Elias I. Franses (81-92).
The effects of the molecular structures for 13 structurally similar chiral solutes on their HPLC retention and enantioresolutions on a commercially important polysaccharide-based chiral stationary phase, cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) are studied. Among these 13 solutes, only methyl ephedrine (MEph) shows significant enantioresolution. The retention factors of these chiral solutes vary significantly from 0.7 to 3.2 in n-hexane/2-propanol (90/10, v/v) at 298 K. The retention factors of some simpler non-chiral solutes having similar but fewer functional groups than their chiral counterparts are also studied under the same conditions and are compared to those of the chiral solutes. The H-bonding interactions between the functional groups of the solute and the C=O and NH functional groups of the polymer are probed with attenuated total reflection-infrared spectroscopy (ATR-IR) for the polymer, for binary sorbent–solute systems. The CDMPC IR amide band wavenumbers change significantly, indicating H-bonding interactions of the polymer C=O and NH groups with the solutes. The elution orders predicted for the enantiomers of these chiral solutes using molecular dynamics (MD) simulations of the polymer–solute binary systems are consistent with the HPLC results. The CDMPC cavity nano-structure and the potential interactions with chiral solutes are proposed based on HPLC data, IR data, and the simulations. The results are consistent with the three-point attachment model and support the hypothesis that significant enantioresolution requires at least three different synergistic interactions which can be a combination of steric hindrance, H-bonding, or π–π interactions.
Keywords: Polysaccharide; Ephedrine; Chiral discrimination; Chiralcel OD; Hydrogen bonding; π–π; Steric interactions;

A proline oligopeptide-derived chiral selector (CS), containing 3,5-dimethylphenylcarbamate residues on the 4 position of the pyrrolidine rings, was bonded to a silica gel chromatographic matrix by the N-terminal group. The chromatographic behaviour of the resulting chiral stationary phase (CSP) was compared with that of a CSP containing the analogous monomeric CS and that resulting from bonding of the polyproline-derived CS by the carboxy-terminal group using several solvents as mobile phase. The CSs were also studied from the conformational point of view in solution using circular dichroism and 13C NMR. A relationship was found between the presence of an ordered conformation in the particular conditions used and increased enantioselectivity.
Keywords: Poly-l-proline oligomers; Chiral selectors; Enantioseparation; Chiral stationary phases;

A residual silanol group-protecting chiral stationary phase (CSP) based on optically active (3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6 was successfully applied to the resolution of racemic cathinone and it analogue aryl α-amino ketones. The separation factors (α) and the resolutions (R S) for 12 analytes were in the ranges of 2.85–16.12 and 6.49–19.64, respectively. The chromatographic resolution behaviors were investigated as a function of the content and type of organic and acidic modifiers and the ammonium acetate concentration in aqueous mobile phase. The practical usefulness of the CSP in the determination of the enantiomeric purity of optically active cathinone and in the preparative resolution of racemic cathinone was demonstrated.
Keywords: Aryl α-amino ketone; Cathinone; Chiral stationary phase; (3,3′-Diphenyl-1,1′-binaphthyl)-20-crown-6; Enantiomer separation; Liquid chromatography;

Cysteine-based chiral selectors for the ligand-exchange separation of amino acids by Benedetto Natalini; Roccaldo Sardella; Antonio Macchiarulo; Roberto Pellicciari (108-117).
Three cysteine-based coated selectors, S-benzyl-(R)-cysteine, S-diphenylmethyl-(R)-cysteine and S-trityl-(R)-cysteine, have been used in the ligand-exchange separation of a selected set of natural and unnatural underivatized amino acids. With only few exceptions, a gain in enantiodiscrimination was obtained when the most lipophilic discriminating agent, characterized by the presence of a trityl moiety, was engaged. Moreover, a new descriptive structure–separation relationship study through molecular surface (Jurs) and shape (Shadow) descriptors provided evidence that specific physico-chemical features of the employed chiral selector result decisive in establishing which property of the analyte is responsible for the enantiorecognition accomplishment.
Keywords: Chiral ligand-exchange chromatography; Enantiomer separation; Molecular descriptors; Classification system; Chiral coated phase;

Serendipitous discovery of a pH-dependant atropisomer bond rotation: Toward a write-protectable chiral molecular switch? by Christopher J. Welch; Mirlinda Biba; Philip Pye; Remy Angelaud; Melissa Egbertson (118-121).
Owing to slow rotation of a sterically constrained dimethylamide substituent, two slowly interconverting enantiomers of a preclinical candidate for pharmaceutical development, 1, (6-(3-Chloro-4-fluoro-benzyl)-4-hydroxy-2-methyl-3,5-dioxo-2,3,5,6,7,8-hexahydro-[2,6]naphthyridine-1-carboxylic acid dimethylamide) are observed by chiral chromatography. Isolation of pure enantiomer by preparative chiral chromatography followed by enantiopurity analysis over time allowed for a study of the kinetics of enantiomer interconversion under a variety of conditions. Relatively slow racemization was observed in alcohol solvents, with a half life on the order of 5–10 h. A dramatic influence of aqueous buffer pH on racemization was noted, with higher pH leading to rapid racemization within a few minutes, and lower pH leading to essentially no racemization for periods up to a week. A hypothesis explaining this unusual effect of pH on carboxamide bond rotation is offered, and some suggestions for potential utility of such a system are considered.
Keywords: Atropisomer; Molecular switch; Chiral HPLC; HPLC-CD; Bond rotation; pH-dependant switch;

Brivanib Alaninate is a novel chiral prodrug possessing two stereogenic centers. Simultaneous HPLC separation of five isomers of Brivanib Alaninate was systematically investigated on a wide variety of polysaccharide-based chiral stationary phases (CSPs) using underivatization and pre-column derivatization methods. The influence of derivatizing groups and mobile phase composition on the enantioseparation and retention behavior of Brivanib Alaninate compounds was studied. To better understand the chiral recognition mechanism, the temperature effect was also evaluated. The results of these studies led to the first complete HPLC resolution of all five isomers of Brivanib Alaninate as carbobenzyloxy (CBZ) derivatives on a cellulose benzoate CSP (OJ-H).
Keywords: Brivanib Alaninate; Chiral separation; Derivatization; Polysaccharide chiral stationary phases; HPLC;

Stereospecific analysis of sakuranetin by high-performance liquid chromatography: Pharmacokinetic and botanical applications by Jody K. Takemoto; Connie M. Remsberg; Jaime A. Yáñez; Karina R. Vega-Villa; Neal M. Davies (136-141).
A stereospecific method for analysis of sakuranetin was developed. Separation was accomplished using a Chiralpak® AD-RH column with UV (ultraviolet) detection at 288 nm. The stereospecific linear calibration curves ranged from 0.5 to 100 μg/mL. The mean extraction efficiency was >98%. Precision of the assay was <12% (relative standard deviation (R.S.D.)%), and within 10% at the limit of quantitation (0.5 μg/mL). Bias of the assay was lower than 10%, and within 5% at the limit of quantitation. The assay was applied successfully to pharmacokinetic quantification in rats, and the stereospecific quantification in oranges, grapefruit juice, and matico (Piper aduncum L.).
Keywords: Sakuranetin; Stereospecific; HPLC; Chiral; Flavonoid;

Stereospecific high-performance liquid chromatographic assay of isosakuranetin in rat urine by Karina R. Vega-Villa; Connie M. Remsberg; Kristy L. Podelnyk; Neal M. Davies (142-147).
A stereospecific method of analysis of racemic isosakuranetin (5,7-dihydroxy-4′-methoxyflavanone) in biological fluids is necessary to study pharmacokinetics. A simple high-performance liquid chromatographic method was developed for the determination of isosakuranetin enantiomers. Separation was achieved on a Chiralpak® AD™-RH column with ultraviolet (UV)-detection at 286 nm. The standard curves in urine were linear ranging from 0.5 to 100.0 μg/ml for each enantiomer. The mean extraction efficiency was >88.0%. Precision of the assay was <15% (CV) and was within 12% at the limit of quantitation (0.5 μg/ml). Bias of the assay was <15% and was within 6% at the limit of quantitation. The assay was applied successfully to stereospecific disposition of isosakuranetin enantiomers in rat urine.
Keywords: HPLC; UV-detection; Stereospecific; Enantiomer; Flavonoid;

Development and validation of a direct enantiomeric separation of pregabalin to support isolated perfused rat kidney studies by Yizhong Zhang; Christopher Holliman; Daniel Tang; Douglas Fast; Steven Michael (148-153).
Pregabalin (Lyrica™) is the first compound approved to treat the neural pain associated with fibromyalgia. Pregabalin is the S-enantiomer of a γ-amino acid analogue and chiral separation from its R-enantiomer must be achieved to support metabolic studies. The direct chiral separation of pregabalin from its R-enantiomer has been developed and HPLC/MS/MS assays have been validated to support isolated perfused rat kidney studies. The separation was developed through serial coupling of various macrocyclic glycopeptide stationary phases until partial separation of the enantiomers was achieved. Identification of the resolving stationary phase followed by optimization of the mobile phase enabled the baseline resolution of the enantiomers using mass spectrometry compatible solvents and modifiers. Assays were developed and validated for quantitation of the enantiomers from rat urine, isolated rat kidney perfusate, and isolated rat kidney perfusate ultrafiltrate to support pregabalin metabolic studies.
Keywords: Pregabalin; HPLC/MS/MS; Chiral separation; Validation;

Development and implementation of a stereoselective normal-phase liquid chromatography–tandem mass spectrometry method for the determination of intrinsic metabolic clearance in human liver microsomes by Yingru Zhang; Christian Caporuscio; Jun Dai; Michael Witkus; Anne Rose; Joseph Santella; Celia D’Arienzo; David B. Wang-Iverson; Adrienne A. Tymiak (154-160).
The stereoselective determination of stereoisomers in biological samples provides vital information on stereospecific metabolism and pharmacokinetic profiles of the drugs. Despite the unique advantage and the great success of normal-phase (NP) HPLC for the separations of drug stereoisomers using polysaccharide-type chiral stationary phases (CSPs), the technique is rarely applied to quantitative HPLC–MS–MS bioanalysis. This is, at least in part, due to the incompatibility between the usual mobile phase (n-hexane or n-heptane) in normal-phase HPLC and the MS ionization sources which poses a potential detonation hazard. An environmentally friendly and nonflammable alternative solvent, ethoxynonafluorobutane (ENFB), was reported previously to potentially provide an ideal solution for combining the powers of stereoselective NP chromatographic separation and MS–MS detection. In this study, a stereoselective NP-HPLC–MS–MS method was developed using ENFB to quantify a pair of Bristol Myers Squibb (BMS) proprietary drug stereoisomers and their ketone metabolite for an in vitro study, which demonstrated, for the first time, the practical applicability and utility of ENFB for bioanalysis in pharmaceutical industry. The effects of different organic modifiers and temperature, as well as the comparison between ENFB and the usual solvent, heptane, for the separation, are discussed. The resolution of the stereoisomers was achieved using 63% of 3:1 mixture of ethanol and methanol with 37% ENFB on a Chiralpak AD-H column at 50 °C. High sensitivity was obtained using the MS–MS detection in the positive ion atmospheric pressure chemical ionization (APCI) mode. The lower limit of quantitation (LLOQ) for the first stereoisomer and the ketone metabolite was 5 ng/mL, and was 10 ng/mL for the second isomer in the human liver microsome–potassium phosphate buffer matrix. The linear dynamic range of 5–1000 ng/mL for both isomers and 10–1000 ng/mL for the metabolite were demonstrated with R 2  ≥ 0.997. The precision of the analysis was <5% R.S.D. at or above the nominal concentration of 80 ng/mL, and <20% R.S.D. at 8 ng/mL. The mean bias was less than 15%. Extraction recovery and acceptable matrix interference were demonstrated using one isomer and the ketone, and better than 75% recovery and less than 25% ion suppression or interference were found. The method was successfully implemented for an in vitro intrinsic metabolic clearance study.
Keywords: Chiral resolution; Enantiomeric separations; LC–MS–MS; NP-HPLC–MS–MS; Ethoxynonafluorobutane (ENFB); In vitro intrinsic metabolic clearance;

Enantioselective analysis of oxybutynin and N-desethyloxybutynin with application to an in vitro biotransformation study by Patrícia da Fonseca; Luis Alexandre Pedro de Freitas; Luis Felipe Ribeiro Pinto; Cezar Rangel Pestana; Pierina Sueli Bonato (161-167).
An enantioselective method using liquid-phase microextraction (LPME) followed by HPLC analysis was developed for the determination of oxybutynin (OXY) and its major metabolite N-desethyloxybutynin (DEO) in rat liver microsomal fraction. The LPME procedure was optimized using multifactorial experiments. Under the optimal extraction conditions, the mean recoveries were 61 and 55% for (R)-OXY and (S)-OXY, respectively, and 70 and 76% for (R)-DEO and (S)-DEO, respectively. The validated method was employed to an in vitro biotransformation study using rat liver microsomal fraction. The results demonstrated the enantioselective biotransformation of OXY.
Keywords: Oxybutynin; N-Desethyloxybutynin; Enantioseparation; Biotransformation;

Circadian changes of d-alanine and related compounds in rats and the effect of restricted feeding on their amounts by Akiko Morikawa; Kenji Hamase; Yurika Miyoshi; Satoru Koyanagi; Shigehiro Ohdo; Kiyoshi Zaitsu (168-173).
The circadian changes of d-alanine (d-Ala), an intrinsic d-amino acid found in mammals, were investigated in rats with diurnal and nocturnal habits, and the profiles were compared to those of l-Ala, other d-amino acids and several hormones. Determination of d-Ala in the rat plasma, pancreas and anterior pituitary gland was carried out using a sensitive and selective two-dimensional HPLC system combining a micro-ODS column and an enantioselective column after fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The amount of d-Ala was high during the sleeping period and low during the active period in rats with both diurnal and nocturnal habits, indicating for the first time that the d-Ala is closely related to the activity rhythm of animals. In contrast, l-Ala and other d-amino acids did not show any clear circadian changes. The circadian change of d-Ala inversely correlated with that of the plasma insulin level in rats with both diurnal and nocturnal habits. Considered together with our previous findings that d-Ala is localized in the insulin secreting beta-cells in the rat pancreas, it is strongly suggested that d-Ala has some functional relationships to insulin in mammals.
Keywords: d-Amino acids; Alanine; Enantiomer separation; Column-switching HPLC; Circadian rhythm; Insulin;

Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals by Yosuke Tojo; Kenji Hamase; Minako Nakata; Akiko Morikawa; Masashi Mita; Yutaka Ashida; Wolfgang Lindner; Kiyoshi Zaitsu (174-179).
A fully automated 2D-HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column has been developed for the simultaneous enantiomer determination of proline, trans-4-hydroxyproline and cis-4-hydroxyproline in mammals. As a first dimension, a monolithic ODS column of 0.53 mm i.d. showed 3–6 times larger theoretical plate numbers than those of particle-packed ODS columns, and the best enantioseparations were obtained by a Chiralpak QN-2-AX column of 1.5 mm i.d. in the second dimension (separation factors: 1.44–1.83). The R.S.D. values for within-day and day-to-day precisions were less than 5.8%, and the lower limits of quantitation for the d-enantiomers were 1 fmol. The present method was successfully applied to the determination of proline and hydroxyproline enantiomers in the serum and collagen-rich skin tissue. Small amounts of d-proline were found both in the serum (1.57 ± 0.19 nmol/mL) and in the skin (0.093 ± 0.015 nmol/mg protein), while the amounts of d-hydroxyproline were smaller than the lower limit of quantitation.
Keywords: Proline; Enantiomer separation; Hydroxyproline; Monolithic column; Two-dimensional HPLC;

On-column epimerization of dihydroartemisinin: An effective analytical approach to overcome the shortcomings of the International Pharmacopoeia monograph by Walter Cabri; Alessia Ciogli; Ilaria D’Acquarica; Michela Di Mattia; Bruno Galletti; Francesco Gasparrini; Fabrizio Giorgi; Silvana Lalli; Marco Pierini; Patrizia Simone (180-191).
We developed a cryo-HPLC/UV method for the simultaneous determination of artemisinin (1), α-dihydroartemisinin (2α), β-dihydroartemisinin (2β), and a ubiquitous thermal decomposition product of 2 (designated as diketoaldehyde, 3), starting from the International Pharmacopoeia monograph on dihydroartemisinin. The method takes for the first time the on-column epimerization process of 2 into consideration. Chromatographic separation was obtained under reversed-phase conditions on a Symmetry C18 column (3.5 μm particle size) with a mobile phase consisting of acetonitrile–water 60:40 (v/v), delivered at 0.60–1.00 ml/min flow-rates, with ultraviolet detection at low wavelength (λ  = 210 nm). Low temperatures (T  = 0–10 °C) were selected on the grounds of a diastereoselective dynamic HPLC (DHPLC) study performed at different temperatures, aimed at identifying the best experimental conditions capable of minimizing the on-column interconversion process.
Keywords: Dihydroartemisinin (DHA); Antimalarials; Epimerization study; Cryo-HPLC; Dynamic HPLC (DHPLC); Computer simulation;

An on-column stopped-flow multidimensional HPLC (sfMDHPLC) procedure using two chiral stationary phases (CSPs) and one achiral C18 column was developed for the determination of rate constants and free energy barriers of enantiomerization of (±)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide. Moreover, a stopped-flow HPLC (sfHPLC) method previously developed was applied to the determination of kinetic parameters of enantiomerization of the above compound in the presence of a CSP. The individual enantiomers of the studied compound were isolated in parallel by preparative HPLC and the rate constants and free energy barriers of enantiomerization were determined in different solvents (off-column method). The data obtained by sfMDHPLC, sfHPLC and off-column methods were compared. The (S) enantiomer of the studied compound (S18986) was prepared by asymmetric synthesis and subsequently purified by preparative HPLC, followed by the determination of rate constants and free energy barriers of enantiomerization in different buffer solutions at pH 2–9.3.
Keywords: S18986; Stopped-flow multidimensional HPLC method; Enantiomerization; Rate constants; Free energy barriers;

Exploring enantiospecific ligand–protein interactions using cellular membrane affinity chromatography: Chiral recognition as a dynamic process by Krzysztof Jozwiak; Ruin Moaddel; Sarangan Ravichandran; Anita Plazinska; Joanna Kozak; Sharvil Patel; Rika Yamaguchi; Irving W. Wainer (200-207).
The chiral recognition mechanisms responsible for the enantioselective binding on the α3β4 nicotinic acetylcholine receptor (α3β4 nAChR) and human organic cation transporter 1 (hOCT1) have been reviewed. The results indicate that chiral recognition on the α3β4 nAChR is a process involving initial tethering of dextromethorphan and levomethorphan at hydrophobic pockets within the central lumen followed by hydrogen bonding interactions favoring dextromethorphan. The second step is the defining enantioselective step. Studies with the hOCT1 indentified four binding sites within the transporter that participated in chiral recognition. Each of the enantiomers of the compounds used in the study interacted with three of these sites, while (R)-verapamil interacted with all four. Chiral recognition arose from the conformational adjustments required to produce optimum interactions. With respect to the prevailing interaction-based models, the data suggest that chiral recognition is a dynamic process and that the static point-based models should be amended to reflect this.
Keywords: Nicotinic acetylcholine receptors; Organic cation transporters; Pharmacophore modeling; Non-linear chromatography; Molecular modeling;

In an effort to simultaneously enantioseparate racemic unfunctionalized alkanes and racemic α-amino acid derivatives by gas chromatography (GC) in forthcoming experiments related to the search for extraterrestrial homochirality, the two versatile modified cyclodextrin (CD) selectors octakis(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin (Lipodex G) and heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin were dissolved in a polysiloxane and the mixed binary chiral selector system was coated onto a 50 m × 0.25 mm i.d. fused silica capillary column. Whereas the former CD selector enantioseparates racemic unfunctionalized alkanes the latter CD selector preferentially resolves N-(O,S)-trifluoroacetyl-α-amino acid alkyl esters. With both CD selectors employed as mixed binary chiral selector system present in one chiral stationary phase (CSP), the simultaneous gas chromatographic enantioseparation of racemic alkanes and of racemic derivatized α-amino acids is achieved in a single temperature-programmed run. Also for other classes of racemic compounds, the scope of enantioseparation could be extended as compared to the conventional use of the single CD selectors in GC.
Keywords: Enantioselective gas chromatography; Mixed binary chiral selector system; Modified cyclodextrins; Racemic unfunctionalized alkanes; Racemic derivatized α-amino acids;

The poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1′-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.
Keywords: Poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column; Supercritical fluid chromatography (SFC); Polysaccharide-based CSPs;

The preparative chromatographic resolution of racemates has become a standard approach for the generation of enantiomers in pharmaceutical discovery laboratories. This paper will discuss the use of preparative HPLC and SFC to generate individual enantiomers for discovery activities. Analytical HPLC and SFC method development to rapidly screen chiral stationary phases and solvent combinations will be presented. The usefulness of preparative chromatographic resolution of racemates will be demonstrated through the presentation of numerous non-routine case studies from the laboratories at Amgen.
Keywords: Chiral separations; Preparative chromatography; Supercritical fluid chromatography; Chiral stationary phase;

2,2,2-Trifluoroethanol (TFE) is evaluated as an alternative modifier for the analysis and purification of alcohol-sensitive chiral compounds using supercritical fluid chromatography (SFC). Four chiral compounds, selected for their sensitivity to alcohols, in addition to a variety of standard chiral compounds were analyzed by SFC using TFE with polysaccharide and Pirkle-type chiral stationary phases (CSPs) to produce selectivities (α) and resolutions (R s) as high as 1.4 and 7.2. A preparative isolation of 2-phenylglutaric anhydride was achieved using TFE as the mobile phase modifier to produce clean enantiomers.
Keywords: SFC; TFE; Fluorinated solvents; Chiral separations; Alcohol-sensitive compounds;

Chiral analysis of amino acids from conventional and transgenic yeasts by Alessandro Giuffrida; Laura Tabera; Ramón González; Vincenzo Cucinotta; Alejandro Cifuentes (243-247).
Autolysis of Saccharomyces cerevisiae yeast is the main source of molecules that contribute to the quality of sparkling wines made by the traditional method. In this work, a genetically modified yeast (LS11) is compared to its isogenic wild type strain (BY4741) after autolysis. Chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC–LIF) is used to identify and quantify the main d- and l-amino acids from both strains after accelerated autolysis. The procedure includes amino acids extraction, derivatization with FITC and chiral-MEKC–LIF separation in a background electrolyte composed of 100 mM sodium tetraborate, 30 mM SDS, 20 mM β-CD at pH 10.0. The d- and l-forms of Arg, Asn, Ala, Glu and Asp, corresponding to the major amino acids found in these samples, are separated in less than 30 min with efficiencies up to 800,000 plates/m and high sensitivity (i.e., LODs as low as 40 nM were obtained for d-Arg for a signal to noise ratio of three). From these results it is corroborated that the genetic modification brings a faster autolysis of the yeast, releasing a higher amount of l-amino acids to the medium in a short time. Interestingly, the pattern of release of d-amino acids was also different between the transgenic and the conventional yeast strains.
Keywords: Capillary electrophoresis; Enantiomer; MEKC; LIF; Transgenic foods; GMOs; Food analysis;

Enantioseparation of pharmaceutical compounds by multiplexed capillary electrophoresis using highly sulphated α-, β- and γ-cyclodextrins by Luis Saavedra; Beverly Nickerson; Ricardo E. Borjas; Frédéric Lynen; Pat Sandra (248-253).
A multiplexed capillary electrophoresis (CE) system equipped with 96 channels was evaluated for high-throughput screening of enantiomers of solutes of pharmaceutical interest. Using highly (DS ∼ 12) sulphated α-, β- and γ-cyclodextrins under acidic conditions (pH 2.5) only 48 channels could be used because of the high conductivity of the chiral selectors. Method transfer from a single channel to a 48 channel CE system is described. Under optimised conditions, the analysis time on the multiplexed 48 channel CE system is ca. five to eight times the analysis time on the single channel CE system. The figures of merit for the multiplexed system are presented as well as performance evaluation including throughput and productivity gain. Intra-day precision (n  = 6) ranged from 2.0 to 16.5% and from 2.2 to 15.5% for migration time and resolution, respectively. These values increased with ca. 10% for intermediate precision.
Keywords: Multichannel capillary electrophoresis; Chiral separations; Pharmaceuticals; High-throughput; System performance;

Enantiomeric separation of ornithine in complex mixtures of amino acids by EKC with off-line derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate by Ana Belén Martínez-Girón; Elena Domínguez-Vega; Carmen García-Ruiz; Antonio L. Crego; Maria Luisa Marina (254-259).
A new analytical methodology was developed by EKC enabling the fast enantiomeric separation of Ornithine in complex mixtures of amino acids. A previous derivatization step with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was achieved to enable the sensitive UV detection of amino acids as well as to make possible their interaction with the CDs employed as chiral selectors. A dual CD system containing an anionic and a neutral CD in phosphate buffer at acid pH showed a high resolving power allowing the enantiomeric separation of 18 protein amino acids and Orn. The method was applied to the analysis of fermented foods to investigate the extent of the presence of Orn enantiomers.
Keywords: Ornithine; Chiral separation; Cyclodextrins; AQC derivatization; Amino acids; Fermented foods; Electrokinetic chromatography;

The first CE method enabling the quantitation of the two enantiomers of bupropion was developed in this work. Electrokinetic chromatography (EKC) mode using cyclodextrins as chiral selectors was employed. A study on the enantiomeric separation ability of different neutral and anionic CDs was carried out. Sulfated-β-CD was shown to provide the highest values for the enantiomeric resolution. The influence of some experimental conditions, such as pH, chiral selector concentration, temperature, and separation voltage on the enantiomeric separation of bupropion was also studied. The use of 10 mM sulfated-β-CD in 50 mM borate buffer (pH 9.0) with an applied voltage of 30 kV and a temperature of 30 °C enabled the separation of the enantiomers of bupropion with high resolution (R s  > 7) and short analysis time (∼3.5 min). Finally, the method was successfully applied to the quantitation of bupropion in two pharmaceutical formulations.
Keywords: Bupropion; Cyclodextrin; Enantiomeric separation; Electrokinetic chromatography; Pharmaceutical formulations; Quantitative analysis;

The present work illustrates possibilities of column-coupling capillary electrophoresis (CE-CE) combined with chiral selector (2-hydroxypropyl-β-cyclodextrin, HP-β-CD) and fiber-based diode array detection (DAD) for the direct quantitative enantioselective determination of trace drug (amlodipine, AML) in biological multicomponent ionic matrices (human urine). Capillary isotachophoresis (ITP) served as an ideal injection technique in CE-CE. Moreover, the ITP provided an effective on-line sample pretreatment prior to the capillary zone electrophoresis (CZE) separation. Enhanced separation selectivity due to the combination of different separation mechanisms (ITP vs. CZE–HP-β-CD) enabled to obtain pure zones of the analytes, suitable for their detection and quantitation. The DAD, unlike single wavelength UV detection, enabled to characterize the purity (i.e. spectral homogeneity) of the analytes zones. A processing of the raw DAD spectra (the background correction and smoothing procedure) was essential when a trace analyte signal was evaluated. Obtained results indicated pure (i.e. spectrally homogeneous) zones of interest confirming effective ITP–CZE separation process. The proposed ITP–CZE–DAD method was characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, selectivity) and successfully applied to an enantioselective pharmacokinetic study of AML.
Keywords: Isotachophoresis; Capillary zone electrophoresis; Column-coupling electrophoresis; Diode array detection; Chiral drug; Amlodipine enantiomers; Cyclodextrin; Human urine;

Enantioseparation of β-methyl-substituted amino acids with cyclodextrins by capillary zone electrophoresis by István Ilisz; Gábor Fodor; Róbert Iványi; Lajos Szente; Géza Tóth; Antal Péter (273-279).
Direct capillary zone electrophoretic methods were developed for the separation of the enantiomers of unnatural β-methyl-amino acids such as erythro- and threo-β-methylphenylalanine, β-methyltyrosine, β-methyltryptophan and β-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Capillary zone electrophoresis was carried out using sulfopropylated-α-CD (SP2-α-CD), sulfopropylated-β-CD (SP2-β-CD) both with a degree of substitution of 2 moles/mole cyclodextrin, and sulfopropylated-β-CD (SP4-β-CD) with a degree of substitution of 4 moles/mole β-cyclodextrin. The effects of selector and buffer concentrations, electrolyte pH and applied voltage were studied on the separation efficiency. Varying the electrophoretic conditions with application of 20 kV, hydrodynamic injection, unmodified silica capillary, three different buffers (borate, phosphate and acetate) and modified cyclodextrins as chiral selectors all compounds investigated are nearly baseline resolved. The elution sequence was determined in most cases.
Keywords: Capillary zone electrophoresis; β-Methyl-amino acids; Native and sulfated cyclodextrins; Cyclodextrin sulfates;

Enantiomeric separation of baclofen by capillary electrophoresis tandem mass spectrometry with sulfobutylether-β-cyclodextrin as chiral selector in partial filling mode by Claudia Desiderio; Diana Valeria Rossetti; Fabio Perri; Bruno Giardina; Irene Messana; Massimo Castagnola (280-287).
Capillary electrophoresis (CE) coupled to tandem mass spectrometry was applied to the chiral separation of baclofen using sulfobutylether-β-cyclodextrin chiral selector in partial filling counter current mode. On-line UV detection was simultaneously used. Method optimization was performed by studying the effect of cyclodextrin and BGE concentration as well as sheath liquid composition on analyte migration time and enantiomeric resolution. The cyclodextrin showed stereoselective complexation towards baclofen enantiomers, allowing chiral resolution at low concentration. The CE capillary protrusion from the ESI needle relevantly affected the chiral resolution and the analyte migration time. Complete enantiomeric separation was obtained by using 0.25 M formic acid BGE containing 1.75 mM of chiral selector and water/methanol (30:70, v/v) 3% formic acid as sheath liquid. The method exhibited a LOD of 0.1 μg/mL (racemic concentration) in MS3 product ion scan mode of detection and was applied to the analysis of racemic baclofen in pharmaceutical formulations.
Keywords: Capillary electrophoresis; Mass spectrometry; Chiral separation; Baclofen; Cyclodextrin;

Separation of nucleoside phosphoramidate diastereoisomers by high performance liquid chromatography and capillary electrophoresis by J.F. Goossens; S. Roux; D. Egron; C. Perigaud; J.P. Bonte; C. Vaccher; C. Foulon (288-295).
Separations of five diastereoisomers of nucleoside phosphoramidate derivatives (pronucleotides) were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and CE method using anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (R s  > 1.5) was readily obtained with all silica-based celluloses and amyloses using in a normal-phase methodology. Capillary electrophoresis was used as an alternative technique to HPLC for the separation of pronucleotides. The diastereoisomers were fully resolved with sulfated cyclodextrins at both BGE pH (2.5 and 6.2). Limits of detection and limits of quantification, calculated for both methods, are up to 200 times higher in CE separations than in HPLC separations. The analytical HPLC method was then applied in a preliminary study for the pronucleotide 1 quantification in cellular extract.
Keywords: Pronucleotides; Diastereoisomers; Chiral HPLC; Chiral CE; Cellular extracts;

Enantioseparations with cellulose tris(3-chloro-4-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography by Salvatore Fanali; Giovanni D’Orazio; Ketevan Lomsadze; Bezhan Chankvetadze (296-303).
Cellulose tris(3-chloro-4-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSPs) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the chiral selector loading onto silica, mobile phase composition and pH, as well as separation variables on separation of enantiomers was studied. It was found that CSPs based on cellulose tris(3-chloro-4-methylphenylcarbamate) can be used for preparation of very stable capillary columns useful for enantioseparations in nano-LC and CEC in combination with polar organic mobile phases.
Keywords: Enantiomers; Capillary electrochromatography; Nano-liquid chromatography; Polysaccharide-based chiral stationary phases;

This work focuses on the simultaneous analysis of β-blockers with multiple stereogenic centers using capillary electrochromatography–mass spectrometry (CEC–MS) with a vancomycin stationary phase. The critical mobile phase variables (composition of organic solvents, acid/base ratios) as well as column temperature and electric field strength, effecting enantioresolution and analysis time were first optimized sequentially. Next, to achieve global optimum a multivariate D-optimal design was used. Although multivariate approach did not improve enantioresolution any further, analysis time was significantly reduced. Under optimum CEC–MS conditions, all stereoisomers were resolved with resolution in the range 1.0–3.1 in less than 60 min with an average signal-to-noise (S/N) greater than 1000. The developed CEC–MS method has the potential to emerge as a screening method for analysis of multiple chiral compounds using a single protocol using the same column and mobile phase conditions, thus reducing the operation time and cost.
Keywords: Packed column CEC–MS; Vancomycin stationary phase; Simultaneous enantioseparations; Multistereogenic center β-blockers; Multivariate approach;

A chiral capillary monolithic column for capillary electrochromatography (CEC) was prepared by covalent bonding of cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) on the silica monolithic matrix within the confine of a 50-μm i.d. bare fused silica capillary. Several pairs of enantiomers including neutral and basic analytes were baseline resolved on the newly prepared chiral capillary monolithic column in CEC with aqueous mobile phases. Fast enantioseparation was achieved due to the favorable dynamic properties of silica monolith. The covalent bonding of CDMPC as the chiral stationary phase for CEC also enabled the use of THF in mobile phase for enantioseparation of prazquantel by overcoming the incompatibility of THF and the physically coated CDMPC on a column.
Keywords: Capillary electrochromatography; Cellulose tris(3,5-dimethylphenylcarbamate); Covalently bonded chiral stationary phase; Enantioseparation; Silica monolithic column;

The use of multilayered capillaries for chiral separation by electrochromatography by Fumihiko Kitagawa; Masato Kamiya; Koji Otsuka (323-328).
Fused-silica capillaries were modified by the successively multiple ionic-polymer layer (SMIL) coating technique for a capillary electrochromatography (CEC) analysis of binaphthyl enantiomers. The SMIL coating capillaries consisting of three different polymers (A+–B–C+ coating) were prepared by the alternative deposition of positively charged chiral or achiral polymers and negatively charged DNA. Previous studies have indicated that DNA–cationic polypeptide or synthetic polymer complexes immobilized onto the inner surface of the capillary worked as the chiral stationary phases for 1,1′-binaphthyl-2,2′-diyl hydrogen phosphate (BNP). In this study, to investigate the chiral recognition mechanism and optimize the CEC separation condition in the DNA–cationic polymer coating, effects of the chirality of the polymer unit, the strand of DNA, and the number of layer pairs on the separation were investigated. It should be noted that, since single stranded DNA (ssDNA) was more suitable to immobilize cationic polymers than double stranded DNA, the ssDNA–cationic polymer immobilized capillaries gave a stable electroosmotic flow and reproducible CEC analyses. As a result, a poly(ethyleneimine)–ssDNA–protamine (Prt) coating provided the best chiral separation of BNP. The high separation performance of the prepared capillary is discussed in terms of DNA/polycations interaction.
Keywords: Open tubular capillary electrochromatography; Successive multiple ionic-polymer layer coating; Chiral separation; DNA;

The present paper demonstrates the enantiomeric separation of omeprazole and its metabolite 5-hydroxyomeprazole performed with open tubular capillary electrochromatography (OT-CEC). The protein avidin was used as the chiral selector. Avidin was immobilized by a Schiffs base type of reaction where the protein was via glutaraldehyde covalently bonded to the amino-modified wall of a fused-silica capillary, 50 μm i.d. Both racemates were baseline resolved. Resolution was 1.9 and 2.3, respectively, using ammonium acetate buffer, pH 5.8, 5% methanol, with UV-detection. These values of resolution using OT-CEC are higher than earlier published results regarding chiral separation of omeprazole and 5-hydroxyomeprazole on packed CEC. The number of theoretical plates also indicated good separation efficiency.
Keywords: Open-tubular capillary chromatography; Enantiomeric separation; Avidin; Omeprazole; 5-Hydroxyomeprazole;