Reversed-phase chiral HPLC and LC/MS analysis with tris(chloromethylphenylcarbamate) derivatives of cellulose and amylose as chiral stationary phases

Three polysaccharide-derived chiral stationary phases (CSP) were evaluated for the resolution of more than 200 racemic compounds of pharmaceutical interest in the reversed-phase (RP) separation mode. The population of test probes was carefully evaluated in order to insure that it covers as completely as possible all structural diversity of chiral pharmaceuticals. RP showed the highest potential for successful chiral resolution in HPLC and LC/MS analysis when compared to normal phase and polar organic separation modes. Method development consisted of optimizing mobile phase eluting strength, nature of organic modifier, nature of additive and column temperature. The newer CSPs, cellulose tris(3-chloro-4-methylphenylcarbamate) and amylose tris(2-chloro-5-methylphenylcarbamate), were compared to the commonly used cellulose tris(3,5-dimethylphenylcarbamate) in regards to their ability to provide baseline resolution. Comparable success rates were observed for these three CSPs of quite complimentary chiral recognition ability. The same method development strategy was evaluated for LC/MS analysis. Diethylamine as additive had a negative effect on analyte response with positive ion mode electrospray (ESI⁺) MS(/MS) detection, even at very low concentration levels (e.g., 0.025%). Decreasing the organic modifier (acetonitrile or methanol) content in the mobile phase often improved enantioselectivity. The column temperature had only a limited effect on chiral resolution, and this effect was compound dependent. Ammonium hydrogencarbonate was the preferred buffer salt for chiral LC with ESI⁺ MS detection for the successful separation and detection of most basic pharmaceutical racemic compounds. Ammonium acetate is a viable alternative to ammonium hydrogencarbonate. Aqueous formic acid with acetonitrile or methanol can be successfully used in the separation of acidic and neutral racemates. Cellulose tris(3-chloro-4-methylphenylcarbamate) and amylose tris(2-chloro-5-methylphenylcarbamate) emerge as CSPs of wide applicability in either commonly used separation modes rivaling such well established CSPs as cellulose tris(3,5-dimethylphenylcarbamate). Screening protocols including these two new CSPs in the preferentially screened set of chiral columns have higher success rates in achieving baseline resolution in shorter screening time.     

Enantiomeric fraction determination of chiral drugs in environmental samples using chiral liquid chromatography and mass spectrometry

When it was recognized that chiral drug residues have stereospecific toxicity towards environmental organisms the attention given to enantiomeric fraction determination of chiral drugs in the environment increased. Among various analytical techniques, chiral liquid chromatography (LC) coupled with mass spectrometry (MS) has been widely used due to its simplicity, wide applicability and high sensitivity. In this review, we aim to provide a comprehensive overview and comparison of the types of chiral stationary phases, elution modes and MS detection techniques employed and address the advances and limitations. The impact of the mobile phase composition on enantioseparation and MS detection are discussed based on the different methods developed. In addition, diverse applications for the enantiomeric fraction determination of chiral drugs in environmental matrices using chiral LC and MS are discussed in depth.     

Enantiomeric separation of proton pump inhibitors on new generation chiral columns using LC and supercritical fluid chromatography

The enantioselectivity of proton pump inhibitors, namely, omeprazole, lansoprazole, rabeprazole, pantoprazole, tenatoprazole, and ilaprazole were studied using new generation chiral packing materials: CHIRALPAK IA, CHIRALPAK IB, and CHIRALPAK IC. Two versatile techniques, HPLC and supercritical fluid chromatography (SFC) were used in this study. CHIRALPAK IC has shown superior selectivity under both LC and SFC conditions, whereas CHIRALPAK IA has shown good selectivity in SFC when compared to LC under primary screening conditions. The chiral recognition ability in LC and SFC modes were found to be in the order CHIRALPAK IC > CHIRALPAK IA > CHIRALPAK IB. In addition to diode array detection, chiral detection was carried out using a laser polarimeter and the elution orders were found to be the same in both LC and SFC elution modes. Mobile phase modifiers and column temperature effects were also studied. In SFC, modifiers (cosolvent) elution strength was found to be in the order ethanol > methanol > 2‐propanol > acetonitrile. In both LC and SFC, a decrease in retention and increase in resolution with an increase in temperature was noticed for all the proton pump inhibitors.     

Simultaneous, sequential quantitative achiral-chiral analysis by two-dimensional liquid chromatography

In general, chromatographic analysis of chiral compounds involves a minimum of two methods; a primary achiral method for assay and impurity analysis and a secondary chiral method for assessing chiral purity. Achiral method resolves main enantiomeric pairs of component from potential impurities and degradation products and chiral method resolves enantiomeric pairs of the main component and diastereomer pairs. Reversed-phase chromatographic methods are preferred for assay and impurity analysis (high efficiency and selectivity) whereas chiral separation is performed by reverse phase, normal phase, or polar organic mode. In this work, we have demonstrated the use of heart-cutting (LC-LC) and comprehensive two-dimensional liquid chromatography (LC × LC) in simultaneous, sequential achiral and chiral analysis and quantitation of minor, undesired enantiomer in the presence of major, desired enantiomer using phenylalanine as an example. The results were comparable between LC-LC and LC × LC with former offering better sensitivity and accuracy. The quantitation range was over three orders of magnitude with undesired D-phenylalanine detected at approximately 0.3% in the presence of predominant, desired L-phenylalanine (99.7%). The limit of quantitation was comparable to conventional high-performance liquid chromatography. A reversed-phase C18 achiral column in the primary and reversed-phase Chirobiotic Tag chiral column in the secondary dimension were used with a compatible mobile phase.     

Evaluation of ethoxynonafluorobutane as a safe and environmentally friendly solvent for chiral normal-phase LC-atmospheric pressure chemical ionization/electrospray ionization-mass spectrometry

Coupling normal-phase LC separation methods to atmospheric pressure ionization (API)-mass spectrometry (MS) for detection can be problematic because of the possible detonation hazard and because nonpolar solvents do not support ionization of the analyte. Unlike achiral separations, enantiomeric separations can be very sensitive to small changes in the separation environment. Thus, completely substituting the main mobile phase component of a normal-phase LC solvent for an environmentally friendly, nonflammable fluorocarbon-ether as a safe and effective solvent must be thoroughly evaluated before it can be recommended for enantioselective separations with API-MS detection. Ethoxynonafluorobutane (ENFB) was used as a normal-phase solvent for the enantioselective separation of 15 compounds on two macrocyclic glycopeptide chiral stationary phases (CSPs) and a new polymeric chiral stationary phase. The chromatographic figures of merit were compared between results obtained with the ENFB mobile phases and traditional heptane-based mobile phases. In addition, the limits of detection (LOD) using the API-MS compatible ENFB were examined, as well as flow rate sensitivities and compatibilities with common polar organic modifier. ENFB is a safe and effective solvent for enantioselective normal-phase/API-MS analyses.     

Enlargement of blue-phase stability for rod-like low-molecular-weight chiral nematic liquid crystal mixtures

In this study, we investigated the enlargement of liquid crystal (LC) blue-phase (BP) temperature range using the rod-like low-molecular-weight cyano phenyl-type chiral nematic LC with various core group and chiral dopant concentrations. Also, the electro-optic response time was investigated for them. We found that the BP temperature range was strongly dependent upon the core structure and the chiral dopant concentration for the chiral nematic LC mixtures having the same terminal group. Also, we found a stable BP with a wide temperature range (more than 6 K), including a BP-isotropic coexistence state over 13.5 K upon heating and cooling processes and very fast response time (less than 1 ms), by using the cyano phenyl-type chiral nematic LC mixture with a high molecular aspect ratio and a high chiral dopant concentration.     

Chiral Separation of Four Piperidinic Benzoxazolinone Compounds by CE and LC

Capillary electrophoresis with negatively charged cyclodextrins and high performance liquid chromatography on polysaccharide chiral stationary phases have been explored for the enantioseparation of chiral piperidinic benzoxazolinone compounds. Operational parameters such as the nature and the concentration of the chiral selectors in CE and the nature of the polysaccharide stationary phase and the composition of the mobile phase in LC were varied in order to achieve the separation of the all four concerning compounds. Limits of detection and quantification of both methods were evaluated under the optimal conditions.     

Enantioseparation of four cis and trans diastereomers of 2',3'-didehydro-2',3'-dideoxythymidine analogs, by high-performance liquid chromatography and capillary electrophoresis

Compounds 1-4 are the four stereoisomers of a synthetic new potential antiviral agent (d4T analog) containing two chiral centers and a base (uracil). Both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) techniques were used to separate and quantify enantiomers with high resolution. The determination of enantiomeric purity of the compounds was developed using both amylose chiral stationary phase by HPLC and anionic cyclodextrins (highly S-CD) as chiral selectors in CE. The HPLC method was found to be superior in sensitivity to the CE method.     

Chiral Separations of Imidazole Antifungal Drugs on AmyCoat RP Column in HPLC

The chiral separations of four racemic imidazole antifungal drugs namely econazole, miconazole, isoconazole and sulconazole were achieved on AmyCoat RP column (150 × 4.6 mm, 3.0 μm particle size). The mobile phases used were different combinations of water and acetonitrile. Besides, few drops of diethylamine and acetic acid were added to optimize chiral separation of sulconazole. The flow rates of mobile phases were 1.00 and 1.5 mL min^?1 with detection at 225 nm. The values of capacity, separation and resolution factors were 0.53–2.10, 1.45–1.98 and 1.29–1.97, respectively. Attempts have been made to describe chiral recognition mechanism at supra-molecular level. The reported chiral LC method is simple, fast and reproducible, and can be used for the resolution of these molecules in biological matrices.     

Chiral liquid chromatography-mass spectrometry for high-throughput screening of enzymatic racemase activity

In finding suitable biocatalysts for processes in chemical industry, expression libraries are constructed containing typically >10,000 clones. Search for a desired activity is done by examination of all the clones in one or more libraries using a high-throughput screening assay. Here we describe a method for the screening of the enzymatic racemase activity of clones from an expression library on alpha-amino-epsilon-caprolactam (ACL) using a fast chiral LC separation and ionspray-MS as the detection technique. After substrate incubation with S-ACL, the 96-well microplates were centrifuged to remove cell material. The conversion of S-ACL to R-ACL was monitored by quantitation of the R-ACL enantiomer. Separation of the two ACL enantiomers was performed on a Crownpak CR+ column within 1 min. A Gilson 215 autosampler with a 889 multiple injection probe was used for injecting the samples into the LC system. The total analysis time for a 96-well microplate was 56 min. The MS was operated in the positive-ion mode using selected ion monitoring at m/z 129 [M+H]+ of ACL. Using this method over 12,000 samples were analyzed without loss in performance of the system. The LC column remained stable without loss of resolution and the MS system did not show loss in sensitivity throughout the screening. Inter-day reproducibility was within 15%.     

Derivatization of chiral carboxylic acids with (S)‐anabasine for increasing detectability and enantiomeric separation in LC/ESI‐MS/MS

A simple and practical derivatization procedure for increasing the detectability and enantiomeric separation of chiral carboxylic acids in LC/ESI‐MS/MS has been developed. (S)‐Anabasine (ANA) was used as the derivatization reagent and rapidly reacted with carboxylic acids [3‐hydroxypalmitic acid (3‐OH‐PA), 2‐(β‐carboxyethyl)‐6‐hydroxy‐2,7,8‐trimethylchroman (γ‐CEHC), and etodolac] in the presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholium chloride. The resulting ANA‐derivatives were highly responsive in ESI‐MS operating in the positive‐ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using selected reaction monitoring; the detection responses of the ANA‐derivatives were increased by 20–160‐fold over those of the intact carboxylic acids and the limits of detection were in the low femtomole range (1.8–11 fmol on the column). The ANA‐derivatization was also effective for the enatiomeric separation of the chiral carboxylic acids; the resolution was 1.92, 1.75, and 2.03 for 3‐OH‐PA, γ‐CHEC, and etodolac, respectively. The derivatization procedure was successfully applied to a biological sample analysis; the derivatization followed by LC/ESI‐MS/MS enabled the separation and detection of trace amounts of 3‐OH‐PA in neonatal dried blood spot and γ‐CEHC in human saliva with a simple pretreatment and small sample volume.     

Crystalline degradation products of vancomycin as chiral stationary phase in microcolumn liquid chromatography

The ability of crystalline degradation products (CDPs) of vancomycin as a chiral stationary phase was reported in a previous study for enantioselective separation of drugs, amino acids and agrochemical toxins by conventional LC column (250 x 4.6 mm). In this work, the potential of CDP of vancomycin for the enantiomeric separation in micro-LC (200 x 1 mm) has been studied. The obtained separation results are better than in our previous study with conventional LC columns. The enantiomers of D,L-phenylalanine, D,L-alanine, methyldopa, atropine and propranolol were used for this evaluation. Experiments have been carried out in a stainless steel tube that was packed with chiral silica particles of 3 and 12 microm diameters. Also, three different ratios of 3 and 12 microm silica particles were used for packing material of chiral columns and the effect on aspect ratio and resolving powers was compared.     

Chiral side-chain liquid crystalline polysiloxanes bearing fluorinated mesogens and cholesteryl groups

A series of chiral side-chain liquid crystalline (LC) polysiloxanes bearing fluorinated mesogens were synthesized with a cholesteric LC monomer and a fluorinated nematic LC monomer. They were characterized by use of various experimental techniques, and effect of fluorinated mesogens on characteristic of LC polysiloxanes was studied as well. In photoluminescence spectra, a narrow and a broad peak occur at around 270-317 nm, originated, respectively, from fluorinated phenyl groups and the conjugated xenene structure. The specific rotation analysis of all polymers showed negative values, but absolute values were lower than those of the chiral monomers. All polymers showed smectic LC phase with very wide temperature ranges on heating and cooling cycles. Especially, only polymers bearing more fluorinated component exhibited smectic-cholesteric phase transition when they were heated. As the polymers contained more fluorinated mesogens, segregation of the fluorinated segment to the surface should occur at mesomorphic temperature. The highly ordered lamellar mesogen-siloxane matrix systems should be disturbed severely by separation of fluorinated mesogens, suggesting mesogenic orders transition from lamellar smectic to cholesteric phase.     

Liquid crystal nose,chiral case: towards increased selectivity and low detection limits

In this paper, we describe a simple prototype of an olfaction system based on chiral liquid crystals (LCs) and suitable for sensing volatile organic compounds (VOCs). The detection of small concentrations of VOCs is based on measuring weak colour fluctuations on the surface of the LC droplet. Detection of larger concentrations is based on measuring colour changes (or shift of the selective reflection band) and isotropisation transition of the whole droplet. Thus, a broad range of VOC concentrations can be detected by this LC nose.     

Simultaneous determination of residues of emamectin and its metabolites, and milbemectin, ivermectin, and abamectin in crops by liquid chromatography with fluorescence detection.

A liquid chromatographic (LC) method was developed for the determination of emamectin and its metabolites (8,9-Z-isomer, N-demethylated, N-formylated, and N-methylformylated emamectin) in various crops. The analytes were extracted with acetone, cleaned up on cartridge columns (C18 and NH2), derivatized with trifluoroacetic anhydride and 1-methylimidazole, and determined by LC with fluorescence detection. Because radish inhibited the formation of the fluorescent derivatives, an additional Bond Elut PRS cartridge was used in the cleanup of Japanese radish samples. During sample preparation, N-formylated emamectin partially degraded to emamectin B1b and emamectin B1a, and the 8,9-Z-isomer partially degraded to N-demethylated emamectin. Therefore, emamectin and its metabolites were determined as total emamectin, i.e., their sum was estimated as emamectin benzoate. Their recoveries from most crops were approximately 80-110% with the developed method. The detection limits for the analytes in vegetables were 0.1-0.3 parts per trillion (ppt). The results for these compounds were confirmed by LC/mass spectrometry (LC/MS; electrospray ionization mode). Because the fluorescent derivative of emamectin was undetectable by LC/MS, the results for the analyte were confirmed by using a sample solution without derivatization. Limits of detection by LC/MS were similar to the fluorescence detection limits, 0.1-0.3 ppt in vegetables. In addition to the emamectins, milbemectin, ivermectin, and abamectin were also determined by the developed method.     

Selective separations of peptides with sequence deletions, single amino acid polymorphisms, and/or epimeric centers using macrocyclic glycopeptide liquid chromatography stationary phases

Separating closely related peptides (those differing by one or two amino acids or the chirality of a single amino acid) can be challenging using reversed-phase liquid chromatography (LC), ion-exchange LC, or using ion-pairing agents. Also, the mobile phases that give the best separations in these modes may not be electrospray ionization mass spectrometry (ESI-MS) compatible. Forty-two peptides from 11 peptide families were separated on three macrocyclic glycopeptide stationary phases in reverse-phase mode using ESI-MS-compatible mobile phases. The peptide classes studied were angiotensin, bradykinin, alpha-bag cell factor, beta,gamma-bag cell factor, beta-casomorphin, dynorphin, enkephalin, leucokinin, lutinizing hormone releasing hormone, neurotinsin, substance P, and vasopressin. High selectivity was observed for single amino acid substitutions (achiral and chiral) regardless of the position of the substitution in the sequence. Mobile phase optimization, its effect on peptide elution behavior, and chromatographic efficiency is also discussed. Using LC-ESI-MS, a 2 ng limit of detection was obtained, two orders of magnitude lower than the UV detection limit.     

Analysis of native amino acid and peptide enantiomers by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

High-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry was used for the separation and detection of amino acid and peptide enantiomers. With detection limits as low as 250 pg, 25 amino acids enantiomers were baseline resolved on a Chirobiotic T chiral stationary phase. APCI demonstrated an order of magnitude better sensitivity over electrospray ionization (ESI) for free amino acids and low molecular mass peptides at the high LC flow-rates necessary for rapid analysis. As the peptide chain length increased (peptides with M(r) > or = 300 Da), however, ESI proved to be the more ideal atmospheric pressure ionization source. A mobile phase consisting of 1% (w/w) ammonium trifluoroacetate in methanol and 0.1% (w/w) formic acid in water increased the sensitivity of the APCI method significantly. A step gradient was then used to separate simultaneously all 19 native protein amino acid enantiomers in less than 20 min using extracted ion chromatograms.     

Molecular Modeling of Enantioseparation of Phenylazetidin Derivatives by Cyclodextrins

β-Lactams are one of the most widely used types of antibiotics. As β-lactams are chiral, the enantiomeric separation of these compounds was investigated using cyclodextrins, frequently used as chiral separators. Molecular modeling methods were utilized in order to predict possible enantioseparation of four model compounds. Our results revealed that permethylated β-cyclodextrin is more likely to chirally separate the phenylazetidin derivates than the parent β-cyclodextrin. LC experiments using cyclodextrin as chiral stationary phase in most cases confirmed our prediction; however, more experiments and statistical evaluation of the results are needed in order to judge the prediction power of the molecular dynamic method.     

Liquid-crystalline polymers: Past, present, and future

The main steps of the evolution in studies related to the design and investigation of the structure and properties of thermotropic LC polymers containing mesogenic groups are discussed. The principal attention is focused on the results of experiments performed at the Laboratory of Chemical Transformations of Polymers, Faculty of Chemistry, Moscow State University, which was established and guided by Academician N.A. Platé from 1966 to 1985 and then supervised by V.P. Shibaev, the author of this review. Historical evidence is presented to demonstrate the contribution of Russian scientists to the development of approaches to the synthesis and study of chiral and electro- and photocontrollable comb-shaped LC polymers and related composites. The concept of preparing multifunctional LC copolymers and LC networks containing mesogenic, chiral, photochromic, and functional (including ionophoric) groups that can undergo hydrogen bonding, form complexes with metal ions, and interact with nanoparticles is scrutinized. Specific features of the structural organization of polymer mesophases are covered. The data on multifunctional comb-shaped LC polymers, LC ionomers, and LC dendrimers are examined, and the problems concerning the design of light-controllable LC copolymers and polymer photochromic composites and networks are reviewed. Some applied aspects of using LC polymers are considered: specifically, approaches to creation of lasers based on cholesterics and photo- and electroactive media in optics and photonics, systems for data recording and storage, holography, display technology, and other fields.     

Determination of proline enantiomers in honey and royal jelly by LC-UV

The chiral separation and quantification of D-proline and L-proline in honey and royal jelly were examined by LC with UV detection. Most of the endogenous compounds existing in honey, such as sugars, were removed by using SPE cartridges containing C18 and strong cation-exchange sorbent. Other components, such as primary amino acids, were also removed by two-step derivatization with o-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC-CI). The components that were derivatized with OPA were separated from proline with a C18 cartridge. Proline was then converted into an FMOC derivative that could be subsequently measured by LC-UV. Sufficient chiral separation of D-proline and L-proline was achieved with an LC chiral column made of a beta-cyclodextrin phase in the polar organic-phase mode. The average recoveries of D-proline and L-proline from honey and royal jelly were in the range of 81.3-98.6% (RSD of < 1.8%). When this method was applied to commercial honey and royal jelly samples, L-proline was detected at concentrations of 369-1930 microg/g, whereas D-proline was not detected.