Choyce A. Weatherly

Water determination in active pharmaceutical ingredients using ionic liquid headspace gas chromatography and two different detection protocols

A rapid, accurate, precise and versatile analytical method was developed for the detection and quantification of water in solid active pharmaceutical ingredients (APIs). The headspace gas chromatography (HSGC) method utilized an ionic liquid (IL) based open tubular capillary GC column to increase sensitivity and ruggedness of this method. ILs are also utilized as the headspace solvent because of their low vapor pressure, unique physiochemical properties and high thermal stability. This method is not affected by side reactions and solubility problems which are common with Karl Fischer Titration (KFT) methods. Nor is it as limited as weight loss on drying approaches. The ability to use either/both modern thermal conductivity or barrier ion discharge GC detection provides flexibility, different dynamic ranges and sensitivity. The developed method also was shown to be broadly applicable.

Analysis of Long-Chain Unsaturated Fatty Acids by Ionic Liquid Gas Chromatography

Four ionic liquid (IL) columns, SLB-IL59, SLB-IL60, SLB-IL65, and SLB-IL111, were evaluated for more rapid analysis or improved resolution of long-chain methyl and ethyl esters of omega-3, omega-6, and additional positional isomeric and stereoisomeric blends of fatty acids found in fish oil, flaxseed oil, and potentially more complicated compositions. The three structurally distinct IL columns provided shorter retention times and more symmetric peak shapes for the fatty acid methyl or ethyl esters than a conventional polyethylene glycol column (PEG), resolving cis- and trans-fatty acid isomers that coeluted on the PEG column. The potential for improved resolution of fatty acid esters is important for complex food and supplement applications, where different forms of fatty acid can be incorporated. Vacuum ultraviolet detection contributed to further resolution for intricate mixtures containing cis- and trans-isomers, as exemplified in a fatty acid blend of shorter chain C18:1 esters with longer chain polyunsaturated fatty acid (PUFA) esters.

Rapid Analysis of Ethanol and Water in Commercial Products Using Ionic Liquid Capillary Gas Chromatography with Thermal Conductivity Detection and/or Barrier Discharge Ionization Detection

Analysis of ethanol and water in consumer products is important in a variety of processes and often is mandated by regulating agencies. A method for the simultaneous quantitation of ethanol and water that is simple, accurate, precise, rapid, and cost-effective is demonstrated. This approach requires no internal standard for the quantitation of both ethanol and water at any/all levels in commercial products. Ionic liquid based gas chromatography (GC) capillary columns are used to obtain a fast analysis with high selectivity and resolution of water and ethanol. Typical run times are just over 3 min. Examination of the response range of water and ethanol with GC, thermal conductivity detection (TCD), and barrier ionization detection (BID) is performed. Quantitation of both ethanol and water in consumer products is accomplished with both TCD and BID GC detectors using a nonlinear calibration. Validation of method accuracy is accomplished by using standard reference materials.

Enantiomeric separation of functionalized ethano-bridged Tröger bases using macrocyclic cyclofructan and cyclodextrin chiral selectors in high-performance liquid chromatography and capillary electrophoresis with application of principal component analysis

The enantiomeric separation of a series of racemic functionalized ethano-bridged Tröger base compounds was examined by high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Using HPLC and CE the entire set of 14 derivatives was separated by chiral stationary phases (CSPs) and chiral additives composed of cyclodextrin (native and derivatized) and cyclofructan (derivatized). Baseline separations (Rs≥1.5) in HPLC were achieved for 13 of the 14 compounds with resolution values as high as 5.0. CE produced 2 baseline separations. The separations on the cyclodextrin CSPs showed optimum results in the reversed phase mode, and the LARIHC™ cyclofructan CSPs separations showed optimum results in the normal phase mode. HPLC separation data of the compounds was analyzed using principal component analysis (PCA). The PCA biplot analysis showed that retention is governed by the size of the R1 substituent in the case of derivatized cyclofructan and cyclodextrin CSPs, and enantiomeric resolution closely correlated with the size of the R2 group in the case of non-derivatized γ-cyclodextrin CSP. It is clearly shown that chromatographic retention is necessary but not sufficient for the enantiomeric separations of these compounds.

Effective methodologies for enantiomeric separations of 150 pharmacology and toxicology related 1°, 2°, and 3° amines with core-shell chiral stationary phases

HIGHLIGHTSRapid method development for optimized enantiomeric separations of 150 amines.Biological compatible MS analysis of 150 amines.Complementary separations with macrocyclic chiral selectors.High‐throughput chiral separations with core‐shell chiral stationary phases.Single LC–MS chiral separation of 18 illicit substances in 35min. ABSTRACT Core‐shell particles (superficially porous particles, SPPs) have been proven to provide high‐throughput and effective separations of a variety of chiral molecules. However, due to their limited commercialization, many separations have not been reported with these stationary phases. In this study, four SPP chiral stationary phases (CSPs) were utilized for the enantiomeric separation of 150 chiral amines. These amines encompass a variety of structural and drug classes, which are particularly important to the pharmaceutical industry and in forensics. This comprehensive evaluation demonstrates the power of these CSPs and the ease of method development and optimization. The CSPs used in this study included the macrocyclic glycopeptide‐based CSPs (VancoShell and NicoShell), the cyclodextrin‐based CSP (CDShell‐RSP), and the cyclofructan‐based CSP (LarihcShell‐P). These CSPs offered versatility for a variety of applications and worked in a complementary fashion to baseline separate all 150 amines. The LarihcShell‐P was highly effective for separating primary amines. VancoShell, NicoShell, and CDShell‐RSP were useful for separating all types of amines. These CSPs are multi‐modal and can be utilized with mass spectrometry compatible solvents. Eighteen racemic controlled substances were simultaneously baseline separated in a single liquid chromatography–mass spectrometry (LC–MS) analysis. Details in high‐performance liquid chromatography (HPLC) parameters will be discussed as well as the improved chromatographic performance afforded by the SPP CSPs.

Hydroxypropyl beta cyclodextrin bonded superficially porous particle-based HILIC stationary phases

ABSTRACT (R,S)-Hydroxypropyl-modified β-cyclodextrin (RSP-CD) is a well-known chiral stationary phase. In this work, hydrophilic interaction liquid chromatographic (HILIC) selectivities of RSP-CD was demonstrated. Further, an evaluation of chromatographic performances of fully porous particles (FPPs)- and superficially porous particles (SPPs)-based RSP-CD stationary phases was performed. The RSP-CD-bonded SPP-based stationary phase showed faster and more efficient HILIC separations compared to the FPP-based stationary phases. In addition, the SPP-based RSP-CD stationary phase showed excellent selectivities for many classes of small polar molecules. Since the SPP-based stationary phase is allowed for separations performed at high flow rates without significant loss of efficiency, ultrafast separations (analysis times under 1 min) also was accomplished utilizing SPP-based RSP-CD stationary phase. GRAPHICAL ABSTRACT

Development and evaluation of gas and liquid chromatographic methods for the analysis of fatty amines.

In contrast to the plethora of publications on the separation of fatty acids, analogous studies involving fatty amines are scarce. A recently introduced ionic-liquid-based capillary column for GC was used to separate trifluoroacetylated fatty amines focusing on the analysis of a commercial sample. Using the ionic liquid column (isothermal mode at 200 °C) it was possible to separate linear primary fatty amines from C12 to C22 chain length in less 25 min with MS identification. The log of the amine retention factors are linearly related to the alkyl chain length with a methylene selectivity of 0.117 kcal/mol for the saturated amines and 0.128 kcal/mol for the mono-unsaturated amines. The sp2 selectivity for unsaturated fatty amines also could be calculated as 0.107 kcal/mol for the ionic liquid column. The commercial sample was quantified by GC with flame ionization detection (FID). An LC method also was developed with a reversed phase gradient separation using acetonitrile/formate buffer mobile phases and ESI-MS detection. Native amines could be detected and identified by their single ion monitoring chromatograms even when partial coelution was observed. The analysis of the commercial sample returned results coherent with those obtained by GC-FID and with the manufacturers data.

A comprehensive methodology for the chiral separation of 40 tobacco alkaloids and their carcinogenic E/Z-(R,S)-tobacco-specific nitrosamine metabolites

The predominant enantiomer of nicotine found in nature is (S)-nicotine and its pharmacology has been widely established. However, pharmacologic information concerning individual enantiomers of nicotine-related compounds is limited. Recently, a modified macrocyclic glycopeptide chiral selector was found to be highly stereoselective for most tobacco alkaloids and metabolites. This study examines the semi-synthetic and native known macrocyclic glycopeptides for chiral recognition, separation, and characterization of the largest group of nicotine-related compounds ever reported (tobacco alkaloids, nicotine metabolites and derivatives, and tobacco-specific nitrosamines). The enantioseparation of nicotine is accomplished in less than 20s for example. All liquid chromatography separations are mass spectrometry compatible for the tobacco alkaloids, as well as their metabolites. Ring-closed, cyclized structures were identified and separated from their ring-open, straight chain equilibrium structures. Also, E/Z-tobacco-specific nitrosamines and their enantiomers were directly separated. E/Z isomers also are known to have different physical and chemical properties and biological activities. This study provides optimal separation conditions for the analysis of nicotine-related isomers, which in the past have been reported to be ineffectively separated which can result in inaccurate results. The methodology of this study could be applied to cancer studies, and lead to more information about the role of these isomers in other diseases and as treatment for diseases.

Variations of l- and d-amino acid levels in the brain of wild-type and mutant mice lacking d-amino acid oxidase activity

Abstractd-amino acids are now recognized to be widely present in organisms and play essential roles in biological processes. Some d-amino acids are metabolized by d-amino acid oxidase (DAO), while d-Asp and d-Glu are metabolized by d-aspartate oxidase (DDO). In this study, levels of 22 amino acids and the enantiomeric compositions of the 19 chiral proteogenic entities have been determined in the whole brain of wild-type ddY mice (ddY/DAO+/+), mutant mice lacking DAO activity (ddY/DAO−/−), and the heterozygous mice (ddY/DAO+/−) using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). No significant differences were observed for l-amino acid levels among the three strains except for l-Trp which was markedly elevated in the DAO+/− and DAO−/− mice. The question arises as to whether this is an unknown effect of DAO inactivity. The three highest levels of l-amino acids were l-Glu, l-Asp, and l-Gln in all the three strains. The lowest l-amino acid level was l-Cys in ddY/DAO+/− and ddY/DAO−/− mice, while l-Trp showed the lowest level in ddY/DAO+/+mice. The highest concentration of d-amino acid was found to be d-Ser, which also had the highest % d value (~ 25%). d-Glu had the lowest % d value (~ 0.01%) in all the three strains. Significant differences of d-Leu, d-Ala, d-Ser, d-Arg, and d-Ile were observed in ddY/DAO+/− and ddY/DAO−/− mice compared to ddY/DAO+/+ mice. This work provides the most complete baseline analysis of l- and d-amino acids in the brains of ddY/DAO+/+, ddY/DAO+/−, and ddY/DAO−/− mice yet reported. It also provides the most effective and efficient analytical approach for measuring these analytes in biological samples. This study provides fundamental information on the role of DAO in the brain and may be relevant for future development involving novel drugs for DAO regulation.

Chiral Liquid Chromatography

Abstract Over the last decade, chiral liquid chromatography has made tremendous advances in selectivity and efficiency. These developments have been beneficial to many scientific disciplines especially in the field of high-throughput research. In this chapter, we review the fundamentals of chiral separations, classes of stationary phases, new particle morphologies, bonding chemistry of chiral selectors and high-efficiency packing procedures. Special detectors for enantiomeric separations are surveyed. The latest developments in chiral chromatography have culminated in sub-second separations of enantiomers. Instrumental considerations to achieve ultrafast enantiomeric separations are discussed. Unique applications of chiral chromatography such as shape selectivity, formation of “Batman” peaks, peak shapes under overloading conditions, and future developments are highlighted.