Masahiko Okamoto

Reversal of elution order during the chiral separation in high performance liquid chromatography

The elution order of the enantiomers is one of the most important topics in the field of chiral separations. The reversal of enantiomeric elution order, although rare, could be observed during the investigations of chiral discrimination in high performance liquid chromatography (HPLC). Scrutinizing these phenomena must be helpful in determining the optical purities because it is favorable to elute the minor enantiomeric impurity before the major component. In this mini-review, several examples of such unusual behavior will be described from the point of mechanistic rationale.

Reversal of elution order during direct enantiomeric separation of pyriproxyfen on a cellulose-based chiral stationary phase

Abstract The enantiomeric separation of pyriproxyfen (Sumilary), a new insect growth regulator, was investigated on several commercially available chiral stationary phases consisting of cellulose esters coated on silica. The mobile phases were composed of n -hexane and alcohols. Resolution was achieved only by using a cellulose tris-(4-methylbenzoate)-coated silica gel. The enantiomeric elution order changed according to the steric bulk of the alcohols. The possible mechanisms of the reversal of elution order are discussed.

Chiral Liquid Chromatography−Circular Dichroism−NMR for Estimating Separation Conditions of Chiral HPLC without Authentic Samples

Chiral separation by high performance liquid chromatography (Chiral HPLC) is one of the most powerful methods for estimating optical and chemical purity of chiral compounds. However, it has a weakness in that much time and effort are required to prepare authentic samples. A novel chiral liquid chromatography-circular dichroism-NMR (LC-CD-NMR) technique, on the other hand, requires only crude chiral compounds that include enantiomers as minor impurities. In this study, chiral LC-CD-NMR was constructed by connecting a conventional LC-NMR system with a CD detector. A pyridylalanine derivative mixture was prepared to mimic technical grade material in an early phase of development. By chiral LC-CD-NMR, the enantiomer peak is identified by an opposite sign of the CD Cotton effect curve and an identical (1)H NMR spectrum to that of the main component. Using NMR as a detector, this method is superior in ability to discriminate enantiomers from other isomers indistinguishable by MS. Furthermore, this method is also applicable for selecting the best separation conditions of chiral HPLC. The degrees of separation (Rs) between the main component and its enantiomer in several chiral columns were compared. Even with modern chromatographic methods, establishing the best chiral HPLC conditions in an early phase of development is difficult: chiral LC-CD-NMR is a suitable solution.

Direct stereochemical resolution of 3,4-dihydroxyphenylserine using a chiral crown ether stationary phase

Abstract The direct stereochemical resolution of the four stereoisomers of 3,4-dihydroxyphenylserine was achieved on a high-performance liquid chromatographic chiral stationary phase based on a chiral crown ether. The effects of pH and temperature were investigated. The role of the crown ether ring in separating the analyte is also described.

A Comparative Study on Structural Elucidation of Sialyl Oligosaccharides by Mass Spectrometry with Fast Atom Bombardment, Electrospray Ionization, and Matrix Assisted Laser Desorption/Ionization

To establish a new protocol for sensitive detection and structural characterization of sialyl oligosaccharides, their sensitivities and structural information from mass spectrometry and tandem mass spectrometry with FAB-, ESI-, and MALDI were evaluated in detail. Among these ionization methods, FAB-MS and FAB-MS/MS gave reproducible and predictable spectra carrying information on sequence and branching of sialyl oligosaccharides after derivatization with 2-aminopyridine (PA). With both positive and negative ion modes, their structural elucidation promises to be straightforward, MS/MS specta being measurable at as low as 200 pmol. Thus, this method consitutes a powerful tool for sensitive detection and structural characterization of limited quantities of sialyl oligosaccharides by FAB-MS and FAB-MS/MS.

Assay validation and technology transfer: Problems and solutions

In the industry of fine chemicals, including pharmaceutical and agricultural chemicals, analytical tests are performed by production departments or contract research organizations at some stage in the research and development of products. These external organizations are required to maintain the capabilities to perform analytical tests using methods that are equivalent to or better than those specified by analytical method validation. For this reason, transfer of analytical procedures to an alternative site becomes necessary. In this review, the relationship between transfer of analytical procedures and assay validation is introduced, focusing on analytical procedures that include HPLC.

Sensitivity Enhancement by Chromatographic Peak Concentration with Ultra-High Performance Liquid Chromatography–Nuclear Magnetic Resonance Spectroscopy for Minor Impurity Analysis

High performance liquid chromatography can be coupled with nuclear magnetic resonance (NMR) spectroscopy to give a powerful analytical method known as liquid chromatography-nuclear magnetic resonance (LC-NMR) spectroscopy, which can be used to determine the chemical structures of the components of complex mixtures. However, intrinsic limitations in the sensitivity of NMR spectroscopy have restricted the scope of this procedure, and resolving these limitations remains a critical problem for analysis. In this study, we coupled ultra-high performance liquid chromatography (UHPLC) with NMR to give a simple and versatile analytical method with higher sensitivity than conventional LC-NMR. UHPLC separation enabled the concentration of individual peaks to give a volume similar to that of the NMR flow cell, thereby maximizing the sensitivity to the theoretical upper limit. The UHPLC concentration of compound peaks present at typical impurity levels (5.0-13.1 nmol) in a mixture led to at most three-fold increase in the signal-to-noise ratio compared with LC-NMR. Furthermore, we demonstrated the use of UHPLC-NMR for obtaining structural information of a minor impurity in a reaction mixture in actual laboratory-scale development of a synthetic process. Using UHPLC-NMR, the experimental run times for chromatography and NMR were greatly reduced compared with LC-NMR. UHPLC-NMR successfully overcomes the difficulties associated with analyses of minor components in a complex mixture by LC-NMR, which are problematic even when an ultra-high field magnet and cryogenic probe are used.

Direct stereochemical resolution of SM-11044, a novel anti-asthmatic drug, and its stereoisomers using a chiral immobilized protein stationary phase

A high-performance liquid chromatographic separation of SM-11044, a novel anti-asthmatic drug, and its antipode and stereoisomers was achieved using a chiral protein column that permits low levels of the antipode to be measured in the SM-11044. The influence of replacing H2O with 2H2O as the mobile phase and the effect of buffer ionic strength, pH, organic modifiers and temperature on the retention times and enantiomeric resolution are discussed.