A magnetic molecularly imprinted polymer (M-MIP) for cyclobarbital (CY) has been prepared by a multi-step swelling and polymerization method using a uniformly-sized magnetic particle as the shape template. Binding experiments and Scatchard analyses revealed that two classes of binding sites, high and low affinity sites, were formed on the M-MIP. The retention and molecular-recognition properties of the M-MIP toward CY, phenobarbital (PB), amobarbital (AM) and phenytoin (PT) were evaluated using a mixture of phosphoric acid and/or sodium phosphate buffer and acetonitrile as a mobile phase by LC. On the M-MIP, retention factors of CY, PB, AM and PT were drastically decreased at mobile phase pH 8.7. This could be ascribable to dissociation of these compounds, whose pKa values are around 8. The imprinting factors were in the order of CY > PB > AM > PT at mobile phase pHs 2.5–6.9, and those are very similar between CY and PB. Furthermore, they were decreased at mobile phase pH 8.7. In addition to shape recognition, hydrogen-bonding and hydrophobic interactions seem to work for the retention and molecular-recognition of CY, PB, AM and PT on the MIP. The M-MIP for CY was applied for the selective extraction of PB in human serum samples.
A two-dimensional (2D) HPLC-MS/MS method has been established for the determination of trace amounts of D-amino acid residues in proteins. D-Amino acid residues are now increasingly recognized as biomarkers of diseases and key moieties for the regulation of protein structures/functions, therefore, a sensitive analytical method is highly required. However, non-negligible amounts of D-amino acids are produced by chemical racemization during the hydrolysis of proteins, and a sensitive determination of D-amino acid residues is normally difficult. In the present study, DCl/D2O hydrolysis is adopted and the produced deuterated D-amino acids are distinguished from naturally-occurring D-amino acids in the proteins using the 2D-HPLC-MS/MS system. D-Ala, D-Asp, D-Glu, D-Pro and D-Ser (frequently observed D-amino acids in higher animals) were selected as the targets, and the sensitive determination (around 1% or less of the L-forms) could be carried out. As an application, the D-amino acid residues in ovalbumin (OVA) were determined, and the presence of a significant amount of D-Ser (1.8% of L-Ser) was demonstrated.
A novel column packing material, chondroitin sulfate-modified silica gel (ChS-silica), was developed for use as a stationary phase or capillary electrochromatography. The sulfonic acid groups on the ChS-silica supported electroosmotic flow at low pH and served as a strong cation-exchanger. Analytes are likely retained on ChS-silica by hydrophilic interactions (HI) and strong cation-exchange; this was verified by investigating the effects of mobile phase composition and buffer concentration on the retention of neutral polar and basic compounds. The baseline separation of seven basic pharmaceuticals was achieved using an applied voltage of 10 kV and a mobile phase consisting of 5 mM phosphate buffer (pH 7.0) and 70% acetonitrile. A column efficiency of 153000-410000 N/m for strongly retained compounds was observed.
A positively charged derivatization reagent, i.e., (S)-pyrrolidine-2-carboxylic acid N-(N'-methylpyridine-2-yl)amide (PCP2-Me), was synthesized and evaluated using non-steroidal anti-inflammatory drugs (NSAIDs), which were selected as the representative chiral carboxylic acids. The separation efficiency and detection sensitivity were compared to (S)-pyrrolidine-2-carboxylic acid N-(pyridine-2-yl)amide (PCP2) which was previously reported as a chiral derivatization reagent for carboxylic acids. The suitable column and mobile-phase composition were different between the PCP2 derivatives and PCP2-Me derivatives. The PCP2-Me has a highly proton-affinitive moiety in its structure, thus the sensitivity was increased as expected (Limit of detection (S/N=3), 15-72 amol for the PCP2-Me derivatives and 49-260 amol for the PCP2 derivatives). Based on these results, PCP2-Me seems to be usable for the determination of chiral carboxylic acids, the same as PCP2.
Preconcentration of aqueous solution of aromatic compounds was successfully carried out with an extraction capillary packed with a bundle of polymer-coated filaments. The extracted analytes were sequentially eluted with a flow of pure water using programmed temperature elevation of the extraction capillary. The results clearly suggest that the polymer-coated fiber-packed capillary could be employed as a sample preparation medium for the analysis of aqueous matrices. The fraction of interest could be transferred to conventional microcolumn liquid chromatography (LC) allowing a heart cutting analysis of aqueous matrices. Stability of the fiber-packed capillary was confirmed with pure water as the mobile phase at elevated temperatures.
A simple, rapid, accurate, precise headspace gas chromatographic (HS-GC) method was developed and validated for simultaneous quantitation of organic volatile impurities (residual solvents) in technical-grade active ingredients (TGAIs) of pesticides and agrochemicals. The HS-GC method utilizes conventional capillary columns. The HS-GC operating conditions were optimized, and 18 organic volatile impurities were efficiently separated. The versatility of the method was confirmed in terms of validation characteristics of specificity, limit of quantitation, linearity, accuracy, and precision. The proposed method was successfully applied to the analysis of pesticide and agrochemical TGAIs.