Sterling A. Tomellini

Non-derivatization approach to high-performance liquid chromatography–fluorescence detection for aminoglycoside antibiotics based on a ligand displacement reaction

An indirect fluorescence detection method has been developed for detecting the aminoglycoside antibiotics following chromatographic separation. This approach to detection is based on a displacement reaction between the aminoglycosides and a copper(II)-L-tryptophan (L-Trp) complex, Cu(L-Trp)2. The aminoglycosides, which contain multiple amino groups, have strong affinities for the Cu(II) ion and displace L-Trp from the Cu(L-Trp)2 complex. The resulting increase in L-Trp fluorescence, which is quenched when coordinated to Cu(II), is indicative of the presence of the aminoglycoside. Fluorescence titration data indicate that there is a stoichiometric ratio of 1:1 between the reaction of the aminoglycosides with Cu(L-Trp)2. This HPLC detection scheme is implemented postcolumn by mixing a buffered Cu(L-Trp)2 solution with the column eluent prior to detection. The aminoglycosides were separated with the use of a column packed with a polymeric strong cation-exchanger. Separation and detection variables were optimized and are discussed. The detection limits for the aminoglycosides tested ranged from 4.2 to 14.5 ng injected (S/N=3). A linear working curve was achieved for amikacin in the range of 29-586 ng for a six point linearity test. The developed separation and detection scheme was further tested by analyzing commercial pharmaceutical formulations of these antibiotics.

Chiral separation of verapamil and related compounds using micellar electrokinetic capillary chromatography with mixed micelles of bile salt and polyoxyethylene ethers

Abstract Micellar electrokinetic capillary chromatography (MECC) using surfactant solutions containing sodium deoxycholate (NaDC), polyoxyethylene ethers, and methanol produced simultaneous baseline separation of the enantiomers of verapamil, norverapamil and gallopamil. Studies were conducted to determine the enantiomeric resolution obtained for verapamil and bi-2-naphthol with NaDC solutions, binary mixtures of NaDC with several polyoxyethylene ethers, and ternary mixtures of NaDC, polyoxyethylene ethers and methanol. Experiments were performed to determine the effect of three variables on chiral resolution: (1) the type of ether; (2) the mole fraction of ether in solutions with bile salt; and (3) the percentage of methanol in the mobile phase. The polyoxyethylene ethers studied included polyoxyethylene-8-decyl ether (C 10 E 8 ), polyoxyethylene-6-dodecyl ether (C 12 E 6 ), and polyoxyethylene-4-dodecyl ether (C 12 E 4 ). Simultaneous baseline separation of the enantiomers of verapamil, norverapamil and gallopamil was obtained using a solution containing the ether C 12 E 4 . The mole fraction of ether was 0.30 and the total surfactant concentration was 50 m M in this solution. The solvent mixture used for this separation was 25% methanol by volume.

Effects of bile salt structure on chiral separations with mixed micelles of bile salts and polyoxyethylene ethers using micellar electrokinetic capillary chromatography

The chiral resolving abilities of micellar solutions of four different bile salts alone and in mixtures with polyoxyethylene-4-dodecyl ether (C12E4) and methanol were investigated using MECC. The four bile salts investigated were the unconjugated sodium salts of cholic, deoxycholic, chenodeoxycholic and ursodeoxycholic acids. The test solutes included verapamil, norverapamil, gallopamil, bi-2-naphthol, atenolol and BAYK8644. The relative hydrophobicities of the micellar aggregates formed in solutions of binary mixtures of each bile salt with C12E4 were investigated by fluorescence spectroscopy using pyrene as a probe molecule. The observed enantiomeric resolution for the test compounds using these binary mixtures as MECC pseudo-stationary phases was determined. Correlations between micellar hydrophobicity for these solutions and chiral resolution of these test solutes are presented. The addition of C12E4 with or without methanol to solutions of sodium cholate and sodium deoxycholate enhanced the chiral resolution observed for compounds containing a longer hydrocarbon chain separating some of the major functional groups from the chiral center. The pure bile salt solutions generally provided better chiral resolution for the compounds where the major functional groups, such as aromatic rings, were closer to the chiral center.

Evaluation of Chloroparaffin/PVC Phases for ATR/FT-IR Analyses

A significant limitation to the use of ATR/FT-IR analysis for aqueous solutions is the relatively high bulk concentration of analyte that is required. One approach to improving the detection of an analyte is to incorporate a thin polymeric phase at the surface of the ATR element. The purpose of the polymeric phase is to extract the analyte of interest and concentrate it within the depth of penetration of the evanescent wave. With the use of a very high molecular weight polyvinyl chloride phase, the time necessary to reach equilibrium for a 0.05% (v/v) nitrobenzene in a 1.5% (w/v) methanol/water solution was over 60 min. A study was undertaken to determine whether incorporating a chloroparaffin plasticizer into the polymeric phase would reduce the time required to reach the maximum level of absorbance achieved for the analyte. Specifically, the ability of phases which are mixtures of PVC and chloroparaffin plasticizers to concentrate the analyte from aqueous solutions has been investigated. The results indicate that incorporating chloroparaffin into a PVC phase reduces the time required to reach the maximum absorbance for the analyte in an aqueous solution containing 1.5% methanol by approximately 45%. The addition of chloroparaffin also results in an increase in the magnitude of absorbance observed for the analytes investigated in this study.

Improving the infrared detection of analytes in aqueous solution using polymer coated ATR elements

Abstract Preliminary results of ATR/FTIR experiments indicate that thinly coating the ATR elements with suitable polymers improves the ability to detect test analytes in aqueous solutions. Experiments were performed using the test analyte, nitrobenzene, in aqueous solutions containing a range of methanol concentrations. the time dependence and effect of methanol concentration on the nitrobenzene IR absorbance was investigated.

Dynamic, computer-assisted interpretation of infrared spectra of condensed-phase mixtures

Abstract A knowledge-based system, MIXIR, designed to assist researchers in interpreting the infrared spectra of condensed-phase mixtures is described. This system overcomes many of the inherent limitations of static-rule-based systems. An iterative problem-solving approach is permitted, making use of information gained during the interpretation process. The dynamic rule selection and significance evaluation alos allow the user to provide information on the sample matrix, or to select a subset of the compounds for rule derivation. The MIXIR system was evaluated with a knowledge base compiled using IRBASE, a complementary program, and a test set of 20 mixtures.

Generation of compound-specific descriptions for interpreting the infrared spectra of condensed-phase mixtures

Abstract A knowledge-based system, IRBASE, has been developed for the rapid creation of compound-specific infrared spectral descriptions. Heuristic rules are used to generate chemically reasonable limits for band shifting effects in mixtures and to perform feature selection. These descriptions form the knowledge base of a second system, MIXIR, used to identify the components of unknown mixtures. Th IRBASE system has been used to generate a knowledge base for 50 common organic compounds.

Expert system for interpretation of the infrared spectra of environmental mixtures

Abstract A program for the identification of the principal components of mixtures through interpretation of the infrared mixture spectrum (IntIRpret) was developed. This program, which was developed as a preliminary screening tool for unknown organic mixtures, has five main subroutines: the interferogram processing and peak-selection subroutine (PUSHSUB), the automated knowledge-acquisition subroutine (AUTOGEN), the system optimization subroutine (STO), the interpretation subroutine (PAIRS), and final processing subroutine to subtract spectral similarity (PAIRSPLUS). Principal advantages of this system compared to earlier systems are speed, flexibility and accuracy.

Zone compressesion effects in high-performance liquid chromatography

Abstract A mathematical description of the post-column compression and acceleration of peaks eluted from a liquid chromatographic column is presented. By using specialized on-column concentration instrumentation for the sequential entrapment, compression and acceleration of individual solute zones, automatic enrichment of long retained solutes is possible. The techniques is useful for preparative separations, where gradient elution cannot always be utilized.

Indirect fluorescence detection of aliphatic biogenic polyamines and diamines following chromatographic separation

Abstract A convenient, sensitive method has been developed for detecting aliphatic biogenic polyamines, 1,2-diamines and 1,3-diamines, following separation by HPLC. The method is based on a postcolumn, non-derivatization reaction using low cost reagents. The basis for this detection scheme is the fluorescence of l -tryptophan ( l -Trp), which is quenched in copper(II)– l -Trp complexes; the fluorescence of l -Trp recovers with the addition of analytes that have a greater copper(II) affinity, such as aliphatic polyamines, 1,2-diamines and 1,3-diamines. Thus, the presence of these compounds in the HPLC eluent can be inferred by monitoring the fluorescence of l -Trp. pH has a strong effect on this detection system and this is discussed in detail. The detection limits following HPLC separation are 5 and 10 pmol injected for spermine and spermidine, respectively, with linear response regions to 1000 pmol.