Mary Lynn Grayeski

Room-temperature phosphorescence, sensitized phosphorescence and fluorescence of licit and illicit drugs enhanced by organized media

Abstract The ability of microscopically organized media, in the form of surfactant micelles and α- and β-cyclodextrins, to enhance the luminescence phenomena of several licit and illicit drugs is discussed. Because physiological samples are not often amenable to direct spectrometric measurements without pretreatment, the applicability of these organized media to liquid chromatography is also considered. Fluorescence enhancements for certain hallucinogenic drugs such as N,N -dimethyltryptamine, mescaline and ibogaine are seen in cyclodextrin media compared to conventional, homogeneous solutions. Heavy-atom substituted sodium dodecyl sulfate micelles induce phosphorescence from cationic and/or hydrophobic drugs at room temperature in fluid solution; drugs such as propranolol, diflunisal, naphalozine, and selected quinoline derivatives can be determined conveniently. Sensitized phosphorescence is observed for several drugs including brethine, cocaine, didrate, estradiol, meprobarbital, methaqualone, phenobarbital, and sulfanilamide; it can be enhanced markedly when micellar solutions are used as the solvent. The energy-transfer step is facilitated by the organizing ability of the micelle; limits of detection can be decreased by over two orders of magnitude compared to homogeneous solvents. Sensitized phosphorescence can also be measured in cyclodextrin solutions, but the detectability is inferior to that in micellar media. Which form of organized medium is superior for determination of drugs is discussed.

New chemiluminescent derivatizing agent for the analysis of aldehydes and ketones by high-performance liquid chromatography with peroxyoxalate chemiluminescence

Abstract Three methods of reductive amination have been investigated for the derivatization of aldehydes and ketones with a new chemiluminescent agent, 3-aminofluoranthene. Use of a borane-pyridine complex was found to give the highest product yield. Analytes were separated by high-performance liquid chromatography (HPLC) and detected by peroxyoxalate chemiluminescence. Detection limits were in the fmol range, chemiluminescence detectability being thirty times better than fluorescence.

Determination of trace levels of steroids in blood plasma by liquid chromatography with peroxyoxalate chemiluminescence detection.

A highly sensitive and specific liquid chromatographic procedure with peroxyoxalate chemiluminescence detection has been developed for the determination of fluocortin butyl, a 3 alpha-ketocorticosteroid, in blood plasma. The technique employs dansylation of the steroid to provide a highly chemiluminescent derivative. After separation by reversed-phase liquid chromatography, reagents necessary for chemiluminescence are added, followed by detection in a conventional fluorimetric detector in which the excitation source is deactivated. The precision is 2.5% relative standard deviation at the 10 ng/ml level, and the response is linear up to at least 4 ng injected steroid. The procedure requires only 1 ml blood plasma and has a limit of detection of 100 pg/ml or 7.5 pg injected steroid. The system is reliably used for routine pharmacokinetic studies and with modifications, is applicable to other steroids as well.

Investigation of Instrument Parameters for the Application of Peroxyoxalate Cheminescence Detection to Microbore Chromatography

Abstract Peroxyoxalate chemiluminescence generated by the reaction of an oxalate and hydrogen peroxide in the presence of a fluorophor was evaluated as a detection system for microbore liquid chromatography. The results of studies on instrumental parameters show that the signal is increased with short reaction lines, increasing flow cell volume, and enhanced response through detectors with improved optics.

Flow-injection chemiluminescent method for an enzyme-labelled DNA probe

Abstract A flow-injection chemiluminescent (CL) method was investigated for the determination of hydrogen peroxide generated from an enzyme-labelled DNA probe. Biotinylated DNA probes were immobilized onto a membrane and labelled with biotinylated glucose oxidase by a streptavidin bridge and placed in a microtitre well. The glucose oxidase-labelled sample was incubated in a solution of glucose and the enzymatically produced hydrogen peroxide was determined using flow injection with a peroxyoxalate chemiluminescence reaction. The peroxide generated is related to the amount of the labelled DNA probe. The important factors for increasing the sensitivity of the CL measurement are to maximize the production of hydrogen peroxide and to reduce the non-specific binding of the enzyme label on the membrane. This CL-flow-injection measurement using biotinylated glucose oxidase-labelled DNA probes was applied to a DNA dot-blot hybridization assay and compared with a visual colorimetric method using 5-bromo-4-chloroindolyl phosphate and nitro blue tetrazolium as a chromogenic substrate for detection of an alkaline phosphatase label. The CL method was quantitative over the range 100 pg-100 ng of target DNA with a precision (r.s.d.) of 4–11%. In comparison, the colorimetric method was useful for the range 10 pg-1 ng.

Quinoxalinone derivatization of biological carboxylic acids for detection by peroxyoxalate chemiluminescence with high-performance liquid chromatography

A quinoxalinone fluorescent tag is evaluated as a carboxylic acid derivatizing reagent for detection by peroxyoxalate chemiluminescence. The synthetic procedure for the quinoxalinone was modified to give a yield that is significantly increased over that reported previously. The new conditions use less hazardous reagents, and produce a final product greater than 97% pure, without the need for intermediate clean-up steps. The derivatization reaction is also modified to give increased yields of greater than 85% compared to 74% obtained previously. The post-column chemiluminescence reaction conditions are optimized to give detection limits of 500 attomole/injection-10 times lower than the fluorescence previously obtained. The reagent is used for the first time on a plasma sample extract. Typical method precision is 4%.

Quantitation of enzymatically generated hydrogen peroxide based on aqueous peroxyoxalate chemiluminescence

Abstract The method involves the reaction of 4,4′-{oxalyl bis[(trifluoromethylsulfonyl)imino]-ethylene}-bis(4-methylmorpholinium trifluoromethanesulfonate) with hydrogen peroxide in the presence of rhodamine-B. Precise measurements, with 1–3% relative standard deviation, can be made in both static and flow systems. In the flow system, the response to hydrogen peroxide is linear from 10 −2 M hydrogen peroxide down to the limit of detection of 7 × 10 −5 M.

Investigation of the quenching of peroxyoxalate chemiluminescence by amine substituted compounds

The role of amine compounds in quenched peroxyoxalate chemiluminescence was investigated. The mechanistic steps examined included (1) fluorescence quenching; (2) base hydrolysis of the oxalate; and (3) a competitive interaction between the quencher and fluorophore for the peroxyoxalate reaction intermediate(s). The results showed no evidence of amines causing fluorescence quenching. Base hydrolysis of the oxalate is significant only at high concentrations of amines. When the concentration of amines is greater than or equal to the level of oxalate, the amines can compete with the fluorophore for reaction with the intermediate(s). Because competitive effects were demonstrated, one analytical implication is that caution must be exercised in applications where more than one fluorophore is present. At low concentrations of fluorophores relative to the oxalate, chemiluminescence emission of both fluorophores will be observed. At higher levels, the fluorophores can compete for reaction with the intermediate(s). Finally, an important analytical implication of this study is that the quenching response can be used to quantify amines without the need for derivatization. The limitation is that the linear response is approximately one order of magnitude.

Investigations of the reversed-phase high-performance liquid chromatographic ligand-exchange retention mechanism on a triamine stationary phase

Abstract A tridentate ligand, trimethoxysilylpropyldiethylenetriamine, was bound to silica and studied as a stationary phase for reversed-phase ligand-exchange chromatography. The tridentate stationary phase “triamine” yielded more stable metal complexes, i.e. , better metal loading efficiencies than previously reported diamine and monoamine stationary phases. This triamine packing with its enhanced metal loading properties was evaluated to identify the key factors that effect solute retention and selectivity. Mobile phase properties like buffer ionic strength and metal concentration greatly affected retention, while type of metal, i.e. , Zn II , Cd II , Hg II , Cu II , or Ni II greatly affected selectivity. The choice of mobile phase organic modifier signigicantly affected both retention and selectivity. Methanol—water mobile phases elicited relatively little metal binding per triamine site, with Hg II , Zn II , Ni II and Cd II occupying 32, 9.5, 7.8 and 5.7, respectively. Changing the organic modifier from methanol to acetonitrile provoked a dramitically different but consistent pattern of metal loading to the triamine. The ratio of loaded metal with the acetonitrile modifier was 2.3 ± 0.1 fold higher than that observed for the methanol modifier in each of the cases tested. Factors found to affect metal loading to the stationary phase were the type of metal and the mobile phase solvent. The loading of metals onto the silica-bound triamine correlated directly to metal basicity. The variety of solute selectivities obtained in this study indicate that the key solute—metal interactions are a function of metal and solute functional group basicities and the number and spacing of solute functional groups possessing lone-pair electrons capable of interacting with the metal. Retention can be greatly enhanced when multiple functional groups possessing donatable electrons are present: functionalities containing nitrogen, sulfur or oxygen. Ring-substituted nitrogen, sulfur or oxygen atoms i.e. piperdines, pyrimidines, purines, xanthines and triazoles, enhance retention more than non-ring-substituted functionalities, such as carboxylic and sulfonic acids. The presence of non-electron-donating functionalities in close proximity to strong interacting groups resulted in significant decreases in retention due to steric hindrance of solute lone-pair electrons to the metal coordination sphere. It is suggested that the mechanism of ligand-exchange retention on this triamine phase involves the reversible binding of a metal to the stationary phase followed by outer-sphere solute—metal complexation.