Kariman Allam

MEASURING DNA ADDUCTS BY GAS CHROMATOGRAPHY-ELECTRON CAPTURE-MASS SPECTROMETRY : TRACE ORGANIC ANALYSIS

Publisher Summary This chapter discusses the practical experience in method development for the determination of trace amounts of DNA adducts, by gas chromatography-electron capture-mass spectrometry (GC-EC-MS). It has detected femtomole amounts of such analytes, by optimizing sample preparation (involving extraction, chemical reaction, and purification steps, starting with a biological sample) and low-attomole amounts of pure, derivatized standards, by GC-EC-MS. Although such methodology is already useful, the concepts and techniques described extend the sample preparation to the attomole level. In this chapter, the work on chemical transformation is emphasized as a part of sample preparation. This is a means to broaden the range of compounds that can be detected by GC-EC-MS. Also, the experience, in operating a GC-EC-MS to achieve attomole detection limits routinely (for standards), is discussed in the chapter. New ionization techniques for MS, such as electrospray and matrix-assisted laser desorption, are increasing the ability of MS to analyze “nonvolatile” substances present even in aqueous samples. Less new but of continuing importance, as a desorption/ionization technique, in this respect is fast atom bombardment, In contrast, this chapter discusses the procedures, in which significant chemical treatment of the sample precedes the “old technique” of GC, to deliver the analyte into the MS. The desorption approaches are attractive, because they can minimize sample preparation. They are also unique in their ability to achieve the direct detection of medium to high molecular weight biopolymers by MS.

Novel synthesis of 2-fluoroestradiol from 19-Cortestosterone: Biomimetic oxidative defluorination to 2-hydroxyestradiol

A new, short synthetic route to 2-fluoroestradiol from 19-nortestosterone is described which gives the target compound in an approximately 25% overall yield. Oxidative defluorination of 2-fluoroestradiol to 2-hydroxyestradiol via treatment with Frémys salt/iodide ion is reported. This process is regarded as biomimetic with respect to cytochrome P-450-dependent oxidative defluorination.

Tresyl activation of a hydroxyalkyl ligand for coupling to a hydrazide gel: Stable immobilization of T-2 toxin for affinity purification of T-2 antibody☆

A stable T-2 hydrazide gel is prepared by activating T-2 toxin with tresyl chloride followed by coupling to agarose-adipic acid hydrazide. Utilized as an affinity chromatography column, this T-2 hydrazide gel purifies a monoclonal antibody for T-2 in high yield directly from ascites fluid. Specific antibody trapped on the column is eluted either with excess T-2 or at pH 11.6. Much less successful are two other T-2 affinity columns that were prepared and evaluated: T-2 bovine serum albumin Affi-Gel 15 and T-2 hexylamine Sepharose.

A Sensitive Bioassay for a Standard of Recombinant Interleukin-1α

Abstract A sensitive bioassay for recombinant interleukin-1α (rIL-1α) has been developed. The assay is based on prostaglandin E2 (PGE2) production by fibroblasts following stimulation with rIL-1α. The stimulated PGE2 levels are determined by radioimmunoassay. Three human fibroblast cell lines, WI38, HEL299 and HS68, were evaluated. The HS68 cells gave the most sensitive and reproducible response. The results from six experiments established that 400 ± 300 femtograms of rIL-1α stimulated a two-fold increase in PGE2 concentration over the background level.