Manasi Saha

High-performance liquid chromatography versus solid-phase extraction for post-derivatization cleanup prior to gas chromatography-electron-capture negative-ion mass spectrometry of N1,N3-bis-(pentafluorobenzyl)-N7-(2-[pentafluorobenzyloxy]ethyl)xanthine, a product derived from an ethylene oxide DNA adduct

N7-(2-Hydroxyethyl)xanthine (N7-HEX), as a standard, has been measured at the low picomole level by the following sequence of steps: (1) derivatization with pentafluorobenzyl bromide; (2) post-derivatization sample cleanup by reversed-phase high-performance liquid chromatography (HPLC) or silica solid-phase extraction; and (3) separation/detection by gas chromatography electron-capture negative-ion mass spectrometry (GC-ECNI-MS). The average yield of product from the two sample cleanup procedures applied to 95 pg (0.48 pmol) of N7-HEX was comparable: 60% for HPLC; 56% for solid-phase extraction. The reaction blanks (0 pg N7-HEX) showed an interfering GC-ECNI-MS peak after HPLC cleanup. This problem was not encountered with solid-phase extraction, which, along with its greater convenience, made it the preferred technique for post-derivatization sample cleanup.

Ester and related derivatives of ring N-pentafluorobenzylated 5-hydroxymethyluracil: hydrolytic stability, mass spectral properties, and trace detection by gas chromatography-electron-capture detection, gas chromatography-electron-capture negative ion mass spectrometry, and moving-belt liquid chromatography-electron-capture negative ion mass spectrometry

One consequence of radiation damage to DNA is the conversion of thymine to 5-hydroxymethyluracil (HMU). In order to sensitively detect this DNA adduct by gas chromatography (GC) or high-performance liquid chromatography (HPLC) with electron-capture detection techniques, it is necessary to derivatize it. This study was designed to select an optimum ester derivative of the aliphatic hydroxyl group on HMU. N1, N3-Bis(pentafluorobenzyl)-HMU was formed as a parent derivative, and from this a series of esters. Also O-pentafluorobenzyl and O-tetrafluorobenzyl ether derivatives were prepared. Of the esters the pivalyl derivative was the best choice because it formed easily, was relatively stable to aqueous hydrolysis (t 1/2 = 9.8 days at pH 11.5, 24 degrees C) and gave a response at fmol levels by GC and LC comparable to that of the ethers. Unanticipated was a good response as well for the parent derivative, a free hydroxyl compound, by GC and LC at this level. The work also demonstrates a high performance by LC-electron-capture negative ion mass spectrometry with a belt interface for the trace detection of derivatives of this type.

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.

4-(Trifluoromethyl)-2,3,5,6-tetrafluorobenzyl bromide as a new electrophoric derivatizing reagent

4-(Trifluoromethyl)-2,3,5,6-tetrafluorobenzyl bromide (TTBB) was synthesized in a single step from alpha,alpha,alpha,2,3,5,6-heptafluoro-p-xylene. The purpose of TTBB is to function as an analogue of pentafluorobenzyl bromide (PFBB) in electrophoric derivatization reactions prior to detection by gas chromatography-electron-capture negative ion mass spectrometry (GC-ECNI-MS). In more detail, it was anticipated that TTBB could be used along with, or as a substitute for, PFBB to help control some interferences and confirm results. This is because a TTBB-product (of an analyte) would have different retention and sometimes m/z characteristics than a corresponding PFBB product in GC-ECNI-MS, while the two products should be similar in their ease of formation and yields. Results demonstrating these expectations were achieved by derivatizing and detecting two analytes with these reagents: N7-(2-hydroxyethyl)xanthine, and 2,3-pyrenedicarboxylic acid.

Primary contribution of the injector to carryover of a trace analyte in high-performance liquid chromatography

When a low-nanogram amount of N1,N3-bis-(pentafluorobenzyl)-N7-(2- [pentafluorobenzyloxy]ethyl)xanthine was subjected to HPLC, low-picogram amounts of the compound could be detected subsequently (off-line by gas chromatography-electron-capture negative-ion mass spectrometry) after injection of pure mobile phase. This was in spite of significant, intermediate washing of the injector and column. It was determined that essentially 99.9% of this analyte contamination came from the injector. Use of two injectors is a practical remedy for this problem.

Oxidation-elimination of a DNA base from its nucleoside to facilitate determination of alkyl chemical damage to DNA by gas chromatography with electrophore detection

Exposure of humans to chemicals can create risks of carcinogenesis and mutagenesis. Since these latter events tend to correlate with chemical damage to human DNA, i.e., with “DNA adducts”, there is interest in measuring this type of damage. High sensitivity may be needed because a trace amount of a DNA adduct might be significant. Here we present a powerful analytical strategy, with particular emphasis on the development of one step, for utilizing gas chromatography (GC) or high-performance liquid chromatography (HPLC) with electrophore detection to determine a small amount of an alkyl damaged base derived from human DNA. The step which is emphasized is an oxidation-elimination reaction, adopted from earlier work by otherslp3, that mildly and efficiently releases the base from a nucleoside.