Page 227 - Tandem Techniques

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chromatographic behavior can be significantly improved and the substances made more volatile. In
particular, peak tailing can be reduced in GC and streaking in TLC. The two popular types of reagent
for acetylation are the acid anhydrides and the acid chlorides. Between 1 and 5 milligrams of the sample
is dissolved in chloroform (about 5 ml) and warmed with 0.5 ml of acetic anhydride and 1 ml of acetic
acid for 2-16 hours at 50°C. Excess of reagent is removed by evaporation in vacuum (assuming the
product is relatively involatile) and taken up in chloroform for subsequent GC analysis. Sodium acetate
is also often used as a basic catalyst for acetylation, particularly for carbohydrates extracted from urine.
The dried residue from the urine is oximated to derivatize the carbonyl groups and then the hydroxyl
groups are acetylated with acetic anhydride in the presence of sodium acetate at 100°C. Acetyl chloride
is not so widely used for acetylation, because hydrochloric acid is evolved in the reaction, so the
presence of an appropriate base is considered necessary to scavenge the hydrochloric acid as it is
produced. The N-acetyl methyl ester derivative of hydroxyanthranilic acid, which is a metabolic product
of tryptophan, is often used for the GC analysis of the material. The sample is first esterified with
diazomethane to give the methyl ester. The product is then evaporated to dryness in a stream of nitrogen
and acetylated with a 50% benzene/acetyl chloride mixture [33]. The reaction mixture is dried in a
stream of nitrogen and taken up in methanol for GC analysis. Only a fraction of the possible
derivatizing reagents have been mentioned, for further details the reader is directed to Blau and Halket
[34].

Chiu et al. [35] employed a derivatization technique to improve the detection limits of O -
6
(hydroxyalkyl)guanines in a GC/MS tandem system, using electron capture as the GC detector. The 0 6
(hydroxyalkyl)-guanines comprise a class of DNA products that appear to be strongly mutagenic as
well as carcinogenic [36,37]. As a consequence these substances are of considerable interest even when
present in trace amounts. In this case, a rather complicated derivatization procedure was employed. The
O -alkylguanines adducts were treated with fluoroboric acid/nitrous acid to form the corresponding 2-
6
fluoro-O -alkylhypoxanthines. The reaction products were then treated with pentafluorylbenzyl
6