One-step conversion of dipicolinic acid to its dimethyl ester using monomethyl sulfate salts for GC-MS detection of bacterial endospores

Abstract

Methyl sulfate (MeSO₄⁻) salts were explored as thermochemolysis–methylation (TCM) reagents for gas chromatographic (GC) analysis of dipicolinic acid (DPA) as its dimethyl ester (Me₂DPA) from bacterial endospores. The reaction was carried out under non-pyrolytic conditions by inserting a small coiled wire filament coated with the sample and reagents directly inside a GC injection port at 290 °C. Above 10 : 1 methyl donor/DPA ratios, alkali metal salts of MeSO₄⁻ effected 80–90% conversion of DPA to Me₂DPA, which was 10–20 times more active than the same amount of tetramethylammonium hydroxide (TMA-OH) at this temperature. A quaternary salt mixture consisting of 1 : 3 : 1 : 3 TMA⁺/Na⁺/OH⁻/MeSO₄⁻ methylated spore DPA with an average conversion of 86% (mean conversion by TMA-OH under the same conditions was 4%). Therefore, the sensitivity for detection of bacterial endospores was increased over 20-fold compared to that observed with the more commonly employed TMA-OH methylating reagent. The limit of detection by this method was 9 × 10⁴ total spores. Mechanisms describing the observed behavior are proposed and discussed. This is the first use of MeSO₄⁻ as a TCM reagent for GC.