A GC–MS method for the quantification of bacterial lipopolysaccharides (LPS, endotoxin) is presented. After hydrolytic cleavage of 3-hydroxy fatty acids (3-OH FAs) from the lipid A region of LPS, derivatisation of both the hydroxyl and the carboxyl group was performed in one step with a mixture of methyl-bis(trifluoracetamide) (MBTFA) and N-methyl-N-(tert-butyldimethylsilyl)trifluoracetamide (MTBSTFA). Using GC–MS in the EI mode with selected ion monitoring (SIM) for analysis, baseline separation of 3-OH FAs (and of possibly interfering 2-OH FAs) was achieved. The sensitivity of the method (LOD 7–50 pg/injection for the different 3-OH FAs investigated) allows for the efficient quantification of LPS in occupational and environmental samples. Degradation of 3-OH FAs as well as of their derivatives during sample preparation and GC–MS separation as a possible source of errors in analytical methods based on 3-OH FA determination is reported for the first time. Thermal elimination of water from the underivatised 3-OH FAs and of trifluoroacetic acid from the derivatives was identified as the cause of degradation. The resulting α,β-unsaturated compounds showing the same mass spectra as the 3-OH FA derivatives were detected as more or less prominent satellite peaks. By using alkaline instead of acidic hydrolysis and cool on-column instead of split/splitless injection, elimination was reduced to an acceptable level.