Gas chromatographic mass spectrometric identification and quantification of aniline after extraction from serum and derivatization with 4-carbethoxyhexafluorobutyryl chloride, a new derivative

Aniline, widely used as an intermediate in the synthesis of dye, is also us ed in the manufacture of pharmaceuticals, photographic developers, shoe pol ish, and other common substances. Exposure to aniline is toxic because it p roduces methemoglobin. Aniline levels are usually not measured in serum; in humans. blood methemoglobin levels are often measured as an index of expos ure to aniline. In this article, we describe a method for the identificatio n and the quantification of aniline by gas chromatography/mass spectrometry (GC/MS) after its extraction from human serum and derivatization with 4-ca rbethoxyhexafluorobutyryl chloride. Aniline, as well as the internal standa rd N-methyl aniline, was extracted from alkaline serum using chloroform. An iline and the internal standard were derivatized with 50 mu L of 1-carbetho xyhexafluorobutyl chloride. After evaporating the excess derivatizing reage nt, the residue was reconstituted in 50 mu L of ethyl acetate and injected into the GC/MS. A positive identification of derivatized aniline can be mad e from the strong molecular ion at m/z 343. Similarly, derivatized internal standard showed a strong molecular ion at m/z 357. The within-run and betw een-run precisions of the assay were 3.8% and 5.8%, respectively, at an ani line concentration of 5 mg/L. The assay was linear for serum aniline concen trations of 0.5 mg/L, to 25.0 mg/L. The detection limit was 0.1 mg/L. The a ssay was not affected by lipemia, hemolysis, or high bilirubin concentratio n in serum, and the assay was applicable to whole blood. We also fed mice ( C57bl/6) with various concentrations of aniline and measured methemoglobin and blood concentrations of aniline. The methemoglobin percentage and anili ne concentrations in blood increased with increasing aniline doses.