High-Performance Liquid Chromatography Identification, Quantification, and Fractionation of a Suspect Allergen, 4-Chloro-3-methylphenol, in an LLNA-Positive Metalworking Fluid

Abstract

A metalworking fluid (MWF) was obtained that produced an allergic reaction in the local lymph node assay (LLNA) with an EC3 = 4%, the EC3 being the estimated concentration needed to provoke a 3-fold allergy response. High-performance liquid chromatography (HPLC) was used to separate, identify, and isolate the suspected allergen. The biocide, 4-chloro-3-methylphenol, was detected in the MWF as a chromatographic peak matching the retention time of an external standard. The technique of standard addition was used to quantify and confirm the presence of 4-chloro-3-methylphenol at about 1% (w/w). Preparative HPLC was used to fractionate 1 gram of MWF separating the 4-chloro-3-methylphenol fraction from the remaining MWF. The two mobile-phase solutions were concentrated back into an MWF and a 4-chloro-3-methylphenol fraction. The original MWF and the reconstituted 4-chloro-3-methylphenol and MWF fractions were also analyzed by gas chromatography–mass spectrometry to confirm the isolation of the biocide.