Determination of amines as pentafluoropropionic acid anhydride derivatives in biological samples using liquid chromatography and tandem mass spectrometry

Abstract

Determination of amines in biological samples as markers of exposure to the amines or the corresponding isocyanates is an important tool for industrial exposure assessment. In this study, a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for determination of amines in biological samples as perfluorofatty amides derivatives is presented. The method enables determination of diamines such as methylene diamine (MDA), toluene diamine (TDA), naphthalene diamine (NDA), hexamethylene diamine (HDA), isophorone diamine (IPDA), methylenedi(cyclohexylamine) (HMDA) and 4,4′-methylene-(2-chloroaniline) (MOCA) in human urine and plasma. The work-up procedure included hydrolysis of the biological samples with 3 M H₂SO₄ at 100 °C for 16 h and extraction of the amines into toluene, where derivatisation of the amines with perfluorofatty acid anhydride was performed. Following removal of excess reagent and the acid formed and an exchange of solvent, the derivatives were analysed using gradient elution with an acetonitrile/water mobile phase composition and electrospray ionisation (ESI) with multiple reaction monitoring (MRM) of [M − H]⁻ → [M − H − 120]⁻ or [119]⁻. Several perfluorofatty acid anhydrides were evaluated as derivatisation reagents, but the LC chromatographic properties of the pentafluoropropionic acid anhydride (PFPA) derivatives were favourable. Quantification of amine–PFPA derivatives was performed using deuterium labelled amine–PFPA derivatives as internals standards with good precision and linearity in the investigated range of 0–20 ng ml⁻¹ urine. The instrumental detection limits for the amine–PFPA derivatives were 0.2–3 fmol for MRM of [M − H]⁻ → [119]⁻ and 0.3–8 fmol for [M − H]⁻ → [M − H − 120]⁻. In 10 urine and 6 plasma samples from workers exposed to isocyanates, determination of TDA and MDA as PFPA derivatives was performed using LC-MS/MS and a reference GC-MS method. No significant difference between the two methods was observed.