Determination of 4,4'-methylenedianiline in hydrolysed human urine using liquid chromatography with UV detection and peak identification by absorbance ratio.

A liquid chromatographic method using multi-wavelength UV detection (258 and 285 nm) is presented for the determination of 4,4'-methylenedianiline (MDA) in hydrolysed human urine. The method is based on hydrolysis under strongly acidic conditions followed by derivatization with pentafluoropropionic anhydride. The perfluoro fatty acid amide derivative formed was analysed on a bonded octadecylsilyl column using isocratic elution with acetonitrile-water (67:33, v/v) as mobile phase. The overall recovery for urine samples containing 115 micrograms/l of MDA was 97 +/- 3%. The calibration graph was linear in the investigated range (12-122 micrograms/l) with a correlation coefficient of 0.998. The precision was 2.3% for urine samples containing 122 micrograms/l and the detection limit was 8 micrograms/l. The chromatograms were evaluated using a combination of retention time data and absorbance ratio by the simultaneous monitoring of the wavelengths 285 and 258 nm. The absorbance ratio (285/258 nm) was virtually constant (0.28 +/- 0.04) in the range 78-10,000 micrograms/l. The precision for the absorbance ratio was 6.1% for urine samples containing 124 micrograms/l and the lowest amount of MDA to give an absorbance ratio was 50 micrograms/l. The procedure for the hydrolysis of urine spiked with MDA and N,N'-diacetyl-MDA and urine from skin-exposed workers was studied under strongly acidic, weakly acidic and basic conditions. MDA was found in hydrolysed urines from skin-exposed epoxy resin workers in the concentration range 8-700 micrograms/l.