Page 210 - Tandem Techniques

Page 193

The urine sample was taken between 6.5 and 8.5 hours after exposure and the blood serum samples 8.5
hours after exposure. The determined 4,4'methylenebisaniline and the deuterated internal standard were
monitored at m/z= 470 (MDA) and m/z=472 (MDDA) respectively, and each chromatogram is shown
separately. The urine and plasma samples were found to contain 19 and 4 nmol of 4,4'-
methylenebisaniline per liter respectively. It is seen that the technique is extremely sensitive with a high
degree of specificity.

Wu et al.[19] developed yet another method for measuring 4,4'-methylenebis(2-chloroaniline) in urine
using a thin layer chromatography stage in the preparation of the sample. A 20 ml sample of urine was
heated to 80°C for 90 minutes and, after cooling, 10 ml of a mixture of diethyl ether-n-hexane (50+50)
and 0.4 ml of 10 M caustic soda was added. The tube was shaken for 2 minutes and 0.5 ml of methanol
was added dropwise across the surface to break up the foam. The mixture was then centrifuged for 10
minutes. The organic layer was removed and the extraction procedure repeated with fresh solvent. The
combined extracts were evaporated to dryness in a stream of nitrogen. The residue was taken up in 2 ml
of the same solvent mixture and 50 µl of pentafluoropropionic anhydride added. The sample was well
mixed and heated to 60°C for 20 minutes. 2 ml of hexane and 4 ml of 0.1 M KH PO buffer (pH=6)
4
2
was added and the mixture well shaken. The mixture was separated by centrifugation and the organic
layer collected. The solvent was removed and the residue taken up 0.5 ml of n-hexane. 0.4 ml of the
solution was progressively loaded onto a thin layer plate and the spot dried. The plate was developed
with an ether n-hexane mixture (25+75). The area associated with the derivatized 4,4'-methylene-bis(2-
chloroaniline) was scraped off and placed in a glass column and washed with n-hexane and the
washings discarded. The sample was then removed with a ether-n hexane mixture (50+50), evaporated
to dryness taken up in 1 ml of ethyl acetate containing 0.3 µg of the standard.

The tandem instrument was similar to that used previously. It consisted of a Hewlett-Packard 5890 gas
chromatograph fitted with a 25 m column 320 µm I.D. carrying a film of stationary phase 0.25 µm
thick, The gas