尿中2,5-ヘキサンジオン(HD)のバックグランド値および作業者尿中HD濃度におよぼす加水分解条件の影響

In order to determine the optimal conditions of acid hydrolysis for urinary 2, 5-hexanedione (HD) measurement, the effects of urine pH or volume of HCl added to urine and hydrolysis period were studied using gas chromatography-mass spectrometry (GC-MS). When 0.3 ml of concentrated HCl was added to 3 ml of urine, complete hydrolysis to liberate HD was attained 2 h after the start of heating at 100°C for urine from non-exposed subjects. On the contrary, in urine from exposed subjects most of HD was liberated in 30 min at 100°C, suggesting that the urinary substrates converted to HD by acid hydrolysis were different between exposed and non-exposed subjects. It was confirmed that hydrolysis for 2 h at 100°C with 0.3 ml of concentrated HCl added to 3 ml of urine gave the most reliable levels and the greatest amounts of HD in both urine from exposed and nonexposed subjects.Reference values were determined by the hydrolysis conditions presented here. In 84 males, 52 females, and 136 with sexes conbined the urinary HD levels were 0.35, 0.49, and 0.39 mg/l (geometric mean), respectively. Urinary HD levels determined without hydrolysis (free HD) were less than 0.006 mg/l in 31 control subjects examined. In urine from exposed subjects the amount of free HD was about one-fifth of the total HD (free plus conjugated HD) as determined with acid hydrolysates, although the percentage of free to total HD varied from 0.4 to 28%.In both urine from exposed and non-exposed subjects, total HD levels determined by GC-FID (flame ionization detector) were in good agreement with those by GC-MS (selected ion monitoring mode), indicating both can be useful for monitoring n-hexane exposure. Two normal urine metabolites, 2-acetylfuran and dimetylthiofene, were found close to HD peak in chromatograms, and were liable to interfere with the HD determination in GC-FID. To avoid the interference, use of non-polar long (50 m) capillary column is recommended for complete separation of HD from the interfering urinary constituents.