Improvement of urinary delta-aminolevulinic acid determination by HPLC and fluorescence detection using condensing reaction with acetylacetone and formaldehyde.

Abstract

We improved the method for determining urinary delta-aminolevulinic acid (ALA) by HPLC-fluorometer after pre-column derivatization with acetylacetone and formaldehyde, and a stable ALA derivative was obtained without any effect from various urinary components as demonstrated by the complete recovery of ALA (100.9 +/- 5.5%, n = 85) from the urine samples. The modified procedure was as follows: Twenty microliters of urine sample, 5 ml of acetylacetone solution (acetylacetone/ethanol/distilled water containing 4 milligrams of NaCl; 15/10/75), and 0.45 ml of 9.3% formaldehyde solution were mixed and boiled for 15 min. The fluorescent derivative of ALA was separated and analyzed by HPLC with the fluorometer at Ex 246 nm and Em 458 nm. Using a gradient program, the retention time of the ALA derivative was 7.3 min and the analysis could be repeated at 13 min intervals. Concentrations of ALA in urine samples measured by this method were significantly correlated with those measured by the Mauzerall-Granick (M-G) method (n = 85, r = 0.993, p < 0.001). The values obtained by our method were, however, lower than those obtained by the M-G method. Urinary ALA concentrations of 40 non-lead workers ranged from 0.1 to 2.3 mg/g creatinine with the mean +/- SD of 1.1 +/- 0.4 mg/g creatinine as measured by the present method.