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A second-derivative scan of an acidified urine sample allows the amplitude of deflection (delta A) and the minimum wavelength of the trough (lambda min) to determine the correct porphyrin concentration and the coproporphyrin:uroporphyrin (copro:uro) ratio, respectively, from a nomogram constructed from calibrator solutions. We measured 24 urine samples for total porphyrin as coproporphyrin equivalents and adjusted the results with factors from the nomogram. The adjusted results (x) (mean +/- SE, 501 +/- 57 nmol/L) compared favorably with the expected results (y) (514 +/- 57). The regression equation and correlation coefficient were: y = 0.993x - 8.9 (r = 0.998, S(y/x) = 16.2). Results of the copro:uro ratio derived by second-derivative spectroscopy and HPLC showed no significant difference (chi 2-test) from samples with various copro:uro ratios. Recovery studies on four urine samples supplemented with known proportions of coproporphyrins and uroporphyrins gave good agreement between the measured and the expected porphyrin ratios. The overall imprecision (CV) of the assay ranged from 3.6% to 6.0% for coproporphyrin and from 3.2% to 9.1% for uroporphyrin.