QUANTITATION OF PLASMA PORPHYRINS:VALIDATION OF LIQUID-LIQUID EXTRACTION METHOD

Erland J. Erlandsen, Peer R Mortensen, Bente Warshawsky, Axel Brock

Department of Clinical Biochemistry, Viborg Hospital, Denmark

We have developed a sensitive and specific method for the measurement of porphyrins in plasma by a one-step liquid-liquid extraction followed by HPLC separation with fluorometric detection. The method has been validated in terms of extraction recovery, linearity, detection limit, analytical impression and sample stability.

Stock solutions of the I or III isomers of uroporphyrin, heptacarboxylporhyrin, hexacarboxyl-porphyrin, pentacarboxylporphyrin and coproporphyrin (Frontier Scientific Inc. Logan Utah, USA) were prepared by dissolving each compound in concentrated HCl and then diluted by water to a final concentration of 1 mol/L HCl. To prepare calibrators the I or III isomer porphyrins were spiked in plasma in concentrations from 0 to 500 nmol/L and pH was adjusted to 7.40.

Porphyrins were extracted by adding 250 μL dimethylsulfoxide and 250 μL 15% thrichloroacetic acid to 500 μL sample (calibrator or patient sample) in a glass tube. The mixture was vortexed for 1 min and then centrifuged at 3000 g for 10 min.

The assay was linear from 0 - 500 nmol/L and the detection limit was 1.0 nmol/L.

Extraction recovery of porphyrins from plasma and albumin solutions (30-50 g/L) was 0.97 (uroporphyrins), 0.94 (heptacarboxylporhyrins), 0.87  (hexacarboxylporphyrins), 0.74 (pentacarboxylporphyrins) and 0.50 (coproporphyrins).

The analytical imprecission (CV) based on duplicate measurements was found to be <5%.

Plasma porphyrins were stable for 3 days at 20°C, 6 days at 4°C and at least one month at –20°C when stored in dark.