Erlandsen E.J., Markussen S., Brock A.
Dept of Clinical Biochemistry, Viborg Hospital, Viborg, Denmark
A highly sensitive and specific method for the measurement of the I and III isomers of porphyrins in plasma by a one-step liquid-liquid extraction followed by HPLC separation with fluorometric detection has been developed and validated. The analytical methodology has been validated in terms of specificity, extraction recovery, linearity, limit of quantitation, stability, between-run and within-run imprecision. Stock solutions of the I and III isomeres of uroporphyrin, hepta-carboxylporhyrin, hexacarboxylpor-phyrin, pentacarboxylporphyrin and coproporphyrin (Frontier Scientific Inc. Logan Utha, USA) were prepared by dissolving each compound in 3 mol/L HCl. To prepare calibrators the I and III isomere porphyrins were spiked in donor plasma from the local blood bank in concentrations from 1 to 1000 nmol/L. Quality controls of porphyrins were prepared by spiking the porphyrin acids chromatographic markers containing the I isomers of porphyrins (Frontier Scientific Inc. Logan Utha, USA) in plasma from the local blood bank in concentration of 10, 50 and 100 nmol/L. To 500 µL sample (calibrator, control or patient plasma) 250 µL dimethylsulfoxide and 250 µL 20% trichloroacetic acid were added in a glass tube. The mixture was vortexed for 1 min and then centrifuged at 3000 x g for 10 min. The clear supernatant was transferred to a brown vial. The Agilent 1100 series HPLC system (Agilent Technologies, Palo Alto, Ca, USA) consisted of a binary pump, a degasser, an autosampler operated at 5 °C, a column oven and a fluorescence detector connected to the Agilent ChemStation Software. Chromatographic separation of porphyrins was achieved by using a reversedphase LiChroCART® RP-18 (7µm) (250 x 4.0 mm) column connected to a LiChrospher® 100 RP-18 (5 µm) guard column (E. Merck, Darmstadt, Germany) operated at 30 °C. The mobile phase consisted of A: 1 mol/L ammonium acetate pH 5.16 and acetonitrile 90:10 (v/v) and B: methanol and acetonitrile 90:10 (v/v). Gradient elutions were performed using a flow rate of 1 mL/min. The initial concentrations were 90% A and 10 % B. During the following 15 min the concentrations were linearly changed to 1 % A and 99 % B. Porphyrins were detected with excitation and emission wavelengths set at 405 and 619 nm, respectively. The sample volume injected was 100 µL. Under these conditions complete separation was obtained for the I and III isomers of uroporphyrins, heptacarboxylporhyrins, hexacarboxylporphyrins, pentacarboxylporphyrins and coproporphyrins. Retention time and fluorescence emission spectra were used for the identification of each porphyrin isomer. The extraction recovery was 90-95%. The assay was linear from 0.5 - 500 nmol/L and the detection limit was < 0.2 nmol/L. Extracted porphyrins were stable for at least one week at 2-8°C. Within run and between run imprecision was < 4 % CV. In conclusion, the method developed and validated in this study is a sensitive, specific, fast and cheap method to measure the I and III isomers of porphyrins in plasma.