Determination of 5,10,15,20-tetra-(m-hydroxyphenyl)chlorin in tissues by high performance liquid chromatography

A procedure for the extraction and high performance liquid chromatographic (HPLC) determination of the photodynamic therapeutic agent 5,10,15,20-tetra(m-hydroxyphenyl)chlorin in human, rat and mouse tissues following intravenous administration of the drug is described. The tissue (tumour, skin, muscle and liver) was homogenized and extracted into a mixture of methanol:dimethyl sulphoxide:water (32:8:1 by vol.) containing, 5,10,15,20-tetra(p-hydroxyphenyl)chlorin as the internal standard. The precipitated proteins were removed by centrifugation and the supernatant was separated by reversed phase HPLC on a Hypersil-ODS column with 77% (v/v) acetonitrile in 0.1% trifluoroacetic acid as the mobile phase. The solute was detected with high sensitivity and specificity by a UV-VIS detector set at 423 nm.