A rapid, new and direct method for isolation and measurement of porphyrins in biological samples by high performance liquid chromatography

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Abstract
Reversed‐phase high performance liquid chromatography on microparticulate columns of octadecylsilane with elution by a gradient of acetonitrile in phosphate buffer, pH 7.5, separated porphyrin free acids directly in urine for the first time. Interfering fluorescence was removed with a precolumn of large particle size at acid pH. Peak area fluorescence by the method of standard addition was related linearly to amount of a porphyrin from 5 to 60 nanograms. The method was reproducible to ± 13%, able to detect 0.3 nanograms of a porphyrin in a 1 ml urine sample in 45 minutes, and capable of detecting the typical clinical patterns of normal and porphyrinuric urines. Modifications make the method applicable to measurements in other biological samples and to research separations. With the added rapidity of analysis, the method could expand porphyrin analysis in routine clinical medicine.