Improved procedures for the determination of lipid phosphorus by malachite green

We have developed two procedures for the measurement of lipid phosphorus based on interaction between phosphomolybdenum and malachite green. One method, the "micro" assay uses 50-200 microliters of HClO4 and has a sensitivity range of 0.01-1.5 micrograms phosphorus. The second method, the "macro" assay, has a sensitivity range of 0.03-5.0 micrograms phosphorus with 100-500 microliters HClO4. Both assays are very reproducible with day to day standard deviations of less than 6% between triplicates irrespective of the HClO4 content used. At different concentrations of HClO4, each method was successfully used to determine the phosphorus content in phosphatidylethanolamine and sphingomyelin standards that covered the proposed sensitivity ranges. The increased range, sensitivity and greater volumes of HClO4 permitted in the procedures represent significant improvements over existing methods.