mobile phase comprising 20 vol methanol and 80 vol acetonitrile the reconstituted eluates were then injected to an lc ms ms for analysis schimazdu hplc connected to an ab sciex api 3000 mass spectrometer the removal rate of the phospholipids is shown in table 4 hereinbelow phosphohpid removal expressed as a percent is determined by dividing the peak area of the phospholipids in the cartridge by the peak area of the phospholipids by protein precipitation thus the results of example 3 showed the cartridge c of the present invention having the combination of the strong cation exchanged silica in a first sorbent zone and the acidified alumina disposed in a second sorbent zone in a single well provided a significantly larger removal of phospholipids 90 wt than the individual components of the cartridge or the inverted arrangement shown as cartridge e the procedure of example 1 was repeated with variations in the ratio of the components of extraction reagent the main components of the extraction reagent of the present invention are methanol and acetonitrile in this example extraction reagent mixtures were evaluated for the volume ratio of the extraction reagent to the sample and the effect of the addition of acid to the extraction reagent was observed as in example 1 the extraction reagent was first loaded into the cartridges and then 0 1 ml of spiked serum sample was added to each cartridge table 5 shows the absolute recovery on a weight basis of 1 25 dihydroxy vitamin d3 for variations in reagent composition reagent to sample ratio and acid content the results of example 4 shown.