Influence of solute polarity in column-switching chromatography for the assay of drugs in plasma and urine.

The polarity of a drug is one of the most important parameters for the elaboration of switching systems. If the polarity of the drug is low or medium, "reversed-phase" chromatography is well adapted. The plasma or urine sample is diluted with water, centrifuged and injected first into a column of medium polarity (C2, CN or diol bonded phases). The compounds of interest are stopped on the top of the column and rinsed with water, then eluted and chromatographed on a C8 or C18 analytical column. A third column of still lower polarity can be added to improve the specificity of the system. In each successive step, the polarities of the mobile phases and columns should be decreased to reconcentrate the sample and reduce the band broadening that occurred in the previous step. Compounds of high polarity show almost no retention on reversed-phase columns, and normal-phase chromatography should be used. Aqueous solutions cannot be injected into polar bonded-phase columns as they lead to excessive band broadening. This problem can be solved by diluting plasma or urine with a large volume of a water-miscible organic solvent and injecting the clear supernatant. The compounds to be assayed are first reconcentrated on a polar column (NH2 or N(CH3)2 bonded phase) and then eluted. The selected "heart cut" of the eluate is chromatographed on another, more polar column. The influence of the polarity of drugs on the choice of switching systems is exemplified by assay methods for drugs of low, medium and high polarity.