Determination of methotrexate in plasma by on-column concentrations and ion-exchange chromatography

A method for the concentration of dilute samples on a column and its application to the analysis of biomedical mixtures is described. Two columns are used, one for the concentration step and the other for the separation. The concentration column is used in the position of the loop in an injection valve. Concentration is effected by eluent switching. The influence of the chromatographic parameters such as capacity ratio and void volume on separation and detectability are described and the optimal choice of conditions is discussed. The method has been applied to the analysis of the cytostatic drug methotrexate. An octyl-modified silica was used as the stationary phase in the concentration column and a chemically bonded anion exchanger on silica in the separation column. The procedure shows good selectivity and precision. The detection limit corresponds to 2.10-8 M (9 ppb) in plasma, which is sufficiently low for therapeutic concentrations to be measured. An example of the use of the same stationary phase in both columns is given for the analysis of phenothiazines in blood, using alkyl-modified silica and electrochemical detection.