A rapid, reliable high-performance liquid chromatographic micromethod for the measurement of cyclosporine in whole blood

The initiation of a liver transplant program in a pediatric hospital prompted the development of a high-performance liquid chromatography method for monitoring cyclosporine, which requires minimal amounts of blood and produces fast, reliable results. A protein-free filtrate is prepared by mixing 250 microliters of whole blood with 750 microliters of organic solvent (acetonitrile and methanol in the ratio of 9:1) containing the internal standard cyclosporine D. After centrifugation the supernatant is subjected to a quick clean-up using two types of disposable cartridges (C18 and silica Bond Elut), and the eluate is dried, reconstituted in equal parts of acetonitrile and water, and subjected to a final heptane wash to remove late eluting peaks. Chromatography is performed at 75 degrees C using a supelcosil C18 column (15 cm X 4.6 mm) with 3 microns packing and a mobile phase of 77/23 of acetonitrile/phosphate buffer at pH 2.5. Flow rate is 0.6 ml/min, providing a chromatography time of 10 min. Absorbance is measured at 214 nm at a sensitivity setting of 0.01 absorbance units full scale. Recovery for cyclosporine A is between 92 and 104%, and the lower level of sensitivity is 15 micrograms/L. Between-day precision provided coefficient of variation values less than 5% for a control serum of 150 micrograms/L. The method is linear to 3,000 micrograms/L. No interfering substances have been found. The method has proved to be consistent, reliable, and simple enough to be performed by all staff members. Column life for a system used daily is at least 3 months.