A Rapid, Reliable High-Performance Liquid Chromatographic Micromethod for the Measurement of Cyclosporine in Whole Blood

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Abstract
Summary: 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 mUl of whole blood with 750 mUl 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 75dGC using a supelcosil C18 column (15 cm x 4.6 mm) with 3 mUm 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 mUg/L. Between-day precision provided coefficient of variation values <5% for a control serum of 150 mUg/L. The method is linear to 3,000 mUg/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.

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