Estimation of Cyclosporin-A in Whole Blood by Simple and Rapid Reversed-Phase HPLC Utilizing a Salting-Out Extraction Procedure

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Abstract
Cyclosporin-A is a fungal cyclic undecapeptide immunosuppressive agent that is potentially active against proliferating T-lymphocytes and therefore helps the body to accept a transplanted organ. In this experiment, the drug is extracted from whole blood with acetonitrile. The extraction solvent (acetonitrile) is mixed with the whole blood at a 1:2 ratio on a vortex mixer and is centrifuged at 2000 × g. The supernatant is transferred into a fresh borosilicate culture test tube and 20 mg of zinc sulfate and 10 mg of cadmium sulfate (for 1.0 mL of whole blood) are added, vortex mixed, and centrifuged at 2000 × g. The supernatant is saturated with anhydrous ammonium sulfate and centrifuged, and salted-out acetonitrile from the aqueous mixture is injected into the HPLC system. A 60- × 4.6-mm, 3-µm, slurry-packed ODS (C18) column is used with an isocratic elution of 66:2:32 (v/v), acetonitrile-isopropanol-water. The column temperature is maintained at 72°C. Cyclosporin-A is detected by UV absorption at 205 nm and 0.10 to 0.002 AUFS. The limit of detection of the method is 15 ng/mL for a 100-×L injection volume, and the completion time for analysis of one sample is less than 15 min. Columns packed with different stationary phases and different dimensions are investigated to determine which column will give the maximum resolution, sensitivity, and selectivity.