Robotic automation of cyclosporine analysis in whole blood

Cyclosporin A (CsA) is currently the most selective immunosuppressant used clinically to prevent organ rejection after transplantation. We used a reprogrammable robot, the Benchmate (Zymark Inc., Hopkinton, MA), to automate much of the sample preparation involved in solid-phase extraction of CsA. Lysed whole-blood specimens containing previously added internal standard were placed on the Benchmate in the specimen-holding area, and a C18 Bond-Elut column was placed in the top of the sample tube. Specimen extraction from this point was handled automatically by the Benchmate, after which we manually injected the sample into the HPLC system for quantification. Analytical recovery of CsA in two concentrations of calibrator was similar for the manual and Benchmate methods. Analytical imprecision for the Benchmate was less than for manual extraction: within-run imprecision (CV) was less than 8% at either concentration. Between-run imprecision, determined by having the Benchmate extract CsA from two specimens each day for 20 days, was less than 9%. Patients' specimens were extracted manually (x) or by using the Benchmate robot (y); the results, compared by linear regression, agreed well: (y = 0.99 (SD 0.02)x + 2.6 (SD 2.8) micrograms/L (Sy[x = 7.59 micrograms/L, n = 27). We conclude that the Benchmate usefully reduces the manual steps necessary to extract specimens before HPLC analysis for CsA.