Pitfalls and solutions in the development of a fully automated solid-phase extraction method for drug screening purposes in plasma and whole blood.

Abstract

A fully automated solid-phase extraction (SPE) method for drug screening is described. The extraction of 19 toxicologically relevant drugs from pretreated plasma and pretreated whole blood was accomplished automatically by a Gilson ASPEC system equipped with disposable 2.8-mL Bond Elut Certify columns. The automated extraction procedure includes 11 fundamental steps: column preconditioning; sample application; column washing; pH adjustment; elution of drugs by two eluents, which were collected into two separated tubes; addition of the chromatographic standard solution; and several SPE column rack movement steps. After evaporation, the drugs were quantitated by gas chromatography. Water was chosen as the transfer liquid in the ASPEC system because it was cheap and, more importantly, because it caused no protein precipitation problem. The effects of the sample and eluent flow rates were investigated, and it was found that low flow rates were necessary to recover the drugs maximally. In the study, the optimal flow rates of sample application, acetone-chloroform elution, and ammoniated ethyl acetate elution were 1.5, 0.72, and 0.33 mL/min, respectively. The absolute recoveries of 19 drugs from whole blood exceeded 82%, with relative standard deviations less than 5% at 2 micrograms/mL.