A novel assay method for the determination of atropine in biological fluids is presented. Atropine is extracted and subsequently hydrolyzed. The generated tropine is them derivatized to heptafluorobutyryl-tropine, which is measured by GLC-MS. Base peaks m/z 124 and m/z 127 are simultaneously monitored. Deuterated atropine serves as internal standard. This specific and sensitive assay permits a delineation of the disposition pharmacokinetics in humans after i.v. administration of the drug. Prepharmacokinetic studies determined pKa values for atropine and tropine of 9.56 and 10.35 respectively at 22 degrees C. Solvent partitioning experiments showed that atropine exerts a significantly larger lipophilicity than tropine. This was consistent with tropine (0%, 0.85) at 37 degrees C and pH 7.4. Plasma protein binding and erythrocyte partitioning of both compounds were concentration independent and invariable in the presence or absence of each other.