THE DETECTION OF TACROLIMUS AND ITS METABOLITES IN WHOLE-BLOOD OF TRANSPLANT PATIENTS BY AN IMPROVED HPLC-ABBOTT TACROLIMUS-II IMMUNOASSAY

A method that combined high performance liquid chromatography (HPLC) a nd microparticle enzyme immunoassay (MEIA) was developed to simultaneo usly measure tacrolimus (T) and its immunoreactive metabolites (TMs) i n whole blood, This method substantially enhanced the analytical recov ery of bath T and TMs, The TMs that ware used as standards were genera ted by a troleandomycin induced microsomal enzyme system, Of the five TMs generated, three were identified as 13-O-desmethyl-T, 15-O-desmeth yl-T, and 31-O-desmethyl-T by mass spectrometry and nuclear magnetic r esonance spectroscopy. The other two TMs were identified as di-desmeth yl-T and hydroxylated-T. All five TMs were tested for cross-reactiviti es with the parent drug. Only 31-O-desmethyl-T and 15-O-desmethyl-T sh owed significant cross-reactivities while the other three TMs showed n o demonstrable cross-reactivities at the concentrations used. In order to detect 31-O-desmethyl-T and/or 15-O-desmethyl-T in whole blood of transplant patients, T and TMs were extracted from blood by solid phas e extraction, After separation of the blood extract by HPLC, the fract ion that contained 31-O-desmethyl-T, 15-O-desmethyl-T, or T was analyz ed by aw automated MEIA performed on the IMx analyzer (Abbott Laborato ries), Our results showed that both 31-O-desmethyl-T and 15-O-desmethy l-T were readily detectable in the blood of these patients. However, t hey were present at relatively low concentrations when compared to tha t of T in the majority of blood samples from hepatic and renal transpl ant patients.