Structural identification of three metabolites and a degradation product of the macrolide immunosuppressant sirolimus (rapamycin) by electrospray-MS/MS after incubation with human liver microsomes.

Sirolimus is a macrolide immunosuppressant that is metabolized by cytochrome P450 3A enzymes to several demethylated and/or hydroxylated metabolites, the exact structures of which have not yet been identified. In addition, sirolimus undergoes degradation in organic solvents and biological fluids. The fragmentation pattern of sirolimus after collision activated dissociation was identified. We used electrospray/MS/MS in combination with collision activated dissociation to elucidate the structures of several sirolimus metabolites and that of a degradation product after incubation of sirolimus with human liver microsomes. The following metabolites were identified: 39-O-demethyl sirolimus, 16-O-demethyl sirolimus, 12-hydroxy sirolimus, as well as the structure of the degradation product 34-hydroxy sirolimus. After incubation with human liver microsomes, 69.7% of the sirolimus derivatives detected were sirolimus, 9.3% 39-O-demethyl sirolimus, 9.3% 34-hydroxy sirolimus, 4.6% 12-hydroxy sirolimus and other hydroxylated metabolites, 2.2% 16-O-demethyl sirolimus, 3% dihydroxylated metabolites (m/z of [M + Na]+ = 968.5), 1.2% trihydroxylated metabolites (m/z of [M + Na]+ = 984.5), and 0.9% tetrahydroxylated metabolites (m/z of [M +Na]+ = 1000.5). Analysis of the fragments of the di-, tri-, and tetrahydroxylated metabolites showed that the hydroxylated sites were located between C(10) and C(27). The intensities of additional fragments was not sufficient to completely identify their structures.