HPLC analysis of 4-hydroxytamoxifen glucuronides formed by human liver microsomes. Human liver microsomes (HLM; 10 μg protein) were incubated at 37°C for 30 min with 25 μM trans-4-hydroxytamoxifen (trans-4-OH-TAM) or 15 μM cis-4-OH-TAM and 4 mM UDP-glucuronic acid prior to analysis by HPLC as described in Materials and methods. Propranolol was added to all assays postincubation as an internal standard for HPLC. (a) No substrate, internal standard (propranolol) only; (b) nonincubated 70:30 mixture of trans-4-OH-TAM:cis-4-OH-TAM (Sigma); (c) nonincubated 98% pure trans-4-OH-TAM (Sigma); (d) nonincubated cis-4-OH-TAM purified from the 70:30 mixture shown in (b); (e) trans-4-OH-TAM incubated with HLM; (f) cis-4-OH-TAM incubated with HLM; (g) trans-4-OH-TAM incubated with HLM and β-glucuronidase; (h) cis-4-OH-TAM incubated with HLM and β-glucuronidase; (i) trans-4-OH-TAM incubated with HLM and 1 N NaOH; (j) cis-4-OH-TAM incubated with HLM and 1 N NaOH. Peak 1, propranolol; peak 2, cis-4-OH-TAM; peak 3, trans-4-OH-TAM; peak 4, trans-TAM-4-O-glucuronide (predicted); peak 5, trans-4-OH-TAM-N
+-glucuronide (predicted); peak 6, cis-TAM-4-O-glucuronide (predicted); peak 7, cis-4-OH-TAM-N
+-glucuronide (predicted). mAU, milli-absorbance units.