No abstract

Metabolism data provided with reduced cycle time has become of increasing importance for the early evaluation of DMPK properties of drugs in discovery. In this regard, quadrupole time-of-flight hybrid mass spectrometers (Q-TOF) can provide very reliable metabolite identification via accurate mass measurement of ions and the consequent access to the elemental composition of the metabolite. However, due to their cost, they are often used for drug metabolism studies on later stage drug candidates or to address challenging metabolism questions. A new prototype, consisting of a five-channel multiplexed electrospray ionization (ESI) source on a Q-TOF with one channel used for lock-mass compound infusion, was evaluated for metabolite identification. The goal was to increase the sample throughput of a single ESI-MS system by a factor of 4, while maintaining efficient metabolite separation in high-performance liquid chromatography (HPLC) as well as adequate sensitivity and mass accuracy, and ultimately improve the speed and quality of metabolism studies supporting drug discovery. The analytical performance of the system was assessed by evaluating the sensitivity and mass accuracy (using real in vitro and in vivo samples), inter-channel differences in retention times, MS/UV response, and cross-talk among channels. The sensitivity using the multiplexed ESI source was on average 2-fold lower than with single ESI, correlating well with previous literature data. The mass accuracy was comparable to that obtained using single ESI in both MS and MS/MS modes, making the metabolite identification process using the multiplexed ESI source as reliable as with single ESI. Compound-dependent differences in ionization efficiencies were observed among channels, and were minimized by analyzing related samples on the same channel. Finally, the level of cross-talk among channels was acceptable (around 0.3%) and comparable to levels previously published for quantitative applications using multiplexed ESI. The paper also focuses on the advantages and disadvantages of this new approach compared to other approaches in the literature in the field of metabolite identification.

1. The glucuronidation of diflunisal to its phenolic (DPG) and acyl glucuronide (DAG) was measured in vitro using microsomes prepared from rat (n = 4) and human (n = 6) liver and kidney tissue. UGT activities towards bilirubin, 4-nitrophenol and (-)-morphine were also determined. 2. beta-Glucuronidase activity towards phenolphthalein glucuronide was much lower in microsomes prepared from human liver (45.2 +/- 3.1 Fishman Units/mg protein), human kidney (22.0 +/- 3.3 FU/mg), and rat kidney (25.1 +/- 2.5 FU/mg) as compared with rat liver (118.7 +/- 8.8 FU/mg). 3. The formation rate of DAG significantly increased when saccharo-1,4-lactone, a beta-glucuronidase inhibitor, was added to the rat liver microsomal incubation medium. beta-Glucuronidase inhibition, however, had little effect on the formation rate of DAG in human liver microsomes, and no effect in rat and human kidney microsomes. The formation of DPG was not affected by the microsomal beta-glucuronidase activity. 4. Unlike rat kidney microsomes, which only formed DAG, human kidney microsomes formed both diflunisal glucuronides. Formation of both diflunisal glucuronides in human kidney microsomes (Vmax = 0.97 +/- 0.21 and 0.27 +/- 0.07 nmol/min/mg for formation of DAG and DPG respectively) represented 60-70% of the activity found in liver microsomes (Vmax = 1.58 +/- 0.32 and 0.40 +/- 0.08 nmol/min/mg for formation of DAG and DPG respectively). 5. These results demonstrate that the in vitro glucuronidation rate of diflunisal may be affected by the microsomal beta-glucuronidase activity particularly when using rat liver microsomes. Our results also demonstrate that the human kidney has an important UGT-activity towards diflunisal.

In vitro glucuronidation was studied in liver microsomes from two patients with Crigler–Najjar type I (CN‐I) disease and compared with the activity measured in microsomes prepared from six control human livers. The UDP‐glucuronosyltransferase (UGT) activity was determined toward the following substrates: 4‐nitrophenol, propofol, (−)‐morphine (formation of the 3‐glucuronide), and diflunisal (formation of the phenolic and acyl glucuronides). Glucuronidation of 4‐nitrophenol was reduced in one of the CN‐I livers (CN‐I No. 1) (0·9 nmol min−1 mg−1) and normal in the other CN‐I liver (CN‐I No.  2) (3.5 nmol min−1 mg−1) compared to the control livers (5·6±2·9 nmol min−1 mg−1, mean±S.D.). Propofol glucuronidation was not detectable (i.e. less than 0·100 nmol min−1 mg−1) in the CN‐I No. 1 liver and normal in the CN‐I No. 2 liver (1·78 nmol min−1 mg−1 against 1·52±0·72 nmol min−1 mg−1 in the control livers). The glucuronidation of (−)‐morphine to the 3‐glucuronide and the formation of the phenolic and acyl glucuronides of diflunisal were normal in both CN‐I livers compared to the control livers. Our results show that CN‐I patients are heterogeneous regarding UGT activity toward the phenolic substances 4‐nitrophenol and propofol.