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. Author manuscript; available in PMC: 2018 Dec 4.
Published in final edited form as: Pharmacogenet Genomics. 2012 Aug;22(8):561–576. doi: 10.1097/FPC.0b013e328354026b

Figure 2. UPLC and MS/MS analysis of CLZ and dmCLZ glucuronides formed by HLM and UGT-over-expressing cell lines.

Figure 2.

Glucuronidation assays were performed using 12.5 μg HLM protein, 250 μg UGT1A1- or 50 μg UGT1A4-over-expressing cell homogenates, and 160 μM CLZ or 320 μM dmCLZ, and incubated at 37°C for 2 h with 4 mM UDP-glucuronic acid prior to analysis by UPLC as described in the Materials and Methods. Panel A, HLM + CLZ; panel B, HLM + CLZ after treatment with 1,000 U of β-glucuronidase for 24h followed by treatment with 3 M HCl for 24 h; panel C, UGT1A1 + CLZ; panel D, UGT1A4 + CLZ; panel E, mass spectra of UPLC peak 3 from panel A; panel F, mass spectra of peak 4 from panel A; Panel G, HLM + dmCLZ; panel H, HLM + dmCLZ after treatment with 3 M HCl for 24 h; panel I, UGT1A4 + dmCLZ; Panel J, mass spectra of UPLC peak 6 from panel G. Peak 1, UDP-glucuronic acid; peak 2, CLZ; peak 3, CLZ-5-N-glucuronide; peak 4, CLZ-N+-glucuronide; peak 5, dmCLZ; peak 6, dmCLZ-5-N-glucuronide. AU, absorbance (arbitrary units); x-axis = retention time (min).