Analytical methods for quantification of glucuronide metabolites in microsome incubations
Substrate | Analytical Methods | Column | Gradient; Flow | TR | HPLC-MS or UV | Internal Standard | Mass Transition | Standard | Standard Concentration |
|---|---|---|---|---|---|---|---|---|---|
| ml/min | min | ||||||||
| Acetaminophen | LC/MS/MS (ESI-) | ODS-AQ 5 μ C18, 50 × 2.0 mm | 5-55% Ba in 1.5′, hold for 0.8′; 0.3 | 1.35; IS: 1.97 | Shimadzu; API3000 | Naphthyl glucuronide (1000 ng/ml) | 326→150 IS: 314→138 | Acetaminophen glucuronide | 1000, 800, 500, 250, 100, 50, 20, 10 ng/ml |
| AZT | LC/MS/MS (ESI-) | ODS-AQ 5 μ C18, 50 × 2.0 mm | 5-55% Ba in 1.5′, hold for 0.8′; 0.3 | 1.82; IS: 1.97 | Shimadzu; API3000 | Naphthyl glucuronide (1000 ng/ml) | 442→125 IS: 314→138 | AZT glucuronide | 1000, 800, 500, 250, 100, 50, 20, 10 ng/ml |
| Bilirubin | LC/UV (445 nm) | Sunfire 3 μ C18, 150 × 2.1 mm | 20-98% Bb in 18′, hold 98% B for 8′; 0.3 (50 μl injection) | Di-G/9.1, mon-G/12.3; IS: 6.0 | Alliance; Finnigan PDA | ACMA (2.812 μM) | N.A. | Bilirubin | 0.167, 0.333, 0.667, 1.667, 3.333, 13.333 μM; r2 = 0.997 |
| Estradiol | LC/MS/MS (ESI-) | Acquity UPLC BEH C18, 1.7 μ 2.1 × 50 mm | 5-30% B in 0.5′, to 40% in 0.5′, to 95% in 0.2′, hold for 0.3′; 0.6 | E3G/0.93, E17G/0.98; IS: 0.86 | ULC-Qtrap | Naphthyl glucuronide (5 μM) | 447→113 IS: 319→113 | Estradiol-3-glucuronide; estradiol-17-glucuronide | 10,000, 7500, 5000, 1000, 500, 100, 50, 10, 5, 1 nM |
| EE | LC/MS/MS (ESI-) | Atlantis 5 μ C18, 50 × 2.1 mm | 2′ at 20% B, 20-40% Bc in 2 min; 1.0 | 3-G/3.27, 17-G/3.61; IS: 2.84 | Shimadzu; Qtrap | Dasatinib (250 ng/ml) | 471→295 IS: 447→297 | EE 3-glucuronide; EE 17-glucuronide | 5000, 1000, 100, 10, 1 for 17-G; 1000, 100, 10, 1 ng/ml for 3-G |
| Lorazepan | LC/MS/MS (ESI-) | Atlantis 5 μ C18, 50 × 2.1 mm | 0.5′ at 10% Bc, 10-40% B in 2′, 40-80% in 0.5′; 1 | 2.43; IS: 2.97 | Shimadzu; Ultima | TFMU (250 ng/ml) | 497→283 IS: 229→201 | Lorazepan glucuronide | 1000, 500, 200, 100, 50, 10, 5 ng/ml |
| Muraglitazar | LC/MS/MS (ESI+) | Luna 3 μ C18, 150 × 4.6 mm | 35% Ac, 65% B; 0.6 | 6.2; IS: 12.24 | Alliance; Quantum | BMS-426707 (100 ng/ml) | 693→292 IS: 531→306 | Muraglitazar glucuronide | 2000, 1000, 500, 100, 50, 25, 10, 5 ng/ml |
| SN-38 | LC/MS/MS (ESI+) | ODS-AQ 5 μ C18, 50 × 2.0 mm | 0.5′ at 10% Bc, 10-95% B in 2.5′, hold for 1.5′; 0.3 | 2.42; IS: 2.70 | Shimadzu; Qtrap | D3-testosterone (100 ng/ml) | 569.1→393 IS: 308.2→272 | No | Multiple volumes injected to ensure the linearity of MS response |
IS, internal standard; HPLC, high-performance liquid chromatography; PDA, photodiode array; UPLC, ultra-performance liquid chromatography; N.A., not applicable; TFMU, trifluoromethyl umbelliferone; ACMA, 9-amino-6-chloro-2-methoxyacridine.
↵ a A: 0.1% TFA in water; B: 0.1% TFA in acetonitrile.
↵ b A: 0.05% TFA in water; B: 0.05% TFA in 1:1 (v/v) methanol:acetonitrile.
↵ c A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile.