HPLC and LC/MS procedures for the identification and/or quantification of TA-COOH and various P450 marker metabolites1-a
| Method | Compound | Mobile Phase | HPLC Column | Flow Rate | Detection Method | MS or MS/MS Ion(s) Monitored (m/z) |
|---|---|---|---|---|---|---|
| A | TA-COOH | 5% ACN in water containing 0.1% formic acid and increased to 70% ACN in 15 min | Waters Symmetry C8 (50 × 2.1 mm) | 300 (μl/min) | (+)ES1-b, MS/MS, SRM | 385 → 2231-c 387 → 223 |
| B | TA-COCH3 (methylated TA-COOH) | 5% ACN in H2O containing 0.1% formic acid and increased to 70% ACN in 15 min | Waters symmetry C8 (50 × 2.1 mm) | 300 (μl/min) | (+)ES, MS/MS, SRM | 399 → 2371-c 401 → 237 |
| C | DCMB-OH (P450 2B6) | 20% ACN in 50 mM NH4OAc (pH 4.8) for 1 min, increased to 80% ACN in 1 min and held for 6.5 min | Phenomenex Luna C8 (150 × 2.1 mm) | 300 (μl/min) | (+)ES, MS/MS, SRM | 397 → 2351-d |
| D | 1′-OH-triazolam (P450 3A4) | 5% ACN and 5% MeOH in 50 mM NH4OAc (pH 7.4), increased to 40% ACN and 13% methanol in 25 min | Supelco Discovery C18 (150 × 2.1 mm) | 250 (μl/min) | (+)ES, MS, SIM | 3591-e |
| E | 4′-OH-diclofenac (P450 2C9) | 15% ACN in 10 mM ammonium formate (pH = 3.0) for 1 min, increased to 80% ACN in 15 min | Phenomenex Prodigy ODS (150 × 3.2 mm) | 750 (μl/min) | UV: 280 nm | |
| F | 4′-OH-mephenytoin (P450 2C19) | A: H2O/MeOH (90:10) with 0.1% 1 M NH4OAc and 0.1% acetic acid; B: (10:90). 10% B in A for 0.5 min, increased to 90% B over 0.3 min and held for 6.8 min | Jones Genesis C8 (100 × 2.1 mm) | 300 (μl/min) | (−)ES1-f, MS/MS, SRM | 233 → 190 |
| G | Dextrorphan (P450 2D6) | A: H2O/MeOH (90:10) with 0.1% 1 M NH4OAc and 0.1% acetic acid; B: (10:90). 10% B in A for 0.5 min, increased to 90% B over 0.3 min and held for 6.8 min | Jones Genesis C8 (100 × 2.1 mm) | 300 (μl/min) | (+)ES, MS/MS, SRM | 258 → 157 |
| H | 6-OH-chlorzoxazone (P450 2E1) | 10% ACN in 10 mM NaHPO4 (pH = 2.6) increased to 20% ACN over 15 min. Increased to 30% ACN over 1 min and held for 5 min | Phenomenex Luna C8 (150 × 3.0 mm) | 700 (μl/min) | UV:295 nm |
↵1-a Incubation conditions for different enzyme assays are given in detail under Materials and Methods and Table 2.
↵1-b Positive ion electrospray.
↵1-c Characteristic fragments produced by the cleavage of the amide bond in TA-COOH or TA-COCH3(methylated TA-COOH) were monitored by SRM. Unambiguous detection of the metabolites was accomplished by monitoring the transition of the chlorine-containing precursor ions to the non-chlorinated product ion.
↵1-d SRM at unit resolution for the transition of precursor ion to the product ion.
↵1-e Single ion monitoring (SIM).
↵1-f Negative ion electrospray.