Sample Preparation: Enzymatic deconjugation with H pomatia glucuronidase followed by protein precipitation with 95:5 Acetonitirile:Methanol

Chromatography
Column: Agilent Poroshell 120 EC-C18 (100 mm x 2.1 mm, 2.7 µm)
Column Temperature: 50 °C
Injection Volume: 2.5 µL
Mobile Phase: A: Ammonium formate (5 mM) and Formic Acid (12.6 mM) in H2O
B: Formic Acid (12.6mM) in acetonitrile
Flow rate: 0.5 mL/min
Elution Profile: Gradient- 95A: 5B initially; 70A:30B from 0.5 to 1.5 min; 30A:70B from 1.5 to 4.5 min; 0A:100B from 4.5 to 7.5min; 95A:5B from 10.0 to 14.0 min
Run Time: 12 min

Mass Spectrometry
Ion Source: Dual Jet Stream Electrospray Ionization
Polarity: Positive in one run; Negative in another run
TOF MS Scan Range: 75-700 Da
MS/MS Scan Range: 50-700 Da
Gas Temperature: 225 °C
Drying Gas Flow Rate: 14L/min
Sheath Gas Temperature: 350 °C
Sheath Gas Flow Rate: 11L/min
Nebulizer pressure: 14psi
Capillary Voltage: 3000 V (positive); -2500 V (negative)
Nozzle Voltage: 500 V (positive); -1500 V (negative)
Skimmer Voltage: 65 V
Octopole RF: 750 V
Fragmentor Voltage: 380 V
Internal Reference Masses: Purine at m/z 121.0509; HP-921 at m/z 922.0098 (positive); Purine at m/z 119.0360; HP-921 at m/z 966.0007 (negative)
Data Acquisition: 2GHz, extended dynamic range
Fragmentation: Auto MS/MS, three maximum precursors (threshold: 500 counts) per cycle with active exclusion after 1 spectrum at a 30s release time