HPLC

Stationary phase

Agilent, Eclipse XDB-C18, 4.6 mm × 50 mm, 1.8 μm

Injection volume, μL

3

Detector

DAD at 250 nm

Mobile phase

A: 10 mM NH₄Ac_(aq) 90%/MeOH 10%

B: 10 mM NH₄Ac_(aq) 20%/MeOH 80%

Degassed in ultrasonic bath for 20 min

Gradient program

0 - 3 min 50% B, isocratic

3 - 12 min up to 100% B, held for 5 min

17 - 23 min down to 5% B, held for 5 min to wash out salts from biomatrices

28 - 31 min up to 50% B, held for 4 min to re-condition column

Flow rate, mL・min⁻¹

0.5

Electrospray mass spectrometry

Temperature (˚C)

400

Detector polarity

Positive ion

Scanning mass range

100 - 1000

Resolution, Q1 and Q3

Unit

Nebulizer gas (p.s.i.)

40

Curtain gas (p.s.i.)

10

Collision gas (p.s.i.)

Medium or high for EMS and EPI scan modes^a, respectively

Ion spray voltage (V)

5000

Declustering potential (V)

40