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2. Column temperature: 30°C
3. Mobile phase A:
10 mM NH
4
OAc and 0.1% FA in H
2
O
4. Mobile phase B:
10 mM NH
4
OAc and 0.1% FA in ACN-MeOH (50:50,
v/v
)
5. Flow rate:
0.3 mL/min
6. Elution gradient
see Table
2015.12B
7. Injection volume:
3 µL
8. Autosampler temp.:
15°C
9. Run time:
25 min
(b)
MS Data Acquisition and Operating Conditions.
—MS data acquisition is performed in full MS-
data dependent product ion scan (dd-MS
2
) and all ion fragmentation (AIF) modes using
parameter settings provided below. Data dependent product ion scan experiment is initiated if a
mass (
m/z
) specified in an inclusion list (see Table
2015.12B
) is detected in correct retention
time window within a mass error of 10 ppm and at the intensity above the set threshold level.
The ion fragmentation in AIF and dd-MS
2
modes is performed at three discrete normalized
collision energy (NCE) values.
1. Ionization mode:
positive ESI
2. Sheath gas flow:
35 arb
3. Aux gas flow:
10 arb
4. Sweep gas flow:
1 arb
5. Spray voltage:
3.5 kV
6. Capillary temperature:
350°C
7. S-lens RF level:
50 V
8. Aux gas heater temp.:
350°C
9. Full MS resolution:
70,000 FWHM
10. Full MS AGC target:
1e6
11. Full MS max IT:
100 ms
12. Full MS scan range:
m/z
200-1100
13. dd-MS2 resolution:
17,500 FWHM
14. dd-MS2 AGC target:
1e5
15. dd-MS2 Isolation window:
1.0 Da
16. dd-MS2 stepped NCE:
40, 70, 100%
17. Intensity threshold:
2.0e4
18. AIF resolution:
70,000 FWHM
19. AIF AGC target:
1e6
20. AIF max IT:
100 ms
21. AIF stepped NCE:
40, 70, 100%
22. AIF scan range:
m/z
50-750
(c)
Inclusion list .—See Table
2015.12C
.
(d)
Positive and Negative Control.
—Analyze a reagent blank (a
negative control
) with each sample
set. Inject the QC Solvent Standard (a
positive control
) at the beginning, after every ten samples
and at the end of the LC-HRMS sequence. The ISTD response in samples should be within 40-
140% of its average response in the QC Solvent Standards.
H.
Data Processing
Candidates for 2016 Method of the Year
140