2. Use a mobile phase consisting of two solvents: A ¼ water + 0.2%
formic acid and B ¼ acetonitrile + 0.2% formic acid, at a flow
rate of 0.4 mL/min and injection volume of 2 μL (see Subheading 2.4, items 1 and 2).
3. Using an isocratic pump, constantly infuse a reference mass
solution to allow for simultaneous mass axis calibration.
4. Run a series of gradients over the course of a 24-min separation
as described in Table 1 (see Note 13).
5. After the run, equilibrate with 85% solvent B for 10 min prior
to the beginning of a new run.
3.6 Ion-Pairing
Reversed-Phase LCMS Run
1. Samples are prepared as in Subheading 3.4 but instead separated on an Agilent Zorbax Extend C18 column linked to an
Agilent Accurate Mass 6220 Time-of-Flight (TOF) spectrometer and coupled with an Agilent 1200 LC system
(or equivalent) (see Note 12).
2. Use a mobile phase consisting of two solvents: A ¼ 97:3 water:
methanol and B ¼ 100% methanol, both containing 5 mM
tributylamine (TBA) and 5.5 mM acetic acid, at a flow rate of
0.25 mL/min (see Subheading 2.6, items 1 and 2).
3. Using an isocratic pump, constantly infuse a reference mass
solution to allow for simultaneous mass axis calibration.
4. Run a series of gradients over the course of a 24-min separation
as described in Table 2 (see Note 14).
5. After the run, equilibrate with 100% solvent A for 10 min prior
to the beginning of a new run.
Table 1
Standard reversed-phase LC-MS gradient
Time (min)
Gradient (% solvent B)
0–2
85
3–5
80
6–7
75
8–9
70
10–11
50
11–14
20
14–24
5
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formic acid and B ¼ acetonitrile + 0.2% formic acid, at a flow
rate of 0.4 mL/min and injection volume of 2 μL (see Subheading 2.4, items 1 and 2).
3. Using an isocratic pump, constantly infuse a reference mass
solution to allow for simultaneous mass axis calibration.
4. Run a series of gradients over the course of a 24-min separation
as described in Table 1 (see Note 13).
5. After the run, equilibrate with 85% solvent B for 10 min prior
to the beginning of a new run.
3.6 Ion-Pairing
Reversed-Phase LCMS Run
1. Samples are prepared as in Subheading 3.4 but instead separated on an Agilent Zorbax Extend C18 column linked to an
Agilent Accurate Mass 6220 Time-of-Flight (TOF) spectrometer and coupled with an Agilent 1200 LC system
(or equivalent) (see Note 12).
2. Use a mobile phase consisting of two solvents: A ¼ 97:3 water:
methanol and B ¼ 100% methanol, both containing 5 mM
tributylamine (TBA) and 5.5 mM acetic acid, at a flow rate of
0.25 mL/min (see Subheading 2.6, items 1 and 2).
3. Using an isocratic pump, constantly infuse a reference mass
solution to allow for simultaneous mass axis calibration.
4. Run a series of gradients over the course of a 24-min separation
as described in Table 2 (see Note 14).
5. After the run, equilibrate with 100% solvent A for 10 min prior
to the beginning of a new run.
Table 1
Standard reversed-phase LC-MS gradient
Time (min)
Gradient (% solvent B)
0–2
85
3–5
80
6–7
75
8–9
70
10–11
50
11–14
20
14–24
5
Metabolomics of Mycobacterium tuberculosis
587