Instruments LC-MS methods Sample preparation Lab photos

LC-MS methods

HILIC-MS Method (Amide12min; MetDNA)

For polar metabolites; 12 min per injection

Column parameter

Waters, UPLC BEH Amide column (1.7 μm, 2.1× 100 mm)

Mobile phase

Positive mode and Negative mode

A：100% H₂O+ 25 mM CH₃COONH₄ + 25 mM NH₄OH

B：100% ACN

Gradient parameter

Time	Flow rate (uL/min)	A %	B %
0.00	500	5.0	95.0
0.50	500	5.0	95.0
7.00	500	35.0	65.0
8.00	500	60.0	40.0
9.00	500	60.0	40.0
9.10	500	5.0	95.0
12.00	500	5.0	95.0

RPLC-MS Method (RP12min; MetDNA)

For nonpolar metabolites and lipids; 12 min per injection

Column parameter

Phenomenex, UHPLC Kinetex C18 column (2.6 μm, 2.1× 100 mm)

Mobile phase

Positive mode and Negative mode

A：100% H₂O + 0.01% acetic acid

B：50%ACN+50%IPA

Gradient parameter

Time	Flow rate (uL/min)	A %	B %
0.00	300	99.0	1.0
1.00	300	99.0	1.0
8.00	300	1.0	99.0
9.00	300	1.0	99.0
9.10	300	99.0	1.0
12.00	300	99.0	1.0

HILIC-MS Method (pHILIC)

For polar metabolites; 25 min per injection

Column parameter

Merck, SeQuant ZIC-pHILIC column (5 μm, 2.1× 100 mm)

Mobile phase

Positive mode and Negative mode

A：100% H₂O 25 mM CH₃COONH₄ + 25 mM NH₄OH

B：100% ACN

Gradient parameter

Time	Flow rate (uL/min)	A %	B %
0.00	200	20.0	80.0
2.00	200	20.0	80.0
17.00	200	80.0	20.0
17.10	200	20.0	80.0
25.00	200	20.0	80.0

RPLC-MS (Lipidomics; Lipid4DAnalyzer)

For untargeted lipidomics analysis; 18 min per injection

Column parameter

Phenomenex, UHPLC Kinetex C18 column (1.7 μm, 2.1× 100 mm)

Mobile phase

Positive mode and Negative mode

A：ACN: H₂O = 6:4; 10mM HCOONH₄

B：ACN: IPA = 1:9; 10mM HCOONH₄

Gradient parameter

Time	Flow rate (uL/min)	A %	B %
0.00	300	60.0	40.0
1.50	300	60.0	40.0
10.50	300	15.0	85.0
14.00	300	15.0	85.0
14.10	300	0.0	100.0
15.00	300	0.0	100.0
15.20	300	60.0	40.0
18.00	300	60.0	40.0

RPLC-MS Method (T3_12min)

For polar metabolites and lipids

1. RPLC-MS Method

Waters, UPLC HSS T3 column (1.8 μm, 2.1× 100 mm)

Mobile phase

Positive mode

A：100% H2O (0.1% Formic acid)

B：100% ACN (0.1% Formic acid)

Negative mode

A：100% H2O (0.5 mM NH4F or 5 mM NH4Ac)

B：100% ACN

Gradient parameter

Time	Flow rate (uL/min)	A %	B %
0.00	500	99.0	1.0
1.00	500	99.0	1.0
8.00	500	1.00	99.0
10.00	500	1.00	99.0
10.00	500	99.0	1.0
12.00	500	99.0	1.0