BAF_Protocol_015_ 3-NPH derivatization of short chain fatty acid

Refer to BAF_Protocol_008 Metabolomics: Soluble Metabolite Extraction for soluble metabolite extraction of a sample of interest and dry the extracted metabolites.

Reagent powder storage: EDC is stored at -20C, all others are at room temperature.

Prepare working solutions on small glass botles.

250 nM 3-NPH solution as derivatization agent in 50% methanol:

--> dissolve 47.4 mg of 3-NPH in 1 mL of 50% methanol

2 mg/mL BHT in 100% methanol:

--> dissolve 2 mg in 1 mL methanol.

150 mM EDC (stored at -20C) in 100% methanol:

--> dissolve 28.75 mg in 1 mL methanol.

7,5% pyridine in 75% methanol.

--> add 750 uL in 9.25 mL of 75% methanol.

Prepare a stock solution of each standard at 10 mg/mL in 75% MeOH. Use the stock solution to generate a 1000 ug/mL

Prepare standard curve:

A	B	C	D	E	F
Workingsolution (ug/mL)	Workingsolution vol (uL)	Diluent vol (uL)	Final Conc. (ug/mL)	ng/mL	Dilution level
1000	1000	0	1000	1E6
100	100	900	100	100,000	L1
10	100	900	10	10,000	L2
1	100	900	1	1000	L3
0.1	100	900	0.1	100	L4
0.01	100	900	0.01	10	L5
0.001	100	900	0.001	1	L6

Solubilize metabolite extracts in 100µL of 75% methanol

Add 50µL of each into a clean new Eppendorf tube

Add 50µL of each standard curve level into a clean new Eppendorf tube

To each sample or standard add:

50µL of 150 mM EDC,

50µL of 7,5% pyridine,

Incubate in thermomixer at 30 ºC

To quench the reaction, add 50µL of BHT

Dilute with 250µL of water making a total of volume of 500µL and to achieve 40% methanol at final sample.

Samples are ready to be analyzed by LC-MS/MS. Use 40% methanol as blank runs.

Column: Acquity UPLC BEH C-18 1.7 um 2.1 x 150 mm (Waters)Solvent A: 0.1% FA in water

Solvent B: 0.1% FA in 90% methanol

Flow = 0.250 ml/min.

Gradient:

0-1 min 50%B; 1-5 min 70% B; 5-8 min 98% B; 8-13 98% B; 13-13.1 min 15%B.

Full scan parameter MS1 Res = 120,000Range = 67-1000RF Lens = 60AGC = 25%Max IT (ms)= 50Polarity = Negative PRM parameters: CE=30Res: 50,000Isolation window: 1.5AGC: 50MaxIT (ms): 86Polarity = NegativeTargeted masses:

Analyze data using Skyline.