Jul 24, 2024
BAF_Protocol_015_ 3-NPH derivatization of short chain fatty acid
- 1University of Virginia Biomolecular Analysis Facility Core
Protocol Citation: Nicholas Sherman 2024. BAF_Protocol_015_ 3-NPH derivatization of short chain fatty acid. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1oz7lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 23, 2024
Last Modified: July 24, 2024
Protocol Integer ID: 103938
Abstract
Short Chain Fatty Acid derivitization before MS analysis.
Materials
- 3-Nitrophenylhydrazine hydrochloride (3-NPH) --> Sigma N21804-5G
- Butylated hydroxytoluene (BHT) --> Sigma PHR1117-1g
- N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (ED) --> Sigma E1769- 5g
- Pyridine --> Sigma 270970 - 100 mL
- Valeric acid --> Sigma 75054-1mL
- Isovaleric acid --> Sigma 78651- 1mL
- Hexanoic acid (caproic acid) --> Sigma 21529 - 1mL
- 4-Methylvaleric acid (Isocaproic acid) --> Sigma 277827 - 1mL
- Butyric acid --> Sigma 19215 - 5 mL
- Isobutyric acid --> Sigma 46935-U - 500mg
- Lactic acid --> Sigma L6661 - 100 mL
- Acetic acid --> Sigma 338826 - 25 mL
- Column: Waters ACQUITY UHPLC BEH C18 1.7 um, 2.1 x 150 mm - 186002353
- Thermo Optima 0.1% FA (formic acid) in water - LS118-4
- Thermo Optima Methanol - A456-212
- Thermo Autosampler vials 0.25 mL – 14-823-136
- Thermo Orange caps – 14-823-380
- Fisherbrand Standard Pipette Tips (200 uL - Yellow) – 2707502
- Thermo Orbitrap ID-X - FETD1-10001
- Thermo Vanquish Duo UHPLC
Samples
Samples
Refer to BAF_Protocol_008 Metabolomics: Soluble Metabolite Extraction for soluble metabolite extraction of a sample of interest and dry the extracted metabolites.
Derivatization reagents: prepare all fresh
Derivatization reagents: prepare all fresh
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.
Standards and standard curve
Standards and standard curve
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 |
Derivatization reaction
Derivatization reaction
Solubilize metabolite extracts in 100 µL of 75% methanol
Add 50 µL of each into a clean new Eppendorf tube
Add 50 µL of each standard curve level into a clean new Eppendorf tube
To each sample or standard add:
50 µL of 150 mM EDC,
50 µL of 7,5% pyridine,
Incubate in thermomixer at 30 ºC
To quench the reaction, add 50 µL of BHT
Dilute with 250 µL of water making a total of volume of 500 µL and to achieve 40% methanol at final sample.
Samples are ready to be analyzed by LC-MS/MS. Use 40% methanol as blank runs.
LC-MS/MS parameters - Vanquish UPLC
LC-MS/MS parameters - Vanquish UPLC
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.
Mass spectrometer parameters - Orbitrap ID-X
Mass spectrometer parameters - Orbitrap ID-X
Full scan parameter
MS1 Res = 120,000
Range = 67-1000
RF Lens = 60
AGC = 25%
Max IT (ms)= 50
Polarity = Negative
PRM parameters:
CE=30
Res: 50,000
Isolation window: 1.5
AGC: 50
MaxIT (ms): 86
Polarity = Negative
Targeted masses:
Analyze data using Skyline.
Protocol references
Han J, Gagnon S, Eckle T, Borchers CH. Metabolomic analysis of key central carbon metabolism carboxylic acids as their 3-nitrophenylhydrazones by UPLC/ESI-MS. Electrophoresis. 2013 Oct;34(19):2891-900. doi: 10.1002/elps.201200601. Epub 2013 Jul 8. PMID: 23580203; PMCID: PMC4033578.