Nov 14, 2025
F5 Targeted profiling method for metabolomics
This protocol is a draft, published without a DOI.
- 1Stanford
Protocol Citation: Megan Danielewicz 2025. F5 Targeted profiling method for metabolomics. protocols.io https://dx.doi.org/
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: November 10, 2023
Last Modified: November 14, 2025
Protocol Integer ID: 91043
Keywords: profiling method for metabolomic, metabolomic, targeted profiling method, f5, preparation of reagent, extraction of plasma, reagent
Abstract
This protocol details the preparation of reagents and samples and extraction of plasma.
Attachments
n295b9mpf.docx
1.8MB
Materials
Analytes, reagents and assay materials:
Details for ordering the appropriate materials for metabolomic analysis are provided in this section. To order materials; the supplier’s name, contact information, and the part number for each reagent or piece of equipment required are indicated below:
Chemicals and reagents:
| A | B | C | |
| Supplier | Description | Part Number | |
| Sigma | Water-HPLC grade | 34877-4L | |
| Sigma | Methanol-HPLC grade | 646377-4L | |
| Sigma | Acetonitrile-HPLC grade | 34998-4L | |
| Thermo Fisher | Formic Acid-LCMS grade | 85178 |
Equivalent reagents from other suppliers can also be used. Assay result might deviate from this optimized method if using reagents other than the ones suggested in this SOP.
Metabolite standards:
| A | B | C | |
| Supplier | Description | Part Number | |
| Cambridge Isotopes | METABOLOMICS QRESS STANDARD 1 | MSK-QRESS1-1 | |
| METABOLOMICS QRESS STANDARD 2 | MSK-QRESS2-1 |
The above listed standards are labeled internal standards recommended to order to cover wide ranges of metabolites during method development. They are not mandatory for the assay, but we recommend a labeled internal standard to track extractions.
HPLC columns:
| A | B | C | |
| Supplier | *Description | Part number | |
| Phenomenex http://www.phenomenex.com/ | Kinetex, 2.6μM F5 100 A (150x 2.1 mm) | 00F-4723-ANY0 |
Preparation of reagents and solutions
Mobile Phase and Sample prep
The instructions for preparing each reagent/solution are provided below:
Mobile phase A (0.1% formic acid in water):
Mix 1 mL of formic acid with 999 mL of water in a 1 L bottle.
Mobile phase B (0.1% formic acid in acetonitrile):
Mix 1 mL of formic acid with 999 mL of methanol in a 1 L bottle.
Needle rinse (1:1:1:1 water/methanol/iso-propanol/acetonitrile):
Mix 250 mL of water, 250 mL of methanol, 250 mL of isopropanol and 250 mL of acetonitrile in a 1L bottle.
Sample and Control Preparation
The instructions for preparing the double blank, blank and QC samples are listed below:
CIL QRESS Internal standard stock solution prep
- QReSS vial 1 Stock: Add 1 mL of 50% methanol via a needle to the stoppered vial to reconstitute (do not remove stopper prior to reconstitute).
- QReSS vial 2 Stock: Add 1 mL of 50% methanol via a needle to the stoppered vial to reconstitute (do not remove stopper prior to reconstitute).
- Take 10.25 µL of each QReSS vial 1 and vial 2 and add 979.5 µL of methanol.
Double blank sample:
Pipet 1 mL 1:1 water/methanol into an autosampler vial.
Blank sample:
Add 50 µL water to 390 µL methanol and 10 µL of internal standard stock solution.
Pooled sample for method development:
Sample extracts containing internal standards can be used.
Extraction protocol
10s
Add sample into 1.5 mL eppendorf tube:
Mouse organ - 100 mg minimum (or closest you can come to that)
Plasma – 100 uL minimum - Make sure the plasma is settled in the bottom of the tube but not on the walls of the tube.
Bacterial supernatant/spent/fresh media – 100 uL -- may need to dilute later
Brine or Food - 100 uL or 100 mg
ALWAYS MAKE – A Double blank – An HPLC vial with 1 mL Mobile phase A (no sample processing)
ALWAYS MAKE – An extraction blank -- process an empty tube alongside your samples
ALWAYS MAKE – An ISTD blank – a blank with only an ISTD spike processed alongside your samples Add 20 uL of “QRESS ISTD spiking stock”. Always want to add it before extraction to normalize for what happens to the sample during extraction
Add 1000 µL of methanol (preferably ice cold)
Add + 20 µL of QReSS IS spiking stock solution
If your sample is a solid, perform beat beating for 1 min, chilling every 30 seconds. After bead beating chill for 30 min.
Vortex the sample/ solvent mixture for 00:00:10 .
10s
Chill for 10+ min in freezer. Can chill overnight.
Centrifuge the sample/ solvent mixture at 10000 rcf, 8°C, 00:10:00 .
10m
Transfer supernatant to fresh labeled eppendorf.
If you are using an aliquot of this for the NPH derivatization, take it now!!!
NPH aliquot: Add 50 uL to a second labeled Eppendorf.
Optional step: Chill 4 °C for 02:00:00 for any further precipitation of proteins.
2h
Dry down supernatant via speedvac or nitrogen evaporation (preferred)
Reconstitute in 100 µL 50% MeOH .
Centrifuge the sample/solvent mixture again at 15000 rcf, 00:10:00 (can use a centrifuge filter).
10m
Transfer 50 uL of solution to fresh labeled HPLC vial with insert.
Inject 5 µL of the supernatant for LC-MS analysis.
Gradient conditions
Source Conditions
MRM list can be provided upon request.
QRESS MRM and RT can be provided upon request.
Protocol references