General minimum amount of material required
- Cell culture: 1-2 million cells
- Microbial pellet: 5-25 mg
- Tissue: 5-25 mg
- Biofluids (plasma/ serum and urine): 50 μL
Recommended extraction solvents
- Metabolites: methanol, acetonitrile, water
- Lipids: methanol, isopropanol, MTBE
Please refer to our protocols for sample preparation for details.
Caution:
- Use only LC/MS-grade solvents, additives, buffers and MilliQ nanopure water
- Avoid contact with any plastic material during lipid extractions. Use glassware and glass syringes during lipid extraction.
- The following plasticware can be used if required:
- Eppendorf Safe-Lock Tubes, 2.0 mL (Part number 0030120094)
- Eppendorf Safe-Lock Tubes, 1.5 mL (Part number 0030120086)
- Eppendorf® twin-tec PCR plates 96 eppendorf (Part number Z651486)
- Use clean containers, sample tubes, Eppendorf tubes for sample processing and storage
- Avoid glassware that has been cleaned with detergent e.g. from the EMBL kitchen, or clean them several times with MilliQ water and MS-grade organic solvents prior to usage.
Samples can be submitted as:
- Dried extracts obtained using speedvac / under nitrogen (preferred at cool conditions or room temperature)
- Aqueous methanolic extracts
- For outstations: please ship samples in sufficient dry ice.
Recommended reconstitution solvents
- Water (50─80%)
- Aqueous methanol/acetonitrile (20─80%)
- Isopropanol
- Methanol (caution: volume changes due to solvent evaporation at room temperature)
- Minimum reconstitution volume 50-100 μl
Quality control
- Do include pooled QC samples with each batch of samples for bracketing during analysis.
- Internal standards spiking during sample preparation: Deuterated or 13C internal standards are an ideal choice.
- Prepare replicates, preferentially biological and preparation replicates
Note: Please consult with MCF staff for the appropriate selection of the replicates, quality control samples, and internal standards that are suitable for your desired LC-MS analysis.
Before sample submission, make sure that:
- extracts are completely soluble reconstitution solvents
- samples are centrifuged above 10,000 g
- there is no insoluble matter in samples
- extracts are filtered if necessary (make sure to wash filters prior to usage, as some filter membranes contain stabilizing reagents)
Storage
- Dried metabolite extracts should be stored at -80°C (optional filling with argon/nitrogen would help to prevent oxidation)
- During processing, samples should be kept on ice
- Pack samples in dry ice for shipment times of more than 1 hour
- Samples will be kept at 4°C during LC-MS analysis