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MB Sample ID: SA257309
| Local Sample ID: | B4_RP_pos |
| Subject ID: | SU002659 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 82 |
| Gender: | Female |
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Sample Preparation:
| Sampleprep ID: | SP002665 |
| Sampleprep Summary: | The bone piece was further powdered using a Spex SamplePrep 6775-115 Freezer/Mill Small Cryogenic Grinder operated in liquid nitrogen at speed 10 with 3min pre-cooling, 2min run and 2min cooling protocol. The powder was stored in a cryovial at -80◦C until further processing. Biphasic Extraction 50mg of bone powder was placed in a 2mL Pre-Filled Bead Mill Tubes (ceramic 1.4 mm in diameter) and 900µL of 2:1 (% v/v) Chlor:MeOH were added. Samples were vortexed for 30s and and homogenised (4x20s bursts at 7200RPMs, pause 2min between bursts) in a Precellys evolution. To induce phase separation, 400µL of LC‐MS grade water was added and homogenised (4x20s bursts at 7200RPMs, pause 2min between bursts). The samples were then centrifuged at 4°C for 10min at 2000RPMs and rest in ice for 5min. 600µL lower fraction (organic) was collected and transferred to fresh Eppendorf tubes and the samples were re‐extracted for a second time using 500µL of 2:1 (% v/v) Chlor:MeOH and the tube homogenised (4x20s bursts at 7200RPMs, pause 2min between bursts). The two respective fractions were combined and concentrated. 600µL lower fraction (organic) was collected and transferred the previous Eppendorf tube. This was centrifuged at 13000rpm, 4°C for 10min and 1ml of the supernatant was collected and dried under nitrogen flow. 350µL of aqueous phase in a fresh Eppendorf tube and centrifuge at 13000rpm, 4°C for 10min and 300µL transferred to a fresh tube and dried under nitrogen flow. Dry extracts were store at -80°C until testing. Methanol-Water Extraction 50mg of bone powder was placed in a 2mL Pre-Filled Bead Mill Tubes (ceramic 1.4 mm in diameter) and 750µL of 8:2 (% v/v) MeOH:Water were added. Samples were vortexed for 30s and and homogenised (4x20s bursts at 7200RPMs, pause 2min between bursts) in a Precellys Evolution. The homogenisation tube was centrifuged at 13000RPMs, 4°C for 10min, 700µL were moved to a fresh tube. 750µL of 8:2 (% v/v) MeOH:Water were added and the homogenisation step was repeated. The homogenisation tube was centrifuged at 13000RPMs, 4°C for 10min, 700µL were moved to the same collection tube. The tube with the two extracts was centrifuged at 13000RPMs, 4°C for 10min and 1.2mL of supernatant were transferred in a fresh tube and dried under nitrogen flow. Dry extracts were store at -80°C until testing. Methanol-Acetonitrile-Water Extraction 50mg of bone powder was placed in a 2mL Pre-Filled Bead Mill Tubes (ceramic 1.4 mm in diameter) and 750µL of 2:2:1 (% v/v/v) MeOH:AC:Water were added. Samples were vortexed for 30s and and homogenised (4x20s bursts at 7200RPMs, pause 2min between bursts) in a Precellys Evolution. The homogenisation tube was centrifuged at 13000RPMs, 4°C for 10min, 700µL were moved to a fresh tube. 750µL of 2:2:1 (% v/v/v) MeOH:ACN:Water were added and the homogenisation step was repeated. The homogenisation tube was centrifuged at 13000RPMs, 4°C for 10min, 700uL were moved to the same collection tube. The tube with the two extracts was centrifuged at 13000RPMs, 4°C for 10min and 1.2mL of supernatant were transferred in a fresh tube and dried under nitrogen flow. Dry extracts were store at -80°C until testing. |
| Processing Storage Conditions: | Described in summary |
| Extract Storage: | -80℃ |
