Transfer the sample (plasma, , cell suspension) into a glass centrifuge vial .
{"action": "transfer", "output": "", "reagent": ["the sample (plasma, , cell suspension)"], "container": ["a glass centrifuge vial"]}
adjust the volume to 1 ml with PBS #####%%%%% output "heparinized blood.1 ml medium" are used in line6
{"action": "modify", "output": "heparinized blood.1 ml medium", "volume": ["1 ml"], "length": [""], "device": [""]}
follows:50-200 µl plasma was taken from heparinized blood.1 ml medium .
{"action": "take", "output": "", "reagent": ["heparinized blood.1 ml medium"]}
was directly taken from cell culture #####%%%%% output "a plasma sample" are used in line12
{"action": "take", "output": "a plasma sample", "reagent": [""]}
Add 10 µl of the internal standard (10 μM C17- S1P in MeOH).Add 300 µl of 18.5% HCl.
{"action": "add", "output": "", "reagent": ["18.5% HCl"], "volume": ["300 µl"]}
As an example, S1P extraction from a plasma sample is shown in step A7. #####%%%%% output "step A7" are used in line14
{"action": "show", "output": "step A7", "reagent": ["a plasma sample"]}
The CHCl3-phase is extracted by directly pipetting through the upper aqueous phase. #####%%%%% output "the CHCl3" are used in line18
{"action": "extract", "output": "the CHCl3", "container": ["the upper aqueous phase"], "reagent": ["step A7"]}
Add this CHCl3-phase to the transferred CHCl3-phase of step A7.
 {"action": "add", "output": "", "reagent": ["this CHCl3-phase"]}
Vacuum-dry the CHCl3 in the vacuum rotator at 60 °C for 45 min.
{"action": "rinse", "output": "", "reagent": ["the CHCl3"], "temperature": ["60 \u00b0C"], "time": ["45 min"]}
Alternatively, the samples can be dried under nitrogen gas flow.
{"action": "dry", "output": "", "reagent": ["samples"], "time": ["1-20 min"]}
1-20 min: Re-equilibrate with 90% solution A #####%%%%% output "S1P" are used in line24
{"action": "equilibrate", "output": "S1P", "concentration": ["90% solution"], "volume": [""]}
S1P is analyzed with the mass transition 380 m/z -> 264 m/, For quantitative analysis a standard curve with S1P amounts of 1 pmol to 100 pmol as the internal standard is generated. #####%%%%% output "quantitative analysis" are not used
{"action": "examine", "output": "quantitative analysis", "reagent": ["S1P"]}