Hello everyone,
I am currently working on developing a method to quantify butyric acid in plasma on single quad-GCMS using electron impact mode.
For this purpose, I am currently following this protocol:
- Thaw the plasma on ice, take 50 uL of plasma and add 50 uL of 100% acetonitrile and 100 uM of Internal standard (d7-butyric acid)
- Centrifuge at 4*C for 10 min at 2000 g.
- Collect 100 uL of supernatant and add 10 uL of 20% PFBBr. Vortex and heat at 60*C for 1 hour.
- Rest the sample for 15 min.
- Add 0.3g/mL of sodium thiosulphate. Heat for 2 min at 60*C and rest for 15 min.
- Add hexane to extract the sample
I also prepare a blank with MQ water instead of plasma, and follow the same protocol as above for the plasma samples.
The problem however is the result I am getting!! In my plasma samples, I am getting no peaks for Butyric acid and an unusually tall peak for the internal standard (IS). Whereas, in my blank, I am getting reasonable peaks for both butyric acid and the internal standard -- though this result is not reliably reproduced (sometimes it is, sometimes it isn't).
Could you please tell me why this is happening? Does anyone have any speculation about why my plasma is reading no butyric acid, even though the blank seems to sometimes appropriately picks up the d7-butyric acid in the internal standard?
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