I am determining butyric acid in plasma.
For this, I am developing a method.
I will be giving a brief on how I prepare my highest concentrated calibration standard.
I prepare my internal standard (d7- butyric acid) in 100% acetonitrile having a concentration of 100 uM. From which, I transfer 10 uL to a cold glass vial along with 10 uL of 10 mM of Butyric acid as my highest concentrated calibration standard. I use 10 uL of 20% PFFBr in 100% acetonitrile to derivatise my standard and heat it for 1 hour at 60*C.
The problem I am facing is low signals of internal standard to an extent that it cannot be even detected within the given retention time.
Also, I am receiving signals for butyric acid in my blank and no peak for internal standards.
Could you please help me?