I'm having difficulties getting an efficient extraction of deuterated PGF1-alpha and TXB2 from culture media using both hexane/isopropanol and Bligh and Dyer extraction procedures. I've been able to recover these in the past and measure them using our triple quad mass spec, but I'm no longer able to detect these on our MS using identical methods. I'm using a waters ODS2 column, with mobile phase of A: Water/acetonitrile/glacial acetic acid 750:250:1, and B: Methanol/acetonitrile/glacial acetic acid (600:400:1). Gradient goes from 50:50 A:B to 90%B over 10 mins. I'm struggling to identify what the problem is; am I not able to extract the standards, or do I have issues detecting them?
1. Are you sure PGF1 and TXB2 are soluble in the HPLC solvent system (acidified by acetic acid for ionization yield)? Just try without the acid.
2. Try infusion of the PG/TXB2 standard in the MS source. Try +/- acid.
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