Adenine nucleotide HPLC assay

David W. Green Green_david_w.prilvms3 at
Tue Apr 18 09:23:51 EST 1995

We have been using a reversed-phase, C18 HPLC assay to determine adenine
nucleotide levels in tissue samples.  The samples are extracted with
perchloric acid, neutralized and filtered before running in a
KPO4/CH3CN/TBAP mobile phase (CH3CN gradient) at pH 5.0 on a 0.46 x 25 cm
column at 1.5 ml/min.  This gives a beautiful separation of AMP, ADP and
ATP, but a significant backpressure builds up over just a few repetitive
runs.  We have localized this to the solvents, which have been extensively
filtered and degasssed.  While the column can be washed with water to
relieve the back-pressure, we would just as soon try another solvent
system, resin or chemistry that can give us the same or better resolution
without the backpressure problems.
Does anyone have any suggestions?
David W. Green, PhD
Sr. Research Investigator
Bristol-Myers Squibb Pharmaceutical Research Institute
Princeton, NJ

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