Adenine nucleotide HPLC assays

David W. Green green_d at
Mon Apr 17 14:25:59 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.  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 assume 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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