An improved "ion pairing agent free" HPLC-RP method for testing cAMP Phosphodiesterase activity

Talanta. 2019 Jan 15:192:314-316. doi: 10.1016/j.talanta.2018.09.058. Epub 2018 Sep 18.


Current HPLC methods for analyzing cAMP Phosphodiesterase activity (PDE) use salts, limiting the life of the columns. For this reason, we have developed an improved "ion pairing agent free" method, using a simple 150 mm C18-hydro column at 30 °C and two phases: (a) water with 0.1% acetic acid and (b) 85/15 w/w MeOH/tetrahydrofuran with 0.1% acetic acid. Using this method the peaks for cAMP and AMP were obtained with good resolution (R ≈ 1.35) and sensitivity (5·10-9 mols) in only 15 min. Moreover, the method was applied to the GMP/cGMP pair obtaining the same sensitivity and resolution (R ≈ 1.38). The precision and accuracy were tested and the method was verified with a Type IV Phosphodiesterase reaction, which produced AMP from cAMP. The method is cleaner and less aggressive, and represents an interesting alternative to currently used methods.

Keywords: HPLC; PDE; cAMP/AMP; cGMP/GMP.

MeSH terms

  • 3',5'-Cyclic-AMP Phosphodiesterases / chemistry*
  • Acetic Acid / chemistry
  • Adenosine Monophosphate / analysis
  • Chromatography, High Pressure Liquid / methods
  • Cyclic AMP / analysis
  • Cyclic AMP / chemistry
  • Cyclic GMP / analysis
  • Cyclic GMP / chemistry
  • Enzyme Assays / methods*
  • Furans / chemistry
  • Guanosine Monophosphate / analysis
  • Limit of Detection
  • Methanol / chemistry
  • Water / chemistry


  • Furans
  • Water
  • tetrahydrofuran
  • Adenosine Monophosphate
  • Guanosine Monophosphate
  • Cyclic AMP
  • 3',5'-Cyclic-AMP Phosphodiesterases
  • Cyclic GMP
  • Acetic Acid
  • Methanol