A LC-MS/MS method for the diagnostic measurement of cAMP in plasma and urine

Isabelle Piec (Lead Author), John Dutton, Sulaiman Al Riyami, Christopher Washbourne, Inez Schoenmakers, Hillel Galitzer, Gregory Burshtein, Philip Schwartz, Jonathan C Y Tang, William Fraser

Research output: Contribution to conferencePosterpeer-review

19 Downloads (Pure)


Background: Parathyroid hormone (PTH) plays a key role in calcium and phosphate homeostasis. Upon binding to its receptor, it signals via a second messenger, cyclic adenosine 3, 5’ monophosphate (cAMP). Lack of increase in plasma and urinary cAMP concentrations in response to PTH are used as diagnostic markers for pseudohypoparathyroidism (PHP), a condition primarily associated with resistance to PTH (Ellsworth-Howard Test). 
Aims: 1) Develop and validate a LC-MS/MS method for the quantification of cAMP in plasma and urine. 2) Investigate assay performance in a rat pharmacokinetic study investigating the response to an oral dose of PTH (1-34) and the response to subcutaneous (sc) PTH administration in a patient with suspected PHP. 
Method: cAMP and 13C5-cAMP internal standard were extracted from EDTA plasma using a weak anion exchange solid phase extraction. Chromatography was performed in positive electrospray ionisation mode, using a pentafluorophenyl column with a 10 mins 2% formic acid water:acetonitrile gradient. Transitions were m/z 330/136 for cAMP and 335/136 for 13C5-cAMP. Over concentrations ranging from 4.6 (lower limit of quantification) to 293.5 nmol/L, the calibration curve was linear (mean curve fits of >0.95, 5 repeats) and intra- and inter-assay precisions were <12% and <8%, respectively. Spiked recovery was 98±5%. 
Application: A single oral dose of 5 mg/kg PTH (1–34) or placebo was administered to Sprague-Dawley rats after an overnight fast. cAMP was analysed in EDTA samples obtained at baseline, prior to dosing and every 15 min for 1h and then hourly for another 3h after dosing. In the suspected PHP patient, urinary cAMP was measured after a standard 20µg sc injection of teriparatide (Forsteo). 
Results: In rats, plasma PTH (1-34) and cAMP increased significantly within 15min of dosing, reaching peak values between 15 and 30 mins. PTH (1–34) concentration increased significantly by up to 6770-fold, although response to PTH (1-34) varied between animals. Plasma cAMP typically tripled, from 36.5±3.7 nmol/L at baseline to 108.9±26.3 nmol/L. Placebo had no effect.Urine cAMP from the suspected PHP patient did not change significantly reflecting a lack of biological response to sc PTH (1-34) despite a significant increase in plasma PTH (1-34) (27.8 to 101.1 pmol/L). 
Conclusion: The present method was robust and selective. It also showed utility in determining cAMP in biological systems and the ability to study the effect of drugs such as Forsteo.
Original languageEnglish
Publication statusPublished - 9 Sep 2017
EventASBMR 2017 Annual Meeting - Colorado Convention Center, ASBMR Discovery Hall, United States
Duration: 8 Sep 201711 Sep 2017


ConferenceASBMR 2017 Annual Meeting
Country/TerritoryUnited States

Cite this