Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of opiorphin in human saliva.

Opiorphin, QRFSR-peptide, is a mature product of the PROL1 (proline rich, lacrimal 1) protein that showed beneficial effects in pain management, antidepressant-like actions as well as involvement in colonic motility and erectile physiology. Using opiorphin as a potential biomarker of different pathological states requires the development of robust and sensitive methods. We report a highly sensitive and specific liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) analytical method for the analysis of opiorphin in human saliva. Quantification was based on multiple reaction monitoring using characteristic transitions (m/z 347/120 - as quantifying ion; 347/175 and 347/268 as qualifying ions). The assay was linear in the range of 0-110 ng/ml and the lower limit of quantification reached was 1.0 ng/ml. The intra-day precision and accuracy were between 2.7-5.6% and -2.3 to 3.2%, respectively. The inter-day precision and accuracy were between 10.8-13.7% and -11.0 to 52%, respectively. Mean recovery was 106% and mean matrix effect was 0.97. Opiorphin in TFA treated saliva samples was stable for at least 12h at room temperature and up to 30 days at -20°C. Opiorphin levels in human saliva samples collected from young healthy individuals ranged from 2.8 to 25.9 ng/ml.