A New Extraction Method for the Determination of Oxytocin in Milk by Enzyme Immune Assay or High-Performance Liquid Chromatography: Validation by Liquid Chromatography–Mass Spectrometry

Abstract

There has been a controversy regarding the use of exogenous oxytocin (OT) in milking cattle which may have toxicological consequences during nonphysiological exposure. In the present study, a new sensitive extraction method for OT was developed followed by enzyme immune assay (EIA) or high-performance liquid chromatography (HPLC) analysis. The extraction of OT in milk involves two steps: (1) TCA precipitation of milk proteins and (2) solid-phase extraction (SPE) cleanup process. Without these steps, analysis of OT in milk was not possible. Utilizing EIA as a quantitative tool the limit of detection (LOD) and limit of quantitation (LOQ) were found to be 7.74 and 10.3 pg ml−1, precision in terms of intra- and interday coefficient of variation was below 13 % (%RSD, N = 8), while percent recoveries were between 85 and 92 %. Utilizing UV-HPLC, the LOD, LOQ, precision, and recovery values were found to be 4.1 ng ml−1, 9.8 ng ml−1, 2–10 %, and 84–91 %, respectively. OT was found to be stable against adverse temperature (up to 100 °C) and pH (2 to 10) and simulated gastric fluid digestibility assay. Four milk samples collected from the market were analyzed, which showed that TCA precipitation and SPE steps are mandatory and the results were validated by LC-MS showing mass ion peak at 1 kD.