Use of Immunocapture Techniques to Enable Efficient Sample Preparation for LC-MS/MS Analysis of Oxytocin in Human Plasma

Abstract: Oxytocin (OT) is a small neuropeptide, which is present in human plasma at extremely low endogenous levels of only a few pg/ml. It is mainly involved during labour and lactation but it has recently also been found that OT is involved in social behaviours like bonding and feeling empathy. During the last couple of years, the interest in OT has been raised and due to the matrix complexity, it is very challenging to develop a highly sensitive assay to be able to measure endogenous OT. An established quantitative tool for the detection of peptides in biological samples is liquid chromatography (LC) combined with tandem mass spectrometry (MS/MS). However, when working with complex matrices and concentrations in the low pg/ml range, an efficient sample preparation is required to remove endogenous matrice components which interfere with the detection of the analyte by LC-MS/MS analysis. In this project the potential of using immunocapture as sample preparation was investigated. The immunocapture techniques investigated where Dynabeads® and MSIATM pipette tips. To evaluate the success of the developed immunocapture technique a SPE method for OT, used for comparison, was optimized. Using commercially available antibodies, the Dynabeads® showed a great potential whereas the results for MSIATM were inconclusive. The Millipore antibody MAB5296 used together with Dynabeads® Protein G was found to generate the highest recovery. To increase the recovery during optimization of the immunocapture method, parameters like elution, incubation time and sample volume were investigated. It was found that these parameters did not have a great impact on recovery but an increase in sample volume, without changing the eluate volume, did generate approximately a twofold increase in signal, making it possible to obtain a LLOQ of 10 pg/ml. The final immunocapture method showed a good linearity in the concentration range of 10 to 100 pg/ml using seven calibration points. The precision and accuracy were qualified using four quality control (QC) samples (10, 30, 45 and 85 pg/ml). The precision ranged from 12 to 15% and the accuracy ranged from -8 to 2% except for the HIGH QC-sample where it ranged from +23 to 33%. This was probably due to the use of wrongly spiked QC-samples (100 pg/ml). When comparing the final method with the SPE it was found that a much cleaner extract was obtained with the immunocapture method where endogenous OT was detected whereas no endogenous OT could be seen when using SPE. The S/N for 5 and 10 pg OT/ml plasma was 4.01 and 0.97 for the SPE prepared samples compared to 25.3 and 41.4 for the immunocapture method. These observations show that immunocapture is an efficient technique for selective extraction of a target peptide enabling LC-MS/MS detection at very low concentrations in complex matrices such as plasma.