The Application and Validation of HybridSPE-Precipitation Cartridge Technology for the Rapid Clean-up of Serum Matrices (from Phospholipids) for the Clinical Analysis of Serotonin, Dopamine and Melatonin

Phospholipids have been shown to cause matrix effects particularly in liquid chromatography-mass spectrometry (LC-MS) analysis of small molecules. This results in suppression of the analyte signal. This study provides a versatile validated method for the analysis of serotonin in serum along with dopamine and melatonin using LC-MS/MS. It utilises HybridSPE-Precipitation cartridges for the clean-up of serum samples. This technology involves a simple protein precipitation step together with a fast and robust SPE method that is designed to remove phospholipids. Serotonin and dopamine are major neurotransmitters in the brain which affect various functions both in the brain and in the rest of the body. Melatonin plays an important role in the regulation of circadian sleep-wake cycle. Good linear calibrations were obtained for the multiplex assay of analytes in serum samples (0.021-3.268 mu mol L-1; R (2) = 0.9983-0.9993). Acceptable intra- and inter-day repeatability was achieved for all analytes in serum. Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with LODs of 3.2-23.5 nmol L-1 and the LOQs of 15.4-70.5 nmol L-1 for these analytes in serum. The sample clean-up procedure that was developed provided efficient recovery and reproducibility while also decreasing preparation time and solvent use. A sample storage protocol was established, this was achieved by investigation of sample stability under different storage conditions. Evaluation of matrix effects was also carried out and the influence of ion suppression on analytical results reported. This clean-up protocol was then applied to the analysis of clinical serum samples.