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

Abstract

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 lmol 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.

Improved Voltammetric Determination of Kynurenine at the Nafion Covered Glassy Carbon Electrode – Application in Samples Delivered from Human Cancer Cells

“…The amount of these metabolites in the different biological samples might vary depending on cellular conditions and pathological state, that is, in human serum (healthy donors) IS, IA 5-HT, ME are present at similar or below Kyn level. 51,55,56 In human plasma, 5HT, 5HIAA, ME amounts are much lower than Kyn. 56,57 Therefore, the method accuracy in the biological matrix of interest needs to be verified using a comparative method.…”

“…40,[52][53][54] In some conditions, that is, IFN-γ stimulation, Trp metabolism is accelerated generating both Kyn and downstream kynurenines secretion. 52,54 In addition to oxidation occurring within the kynurenine pathway, in a cell, Trp might undergo hydroxylation, decarboxylation, or transamination to yield other biologically important 51,55,56 In human plasma, 5HT, 5HIAA, ME amounts are much lower than Kyn. 56,57 Therefore, the method accuracy in the biological matrix of interest needs to be verified using a comparative method.…”

Recent advances in sample preparation techniques to overcome difficulties encountered during quantitative analysis of small molecules from biofluids using LC-MS/MS

“…The use of hybrid precipitation/SPE plates for selective removal of phospholipids and precipitated proteins has been increasing over the past few years. 4,38,69,70 Several types of these plates are now commercially available, e.g. Hybrid SPE™ (Sigma Aldrich), Ostro™ (Waters), Captiva™ ND (Agilent) and Phree™ (Phenomenex).…”

Novel strategy for the facile enrichment of isopentenyl pyrophosphate in rat plasma via Ti4+ -immobilized polydopamine@Fe3 O4 core-shell microspheres

“…Effective extraction methods before LC-MS/MS analysis have played an important role in the trace-level quantitation of IPP and have served to increase the detection sensitivity. Hybrid® SPE, which is the only reported method in the literature to date, is expensive and time-consuming and requires complicated operations [4][5][6]. Thus, in this paper, a novel pretreatment method based on magnetic microspheres was proposed for IPP extraction in vivo because of the low abundance and low ionization efficiency of IPP.…”