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Neuropeptide Y (NPY) is a 36-amino acid peptide circulating at a subpicomolar concentration participating in multiple physiological and pathological processes. NPY is prone to peptidolysis, generating metabolites with modified affinity for the five known receptors of NPY that mediate distinct effects. It is, therefore, crucial to distinguish each metabolite to understand the multiple functions of NPY. Since immunoassays are not able to distinguish NPY from its metabolites, we have validated a microliquid chromatography tandem mass spectrometry (micro-LC-MS/MS) assay for the quantification of endogenous NPY, NPY2-36, NPY3-36, NPY1-35, and NPY3-35 in human plasma. Sample preparation relies on immunoextraction in 96-well plates, followed by solid-phase extraction prior to micro-LC-MS/MS. The LLOQ ranged from 0.03 to 0.16 pM, intra- and inter-assay precision were <27% and trueness <22%. We determined reference intervals in 155 healthy volunteers and 40 hypertensive patients. We found that NPY3-36 is the main circulating peptide in resting conditions and that NPY and catecholamines are simultaneously increased during orthostasis. We also showed that the concentrations of NPY and its metabolites are similar in healthy volunteers and hypertensive patients. NPY is the prototype peptide that circulates in concentrations expected to be beyond instrumental capacities. We have been successful in developing a high-throughput specific and sensitive assay by including a deep knowledge of the physicochemical properties of these peptides to an efficient multistep sample preparation, and a micro-LC chromatography. We believe that our methodological approach opens the possibility to selectively quantify other endogenous peptides cleaved by peptidases whose concentrations are below 1 pM.

Plasma catecholamines provide a reliable biomarker of sympathetic activity. The low circulating concentrations of catecholamines and analytical interferences require tedious sample preparation and long chromatographic runs to ensure their accurate quantification by HPLC with electrochemical detection. Published or commercially available methods relying on solid phase extraction technology lack sensitivity or require derivatization of catecholamine by hazardous reagents prior to tandem mass spectrometry (MS) analysis. Here, we manufactured a novel 96-well microplate device specifically designed to extract plasma catecholamines prior to their quantification by a new and highly sensitive ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Processing time, which included sample purification on activated aluminum oxide and elution, is less than 1 h per 96-well microplate. The UPLC-MS/MS analysis run time is 2.0 min per sample. This UPLC-MS/MS method does not require a derivatization step, reduces the turnaround time by 10-fold compared to conventional methods used for routine application, and allows catecholamine quantification in reduced plasma sample volumes (50-250 μL, e.g., from children and mice).