Development of novel liquid chromatography mass spectrometric assays for diagnosis of neuroendocrine tumors, sepsis and sleeping disorders

Biomarkers are naturally occurring molecules that have different functions in the human body but can also be indicative of various clinical conditions. In the field of clinical chemistry, liquid chromatography mass spectrometry (LC-MS/MS) has quickly become the gold standard in measurement of many of these molecules. One of the key benefits of MS is excellent specificity and accuracy of detection. Mass spectrometry is exceptionally useful in studying small analytes in biological matrices, which makes it an essential tool in clinical and medical research and diagnostics. The aim of the study was to develop new LC-MS/MS assays to complement and improve existing methods, or to use mass spectrometry to measure recently recognized biomarkers in new ways. We developed and validated LC-MS/MS assays for three different clinical biomarkers, which included serum serotonin for diagnosis of neuroendocrine neoplasms (NEN), plasma bradykinin (BK) for monitoring septic shock progression and cerebrospinal fluid (CSF) orexin-A and -B for narcolepsy diagnostics. Many NENs are currently diagnosed by measuring serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) from either urine or serum, which is sensitive to dietary factors. Septic shock is a rapidly progressing life-threatening condition, for which medication exists, but it´s efficiency is highly dependent on the timing of the administration. We studied a potential sepsis biomarker BK by comparing samples from healthy volunteers to patients during different phases of the shock progression. Low CSF orexin-A is a currently agreed key diagnostic criterion for diagnosing narcolepsy, but the currently used radio immunoassay (RIA) may suffer from interferences and usually shows greater variability and imprecision between analysis batches than LC-MS/MS. The knowledge about the exact functions of a similar peptide orexin-B is still scarce, so this analyte was included in the assay as well. Of the developed assays, serum serotonin proved to be of equal performance compared to serum 5-HIAA assay and is a good complement for e.g. 5-HIAA in NEN diagnostics, both detecting NENs that the other had missed. BK was not found to be elevated during septic shock, and thus not a good biomarker to monitor sepsis, but the analytical method was sensitive and suitable for clinical research purposes. Orexin assay was successful in distinguishing narcoleptics from the healthy based on their CSF orexin-A concentration, but additional tests are needed to bring the assay to routine use due to the very limited sample quantities available to us during validation. Orexin-B concentrations in CSF samples were below our assay range. However, these results can be used as is, along with the few published LC-MS/MS assays for orexins, to determine the clinically relevant concentrations expected for both healthy and narcoleptic patients.