MSACL 2019 EU Abstract

Development and Validation of an LC-MS/MS Method for the Measurement of Plasma Tryptophan, Kynurenine, Serotonin and 5-Hydroxyindoleacetic Acid

Leanne Sheppard
Liverpool Clinical Laboratories

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Leanne Sheppard (Presenter) Liverpool Clinical Laboratories

Presenter Bio: After obtaining a BSc in Life Sciences Applicable to Medicine and distance-learning Graduate Certificate in Biomedical Sciences I was fortunate enough to gain a place on the NHS Scientist Training Programme. Following completion of my final year of training in September 2019, I will progress to the role of Senior Clinical Scientist within Liverpool Clinical Laboratories.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Honorarium/Expenses N/A Grant/Research Support N/A Committee/Board/Advisory Board N/A Stock/Bonds N/A Salary N/A Royalty / IP / Other Income N/A Conflict mitigation NOT REQUIRED.

INTRODUCTION: Carcinoid tumours are rare neoplasms arising from the enterochromaffin cells of the neuroendocrine system. Midgut carcinoids are often associated with hyper-secretion of serotonin and can result in carcinoid syndrome; with symptoms including wheezing, diarrhoea and flushing. Measurement of urinary excretion of serotonin metabolite 5-HIAA is widely recommended for both the diagnosis and monitoring of midgut carcinoid tumours. However, collection of 24-hour urine samples can be burdensome, error prone and subject to dietary and medication induced fluctuations. Substitution with a single plasma sample would prove more convenient for patients. Combined measurement of related compounds may also allow a greater understanding of tryptophan metabolism in the carcinoid patient. AIMS: Development of a LC-MS/MS assay for the simultaneous quantification of plasma serotonin, its precursor tryptophan, 5-HIAA and associated compound kynurenine in accordance with CLSI C62A guidelines and establishment of healthy population reference ranges for all analytes. METHOD: Following protein precipitation, chromatographic separation of analytes and their respective deuterated internal standards was achieved following a 10µL injection onto a C18 Fortis column (4.6 x 150mm, 5.0µm). Analysis was carried out using a Waters Acquity® UPLC system coupled to a Xevo TQD® tandem mass spectrometer operating in ESI-positive mode. Multiple reaction monitoring was used to detect analyte and corresponding internal standard transitions. Matrix effects together with assay linearity, imprecision, accuracy, recovery, stability and sensitivity were assessed. Sample comparisons for both 5-HIAA (n=38) and tryptophan (n=12) were carried out using local established assays.
RESULTS: Co-elution of tryptophan, kynurenine, serotonin and 5-HIAA with their deuterated internal standards at respective retention times of 4.93, 4.51, 4.12 and 5.10 minutes was achieved using gradient elution. The assay was linear to 375μmol/L for tryptophan (R2=0.995), 17.5µmol/L for kynurenine (R2=0.999), 12.5µmol/L for serotonin (R2=0.999) and 10µmol/L for 5-HIAA (R2=0.999). Despite profound matrix effects for serotonin and tryptophan (MF 0.1-0.8), the assay was both adequately accurate and precise with inter-assay precision of <10% for all analytes across the working concentration range. Acceptable assay sensitivity was demonstrated with lower limits of the measuring interval defined as 5μmol/L, 250nmol/L, 200nmol/L and 30nmol/L for tryptophan, kynurenine, serotonin and 5-HIAA, respectively. Good correlation (R2=0.99) but a mean difference of 21.9% (95% CI of -59.8-16.1%) was observed between the two 5-HIAA assays. Difference of 33.7% (95% CI of 9.1% to 58.3%) was noted between new tryptophan assay and the referral laboratory method. CONCLUSION: Despite further planned work to reduce impact of endogenous sample matrix, a simple LC-MS/MS method has been developed with potential clinical utility.