Volumetric Absorptive Microsampling (VAMS) for Targeted LC-MS/MS Determination of Tryptophan-Related Biomarkers

Abstract

L-Tryptophan (TRP) metabolites and related biomarkers play crucial roles in physiological functions, and their imbalances are implicated in central nervous system pathologies and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, schizophrenia and depression. The measurement of TRP metabolites and related biomarkers possesses great potential to elucidate the disease mechanisms, aid preclinical drug development, highlight potential therapeutic targets and evaluate the outcomes of therapeutic interventions. An effective, straightforward, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of 24 TRP-related compounds in miniaturised murine whole blood samples. Sampling and sample pretreatment miniaturisation were achieved thanks to the development of a volumetric dried blood microsampling approach. Volumetric absorptive microsampling (VAMS) allows the accurate sampling of microvolumes of blood with advantages including, but not limited to, minimal sampling invasiveness, logistical improvements, method sustainability in terms of solvents and energy consumption, and improvement of animal studies in the framework of the 3Rs (Replacement, Reduction and Refinement) principles on animal welfare. The VAMS-LC-MS/MS method exhibited good selectivity, and correlation coefficient values for the calibration curves of each analyte were >0.9987. The limits of quantitation ranged from 0.1 to 25 ng/mL. The intra- and inter-day precisions in terms of RSD were <9.6%. All analytes were stable in whole blood VAMS samples stored at room temperature for at least 30 days with analyte losses < 14%. The developed method was successfully applied to the analysis of biological samples from mice, leading to the unambiguous determination of all the considered target analytes. This method can therefore be applied to analyse TRP metabolites and related biomarkers levels to monitor disease states, perform mechanistic studies and investigate the outcomes of therapeutic interventions.

Blood microsampling technologies: Innovations and applications in 2022

“…Microsampling technologies have been evaluated in biomarker research studies for the diagnosis and prognosis of various conditions and the prediction and monitoring of response to an intervention 24,41,42,132–144 . Omics‐based approaches, such as metabolomics and proteomics, are increasingly employed for the generation of large amounts of data to facilitate the rapid discovery of biomarkers.…”

“…Based on a Net Promotor Score, upper arm sampling was found to be preferred over finger prick sampling by patients 24 . Other studies that have implemented the VAMS technology include research for (i) the human biomonitoring of mercury concentrations for exposure assessment, 41,145 (ii) the determination of tryptophan‐related biomarkers for understanding pathologies of neurodegenerative diseases such as Alzheimer's or Parkinson's disease 138 and (iii) the measurement of endocrine and bone biomarkers 139 . VAMS technology was also capable of detecting carcinogens, such as acrylamide and aflatoxin B 1 140,141 …”

“…Microsampling technologies have been evaluated in biomarker research studies for the diagnosis and prognosis of various conditions and the prediction and monitoring of response to an intervention. 24 , 41 , 42 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 Omics‐based approaches, such as metabolomics and proteomics, are increasingly employed for the generation of large amounts of data to facilitate the rapid discovery of biomarkers. A study assessed the DBS and DPS technology for non‐targeted lipidomic analysis as high throughput profiling and quantification of lipids could expand knowledge of underlying disease pathologies.…”

VAMS-Based Blood Capillary Sampling for Mass Spectrometry-Based Human Metabolomics Studies

“…Among them, VAMS facilitates participants’ self-collection of blood, hence supporting remote health monitoring and avoiding unnecessary extra hospital visits [ 9 ], which can be a burden, especially for the elderly. Moreover, it minimizes volume collection and does not, generally, require refrigerated transport and storage conditions [ 9 , 11 ].…”

Profiling Analysis of Tryptophan Metabolites in the Urine of Patients with Parkinson’s Disease Using LC–MS/MS

“…Of the tryptophan metabolism pathways, since indole metabolites on the indole metabolism pathway can only be produced via gut microbiota and influence the immune system and CNS in the human body as ligands for the aryl hydrocarbon receptor (AhR), indole metabolites have been speculated to play a key role in the gut-brain axis [14]. For this reason, to find potential biomarkers for several diseases, analytical approaches to detect gut microbial metabolites, including tryptophan metabolites, have been developed [15][16][17]. Furthermore, since it was reported that indole-3-propionic acid and other microbial metabolites were changed in the plasma of PD patients [18], the interest in changes in indole metabolites in PD patients has been continuously increasing.…”