|
Abstract INTRODUCTION:
Exposomics, the study of environmental exposures, life style, diet and their effects on health, and metabolomics, the analysis of small molecules (metabolites) in biological samples, are increasingly used together to understand how environmental and other factors influence human health. Metabolomics can provide insights into the impact of environmental exposures on metabolic processes, helping to identify potential biomarkers and mechanisms of disease.
OBJECTIVE:
In this quest, we have developed a custom made, comprehensive, quantitative LC-MS/MS-based assay for targeted exposomic (environmental and diet) compounds analysis of biospeimens such as serum and urine. This assay allows for the identification and quantification of up to 265 exposomic compounds.
METHODS:
Our method uses a reverse phase LC-MS/MS in both positive and negative ionization modes to separate metabolites. It combines the derivatization and extraction of analytes, and the selective mass-spectrometric detection using multiple reaction monitoring (MRM) pairs. Two separate panels involving two different precolumn derivatization reactions were developed for this assay: Panel A - Phenylisothiocyanate (PITC) derivatization targeting amine-containing compounds and Panel B - 3-nitrophenylhydrazine (3-NPH) derivatization targeting keto- and carboxyl-containing compounds. Isotopically-labeled internal standards are used for metabolite quantification. Calibration of metabolite concentration ranges in both panels was adjusted for different biofluid types.
For panel A, a 96 deep-well plate with a filter plate attached via sealing tape, containing the required reagents and solvents, was used to prepare the plate assay. The first 14 wells of each plate are used for calibration and quality control purposes. For all metabolites, except organic acids, samples are first thawed on ice and then vortexed and centrifuged at 13,000x g. 10 µL of each sample are loaded onto the center of the filter on the upper 96-well plate and dried in a stream of nitrogen. Subsequently, PITC is added for derivatization. After incubation, the filter spots are dried again using an evaporator. Extraction of the metabolites is then achieved by adding 300 µL of extraction solvent. The extracts are obtained by centrifugation into the lower 96-deep well plate, followed by a dilution step with MS running solvent.
For organic acid analysis (panel B), 90 µL of ice-cold methanol is added to 30 µL of each sample for overnight protein precipitation. The sample is centrifuged at 13000x g for 20 min. 50 µL of supernatant was loaded into the center of wells of a 96-deep well plate, followed by the addition of NPH. After incubation for 2h, isotope-labeled internal standards, BHT stabilizer and water are added before LC-MS injection.
Mass spectrometric analysis was performed on Sciex 5500 QTrap® tandem MS instrument equipped with an Agilent 1290 series UHPLC system. The samples are delivered to the MS by a standard LC method. Data analysis was done using Analyst 1.6.2. Calibration regression, accuracy and precision of QC standards, and spiked recovery of each targeted metabolite were used for method validation.
RESULTS:
This custom assay can be used for the targeted identification and quantification of up to 265 metabolites across 16 chemical classes including amino acids and derivatives, biogenic amines, acylcarnitines, organic acids, fatty acids, nucleotides/nucleosides, bile acids, uremic toxins, parabens, plasticizers, pharmaceutical intermediates and other exoposomic compounds. The accuracy of QC standards with 3 different concentration levels are in the range of 80% to 120% with satisfactory precision values of less than 20%. The recovery rates of spiked serum, urine and fecal extract samples with three different concentration levels are in the range of 80% to 120% with satisfactory precision values of less than 20%.
CONCLUSIONS:
We have developed a comprehensive, sensitive, high-throughput, low-volume, quantitative targeted LC-MS/MS assay for the analysis of up to 265 exposomic compounds, across 16 chemical classes for serum and urine samples. Only 40 µL of a given sample are required for the entire analysis. We have adapted this assay into a 96-well plate format to enable high-throughput analysis.
|
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
