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Abstract INTRODUCTION:
Bacterial metabolic processes are an increasingly important area of study to better understand host-microbe interactions. To better understand these metabolic processes, we are in need of reliable approaches to identify and quantify these metabolic byproducts using untargeted and targeted metabolomics. One challenge of untargeted metabolomics, however, is the lack of normalization across samples and metabolites, which makes it difficult to draw conclusions confidently without appropriate internal standards. An additional challenge faced in this realm is the complexity of bacterial media and other biological matrices. We aim to determine optimized parameters for both targeted data analysis of untargeted metabolomics assays and targeted metabolomics using a deuterated amino acid library as a reliable internal standard set across all four instrument modes: C18 and HILIC in both positive and negative ion modes. These methods will then be optimized for complex biological matrices as well. This work will improve in-house metabolomics approaches for various applications and increase data quality and interpretability
OBJECTIVE(S):
The objective of this study is to develop an internal standards library for HILIC and C18 chromatography in both positive and negative ion modes, for a total of four methods, using a quadrupole time-of-flight mass spectrometer (QTOF). This library will then be applied and further optimized on a triple quadrupole mass spectrometer (QqQ).
METHODS:
Building the Deuterated Amino Acid Internal Standards Library
Deuterated amino acids were diluted at a range of concentrations and analyzed using HILIC positive and negative ion modes, as well as C18 positive and negative ion modes. One internal standard library consisting of deuterated amino acids was constructed for each of the four modes by recording and averaging the retention times of each amino acid that displayed consistent peak quality across the tested concentrations.
Spiking Deuterated Amino Acids as Internal Standards
For bacterial supernatants and media samples, 200 µL of sample, including Brain Heart Infusion (BHI) media and nectar, was mixed with 1 mL methanol to precipitate proteins and reduce background signal. After a 5 min incubation at room temperature, samples were centrifuged for 10 min to pellet precipitated material. A total of 880 µL of clarified extract was transferred into two tubes, spiked with 1, 2, 5, 7.5, or 10% (v/v) deuterated amino acid standards, and dried to completion in a SpeedVac. The dried sample was resuspended in 200 µL of 100% HPLC-grade water, vortexed, centrifuged, and the final supernatant was transferred to an MS vial for analysis. In MS-Dial, these samples were run against the corresponding internal standard library constructed previously to determine efficacy.
RESULTS:
In HILIC mode, many of the 24 deuterated amino acids showed consistent retention times across different concentrations. Positively charged amino acids, such as histidine and arginine, ionized more effectively in positive ion mode, while negatively charged amino acids, such as glutamic acid and tyrosine, ionized more effectively in negative ion mode. More than five amino acids were consistently identified in HILIC mode in plant nectar as the matrix, which is a highly diluted sugar solution. In the more complex bacterial growth medium, BHI, approximately two deuterated amino acids were consistently identified at a minimum spike concentration of 5% in both HILIC positive and negative modes. In C18 mode, most deuterated amino acids eluted within the first one to two minutes, with only a few exceptions such as methionine. This suggests that these polar amino acids are not effective internal standards for C18 chromatography.
CONCLUSION / DISCUSSION:
Overall, this study shows that deuterated amino acids are promising internal standards for HILIC-based metabolomics workflows. However, in C18 chromatography, many deuterated amino acids elute close to the void volume, indicating that they have very low affinity for the nonpolar stationary phase. As a result, these amino acids are unlikely to experience the same chromatographic environment as analytes that interact more strongly with the C18 column, suggesting that they may not be effective internal standards for C18-based assays.
Currently, we are testing chlorpropamide as a new candidate internal standard for constructing an internal standards library in C18 mode. We are also investigating the effect of solvent composition on chromatogram quality. Previously, we used water as the sample solvent, and we are now testing whether changing the solvent to acetonitrile:water (50:50) or 100% acetonitrile affects how samples interact with the polar HILIC column and the nonpolar C18 column. After constructing the internal standard library on the quadrupole time-of-flight mass spectrometer (QTOF), we will apply and further optimize the method on a triple quadrupole mass spectrometer (QqQ), where identified metabolites can be quantified more confidently. |
= Discovery stage. (57.21%, 2026)
= Translation stage. (23.38%, 2026)
= Clinically available. (19.40%, 2026)
