(M0930-06-31) Parallel Accelerator and Molecular Mass Spectrometry for Tracing ¹⁴C-Labeled Metabolites

Purpose: Accelerator mass spectrometry (AMS) is the method of choice for quantitation of low amounts of ¹⁴C-labeled biomolecules. Despite sensitivity as low as 50 zmol of ¹⁴C, an important limitation of AMS is its inability to provide structural information about the analyte. This limitation is not critical when the labeled compounds are well-characterized prior to AMS analysis. However, analyte identity is important in other experiments where, for example, a compound is metabolized and the structures of its metabolites are unknown. We previously described a moving wire interface that enables direct AMS measurement of liquid sample in the form of discrete drops or HPLC eluent without the need for individual fraction collection, termed liquid sample-AMS (LS-AMS).^(1-3) Here, we report the coupling of LS-AMS with molecular mass spectrometry, providing parallel accelerator and molecular mass spectrometry (PAMMS) detection of analytes separated by liquid chromatography.

Methods: A simplified schematic of PAMMS instrumentation is shown in Figure 1. The liquid sample is separated chromatographically using an Acquity H-Class UHPLC (Waters Corporation, Milford, MA). A post-column flow splitter with a split ratio adjustable from 1:1 to 20:1 was used to split LC eluent between a Waters Xevo G2-XS QTOF-MS and the moving wire interface. The moving wire interface collects the LC eluent on a nickel wire, which passes through an oven where the sample is combusted. The resulting CO₂ is directed into the AMS ion source. The AMS instrument (National Electrostatics Corporation; Middleton, WI) provides time-resolved and quantitative signal from stable carbon and radiocarbon. These data are used to determine carbon ratio and radioactivity. Saccharomyces cerevisiae strain S288C obtained from the American Type Culture Collection (Manassas, VA) was grown in synthetic complete medium (SCM) containing 2% glucose and 6.11 pCi/mL of ¹⁴C Trp (prepared as previously described).⁴ Cell extracts for AMS analysis were obtained at time zero and after 21 hours. Trp and its metabolite kynurenine (Kyn) in the cell extracts were chromatographically separated using previously developed methods.^5,6 An Acclaim Mixed-Mode WAX-1 column (4.6 x 150 mm, 5 µm, 120 Å; Thermo Fisher Scientific, Waltham, MA) was used with a 400 µL/min flow rate and a 30 min run time. Elution was isocratic with a mobile phase consisting of 10 mM ammonium formate + 2.7% acetonitrile at pH 6.4. The flow splitter delivered 125 µL/min to the moving wire. UV absorbance data was collected at 286 and 360 nm, and molecular MS was performed in negative ion mode.

Results: Following cell culture in the presence of ¹⁴C Trp, polar metabolites were extracted and analyzed by PAMMS. Figure 2 shows the overlaid chromatograms generated from a single PAMMS injection. Two peaks detected by LS-AMS and UV absorbance (286 and 360 nm wavelengths) were identified as Trp and Kyn using high-resolution molecular MS. No other ¹⁴C peaks higher than 3.4% relative intensity demonstrates absence of other abundant metabolites. It is possible that other ¹⁴C-labeled metabolites coeluted with Trp or Kyn, which is an inherent feature of liquid chromatography. In this case, LS-AMS would not distinguish between the compounds, but they would still be distinguished by molecular MS. If it was suspected that two radiolabeled compounds were coeluting, the chromatography could be modified to separate those peaks. The separation and identification of ¹⁴C-labeled Trp and Kyn in a biological system demonstrates the utility of PAMMS for tracing a ¹⁴C precursor and providing definitive identification of the precursor and its metabolites by alignment of chromatographic, UV absorbance, molecular MS and AMS data.

Conclusion: The low limits of detection of AMS affords possibilities for detection of isotopically labeled compounds that is not feasible using stable isotopes. The major limitation of AMS is its inability to provide structural identification of analytes. PAMMS is a breakthrough which enables simultaneous identification and quantitation of ¹⁴C-labeled analytes in real time while drastically reducing sample preparation. Experiments are ongoing to characterize LS-AMS signal response and improve reliability. The identification of kynurenine originating from ¹⁴C-labeled Trp demonstrates the advantages of a combined platform of molecular mass spectrometry and LS-AMS for the analysis of biological molecules. Chromatographic separation and structural identification are not possible with AMS. This novel capability allows for previously intractable biological experiments in a variety of research areas, including microdosing experiments for ADME, toxicokinetic and pharmacokinetic studies.

References: 1. A.T. Thomas, T. Ognibene, P. Daley, K. Turteltaub, H. Radousky, G. Bench, Ultrahigh Efficiency Moving Wire Combustion Interface for Online Coupling of High-Performance Liquid Chromatography (HPLC), Anal. Chem., 83 (2011) 9413-9417.
2. A.T. Thomas, B.J. Stewart, T.J. Ognibene, K.W. Turteltaub, G. Bench, Directly Coupled High-Performance Liquid Chromatography–Accelerator Mass Spectrometry Measurement of Chemically Modified Protein and Peptides, Anal. Chem., 85 (2013) 3644-3650.
3. T.J. Ognibene, A.T. Thomas, P.F. Daley, G. Bench, K.W. Turteltaub, An Interface for the Direct Coupling of Small Liquid Samples to AMS, Nucl. Instrum. Methods Phys. Res., Sect. B, 361 (2015) 173-177.
4. B.J. Stewart, A. Navid, K.W. Turteltaub, G. Bench, Yeast Dynamic Metabolic Flux Measurement in Nutrient-Rich Media by HPLC and Accelerator Mass Spectrometry, Anal. Chem., 82 (2010) 9812-9817.
5. J. Kim, B. Stewart, R.H. Weiss, Extraction and Quantification of Tryptophan and Kynurenine from Cultured Cells and Media Using a High Performance Liquid Chromatography (HPLC) System Equipped with an Ultra-Sensitive Diode Array Detector, Bio-Protoc., 6 (2016) e1781.
6. A. Laich, G. Neurauter, B. Widner, D. Fuchs, More Rapid Method for Simultaneous Measurement of Tryptophan and Kynurenine by HPLC, Clin. Chem., 48 (2002) 579-581.

Acknowledgements: The authors wish to thank Esther Ubick and Kurt Haack for preparation of the solid graphite AMS targets. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this abstract. This work was performed in part at the National User Resource for Biological Accelerator Mass Spectrometry, which is operated at LLNL under the auspices of the U.S. Department of Energy under contract DE-AC52-07NA27344. The User Resource is supported by the National Institutes of Health (NIH), National Institute of General Medical Sciences (NIGMS) under grant R24GM137748. Reviewed and released as LLNL-ABS-863697.


Figure 1. Simplified schematic of PAMMS instrumentation. The sample is injected by HPLC and chromatographically separated. The eluent is split using a flow splitter between the QTOF-MS and the LS-AMS interface for simultaneous measurement by both instruments.


Figure 2. PAMMS chromatogram overlay of tryptophan metabolism in yeast. 14C-labeled Trp (tR = 16.3 min) and Kyn (tR = 10.4 min) were detected using LS-AMS and UV absorbance (286 and/or 360 nm), and were identified using molecular MS extracted-ion chromatograms. Chromatograms are scaled and shifted to align retention times.