(T1130-11-63) Optical Radiocarbon (¹⁴C) Tracing for Biological and Pharmaceutical Applications

Purpose: Radiocarbon (¹⁴C) is a universally applicable, long-lived (half-life of ~5300 years) isotope tracer for biological and pharmaceutical investigations. Given its natural rarity (¹⁴C/C ≈ 1.2 parts-per-trillion), the distribution and metabolic fate of a ¹⁴C-labeled compound in the system of interest can be traced with a very high signal-to-background ratio. Currently, the accelerator mass spectrometry (AMS) technique is used for studies that require very low dosages of the ¹⁴C-labeled chemicals. However, the high investment and operational cost of AMS (multi-million dollar in equipment and staff) and its long sample turnaround time with conventional analysis of graphitized samples have limited the wider adoption of the technique in biological and pharmaceutical applications. In the past decade, laser-based ¹⁴C quantitation has been proposed as a cheaper, higher-throughput, table-top alternative to AMS. Here, we demonstrate the feasibility of a mid-infrared ¹⁴C detector, based on two-color cavity ringdown spectroscopy (2C-CRDS) [1-3] (Figure 1), for low-level ¹⁴C isotope tracing studies. With 2C-CRDS detection, we have previously demonstrated the most sensitive and quantitatively accurate optical measurements of ¹⁴CO₂ [3]. The current quantitation limit of the instrument, at a few per mille of ¹⁴C natural abundance, is competitive against AMS. By measuring the mole fraction of ¹⁴CO₂ in the combusted CO₂ samples from two series of complementary experiments relevant to biological and pharmaceutical studies, we demonstrate here sub-fCi level quantitation of sample ¹⁴C activity, with a minimum sample-size requirement of 3 mg carbon.

Methods: In the first series of experiments, the 2C-CRDS sensor was used to analyze the ¹⁴C activities in plasma samples from a multi-dose drug metabolism animal study. The measurements covered the entire time sequence, from pre-dose to 168 hours. The second series of experiments mimics the chemical separation, identification, and analysis of metabolites from ¹⁴C-labeled compounds used for pharmacokinetic studies. Specifically, following injection of a mixture of ¹⁴C-labeled amino acids (tryptophan and alanine) into a high-performance liquid chromatography (HPLC) instrument, the ¹⁴C content in the collected fractions of the HPLC eluent was quantified by 2C-CRDS. For the analysis of these HPLC fractions, the samples were combusted in a flash pyrolysis oven, which is directly coupled to the 2C-CRDS instrument. Each sample measurement currently takes ~25 minutes, which is predominantly spent on CO₂ gas trapping and processing for the 2C-CRDS instrument.

Results: In Figure 2, we show a comparison of the 2C-CRDS and AMS measurements of the ¹⁴C specific activity (in the unit of fCi/mg C) in the plasma samples from the multi-dose animal study. The 2C-CRDS results agree within a few percent of the AMS values. Such discrepancy is within the range of sample preparation errors typical of biological samples in our laboratory. In Figure 3, we show the 2C-CRDS measurements of the total ¹⁴C activity in the collected fractions of the HPLC eluent of a two-component amino acid mixture. Thanks to the sub-fCi quantitation uncertainty, clear ¹⁴C signals from the HPLC-separated tryptophan and alanine are observed in our experiment. The relative intensity of the two corresponding ¹⁴C peaks from the 2C-CRDS measurements reproduces that observed in the AMS moving wire experiment.

Conclusion: In this work, we demonstrate laser-based ¹⁴C tracing for biological applications using the 2C-CRDS technique. In terms of both ¹⁴C quantitation limit (70 aCi) and the sample size requirement (minimum ~3 mg C), the measurement capability of the current 2C-CRDS ¹⁴C sensor is competitive against AMS analysis of static graphitized biological samples (20 aCi and minimum ~1 mg C). Furthermore, the sample throughput of the current setup (25 minute/sample) is much higher than AMS that requires sample graphitization (at best a few hours/sample, even with full automation). Preliminary results of flow-through detection of ¹⁴CO₂ from flash pyrolysis suggest the possibility of an even higher measurement throughput of a few minutes per sample. This measurement rate, if realized in the future, will be highly valuable for routine application of low-level ¹⁴C quantitation in pharmaceutical and clinical studies, especially considering the much lower cost of this optical-based sensor compared to an AMS instrument.

References: [1] J. Jiang and A. D. McCartt, "Two-color, intracavity pump–probe, cavity ringdown spectroscopy," The Journal of Chemical Physics, vol. 155, no. 10, pp. 104201-104201, 2021.
[2] A. D. McCartt and J. Jiang, "Room-temperature optical detection of ¹⁴CO₂ below the natural abundance with two-color cavity ring-down spectroscopy," ACS Sensors, vol. 7, no. 11, pp. 3258-3264, 2022.
[3] J. Jiang and A. D. McCartt, "Mid-infrared detection with parts-per-quadrillion quantitation accuracy: Expanding frontiers of radiocarbon sensing," Proceedings of the National Academy of Sciences, vol. 121, no. 15, pp.e2314441121, 2024.

Acknowledgements: Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (Award Number R01GM127573). 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 DE548 AC52-07NA27344. The User Resource is supported by the National Institutes of Health, National Institute of General Medical Sciences under grant R24GM137748.


Figure 1. 2C-CRDS measurements of ¹⁴CO₂. (a) Simplified experimental schematics, with two lasers (pump and probe), an optical cavity that contains the test gas, and selected optical components. (b) Time traces of the pump and probe signals. Note that the pump is switched off during alternative probe signals. (c) Principle of 2C-CRDS detection. The v=0, 1, and 2 are energy levels of the target analyte. The pump laser moves population from v=0 to an initially unpopulated v=1 level. The probe detects a transition from v=1 to an even higher-energy v=2. (d) Background compensation. The unwanted background signal and its fluctuation are cancelled out in the net 2C-CRDS spectrum (i.e., the difference between the “Pump-on” and “Pump-off” signals).


Figure 2. Comparison of 2C-CRDS and AMS measurements of ¹⁴C specific activities of plasma samples from the multi-dose drug metabolism study. The first (pre-dose) and last samples were only analyzed by 2C-CRDS due to limited sample quantity. Note that sample preparation error led to the loss of one of the 2C-CRDS samples (at Time=100 hr).


Figure 3. Measurements of the total ¹⁴C activity in the HPLC eluent of a mixture solution of ¹⁴C-labeled tryptophan and alanine, by 2C-CRDS (fraction collection) and AMS (moving wiring). The extracted ion chromatograms (EIC) of the two amino acid components (which are predominantly unlabeled in the mixture solution) are also shown here. For the 2C-CRDS measurements, the total ¹⁴C activity from the added sucrose (~28 fCi) to each HPLC fraction has been subtracted from each measurement.