¹³C-Radiolabeling Service

The ¹³C stable isotope labeling service provides custom synthesis of non-radioactive tracers, enabling precise quantification of metabolic pathways and pharmacokinetics via mass spectrometry and nuclear magnetic resonance (NMR), and other usable technologies. Due to their expertise and competence in advanced organic synthesis, Alfa Cytology can offer high purity, custom labeling of ¹³C-compounds, providing the critical tools required for definitive research outcomes.

Introduction to ¹³C Isotopes

Carbon-13 is an isotope, nonradioactive and more stable, which is used as a tracer candidate in biological and chemical research. Because ¹³C is chemically identical to ¹²C, it can be incorporated into any organic compound without changing its function, thus allowing it to be tracked anytime. Unlike its radioactive counterparts, ¹³C is detected by its unique mass via mass spectrometry or its nuclear spin in NMR spectroscopy.

This is immensely beneficial for quantitative applications such as metabolic flux analysis and pharmacokinetic studies, as it gives detailed information without the use of ionizing radiation.

Overview of ¹³C-Labeling for Radiopharmaceuticals

In pharmaceutical development, synthesizing stable, non-radioactive isotopic tracers is done using stable ¹³C-labeling. These isotopic tracers, which are chemically identical to the active pharmaceutical, enable the radiation-free tracking of the drug's biological fate. Consequently, the ¹³C-labeled compounds serve as definitive tracers for elucidating metabolic pathways, microdosing studies for assessing absolute bioavailability, and as gold-standard internal standards in quantitative bioanalysis to achieve high-accuracy and high-precision measurements.

Fig.1 Overview of ¹³C isotope tracing of metabolism in TBI. (PEPER C J, et al., 2024)

Our Services

Alfa Cytology has a professional team that works on complex and multi-step organic synthesis, which aims at incorporating excels at incorporating Carbon-13 at any desired position within a molecule. This ensures the delivery of high-purity and stable isotope-labeled compounds, and provides tracers and the critical internal standards for mass spectrometry and advanced DMPK studies.

Custom ¹³C Conjugation Service

¹³C conjugation service employs advanced bioconjugation chemistry to stably attach ¹³C-labeled probes to larger macromolecules and forge intricate tools for structural analysis by NMR and for quantitative tracking by mass spectrometry.

Small-Molecule Radionuclide Conjugate (SMRC)

Antibody-Radionuclide Conjugate (ARC)

Peptide-Radionuclide Conjugate (PRC)

siRNA-Radionuclide Conjugate

Workflow for Developing ¹³C-Radiolabeled Small Molecule Conjugates

¹³C Custom Radiosynthesis

• Design Labeling Position: A well-thought-out labeling strategy is the key to successful radiosynthesis. Our process starts with a consultative collaboration and an initial detailed analysis of your target molecule. Examine the drug’s chemical structure closely to select the most metabolically stable position for the ¹³C label. The ¹³C should be placed on the central scaffold so that it is retained after metabolic breakdown. This will help in tracking as many metabolites as possible.
• Design Synthesis Route: After defining the target position, we move on to thoughtful, practical planning and preparation. This stage is crucial to de-risking the project and ensuring smooth synthesis. We develop an efficient and reliable chemical synthesis route to fix the position of the ¹³C atom in the molecule at the predetermined position.
• Custom Radiosynthesis: The next step is performing the synthesis to generate your high-specific-activity ¹³C-labeled compound under controlled conditions. The synthesis is performed by our expert radiochemists in specialized laboratories equipped with modern technology. For safety, reproducibility, and efficiency, we observe the prescribed protocols.

Purification & Quality Control

• Purification & Isolation: Isolate and purify the target ¹³C-labeled product from the reaction mixture using methods such as high-performance liquid chromatography (HPLC).
• Structure Confirmation: The chemical structure is typically confirmed by comparing the product to a "cold" (non-radioactive) reference standard using mass spectrometry and NMR.
• Quality Control (QC) Certification:
  • Chemical Purity: We use analytical HPLC with UV/VIS or CAD (Charged Aerosol Detection) to assess the amount of non-radiolabeled chemical impurities, which include solvents, buffers, and excess linker molecules.
  • Radiochemical Purity: Ascertained via radio-HPLC to confirm the radioactivity signal comes solely from the target molecule.
  • Specific Activity: This is a calculated parameter that defines the radioactivity per unit mass of the compound, determined by total radioactivity and the accurate concentration of the chemically conjugated compound.

In Vivo Study

Systematically collect a complete set of biological samples at various time points post-dose, including:

• Blood/Plasma: For pharmacokinetic (PK) analysis.
• Urine & Feces: For mass balance and determination of excretion pathways.
• Expired Air: To trap ¹³CO₂ if the label is in a metabolically labile position.
• Bile: For investigating hepatobiliary excretion in certain animal models.
• Tissues: At the terminal timepoint in preclinical studies for quantitative whole-body autoradiography (QWBA).

Bioanalytical Sample Analysis

• Total Radioactivity Measurement: The entirety of ¹³C radioactivity within the biological samples can be gauged using a scintillation counter (LSC). This will be used to calculate mass balance (recovery) as well as to define PK profiles.
• Metabolite Profiling: Analyze extracts from plasma, urine, and feces using HPLC with inline radio-detection. This process yields a "radiochromatogram," quantifying the parent drug and depicting the abundance of all major metabolites.
• Metabolite Identification: Couple the radio-chromatography data with high-resolution mass spectrometry (LC-MS/MS) to determine the chemical structures of the major metabolites detected.
• Quantitative Whole-Body Autoradiography (QWBA): In a preclinical study, animal carcasses are frozen, prepared into thin whole-body sections, then the ¹³C signal from the drug is visualized and quantified by means of phosphor imaging, and its distribution of all the tissues and organs is mapped out.

Applications of ¹³C-Radiolabeling

As a non-radioactive stable isotope tracer, ¹³C-labeling is primarily used with mass spectrometry and NMR spectroscopy to quantify metabolic fluxes, trace pathway dynamics, and explain mechanisms of biotransformation.

The team of synthetic chemists at Alfa Cytology has perfected the intricate organic synthesis involving multiple steps for custom ¹³C stable isotope labeling, thus enabling us to incorporate the label at any chosen position within the compound while maintaining utmost isotopic purity. Please get in touch with us to discuss the details of your project, and allow our specialists to provide you with the critical and high-purity tracer to accelerate your DMPK and metabolic research program.

Reference

1. PEPER C J, KILGORE M D, JIANG Y, et al. Tracing the path of disruption: (¹³)C isotope applications in traumatic brain injury-induced metabolic dysfunction [J]. CNS neuroscience & therapeutics, 2024, 30(3): e14693.

For research use only. Not intended for any clinical use.