Metabolite profiling with HPLC-ICP-MS - a rapid and simple way to assess in vivo behavior of imaging probes.

Objectives: Determination of pharmacokinetics and quantification of metabolites is a critical step in the development of new drugs including imaging probes. The gold standard for radiolabeled probes is radio-HPLC analysis, but in-line gamma detectors lack sensitivity. This can be overcome by using high amounts of radioactivity with concomitant exposure risk, or collecting fractions and analyzing off-line which is tedious and provides limited chromatographic resolution. ICP-MS is a very sensitive technique for detection of metal ions and metalloids. Here we used HPLC-ICP-MS to measure the pharmacokinetics and metabolism of peptide-DOTA conjugates labeled with stable isotopes of Ga and In at ligand concentrations used in nuclear medicine. We also assessed the ability to simultaneously evaluate In and Ga labeled probes. By using both reverse phase and size exclusion columns we sought to quantify peptide-based metabolites as well as protein-bound transmetallation products.

Methods: Two previously reported peptide-DOTA conjugates, FBP2 and FBP3, chelated with ^natGa and ^natIn were studied using an Agilent 8800 ICP-MS interfaced to an Agilent 1260 HPLC. Ga and In complexes were synthesized and purified using preparative HPLC methods. Probes (150 nmol, 0.6 mL) were injected either individually or as a 1:1 mixture of Ga-FBP2/In-FBP2, or Ga-FBP3/In-FBP3 into Wistar rats via femoral vein catheter. Blood (0.2 mL) was sampled via femoral artery catheter at 5, 10, 15, 30 and 60 minutes post injection and the plasma was taken for two HPLC-ICP-MS analyses. The first used a C18 column (Kromasil 5um C18, 250 x 4.60 mm), 0.8 mL/min, with a water/0.1% TFA/acetonitrile mobile phase and a 5 - 50% gradient of acetonitrile. The second method used a size exclusion column (Phenomenex BioSep-SEC-s2000, 300 x 7.80 mm), 1 mL/min, with a 150 mM NaCl, 50 mM Tris, 5 mM HCO₃ mobile phase. Quantification was performed by comparing peak areas to standard curves of pure compounds.

Results: The probe quantification limit using LC-ICP-MS was 10 pmol for ⁶⁹Ga and 4 pmol for ¹¹⁵In and therefore suitable to detect in vivo probe and metabolite quantities equivalent to < 0.1 % of injected dose. The four conjugates exhibited different behavior. Both Ga-FBP2 and In-FBP2 remained largely intact over the course of the experiment, while Ga-FBP3 and In-FBP3 both showed more probe degradation. Similar results were obtained when we injected a mixture of Ga-FBP2/In-FBP2 or Ga-FBP3/In-FBP3 into rats and simultaneously analyzed for both Ga- and In-containing species. As an example, Panel A shows the plasma pharmacokinetics of intact Ga-FBP3 which has a blood half-life of 5.95 min. We observed a number of Ga-containing metabolites by reverse phase HPLC (Panel C) and these were grouped based on their retention times and quantified (Panel B). The fraction of detected Ga as intact probe decreases with time and the fraction of metabolites eluting from 3-5 min increases with time. Panel D shows a size exclusion chromatogram indicating the presence of low molecular weight metabolites as well as the formation of Ga-transferrin, confirmed by comparison with a Ga-transferrin standard.

Conclusion: HPLC-ICP-MS is a sensitive and radiation-free technique to characterize the pharmacokinetics and metabolism of imaging probes at the low concentrations used in nuclear imaging. Probe-based metabolites as well as transmetallation products can be identified by reverse phase and size exclusion chromatography. Mixtures of probes with different labels can be administered to an animal and analyzed in a single run. Ga and In were evaluated here but the technique is applicable to probes containing other radiometals or metalloids like Sc, Zr, Y, Lu, Re, Bi and I. Research Support: HL109448 (Caravan) and K99HL125728 (Boros) from the NHLBI and an Agilent Technologies, Inc. Research Project Grant.

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