(T1230-10-69) Metabolite Identification of Nucleoside Analog Prodrugs in Biological Matrix by Derivatization Coupled with Radio-Detector and Mass Spectrometry

Purpose: Prodrugs of nucleoside analog are widely designed for the treatment of antiviral infections and anticancer chemotherapy. Metabolite profiing and identification is of utmost importance to understand the pharmacokinetics characters of the prodrugs. However, this task is often very challenging due to their strong polarity of the metabolites, especially for the mono-/di-/tri-phosphate metabolites, and endogenous interference from matrix. In addition, some metabolites are endogenous substances hard to find and distinguish. Using radio-labelled compound as a tracer has a significant advantage of detecting the metabolites from endogenous matrix. This abstract, provides a method of acetylation derivatization coupled with radioactivity determmination to identify the metabolites of nucleoside analog prodrugs in biological matrix.

Methods: In this study, ¹⁴C-labeled DF-006 (adenosine monophosphate derivatives, labelled at adenine moiety), an ALPK1 agonist, was orally dosed to mice. Plasma, urine, feces, and bile samples were collected from the mice. Radiolabeled metabolite profiles of mouse plasma, urine, bile, and feces samples were determined using liquid chromatography coupled with radioactivity monitor (LC-RAM). The structures of detected metabolites were characterized by liquid chromatography coupled with Thermo Q Exactive high-resolution mass spectrometry (LC-HRMS).

Results: A major metabolite, designed as M1, was detected in the radio-profile of urine samples. An ion of m/z 157.0360 at the retention time of the radio-peak M1 was observed in MS spectrum, which potentially corresponds to the [M-H]^- ion peak of allantoin. Meanwhile, the ion peak was also detectable in mouse blank urine samples with similar ion intensity. To investigate further, an acetylation derivatization was conducted for urine samples. 1 mL urine sample collected over 0-24 h postdose was dried under nitrogen stream. After adding 50 μL of pyridine and 400 μL of acetic anhydride, the vial was sealed and heated at 50 ℃ for 4 h. The reaction was terminated by adding methanol followed by drying. The sample was reconstituted and submitted to LC-RAM/HRMS system to obtain radio-profiles and HRMS data. After acetylation, the original radio-peak M1 in postdose urine samples disappeared. Instead, one major and three minor radio-peaks (D1~D4) were observed. The ion peak of m/z 241.0578 at the retention time of the radio-peak D1 was proposed to be the [M-H]^- ion peak of acetylation product of allantoin (allantoin + 2 COCH₂) , and the ion peaks of m/z 265.0578 at the retention time of the radio-peak D2~D4 were proposed to be the [M-H]^- ion peaks of acetylation products of allantoin (allantoin + 3 COCH₂ – H₂O). Therefore, M1 in urine samples was identified as allantoin.

Conclusion: Adenosine monophosphate derivatives may undergo a purine metabolic pathway, leading to the formation of xanthine, uric acid, and allantoin, which can be detected by MS both in postdose samples and blank samples posing challenges in metabolite profiling and identification. Derivatization coupled with radioactivity determination proved to be a useful/valuable method for the identification of the metabolites, such as purine, pyrimidine and their analogs, particularly in the presense of endogenous matrix interference or when the metabolites have poor retention in HPLC systems.

Acknowledgements:We thank Drug Farm team for supporting this work.