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Abstract INTRODUCTION:
5-Fluorouracil (5-FU) is an antimetabolite and one of the most frequently used cytostatics in oncology for systemic tumor therapy (chemotherapy). It can be either administered directly by transfusion or as prodrug (capecitabine, tegafur). The prodrugs are metabolized into 5-fluorouracil after absorption in the liver. 5-FU is metabolized into Dihydro-5-Fluorouracil (5-DHFU) by the dihydropyrimidin-dehydrogenase (DPD), which under normal physiological conditions metabolizes uracil (U) into 5,6-dihydro-uracil (DHU), in the liver as well as mononuclear cells of the immune system. To exert a substantial anti-tumor effect, it is necessary to maintain high levels of 5-FU in the blood and tissues. 5-FU is associated with potential severe adverse events leading in some cases to substantial morbidity and mortality. DPD deficiency is thought to be the main mechanism leading to 5-FU accumulation and toxicity. Identifying patients with DPD deficiency and notably patients with complete deficiency, is then a relevant approach to limit the risk of severe adverse event (Tron, 2021). A side from PCR diagnostics of DPD mutations, LC-MS/MS has been shown great potential for initial phenotyping of DPD deficient patients by measuring U and DHU as surrogate markers for DPD activity as well as measuring the prodrugs, 5-FU and 5-DHFU as therapeutic drug monitoring (TDM) approach. We present here a prototype of a commercial diagnostic kit for the phenotyping and TDM of the aforementioned drugs and metabolites.
METHODS:
For the measurement of capecitabine, tegafur, U, DHU, 5-FU, 5-DHFU a prototype of the commercial LC-MS/MS kit (Immundiagnostik AG, Germany) was used. Analytes were extracted from 50 µl sample (serum, EDTA, citrate and lithium-heparin) or CAL/CTRL (6 CALs/ 3 CTRLs) by protein precipitation after application of 13C/15N/D labelled internal standards. Subsequently, supernatants were collected, evaporated, reconstituted and 5 µl injected into an Exion/ QTRAP 5500 system (Sciex). Total cycle time between injections was 10 mins.
RESULTS:
The chromatographic run allowed baseline separation of all analytes in all matrices in a run of 10mins. EDTA plasma gave the highest signal responses in comparison to all other matrices. The measuring interval covered the complete physiological and treatment related range (5-2500ng/mL 5-FU r²=0.998; 5-5000ng/ml 5-DHFU r²=0.996; 2-500ng/mL U r²=0.996; 50-5000ng/ml DHU r²=0.998; 2-1250ng/ml capecitabin r²=0.995; 10-5000ng/mL Tegafur r²=0.997). Precision and repeatability was <15/20% as stated by the FDA guidelines.
CONCLUSION:
The prototype of the commercial kit allowed robust quantification from all matrices for the physiological and treatment related ranges of all analytes. While the developed chromatographic run allowed baseline separation of all analytes, the final runtime has further room for improvement. Moreover, stability of real clinical samples, due to prolonged enzymatic activity in the given matrices needs to be firmly investigated, in order to achieve robust area under the curve determination for the given drugs. After the final adaptation of the chromatographic separation full validation under the IVDR and a clinical study for the TDM purpose as well as phenotyping of patients is planned.
REFERENCES:
Tron C, Lemaitre F, Boisteau E, Sourd SL, Lièvre A. When helping the minority of patients may hurt the majority: The case for DPD phenotyping and 5-fluorouracil therapeutic drug monitoring. Dig Liver Dis. 2021 Feb;53(2):258-260. doi: 10.1016/j.dld.2020.11.004. Epub 2020 Nov 20. PMID: 33229275.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
