Fully automated LC-MS/MS method to assess DPD deficiency in Cancer treatment with 5-FU

Importance of the Topic

The activity of dihydropyrimidine dehydrogenase (DPD) is a critical determinant of patient safety during fluoropyrimidine chemotherapy. Deficiency in DPD can lead to severe or life-threatening toxicities when administering drugs such as 5-fluorouracil or capecitabine. Monitoring the plasma ratio of uracil to dihydrouracil (UH2/U) provides an indirect but comprehensive phenotyping approach that captures all functional deficits of DPD. A rapid, robust and fully automated analytical workflow is essential to ensure high-throughput screening, timely clinical decision-making and minimized workload in busy diagnostic laboratories.

Objectives and Study Overview

This work aimed to develop and validate a fully automated liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of uracil and dihydrouracil in human plasma. The goal was to integrate sample preparation with direct coupling to LC-MS/MS instrumentation, reducing manual steps and improving reproducibility. The method was benchmarked against an existing routine assay at a university hospital to confirm its accuracy and clinical applicability.

Methodology and Instrumentation

Sample preparation was performed on a CLAM-2030 robotic liquid handler, directly coupled to a Shimadzu LCMS-8060 triple quadrupole mass spectrometer. Key steps included:

• Addition of isopropanol, sample and stable isotope-labeled internal standards
• Protein precipitation with acetonitrile and formic acid
• Automated mixing, filtration and dilution

Chromatographic separation used a Hypercarb 2.1 × 150 mm column with water and acetonitrile both containing 0.5% acetic acid, at a flow rate of 0.25 mL/min and 25 °C. Detection employed positive electrospray ionization with multiple-reaction monitoring transitions for native and labeled analytes.

Key Results and Discussion

The calibration range covered 5–320 ng/mL for uracil and 10–640 ng/mL for dihydrouracil, with linearity (R2 > 0.99). Intra- and inter-assay precision showed relative standard deviations below 9% around critical clinical thresholds of 16 and 150 ng/mL for uracil. Accuracy ranged between 85% and 104% across quality controls. Application to 64 patient samples revealed excellent correlation (R2 ≈ 0.998) with the routine method, and Bland-Altman analysis confirmed agreement within ±2 standard deviations.

Benefits and Practical Applications

The fully automated workflow delivers:

• High throughput with parallel sample preparation and analysis
• Reduced manual handling and potential errors
• Robust quantification meeting ISO-15189 validation criteria
• Timely and reliable DPD status determination to guide personalized dosing

Implementation in clinical laboratories can improve patient safety by ensuring accurate assessment of DPD deficiency before fluoropyrimidine therapy.

Future Trends and Applications

Advances may include:

• Integration of additional pyrimidine metabolites or pharmacogenetic markers
• Cloud-based data processing and AI-driven quality control
• Miniaturized and point-of-care LC-MS platforms
• Expansion to other therapeutic drug monitoring applications

Such developments will further streamline personalized oncology and broaden the scope of automated bioanalysis.

Conclusion

A fully automated LC-MS/MS method for uracil and dihydrouracil quantification has been established and validated. It offers high accuracy, precision and throughput, with direct benefits for clinical management of cancer patients receiving fluoropyrimidines. The workflow’s automation and robust performance support its adoption in diagnostic and pharmaceutical settings.

Instrumentation Used

• CLAM-2030 robotic liquid handler
• Shimadzu LCMS-8060 triple quadrupole mass spectrometer
• Hypercarb 2.1 × 150 mm, 3 µm analytical column
• Autosampler and Nexera UHPLC system

References

• Rexroth S, Toinon D, Robin T, Moreau S, Franck-Saint-Marcoux F. Fully automated LC-MS/MS method to assess DPD deficiency in cancer treatment with 5-FU. Shimadzu Application Note.