Monitoring 5-Fluorouracil Levels in Dried Blood Spots with UHPLC-MS/MS


Researchers have developed a method to monitor 5-Fluorouracil levels in dried blood spots (DBS) using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This study, completed by the Faculty of Pharmacy of Universitas Indonesia was featured in a recent issue of Heliyon (1).

© Denis-

© Denis-

5-Fluorouracil is a pyrimidine analog compound commonly used to treat stomach, pancreatic, breast, and colorectal cancer. The drug is frequently used during chemotherapy. 5-Fluorouracil has a narrow therapeutic window, and its necessary to monitor its levels in patients. According to the researchers in previous studies, the monitoring of 5-Fluorouracil levels in blood was mostly done in plasma matrices. However, this method required the blood sample to be placed on ice, centrifuged under cold conditions, and the separated plasma had to be frozen immediately, due to 5-Fluorouracil’s instability and rapid metabolism by enzymes in the blood. Utilizing DBS rather than plasma can simplify sample preparation because the DBS samples do not need to be centrifuged, and they can be prepared at room temperature. Furthermore, smaller blood samples are required when sampling with DBS (10–100 μL) as opposed to the plasma method (5–15 mL).

The smaller blood samples mean that there is less chance of a biological hazard occurring, and the ability to be more in compliance with green chemistry principles. Sampling with DBS can also increase the comfort of the patient, as it only requires a finger prick compared to the venipuncture necessary with plasma sampling. DBS is also easier to transport during the testing process, since the blood is already dried; this minimizes the risk of spillage. Taking these factors into account, the researchers aimed to create and support an analysis process for 5-Fluorouracil in DBS utilizing UPLC-MS/MS, with the goal of providing an easier, more time-efficient method of bioanalysis.

The authors of the paper state that the optimization of the analysis conditions was carried out by using standard solutions of 5-Fluorouracil and internal standard propylthiouracil, each at a concentration of 1 μg/mL. The optimization was conducted for MS detection, mobile phase composition and combination, flow rate, and column temperature. The optimization of mass spectrometry detection was performed by infusing a mixture of 5-Fluorouracil standard solution and propylthiouracil internal standard solution into the mass spectrometry equipment. During this process, the voltage on the capillary tube, temperature, desolvation gas flow rate, voltage at the entrance, and voltage in the collision chamber for negative electrospray ionization which gave the highest 5-Fluorouracil and propylthiouracil response were recorded, and these voltages were used during analysis, the researchers wrote in the study.

This method could be used for therapeutic drug monitoring of 5-Fluorouracil in cancer patients, the authors wrote. According to the paper, the method has proven to be accurate, precise, and selective, with a calibration curve range of 0.1 μg/mL to 60 μg/mL. Further clinical research can be conducted to demonstrate that plasma matrix can be substituted with DBS.

Similar research was conducted in a collaboration between Feevale University and the University of Santa Maria (Brazil); findings were published in a recent article in the Journal of Pharmaceutical and Biomedical Analysis (2). These authors concluded that, due to the lack of agreement between the plasma and capillary concentrations in their research, there was a necessity for larger sample sizes in subsequent studies.


  1. Harahap, Y.; Amarta, V.; Saputri, F. A. Development and Validation of UPLC-MS/MS Method for 5-Fluorouracil Quantification in Dried Blood Spot. Heliyon 2024, 10 (9), e29990. DOI: 10.1016/j.heliyon.2024.e29990.
  2. da Silva, L. C.; Ana P. Grando, A. P.; Lucas S. de Baco, L. S.; Roberta Z. Hahn, R. Z.; Antônio F. Ferreira Filho, A. F.; Natália Brucker, N.; Rafael Linden, R.; Marina V. Antunes, M. V. Evaluation of Dried Blood Spots as an Alternative Sampling Strategy for 5-fluorouracil Monitoring: From Method Development to Clinical Application. J. Pharm. Biomed. 2023, 235, 115539. DOI: 10.1016/j.jpba.2023.115539.
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