microsampling to measure anticancer drugs and their metabolites

An article published by Mirjana Radovanovic et al from multiple institutions in Australia, in the January 2022 issue of the Journal of Chromatography B, reported on the development and validation of an LC-MS/MS method of anticancer drugs and metabolites using Mitra^® microsampling devices with VAMS^® technology. The paper is entitled “Measurement of 5- fluorouracil, capecitabine and its metabolite concentrations in blood using volumetric absorptive microsampling technology and LC-MS/MS.”

It describes the development of 5-fluorouracil, capecitabine and its metabolite concentrations bridging capillary and venous blood collected from 20 patients in a study. The researchers were able to conclude that blood collection using volumetric absorptive microsampling proved to be as reliable as conventional blood collection for 5-FU and capecitabine.

It is important to know, however, that the half-life of a 5-FU in wet blood makes wet sampling a challenge, which led to a challenging bridging study. The research group proposed further work to continue to explore if dried blood capillary samples could replace standard venous draws in the future for these analytes.
Background on 5-FU and Other Chemotherapy Drugs

5-Fluorrouracil (5-FU) is a 60-year-old chemotherapy drug used alone or combination with other drugs to treat a variety of cancers such as those found in the gastric tract, pancreas, head and neck. Fluorouracil is antineoplastic anti-metabolite, which blocks an enzyme involved with converting cytosine into the nucleotide cytosine monophosphate. This is an important step in the incorporation of the nucleotide into DNA, and it disrupts DNA synthesis.

Dosing of 5-FU is currently based on body surface area, however, reports in other studies show high interpatient variability. In their study paper, Mirjana Radovanovic et al commented that dose individualization would be a logical approach to reduce toxicity and improve cancer outcomes. Indeed, the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT) recommends TDM of IV 5-FU for colorectal, head and neck cancers. Currently, this is achieved by sampling blood around 18 hours after commencement of treatment.

Capecitabine, an orally administered prodrug of 5-FU, was developed to mimic continuous infusion of 5-FU. Capecitabine can be self-administered but is not routinely monitored via therapeutic drug monitoring (TDM) programs. The research group noted that, perhaps for this reason, serious adverse events associated with capecitabine are less frequently observed in the literature. However, Mirjana Radovanovic et al commented that drug monitoring studies of capecitabine would be of interest for investigating the significance of the drug and its metabolites during long-term administration.

Both 5-FU and capecitabine are easily metabolized, so cold chain shipping and storage of wet blood samples and blood products is critical to achieving reliable measurements. However, cold chain shipping and storage of wet venous blood samples may be impractical in low-resource regions or at some facilities.

Another drawback of venous sampling, as highlighted in the study paper reviewed here, is when PK measurements are needed. PK sampling to obtain individual dosing needs, for example, requires more than one sampling event. This is a concern for critically ill or pediatric populations from both an interventional and a blood volume perspective.

The researchers commented that both issues could be solved by employing remote microsampling, where samples could be collected at home by the patient or study volunteer. Remote microsampling using Mitra devices produces dried blood microsamples for lab analysis. An additional benefit of dried blood microsamples is that these anticancer drugs are stable in dried blood, so cold chain shipping may be eliminated.

Remote microsampling has budgetary benefits as well. The research group reported that their overall cost involved in collecting, sample processing, storage and transport was significantly reduced with microsampling methods. Furthermore, the reduced volume of microsamples is beneficial for pediatric populations, where high-volume blood draws can negatively impact health.

Finally, they highlighted that VAMS was preferable over DBS as it overcomes sample homogeneity concerns with post punch protocols and spot area hematocrit bias. For these reasons, the group embarked on developing an LC-MS/MS assay for 5-FU, capecitabine and metabolites.

Anticancer TDM Study Methods

• Calibrators and QCs were spiked into blood on day of analysis and sampled onto Mitra devices and stored for at least 3h.
  
  
• An organic extraction method was conducted in ACN: water with IS using both sonication and vortexing, dried down and then reconstituted in 0.1% formic acid before analysis.
  
  
• Validation was conducted in accordance with FDA and NATA guidelines. Specifically, linearity, accuracy & precision, specificity & selectivity (including Matrix effect, recovery, and process efficiency assessment), carryover and stability (-30° C, RT, 3 days, 9 months, 2years) was conducted.
  
  
• Both capillary and venous blood samples were collected from cancer patient volunteers. These were both sampled onto Mitra devices for the bridging study.

Study Authors’ Results and Discussion

• The LC-MS/MS was successfully developed.
  
  
• Mixed results were observed for matrix effect, recovery, and process efficiency from the dried blood samples. Indeed, lower than ideal recovery was seen from one lot of Mitra. Interestingly, optimizing extraction time did not improve the results.
  
  
• Stability was excellent at 9 months at RT, 2 years at –30° C, and 3 days at 50° C
  
  
• When measuring 5-FU from IV administered patients, a large variability was observed between paired capillary samples and venous samples collected on Mitra. This was, in part, attributed to the short half-life of 5-FU (15min) and the differences in the sampling times. The quality of the sample collection by the end-user, such as insufficient sampling volume, may also have contributed.
  
  
• For the oral study, where patients were administered capecitabine, the authors stated, “[A] good relationship was observed between the VAMS samples prepared from a capillary blood and those prepared from a venous blood for all the metabolites with slightly weaker relationship for 5′d-FCR (y = 0.6282*X + 194).” However, the small sample size (n-20) contributed, in part, to wide upper and lower confidence intervals from a Bland-Altman bias plot.
  
  
• Further studies are ongoing to explore the feasibility of using microsampling for capecitabine and 5-FU TDM. In future studies, the group wishes to compare plasma levels and capillary blood.

Study Authors’ Conclusions

• An LC-MS/MS method was developed for VAMs extracts to measure capecitabine, 5FU and related metabolites.
  
  
• Further work needs to be conducted to build a relationship between plasma and dried blood.
  
  
• The observed stability of venous blood samples collected onto Mitra devices highlights the potential for their use in transporting blood samples at ambient temperatures versus current methods, which require keeping blood samples cold.

Final Thoughts from Neoteryx

When developing assays on dried blood, analyte stability is absolutely key to good validations. An interesting observation with this study is that when the analytes were dried on Mitra devices with VAMS, they were shown to be very stable. However, when in wet blood, analyte instability made comparing capillary and venous sampling a challenge.

The study authors proposed that the main cause of this was the difference in the timings of the collection between venous and capillary blood of the paired samples, which impacted the reported 5-FU levels as a result of its short half-life. This illustrates again how critical it is to conduct capillary to venous bridging and to design any potential study to sample onto the devices concurrently, especially when analyte stability in the wet matrix is an issue.

One further point to raise was the inconsistency of the absolute recoveries from the microsampling devices. As reported in a previous blog, scientists from Merck demonstrated that moving from an organic extraction method to an aqueous extraction method followed by liquid liquid extraction vastly improved extraction efficiency.

A similar approach may help with improving the accuracy of 5-FU and its related components. We wish the group all the best with the critical work they are conducting in cancer TDM research.

This study paper was summarized for our readers by James Rudge, PhD, Neoteryx Technical Director. This is curated content. To learn more about the important research outlined in this review, visit the original article published in the Journal of Chromatography B.

Image Credits: capecitabine image iStock, Neoteryx

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