An article by Stanislas Grassin-Delyle et al at three institutions in the United Kingdom and France, published in the June 2020 issue of Bioanalysis, reported on the successful validation of the antifibrinolytic drug tranexamic acid (TXA) from whole blood samples and dried blood microsamples. Tranexamic acid is commonly used to help stop bleeding by increasing blood clotting, typically for patients prone to heavy menstrual bleeding or frequent nose bleeds.
The paper summarized here is entitled “Tranexamic acid quantification in human whole blood using liquid samples or volumetric absorptive microsampling devices.” It describes a thorough method optimization and validation of this drug from both matrices, using Mitra® devices with VAMS® technology for the volumetric dried blood sampling.
It also discusses testing the method on patients in a phase 1 clinical trial. The research group concluded that these methods can be used in subsequent studies for a better understanding of the pharmacokinetics and pharmacology of tranexamic acid.
A review in the New England Journal of Medicine (2018) reported that around 60,000 Americans die from blood loss every year. Worldwide that amount is nearly two million, and 75% of these cases occur as a result of trauma. Drugs that can safely improve blood clotting to stop life-threatening blood loss are vital to reducing the mortality rate from hemorrhage.
One of the most effective drugs (tranexamic acid) to treat hemorrhage dates to the 1960s, from Utako and Shosuke Okamoto's laboratory in Japan. The drug was developed in an effort to reduce death by blood loss, especially in postpartum hemorrhage – a major killer of Japanese women at the time. Tranexamic acid is a more potent form of Epsilon-aminocaproic acid (EACA), a chemical entity that had been tested by Okamoto's group in the 1950s.
Tranexamic acid is synthetically derived from the amino acid lysine and its key mode of action is to inhibit the enzyme plasmin, which then acts to displace the zymogen plasminogen from the surface of another protein fibrin. Plasmin is a serine protease, which acts to dissolve fibrin blood clots. If plasmin is inhibited, the clots are prevented from being dispersed, thus minimizing blood loss through this mechanism.
Like methotrexate (discussed in a recent blog), another drug that dates back to the 1960s, there is still more to learn about tranexamic acid. Typically, tranexamic acid is given intravenously, especially in patients with acute life-threatening hemorrhage, because IV dosing is a reliable method to rapidly administer the drug.
The pharmacokinetics of other drug administration routes is poorly understood. Due to this lack of pharmacokinetic knowledge, the research group that co-authored the paper summarized here embarked on a feasibility study to investigate alternative dosing routes. The group chose to use Mitra® devices based on VAMS® technology because these are based on volumetric capillary microsampling.
This type of device can be used remotely, which negates the need for phlebotomists since samples can be collected by practically anyone, anywhere and at any time. The researchers commented that processing dried blood VAMS samples would be more efficient during lab extraction as compared to producing serum or plasma before extraction.
Optimizing extraction conditions is critical to delivering successful validations. Stanislas Grassin-Delyle et al, evaluated several conditions and found an optimal procedure that yielded a very high extraction recovery. In terms of choice of extraction solvent, acetonitrile gave poor peak shapes, which was possibly due to injection solvent mobile phase mismatch. However, in contrast, the weaker Methanol / Water extraction solvent yielded very acceptable peak shapes.
It must be noted that if there is a situation where extracting in a strong solvent provides optimal extraction conditions but delivers poor peak shapes, there are several strategies to solve this. The first is to simply dilute the sample with water, post extraction. However here it is important to check for sample solubility. and that there is enough sensitivity for the method. If sensitivity is an issue, and if the sample is weak enough, injecting a larger volume sometimes helps.
Another solution is to inject a smaller volume of the undiluted extract on the LC-MS/MS but, again, you may face sensitivity issues. Finally, a common solution is to evaporate the same to dryness and reconstitute into mobile phase A. This solution can be highly successful, though losses can be observed, with dried analyte sticking to the side of the vessel.
This article 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 blog, visit the original article in Bioanalysis.
Image Credits: iStock, Neoteryx, Trajan Scientific and Medical
Access this microsampling article and others in our Technical Resource Library.