measuring tranexamic acid from microsamples to control hemorrhage
by James Rudge, PhD, Technical Director, Neoteryx on Aug 29, 2022 9:00:00 AM
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.
Impact of Uncontrolled Bleeding, or Hemorrhage
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.
Research into Tranexamic Acid
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.
Tranexamic Acid Study Method and Findings
- Liquid whole blood and dried blood Mitra-VAMS devices (both 10 µL) were compared.
- Both matrices were extracted in Water / Methanol (20/80, v, v) plus Internal standard (IS).
- Extraction from VAMS was optimized requiring increased shaking time and sonication. Extraction recoveries were high (93-101%).
- A hematocrit range (30-70%) was then tested; researchers concluded that hematocrit did not affect tranexamic acid measurements with VAMS devices. When acetonitrile was evaluated, it yielded very poor peak shapes.
- Analysis was conducted using LC-MS/MS.
- Successful method validation was carried out in accordance with European Medicines Agency and US Food and Drug Administration (FDA) guidelines for bioanalytical method development.
- Highlights of the method validation are listed as follows:
- No carryover occurred even after ULOQ.
- LLOQ was set to 0.1 mg/L where both matrices passed both intra- and inter-day accuracy and precision.
- Matrix effects of 19.2-38.7% were observed at two concentration levels but the IS was able to compensate and the CVs of the IS normalized method were lower than 10%.
- The VAMS samples were stable for 1 month at 50° C
- No carryover occurred even after ULOQ.
- Suitability of the method was piloted on 5 volunteers who were participating in a phase 1 clinical trial entitled “Pharmacokinetics of Tranexamic Acid After Oral, Intramuscular or Intravenous Administration: A Prospective, Randomised, Cross-over Trial in Healthy Volunteers. (PharmacoTXA).”
- After each dosing event, both venous samples and capillary VAMS samples were collected. Venous samples were stored at -80° C and VAMS samples were stored at room temperature before analysis. Excellent agreement was observed between both matrices and the group commented that this demonstrated the feasibility of measuring TXA in those whole blood samples.
- A clinical validation study is ongoing.
Tranexamic Acid Study Authors’ Conclusions
- This was the first method to quantify tranexamic acid in liquid whole blood and dried VAMS samples.
- The assay was successfully validated and even with only 10 µL microsamples, the method showed a high dynamic range.
- Both matrices showed excellent agreement, and both can be used in studies to understand the pharmacokinetics and pharmacology of tranexamic acid (TXA).
- Tranexamic acid is highly stable at 50 °C on VAMS devices, which allows researchers to perform further pharmacokinetic studies to improve the dosing regimens of all patient populations.
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
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