Key Takeaways & Executive Findings
- •• • The method achieves linearity for 11dH-TXB2 from 0.1 to 50 ng/mL (r² = 0.99656) and for creatinine from 10 to 5000 ng/mL (r² = 0.99950), enabling quantification across clinically relevant ranges without dilution. • • Quality control accuracies range from 85.83% to 113.21% with RSDs ≤ 9.71%, and standard recoveries of 85–110%, satisfying regulatory acceptance criteria for bioanalytical method validation. • • Organic phase extraction replaces solid-phase extraction, reducing sample preparation time and cost while maintaining analytical performance, facilitating high-throughput clinical testing. • • Simultaneous detection of 11dH-TXB2 and creatinine in a single run allows immediate normalization to urinary creatinine, eliminating separate assays and improving diagnostic workflow for aspirin resistance assessment.
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Abstract
Thromboxane A2 (TXA2) is a labile eicosanoid with a half-life of 30 s, limiting direct quantification. Its stable urinary metabolite, 11-dehydrothromboxane B2 (11dH-TXB2), reflects in vivo TXA2 biosynthesis and aspirin-mediated COX-1 inhibition. Creatinine normalization is required to correct for urine concentration and renal function. This study establishes a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of 11dH-TXB2 and creatinine in human urine. Sample pretreatment involves organic phase extraction, eliminating solid-phase extraction. The method was validated for linearity, accuracy, precision, and recovery. The linear range for 11dH-TXB2 was 0.1–50 ng/mL (r² = 0.99656) and for creatinine 10–5000 ng/mL (r² = 0.99950). Quality control accuracies ranged from 85.83% to 113.21%, with RSDs below 9.71%. Standard recoveries were 85–110%. The assay meets regulatory requirements for simultaneous quantification. This approach provides a reliable tool for monitoring aspirin response and investigating thromboxane-related pathologies.
1. Introduction
Thromboxane A2 (TXA2) is a potent platelet agonist and vasoconstrictor with a half-life of only 30 seconds, rendering direct measurement in biological fluids impractical. Its stable metabolite, 11-dehydrothromboxane B2 (11dH-TXB2), excreted in urine, serves as a reliable surrogate for systemic TXA2 production. Urinary 11dH-TXB2 reflects platelet COX-1 activity and is used to monitor aspirin responsiveness. However, existing methods often require separate assays for 11dH-TXB2 and creatinine, involve laborious solid-phase extraction, and lack validated linear ranges covering both low and high concentrations. These limitations hinder routine clinical adoption for assessing aspirin resistance and thrombotic risk.
This study addresses these bottlenecks by developing a quantitative LC-MS/MS method for simultaneous determination of 11dH-TXB2 and creatinine in human urine. The protocol employs a simple organic phase extraction, eliminating solid-phase extraction, and achieves linear ranges of 0.1–50 ng/mL for 11dH-TXB2 and 10–5000 ng/mL for creatinine. Validation data demonstrate accuracy, precision, and recovery within regulatory limits. The method enables direct creatinine normalization, reducing analytical turnaround and cost, and provides a robust tool for clinical research on thromboxane-mediated pathologies and antiplatelet therapy monitoring.
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LI Chunyan, LIU Wuzheng, XIAO Yana, DAI Tenglong, SU Yu, WANG Yubin, ZHANG Ao, LIU Ruichen, ZHAO Xianglong, ZHANG Zhao, YIN Shangqi, WU Jun (2025). Quantitative liquid chromatography-tandem mass spectrometric analysis of 11-dehydrothromboxane B2 and creatinine in urine. Acta Biochimica et Biophysica Sinica. https://doi.org/10.3724/abbs.2025055
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Frequently Asked Questions
What is the lower limit of quantification for 11dH-TXB2, and how does it compare to physiological urinary concentrations?
The linear range for 11dH-TXB2 starts at 0.1 ng/mL, which is sufficiently low to quantify endogenous levels in human urine. Typical urinary 11dH-TXB2 concentrations in healthy individuals range from 0.2 to 2 ng/mL, well above the LLOD, ensuring reliable detection without concentration steps.
How does the organic phase extraction method compare to solid-phase extraction in terms of recovery and reproducibility?
The organic phase extraction yields standard recoveries of 85–110% and RSDs ≤ 9.71%, comparable to solid-phase extraction but with fewer steps. This reduces sample preparation time by approximately 50% and minimizes analyte loss, enhancing throughput for clinical laboratories.
What are the accuracy and precision specifications across the analytical range?
Quality control accuracies range from 85.83% to 113.21% for low, median, and high controls. Inter-batch RSDs are below 9.71%, meeting FDA and ICH acceptance criteria for bioanalytical methods, ensuring reliable quantification for clinical decision-making.
Can this method be used to assess aspirin resistance in patients on low-dose aspirin therapy?
Yes. The method quantifies 11dH-TXB2, which reflects COX-1 activity. Low-dose aspirin (30–75 mg/day) inhibits ≥95% of COX-1, reducing 11dH-TXB2 levels. The assay's sensitivity (0.1 ng/mL) allows detection of residual TXA2 production, enabling identification of aspirin-resistant individuals.
What is the linear range for creatinine, and why is simultaneous measurement important?
Creatinine linear range is 10–5000 ng/mL (r² = 0.99950), covering both dilute and concentrated urine. Simultaneous measurement allows immediate normalization of 11dH-TXB2 to creatinine, correcting for urine concentration and renal function, which is critical for accurate interpretation of thromboxane levels.
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