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Journal of separation science2025; 48(10); e70308; doi: 10.1002/jssc.70308

Bioanalytical Uncertainty Assessment of Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry Method for Caffeine and Lidocaine in Equine Antidoping: A Dual Perspective on Bottom-up and Top-Down Approaches.

Abstract: The quality of quantitative results in bioanalysis requires not only a validated analytical method but also a rigorous estimation of measurement uncertainty. This study examines the challenges associated with the implementation of two distinct approaches in equine anti-doping control for the assessment of uncertainty associated with an ultra-high-performance liquid chromatography-high resolution mass spectrometry quantitative method for caffeine and lidocaine in horse urine. The bottom-up approach, based on the ISO Guide to the Expression of Uncertainty in Measurement (ISO GUM), was compared to the top-down approach using β-content, γ-confidence tolerance intervals (β,γ-CCTI) via F-test. The key limitation of the ISO GUM method was accurately quantifying the various uncertainty components; it gives standardized uncertainty estimates but requires detailed assumptions and modeling about error sources. The direct application of the GUM method imposes the beforehand correction of the matrix effect to provide reliable results. Parallelly, the chemometric approach β,γ-CCTI offers more flexible and realistic estimations. Four combinations of β and γ were investigated to assess their influence on uncertainty interval width: β = 66.7% and 80%; γ = 90% and 95%; and the method was evaluated under repeatability and intermediate precision conditions through the use of advanced computation that adjusts for matrix effects and proves more straightforward for capturing variability inherent in experimental data. The top-down approach is a reliable alternative for routine use and, particularly, for ensuring compliance with regulatory requirements, with the fact that a known proportion β of future results will be within predefined acceptance limits.
© 2025 Wiley‐VCH GmbH.
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Publication Date: 2025-10-23 PubMed ID: 41126565DOI: 10.1002/jssc.70308Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) was used to measure caffeine and lidocaine levels in horse urine within an anti-doping context, and the study compared two methods for assessing the uncertainty in these measurements to ensure result reliability.

Research Context and Objective

  • Ensuring high-quality quantitative outcomes in bioanalysis, particularly in equine anti-doping, requires not only validated methods but also an accurate estimation of measurement uncertainty.
  • This study focused on assessing uncertainty in an UHPLC-HRMS method for determining caffeine and lidocaine concentrations in horse urine samples.
  • The main objective was to compare two different uncertainty estimation approaches—a bottom-up approach based on the ISO Guide to the Expression of Uncertainty in Measurement (ISO GUM), and a top-down approach using β-content, γ-confidence tolerance intervals (β,γ-CCTI) with an F-test.

Methods Compared

  • Bottom-up Approach (ISO GUM):
    • Relies on identifying and quantifying individual sources of uncertainty, such as instrument precision, sample preparation, and matrix effects.
    • Gives standardized uncertainty estimates but requires detailed assumptions and models about all possible error sources.
    • Needs correction for matrix effects beforehand, which can complicate obtaining reliable uncertainty values.
  • Top-down Approach (β,γ-CCTI):
    • Based on analyzing observed experimental variation directly without modeling individual sources of errors.
    • Uses β-content, γ-confidence tolerance intervals to define uncertainty, where β is the proportion of future measurement results expected to fall within the interval, and γ is the confidence level of this estimation.
    • Employs an F-test to calculate tolerance intervals that capture overall variability under repeatability and intermediate precision conditions.
    • Handles matrix effects more flexibly and realistically using advanced computation.
    • More straightforward in capturing actual variability present in repeated experimental data.

Evaluation Parameters and Findings

  • The study tested four combinations of β and γ values to examine uncertainty interval width and behavior:
    • β = 66.7% and 80% (proportion of future results contained)
    • γ = 90% and 95% (confidence level in the tolerance interval)
  • Results showed that the top-down approach offered more flexible and realistic uncertainty estimates adaptable to regulatory compliance needs.
  • The bottom-up (ISO GUM) method’s main limitation was its complexity and reliance on accurate quantification of all individual uncertainty sources with necessary matrix effect corrections.
  • The top-down approach was recommended as a reliable alternative particularly suited for routine anti-doping control where known proportions of future results should fall within specified acceptance limits.

Implications for Equine Anti-Doping and Bioanalysis

  • Provides critical insights into improving measurement uncertainty assessment for high-stakes analyses such as anti-doping testing in horses.
  • Suggests that top-down methods like β,γ-CCTI could enhance traceability and confidence in quantitative bioanalytical results with potentially less workload and assumptions compared to traditional bottom-up techniques.
  • Supports regulatory compliance by enabling laboratories to set scientifically justified and statistically sound acceptance criteria for future measurement results.

Summary

  • The paper contributes to methodological improvement in measuring caffeine and lidocaine in equine urine via UHPLC-HRMS.
  • It highlights the advantages of top-down uncertainty evaluation approaches over conventional bottom-up modeling for bioanalytical methods affected by complex matrices.
  • The findings encourage adoption of tolerance interval methods to ensure measurement reliability in routine doping control analyses.

Cite This Article

APA
El-Ghaly W, El Kamli T, Zaari Lambarki L, El Hamdani M, Lahkak FE, Benmoussa A, Balouch L, Bakkali F, Saffaj T, Jhilal F. (2025). Bioanalytical Uncertainty Assessment of Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry Method for Caffeine and Lidocaine in Equine Antidoping: A Dual Perspective on Bottom-up and Top-Down Approaches. J Sep Sci, 48(10), e70308. https://doi.org/10.1002/jssc.70308

Publication

Journal of separation science
ISSN: 1615-9314
NlmUniqueID: 101088554
Country: Germany
Language: English
Volume: 48
Issue: 10
Pages: e70308

Researcher Affiliations

El-Ghaly, Wafaa
  • Drug Sciences Research Laboratory, Faculty of Pharmacy, Mohammed VI University of Sciences and Health (UM6SS), Casablanca, Morocco.
El Kamli, Taha
  • Department of Veterinary Biological Sciences and Pharmaceuticals, Hassan II Agronomic and Veterinary Institute, Rabat, Morocco.
Zaari Lambarki, Lamia
  • Applied Organic Chemistry Laboratory, Faculty of Sciences and Technology, Sidi Mohamed Ben Abdallah University, Fes, Morocco.
El Hamdani, Maha
  • Faculty of Science, Ibn Tofail, Kenitra, Morocco.
Lahkak, Fatima-Ezzahra
  • Department of Veterinary Biological Sciences and Pharmaceuticals, Hassan II Agronomic and Veterinary Institute, Rabat, Morocco.
Benmoussa, Adnane
  • Drug Sciences Research Laboratory, Faculty of Pharmacy, Mohammed VI University of Sciences and Health (UM6SS), Casablanca, Morocco.
Balouch, Lhousaine
  • Drug Sciences Research Laboratory, Faculty of Pharmacy, Mohammed VI University of Sciences and Health (UM6SS), Casablanca, Morocco.
Bakkali, Fadil
  • Drug Sciences Research Laboratory, Faculty of Pharmacy, Mohammed VI University of Sciences and Health (UM6SS), Casablanca, Morocco.
Saffaj, Taoufiq
  • Applied Organic Chemistry Laboratory, Faculty of Sciences and Technology, Sidi Mohamed Ben Abdallah University, Fes, Morocco.
Jhilal, Fayssal
  • Department of Chemistry and Brewing Science, Bishop's University, Sherbrooke, Canada.

MeSH Terms

  • Chromatography, High Pressure Liquid
  • Animals
  • Horses
  • Uncertainty
  • Lidocaine / urine
  • Doping in Sports
  • Caffeine / urine
  • Mass Spectrometry

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