Development and Validation of an LC-MS/MS-Based Screening Method for Determination of Salbutamol in Urine for Doping Control Using a Surrogate Analyte Approach with Stable Isotope-Labeled Salbutamol (Salbutamol-d9).

Authors

Zeinab Srour, Farzad Kobarfard, Zahra Sheikholislam, Mehrdad Faizi, Bahram Daraei

Published in

Iranian journal of pharmaceutical research : IJPR. Volume 25. Issue 1. Pages e169531. Epub Aug 19, 2026.

Abstract

Athletic doping has long received attention in the sports community. Salbutamol is prohibited by the World Anti-Doping Agency (WADA) both in and out of competition, with a urinary concentration threshold of 1000 ng/mL defining the legal limit. Traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical methods that use an internal standard cannot fully overcome matrix effects and biological variation, requiring revalidation for different biological samples; this process can be time-consuming and can remain prone to matrix-related errors.
To develop a novel approach for screening salbutamol in urine that overcomes the limitations of traditional LC-MS/MS methods and improves analytical reliability and efficiency across biological matrices.
A surrogate analyte-based LC-MS/MS method was developed for the quantitative determination of salbutamol in human urine, using salbutamol-d9 as a stable isotope-labeled surrogate analyte. During method optimization, concentrations ranging from 1 ppm to 0.1 ppb were analyzed in aqueous and urine samples to evaluate extraction efficiency, purification, and matrix effects. Molar-equivalent concentrations of unlabeled salbutamol (1000 ng/mL, the WADA legal limit) and labeled salbutamol (1039 ng/mL) were analyzed in both matrices to validate the surrogate analyte approach and confirm comparable peak areas. To evaluate the applicability of the method, urine samples were collected from healthy volunteers after oral administration of 4 mg salbutamol. All samples were extracted under alkaline conditions using liquid-liquid extraction and analyzed by LC-MS/MS in positive-ion mode with multiple reaction monitoring (MRM). The labeled compound served as a reference for interpretation of the results. Method validation was performed according to international guidelines, including assessments of linearity, precision, accuracy, and sensitivity.
The method demonstrated excellent linearity (R² > 0.99), with intra- and inter-day precision within 8% RSD and acceptable accuracy. The limits of detection (LOD) and quantification (LOQ) were 0.3 and 1 ng/mL, respectively. Analysis of urine from healthy volunteers following a 4 mg oral dose confirmed reliable quantification for up to 24 hours, demonstrating applicability to biological matrices.
These findings indicate that this isotope-based surrogate analyte approach eliminates the need for calibration curves, compensates for matrix effects and sample preparation errors, and improves analytical reliability and efficiency across biological matrices.

PMID:
42763648
Bibliographic data and abstract were imported from PubMed on 20 Sep 2026.

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