Development of biomatrix-based reference materials for accurate quantification of ephedrine in urine using LC-MS/MS

Abstract

Ephedrine has been defined as a threshold substance by the World Anti-Doping Agency (WADA) at a concentration of 10 μg/mL. An Adverse Analytical Finding (AAF) is defined as a concentration of a prohibited substance in a sample that exceeds the threshold concentration within at least a 95% confidence interval. Validated reference materials can be utilized for accurate and quantitative determination of the concentration level. Specifically, biomatrix-based reference materials can improve the quality of results in laboratory testing by better representing actual samples. The developed biomatrix-based reference material was formulated by spiking a mixture of five volunteers&apos; urine with ephedrine at a concentration of 10 μg/mL. Quantitative analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a 20 μL sample. The validation of the analysis method was verified for linearity, accuracy, precision, selectivity, matrix effect, and carryover. Additionally, homogeneity and stability studies were necessary to verify the reference material. To validate this method, seven categories of validation testing were applied with the following results: linearity, R2=0.9997; limit of quantification, 0.5 μg/mL; accuracy, >95%; matrix effect, >99%; carryover, < 10% and %CV < Tcrit). For the homogeneity study, 10 batches were randomly selected from 200 batches and evaluated for within- and between-bottle homogeneity. Short-term stability was evaluated for 4 weeks at 4 ℃, -20 ℃, and ？80 ℃. This study developed a biomatrix-based reference material to accurately quantify ephedrine in urine. This material provides a reliable and stable alternative in quality control when quantifying ephedrine, contributing to accurate and reliable quantitative results.