Development and validation of an LC–MS/MS method for the quantification of ephedrines in urine

The objective of this study was to develop a simple and robust LC–MS/MS method for the quantification of ephedrine type substances in urine. Sample preparation consisted of a 10-fold dilution step of the samples into the internal standard solution (ephedrine-d3, 4 μg/mL in water). Baseline separation of the diastereoisomers norpseudoephedrine–norephedrine and ephedrine–pseudoephedrine was performed on a C8-column using isocratic conditions followed by positive electrospray ionisation and tandem mass spectrometric detection. The mobile phase consisted of 98/2 (H2O/ACN) containing 0.1% HAc and 0.01% TFA. Calibration curves were constructed between 2.5 and 10 μg/mL for norephedrine and norpseudoephedrine and 5 and 20 μg/mL for ephedrine, pseudoephedrine and methylephedrine. The bias ranged from −5.5 to 12% for norephedrine, −4.1 to 8.0 % for norpseudoephedrine, 0.3 to 2.1 % for ephedrine, 1.6 to 2.6 % for pseudoephedrine and 2.9 to 5.0 % for methylephedrine. Precision of the method varied between 2.8 and 10.4% for all compounds and the matrix effect was less than 15%. The applicability of the method has been checked by the analysis of 40 urine samples. The results were compared with those obtained with the common GC–NPD method. Results show a good correlation between both methods with correlation coefficients higher than 0.95 for all analytes.