Elsevier

Journal of Chromatography B

Volume 877, Issue 4, 1 February 2009, Pages 369-374
Journal of Chromatography B

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

https://doi.org/10.1016/j.jchromb.2008.12.032Get rights and content

Abstract

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.

Introduction

Stimulants are synthetic derivatives of the endogenous stimulant adrenaline and have similar pharmacological effect on mental function and behaviour, producing excitement and euphoria and increase motor activity. One of the oldest therapeutically applied stimulants are the group of the ephedrines. These substances can be found as natural source in Ephedra or Ma Huang (Ephedra sinica). Extracts of these plants are still used nowadays in nutritional supplements [1]. The major active compounds are five optical active compounds including ephedrine (EP), pseudoephedrine (PEP), norephedrine (NEP), methylephedrine (MEP) and norpseudoephedrine (NPEP) also known as cathine (Table 1). PEP is frequently applied therapeutically for the treatment of allergic rhinitis. Preparations containing EP are used for the treatment of cough. Because of the (frequent) therapeutical use of ephedrines the regulations of the World Anti-Doping Agency (WADA) [2] are complicated: PEP and NEP are not on the list and can be used unrestricted. For NPEP a threshold of 5 μg/mL and for EP and MEP a threshold of 10 μg/mL is applied. In anti-doping analysis ephedrines used to be quantified in urine by GC–NPD, GC–MS or HPLC-UV after preconcentrating the sample by liquid–liquid extraction (LLE) [3], [4], [5] or after an online clean-up [6]. The sensitivity of liquid chromatography coupled to mass spectrometry (LC–MS) and the compatibility of the system with the aqueous matrix of urine has allowed quantitative analysis of ephedrines without preconcentration step by direct injection [7], [8]. The aim of this work is to develop and validate a quantitative method for the direct urinary detection of ephedrines by LC–MS/MS. Several chromatographic as well as mass spectrometrical problems encountered during method development are discussed. Finally, the applicability of the developed methods is tested by comparing results with those obtained by the GC–NPD method routinely applied in our laboratory.

Section snippets

Standards and reagentia

EP, PEP, NEP, MEP, ephedrine-d3 (EP-d3) and trifluoroacetic acid (TFA) (LC–MS grade) were purchased from Sigma–Aldrich (Bornem, Belgium). NPEP was purchased from National Measurement Institute (Sydney, Australia). Methanol (MeOH) (HPLC-grade) was purchased from Acros-Organics (Geel, Belgium), acetonitrile (ACN) (HPLC-grade) from Biosolve (Valkenswaard, The Netherlands), acetic acid (HAc) and HPLC-grade water from Fischer Scientific (Loughborough, United Kingdom). Gases used for the mass

Mass spectrometry

Amphetamines contain an amine function which can be easily protonated. Hence, very abundant protonated molecular ions [M+H]+ were observed for all compounds with both APCI and ESI. Deprotonated molecular ions were not detected in negative ionization mode. ESI is routinely used at our laboratory and was further preferred as interface. Because tandem mass spectrometry often results in improved sensitivity this technique was applied. All substances show similar fragmentation behaviour starting

Conclusions

A simple and sensitive method was developed and validated for the quantification of five ephedrine type stimulants. Chromatography was investigated using several C8 type columns.

The Zorbax RX C8 exhibited excellent selectivity and specificity for the investigated substances. Adding TFA to the mobile phase showed to be a useful strategy to improve both peak shape as well as resolution. Sample preparation was minimised by a direct 10-fold dilution of the urine directly into the autosampler vial

Acknowledgements

Postdoctoral grants by the Flemish Ministry of Culture, Youth, Sports and Brussels (PVE and KD) and the Spanish Ministerio de Educacion y Ciencia (OJP) are gratefully acknowledged. The technical assistance of Kris Roels was greatly appreciated.

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