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