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LCMS methods

Liquid chromatography-mass spectrometry (LC-MS) provides an alternative method for doping control. Presently the AAS are determined after hydrolysis of AAS conjugates by GC-MS. However, the GC-MS method is time-consuming and faster methods are required. Furthermore, the sensitivity with GC-MS in analysis of polar AAS may not suffice. LC-MS provides for polar compounds such as AAS glucuronides fast and highly sensitive method.

The aim of this work was to develop and evaluate screening and confirmation LC-MS/MS method for twelve AAS glucuronides in urine samples. AAS glucuronides were extracted from urine samples by solid phase extraction (SPE). Extraction method, as well as the HPLC-separation method was optimised and validated. The detection is performed by using tandem mass spectrometer combined with electrospray ionisation (ESI). Novel LC-MS/MS methods were validated with respect to specificity, accuracy, precision and limit of detection. Recovery of the SPE extraction varied between 89-100 %.

The quantitative repeatability varied within day from 2 to 10 % (relative standard deviation) and between days from 8 to 32 %. The repeatability of retention time was below 0.1% within day and below 1 % between days. Detection limits in urine were from lng/ml to 40ng/ml, which corresponds to 20-800ng/ml in injected solution. Detection limits in buffer were from 0.1 to 15ng/ml, which corresponds to 2-300ng/ml in injected solution. The results obtained show that the developed LC-MS method provides reliable method for the detection of AAS glucuronides.

However, the limits of detection measured with standards prepared in pure solvent were one-two orders of magnitude lower than those obtained with urine samples. This shows that the selectivity of the method is limited due to endogenic compounds in urine samples. This is true in spite of use of tandem mass spectrometry.

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University of Helsinki
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00014 Helsinki
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