SULM – Schweizerische Union für Labormedizin | Union Suisse de Médecine de Laboratoire | Swiss Union of Laboratory Medicine

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C ROUX1, M VAGLIO1, S.J. BRUCE1, O BRAISSANT1, H.A. BROWN2, J.P. HOPKINS2, P.-A. BINZ1

1Service de Biomédecine, CHUV, Lausanne, 2Waters Corporation, Wilmslow, UK

Molecular diagnostics and patient follow-up of inborn errors of metabolism generally signifies quantitative measurements of amino acids, organic acids and acylcarnitines. For amino acid quantification, the Biochrom method is still regarded as the gold standard approach for the analysis of a wide panel of target analytes. Following a simple protein precipitation step with sulfosalicylate, amino acid extracts undergo a complex pH and temperature varying HPLC gradient. Post-column derivatisation with ninhydrin produces chromophores detected at 2 separate wavelengths.This gradient (~3 hours long) currently permits the quantification of around 55 analytes and the qualitative assignment of 10 additional ones.
With an ever increasing need to improve sample throughput, the challenge is to replace this well established method with an alternative approach that still allows for a simple sample preparation step, but one that would greatly reduce analysis time (i.e. a shortened chromatography), keep within the robustness, detection and sensitivity constraints of the Biochrom approach and also allow for an accurate and efficient post-analytical processing step. For these reasons, a preliminary evaluation of an AccQ•Tag™ based derivatisation method followed by a rapid UPLC® separation using a single quadrupole MS detector has been performed.
Two sample pools (plasma and urine) were selected in order to evaluate the robustness of the method (intra and inter-series, n = 30) on 55 target compounds. A comparison of quantitative data from both the Biochrom and LC-MS methods was performed on a selection of plasma, urine and CSF samples, and compared with results from an external quality control survey.
Preparative and analytical performances were evaluated in terms of compatibility with diagnostic and patient follow-up requirements defined from a clinical perspective.

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