Presenter Bio: Steve Bruce has previously worked at Imperial College London (where he obtained his PhD), Umea University, Sweden, Nestle Research Center, Lausanne, Switzerland. Currently based within the clinicial chemistry department at the Univerity Hospital, Lausanne (CHUV) where he is responsable/the scientific expert for development and routine application of LC-MS and HPLC clinical methods at CHUV.
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
INTRODUCTION: Acylcarnitine profiling is routinely performed in order to quantitatively screen for disorders related to β-oxidation and / or organic acid metabolism. Today many laboratories still perform acylcarnitine profiling using LC-MS/MS based methodology that has remained almost unchanged for around 2 decades. This “classical” approach normally involves a direct infusion (no chromatographic separation) into the MS system following a butylation based sample derivatisation. Although this approach is still widely used today, improvement in LC-MS technology observed over the last 15 years such as UHPLC and higher sensitivity MS systems means that a non-derivatisation approach that can be more informative with a relatively rapid LC separation is feasible.
OBJECTIVES: The primary objective of the work was to replace existing methodologies within our hospital in order to enhance efficiency whilst keeping or improving upon analytical quality.
METHODS: The new method described consists of a simple protein precipitation (isotopic dilution) and relatively large sample dilution followed by a HILIC separation (UHPLC) and subsequently MS detection in the MRM mode. This approach allows the separation and quantification of 42 individual acylcarnitines. Comparisons were made with established, routine methods within our laboratory: the “classical” acylcarnitine profiling approach and a method for the quantification of free and total carnitine also by LC-MS/MS.
RESULTS: Free carnitine (C0) and total carnitine (the sum of all free and acylcarnitines in plasma / serum) compared well with the established routine LC-MS/MS targeted method for C0 (Passing-Bablok: Slope (95% CI): 1.04 (0.99-1.07), n = 57) and total carnitine (Passing-Bablok: Slope (95% CI): 0.96 (0.91-1.06), n = 57). For the evaluation / comparison between methodologies for the acylcarnitine quantitative profiling, 84 patient samples were compared, 23 of which were confirmed cases of either β-oxidation or organic acid metabolic disorders. The new method compared well, successfully identifying all the 23 confirmed cases.
CONCLUSION: The new method described will replace two existing routine methods within our hospital, therefore greatly improving upon laboratory efficiency and significantly increasing the number of samples that can be prepared, run and processed for routine analyses. We estimate that this development saves around 1-1 ½ days per week of both instrumental and technical labour time.
= Discovery stage.
= Translation stage.
= Clinically available.
