Abstract
The need for rapid and efficient high throughput metabolic phenotyping (metabotyping) in metabolomic/metabonomic studies often requires compromises to be made between analytical speed and metabolome coverage. Here the effect of column length (150, 75 and 30 mm) and gradient duration (15, 7.5 and 3 min respectively) on the number of features detected when untargeted metabolic profiling of human urine using reversed-phase gradient ultra performance chromatography with, and without, ion mobility spectrometry, has been examined. As would be expected, reducing column length from 150 to 30 mm, and gradient duration, from 15 to 3 min, resulted in a reduction in peak capacity from 311 to 63 and a similar reduction in the number of features detected from over ca. 16,000 to ca. 6500. Under the same chromatographic conditions employing UPLC/IMS/MS to provide an additional orthogonal separation resulted in an increase in the number of MS features detected to nearly 20,000 and ca. 7500 for the 150 mm and the 30 mm columns respectively. Based on this limited study the potential of LC/IMS/MS as a tool for improving throughput and increasing metabolome coverage clearly merits further in depth study.
Highlights
The use of metabolic phenotyping to discover biomarkers of organismal response to environmental and physiological change is widespread
The optimal operation of sub 2-mm LC/mass spectrometry (MS) for small molecule analysis, in gradient elution mode, requires analytical flow rates in the range 0.5e0.9 mL/min, which results in peaks of widths varying between 1.5 and 3 s
To evaluate the effect of column length on the number of features detected the analysis of control human urine samples obtained from 6 volunteers was analysed, both individually and as a pooled sample
Summary
The use of metabolic phenotyping (metabonomics/metabolomics) to discover biomarkers of organismal response to environmental and physiological change is widespread. An obvious way of reducing the problems of co-elution is to use strategies such as 2dimensional separations but this clearly does not solve the problem of maximising throughput Another potential means of maximising metabolite detection without increasing analysis time is to employ ion mobility spectrometry (IMS) prior to MS detection in a hyphenated LC-IMS-MS system. The use of the “drift time” within the ion optics can allow analytes of interest to be separated and detected even in the presence of a co-eluting isobaric species This orthogonal separation provides an increase in peak capacity, in an analogous manner to two-dimensional LC, but without an increase in analysis time. We describe the results of the investigation of the effect of integrating IMS with gradient reversed-phase UPLC, using different column lengths and gradient durations, as a means of enhancing the data obtained for the metabotyping of human urine
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