A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples

Abstract

Liquid chromatography-mass spectrometry (LC-MS) methods using either aqueous normal phase (ANP) or reversed phase (RP) columns are routinely used in small molecule or metabolomic analyses. These stationary phases enable chromatographic fractionation of polar and non-polar compounds, respectively. The application of a single chromatographic stationary phase to a complex biological extract results in a significant proportion of compounds which elute in the non-retained fraction, where they are poorly detected because of a combination of ion suppression and the co-elution of isomeric compounds. Thus coverage of both polar and nonpolar components of the metabolome generally involves multiple analyses of the same sample, increasing the analysis time and complexity. In this study we describe a novel tandem in-line LC-MS method, in which compounds from one injection are sequentially separated in a single run on both ANP and RP LC-columns. This method is simple, robust, and enables the use of independent gradients customized for both RP and ANP columns. The MS signal is acquired in a single chromatogram which reduces instrument time and operator and data analysis errors. This method has been used to analyze a range of biological extracts, from plant and animal tissues, human serum and urine, microbial cell and culture supernatants. Optimized sample preparation protocols are described for this method as well as a library containing the retention times and accurate masses of 127 compounds.

Tandem HILIC‐RP liquid chromatography for increased polarity coverage in food analysis

“…The relative standard deviation (RSD) was <2% for all 4400 detected features derived from the original retention times (none‐aligned retention times). This is in good agreement with other dual‐column methods . Additionally, the majority (>98%) of detected features showed a random distribution of retention time values among the nine replicates and none of the retention times showed a relative deviation greater 3% from its mean, respectively (Fig.…”

“…By comparison, Pyke et al . used only one valve to switch between the three steps . In their setup, effluates of the HILIC and RP column were continuously recombined by a T‐piece before entering the detector.…”

“…showed an increased polarity range covered when a zwitterionic HILIC column was serially coupled to a C18 RP column in the analysis of phenolic compounds . However, serial coupling of columns does not allow independent gradient elution and restricts the selection of columns due to an increase in back pressure. Good summaries of serial couplings have recently been published in comprehensive reviews by Haggarty and Burgess and Alvarez‐Segura et al .…”

“…HILIC and RP). Due to the pre‐fractionation step, analytes are separated either on the HILIC or on the RP column using independent gradient elution . Pyke et al .…”

“…Pyke et al . recently reported a tandem method, which combines aqueous normal phase (ANP) chromatography with RP separation for the analysis of urine metabolites . They showed that the number of detected compounds using the tandem method is comparable to results when the same sample was analyzed in two independent chromatographic runs.…”