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Metabolomic Screening of Human Plasma Using U-HPLC Coupled to a Bench-top Non-hybrid Orbitrap Mass Spectrometer with HCD Fragmentation
Donna L. Wilson (1), Xiaofeng Guo (2), Sucharita Dutta (1), Mark Sanders (1), Jens Hoefkens (3) and Bruce Kristal (2)
(1) Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134
(2) Department of Neurosurgery, Brigham and Women’s Hospital, Boston, MA 02115
(3) Genedata (USA) Inc., 1601 Trapelo Road, Suite 350, Waltham, MA 02451
Metabolomics is the comprehensive analysis of wide arrays of endogenous metabolites in biological samples. These numerous analytes display a diversity of chemistries and polarities and are found at different abundance levels within complex matrices. Metabolomic screening of numerous technical and biological replicates is crucial for results to be statistically and biologically meaningful. Therefore, discovery phase metabolomics relies on rapid full scan analysis to measure as many metabolites as possible. However it is impossible to detect and quantify chemical species which are not adequately mass resolved. One current screening approach utilizes ToF instruments coupled to UHPLC delivering high mass accuracy (~5ppm) at a maximum mass resolution of <15,000. The inability to detect ions with mass resolution of at least 50,000 can lead to inaccurate mass measurements caused by unresolved background matrix interferences.
In this work we highlight a full mass scan screening approach using a novel single stage orbitrap mass spectrometer coupled to U-HPLC, capable of providing high mass accuracy at a range of resolutions: 10,000, 25,000, 50,000 and 100,000. Chromatography was performed using an Accela U-HPLC equipped with a 2.1 mm id Hypersil Gold C18 column packed with 1.9 um particles at a flow rate of 600 uL/min and 40 C column heating.
The analysis focused on the detection and quantification of low molecular weight components of human plasma. U-HPLC coupled with a small particle column afforded a fast analysis time while maintaining very high chromatographic resolution (peak width <3 seconds at half height). The mass accuracy data (mass difference less than 2 ppm with external mass calibration) was used to confirm elemental composition. Identification of several compounds was facilitated by using HCD fragmentation, while also enabling semi-quantitative determinations.
Comparison of the measurements at resolution of up to 50,000 clearly indicated the need for higher resolution for screening applications in complex matrices. This is because interferences may not be resolved from the analytes and in many cases the use of resolutions between 25,000 and 50,000 resulted in poor mass accuracies (> 10 ppm). In contrast, acquisition of data at 100,000 resolution yielded excellent mass accuracies (<2 ppm) for the vast majority of the measured compounds. As a consequence selectivity and also sensitivity is increased. The new single stage orbitrap mass spectrometer is able to perform this high resolution with a fast scan repetition rate making it compatible with fast chromatography. This work shows that a combination from uHPLC and high resolution mass spectrometry provides a rigorous analytical methodology for metabolomic screening and overcomes limitations of using resolution of less than 50,000.
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