A new approach to obtain fragmentation information in liquid chromatography/mass spectrometry (LC/MS) studies of small molecules in complex mixtures is presented using simultaneous acquisition of exact mass at high and low collision energy, MS(E). LC/MS-TOF and LC/MS/MS-TOF are powerful tools for the analysis of complex mixtures, especially those for biological fluids allowing the elucidation of elemental composition and fragmentation information. In this example the composition of rat urine was studied using this new approach, allowing the structures of several endogenous components to be confirmed in one analytical run by the simultaneous acquisition of exact mass precursor and fragment ion data. The spectral data obtained using this new approach are comparable to those obtained by conventional LC/MS/MS as exemplified by the identification of endogenous metabolites present in rat urine.

Statistical heterospectroscopy (SHY) is a new statistical paradigm for the coanalysis of multispectroscopic data sets acquired on multiple samples. This method operates through the analysis of the intrinsic covariance between signal intensities in the same and related molecules measured by different techniques across cohorts of samples. The potential of SHY is illustrated using both 600-MHz 1H NMR and UPLC-TOFMS data obtained from control rat urine samples (n = 54) and from a corresponding hydrazine-treated group (n = 58). We show that direct cross-correlation of spectral parameters, viz. chemical shifts from NMR and m/z data from MS, is readily achievable for a variety of metabolites, which leads to improved efficiency of molecular biomarker identification. In addition to structure, higher level biological information can be obtained on metabolic pathway activity and connectivities by examination of different levels of the NMR to MS correlation and anticorrelation matrixes. The SHY approach is of general applicability to complex mixture analysis, if two or more independent spectroscopic data sets are available for any sample cohort. Biological applications of SHY as demonstrated here show promise as a new systems biology tool for biomarker recovery.

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Ultra-Performance LC (UPLC) utilizing sub-2-mum porous stationary phase particles operating with high linear velocities at pressures >9000 psi was coupled with orthogonal acceleration time-of-flight (oaTOF) mass spectrometry and successfully employed for the rapid separation of lipids from complex matrices. The UPLC system produced information-rich chromatograms with typical measured peak widths of 3 s at peak base, generating peak capacities in excess of 200 in 10 min. Further UPLC coupled with MSE technology provided parent and fragment mass information of lipids in one chromatographic run, thus, providing an attractive alternative to current LC methods for targeted lipid analysis as well as lipidomic studies.

In the original manuscript the references to alternating collision energy were missed out in error. The application of simultaneous acquisition of precursor and production ions in one analytical run was first described by Bateman et al.

The use of a combination of ultraperformance liquid chromatography at approximately 11,000 psi on sub 2-microm particles combined with reversed-phase gradient chromatography at a temperature of 90 degrees C is described as applied to the analysis of endogenous and drug metabolites in human and animal urine. By using elevated temperatures, back pressures can be reduced while maintaining high flow rates and chromatographic efficiency, with peaks 1-3 s wide at the base. Application to urine samples provided a peak capacity of approximately 700 for a 10-min analysis and greater than approximately 1000 in 1 h. Despite the narrow nature of the peaks, good quality mass spectra were also obtained, allowing the identification of typical drug and endogenous metabolites. These ultra-high-resolution chromatograms should be ideal for the analysis of complex samples in, for example, metabolite identification, impurity identification, and metabonomic/metabolomic studies. Applications in natural product analysis and proteomics can also be envisaged.

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Analysis of biological fluids using ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) (metabonomics) can allow new insights to be gained into disease processes, with advances in chromatographic techniques enabling the detection of thousands of metabolites. In this work metabonomics has been used to investigate the metabolic processes involved in type II diabetes in the Zucker obese rat. Plasma was analyzed from three different strains, the Zucker (fa/fa) obese, Zucker lean and the lean/(fa) obese cross. Using UPLC/MS, ca. 10,000 ions were detected due to the narrow peak widths and excellent peak shapes achieved with this technology. Confidence in the chromatographic performance was demonstrated by the use of quality control standards. The positive and negative ion total ion chromatograms obtained from the three strains were readily distinguishable using multivariate statistical analysis. The greatest difference was observed between the Zucker lean and Zucker lean/(fa) rats compared to the Zucker (fa/fa) obese rats. Positive ions m/z 220 (4.36 min), 282(3.78 min), 359 (5.33 min) and 405 (7.77 min) were elevated in the plasma derived from Zucker lean rats whilst ions m/z 385 (6.80 min) and 646 (4.36 min) were at a lower concentration compared to the plasma from the Zucker (fa/fa) obese animals. Negative ions elevated in the Zucker lean rats included m/z 212 (2.30 min), 514 (2.85 min), 295 (4.39 min), 329 (3.11 min), 343 (2.86 min) and 512 (2.86 min) with ions m/z 538 (4.18 min), 568 (4.18 min), 568 (5.09 min) and 612 (4.30 min) being raised in the samples derived from Zucker (fa/fa) obese animals. The ion m/z 514 (3.85 min) was found to correspond to taurocholate, providing further support for an involvement of taurine metabolism in diabetes.