A metabonomic study of strain- and age-related differences in the Zucker rat.

Ultra-performance liquid chromatography (UPLC) coupled to orthogonal acceleration time-of-flight mass spectrometry (oa-TOFMS) in positive electrospray ionization mode was used to obtain metabolite profiles for urine obtained from three strains of Zucker rat. These were the Zucker lean, the Zucker (fa/fa) obese and the Zucker lean/(fa) cross. Clear age- and strain-related differences were noted with the leptin-deficient (fa/fa) obese animal showing significant differences from both the other Zucker rat strains with respect to metabolite profiles.

A gender-specific discriminator in Sprague-Dawley rat urine: the deployment of a metabolic profiling strategy for biomarker discovery and identification.

The use of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) as complementary analytical techniques for open metabolic profiling is illustrated in the context of defining urinary biochemical discriminators between male and female Sprague-Dawley rats. Subsequent to the discovery of a female-specific urinary discriminator by LC-MS, further LC, MS, and NMR methods have been applied in a coordinated effort to identify this urinary component. Thereafter, the biological relevance and context of the identified component, in this case a steroid metabolite, has been achieved. This approach will be deployed in future studies of disease, drug efficacy, and toxicity to discover and identify biologically relevant markers.

Novel application of reversed-phase UPLC-oaTOF-MS for lipid analysis in complex biological mixtures: a new tool for lipidomics.

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.

Statistical search space reduction and two-dimensional data display approaches for UPLC-MS in biomarker discovery and pathway analysis.

A new analytical strategy for biomarker recovery from directly coupled ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC Tof MS) data on biofluids is presented and exemplified using a study on hydrazine-induced liver toxicity. A key step in the strategy involves a novel procedure for reducing the spectroscopic search space by differential analysis of cohorts of normal and pathological samples using an orthogonal projection to latent structures discriminant analysis (O-PLS-DA). This efficiently sorts principal discriminators of toxicity from the background of thousands of metabolic features commonly observed in data sets generated by UPLC-MS analysis of biological fluids and is thus a powerful tool for biomarker discovery.

Statistical heterospectroscopy, an approach to the integrated analysis of NMR and UPLC-MS data sets: application in metabonomic toxicology studies.

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.

A combined (1)H NMR and HPLC-MS-based metabonomic study of urine from obese (fa/fa) Zucker and normal Wistar-derived rats.

(1)H NMR and HPLC-MS were used to generate metabolite fingerprints for the metabonomic analysis of urine obtained from both male and female Zucker obese (fa/fa) rats, used as a model of type II diabetes, and normal male Wistar-derived animals. The resulting data were subjected to chemometric analysis (principal components analysis and partial least squares discriminant analysis) to investigate the effects of strain, diurnal variation is strain, diurnal variation and gender and gender on metabolite profiles. In the case of strain, (1)H NMR spectroscopic analysis revealed increased taurine, hippurate and formate and decreased betaine, alpha-ketoglutarate, succinate and acetate in samples from Zucker-obese compared to Wistar-derived rats. HPLC-MS analysis detected increased hippurate and ions at m/z 255.0640 and 285.0770 in positive, and 245.0122 and 261.0065 in negative electrospray ionisation (ESI), respectively, for the Zucker obese samples. Both techniques enable the detection of diurnal variation in the urine of male and female Zucker rats, marked by increases in taurine, creatinine, allantoin and alpha-ketoglutarate by (1)H NMR, and ions at m/z 285.0753, 291.0536 and 297.1492 (positive ESI) and 461.1939 (negative ESI) using HPLC-MS, in the evening samples. Differences between male and female Zucker rats were also observed. Compared to samples from male rats hippurate, succinate, alpha-ketoglutarate and dimethylglycine ((1)H NMR) were elevated in the urine of female animals together with ions at, e.g., m/z 431.1047, 325.0655, 271.0635 and 447.0946 (positive ESI) and m/z 815.5495 and 459.0985 (negative ESI) by HPLC-MS. Both analytical techniques used in this study were able to detect differences between normal and Zucker obese rats, which may provide markers of metabolic disease.

A rapid screening approach to metabonomics using UPLC and oa-TOF mass spectrometry: application to age, gender and diurnal variation in normal/Zucker obese rats and black, white and nude mice.

The use of Ultra Performance Liquid Chromatography (UPLC), with a rapid 1.5 minute reversed-phase gradient separation on a 1.7 microm reversed-phase packing material to provide rapid "high throughput" support for metabonomic screening is demonstrated. The peak capacity and the number of marker ions detected using these fast UPLC separations and oa-TOF MS was found to be similar to that generated by conventional HPLC-MS methods with a 10 minute separation. The UPLC-MS methodology was applied to the analysis of urine samples from rodents, including normal and Zucker obese rats and three strains of mice (of both sexes), and was found to provide rapid discrimination between age, strain, gender and diurnal variation.

Use of liquid chromatography/time-of-flight mass spectrometry and multivariate statistical analysis shows promise for the detection of drug metabolites in biological fluids.

The process of metabolite identification is essential to the drug discovery and development process; this is usually achieved by liquid chromatography/tandem mass spectrometry (LC/MS/MS) or a combination of liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) spectroscopy. Metabolite identification is, however, a time-consuming process requiring an experienced skilled scientist. Multivariate statistical analysis has been used in the field of metabonomics to elucidate differences in endogenous biological profiling due to a toxic effect or a disease state. In this paper we show how a combination of liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) and multivariate statistical analysis can be used to detect drug metabolites in a biological fluid with no prior knowledge of the compound administered.

Metabonomics: the use of electrospray mass spectrometry coupled to reversed-phase liquid chromatography shows potential for the screening of rat urine in drug development.

The application of liquid chromatography/mass spectrometry (LC/MS) followed by principal components analysis (PCA) has been successfully applied to the screening of rat urine following the administration of three candidate pharmaceuticals. With this methodology it was possible to differentiate the control samples from the dosed samples and to identify the components of the mass spectrum responsible for the separation. These data clearly show that LC/MS is a viable alternative, or complementary, technique to proton NMR for metabonomics applications in drug discovery and development.