Robert Plumb (Presenter)
Imperial College London
Bio: Dr Plumb has published over 100 papers on the subject of HPLC/MS and NMR for bioanalysis, metabolomics and metabolite identification. He is a recognized expert in the use of liquid chromatography with mass spectrometry, capillary scale LC, purifications scale LC and metabonomics, giving many invited papers at international meetings around the world. After obtaining an honors degree in Chemistry from the University of Hertfordshire in 1992, he started work in at Glaxo Research and Development Drug Metabolism Department. During his time at Glaxo and later GlaxoWellcome he continued his research in liquid chromatography combined with NMR and mass spectrometry for metabolite identification and bioanalysis obtaining his PhD in 1999. Dr Plumb continued his work for GlaxoWellcome with the responsibility of metabolite identification using HPLC/MS/NMR and new analytical technology development. In
Authorship: Robert S Plumb, Jeremy K Nicholson,Ian D Wilson
Imperial College, London, UK
| Short Abstract The detection, identification and validation of biomarkers for biomedical research and discovery requires a robust, reliable and information rich analytical platform. Metabolic Phenotyping of large cohort epidemiological and pre-clinical studies provides a metabolic insight into disease mechanism, treatment efficacy and toxicity. Analysis of these large cohort batches by UHPLC-MS is time consuming and often results in batch to batch variations due to subtle analytical batch variation. A high-throughput microbore UPLC-MS method has been developed providing an analytical platform for the metabolic phenotyping of biofluids from large sample cohorts. This approach has demonstrated high throughput reproducibility and the ability to identify biomarkers of toxicity. |
Long Abstract
Introduction
A high-throughput microbore UPLC-MS method has been developed providing an analytical platform for the metabolic phenotyping of biofluids from large sample cohorts. This rapid approach was based on scaling a conventional reversed-phase method from 2.1 to 1 mm scale columns, increasing linear solvent velocity, and decreasing gradient time resulting in an anlysis time of 2.5 min/sample. Comparison the two methodologies indicated the conventional method detected approximately 19,000 features compared to 6000 with the rapid method. However, the statistical analysis showed that both methods delivered the same results and detected the same marker ions. The methodology was demonstrated to be robust, enabling the analysis of up to 700 urine samples and QCs in a single batch with excellent reproducibility and sensitivity.
Methodology
Mass spectrometry was performed on a hybrid IMS-QTof instrument operating in positive ion electrospray accurate mass mode. MS data was collected over the range of 50-1200 m/z. The chromatography was performed on a UHPLC system comprised of a binary solvent manager and autosampler. The chromatography was performed on a 2.1 x 100mm or 1 x 50mm 1.7µm C18 column eluted with an aqueous formic acid-acetonitrile gradient over 10 minutes or 2 minutes respectively. Chip-based nanoelectrospray infusion analysis was performed using the TriVersa NanoMate system. The raw data was processed by Progenesis QI software for the UHPLC or ProteoWizard software for infusion MS. Urine samples were collected from four groups of rats dosed orally with acetaminophen once daily, for seven days.
Results and Discussion
Metabolic Phenotyping of large cohort epidemiological and pre-clinical studies provides a metabolic insight into disease mechanism, treatment efficacy and toxicity. Analysis of these large cohort batches by UHPLC-MS is time consuming and often results in batch to batch variations due to subtle analytical batch variation. A short column narrow-bore UHPLC method with an analysis time of 2.5 min/sample was developed to address these issues, allowing a batch of 768 samples (2 x 384 well-plates) to be analysed in 1.3 days. The LC peak capacity for the conventional and rapid methods was 150 and 50 respectively; delivering a 5-fold increase in throughput, with a 3-fold reduction in resolution. The resulting multivariate statistical analysis of the LC/MS data by principal components analysis (PCA) and orthogonal partial least square discriminate analysis (OPLSDA), with 18,823 features observed using conventional UPLC-MS and 6,188 using the rapid method.
The PCA demonstrated that the rapid microbore analysis gave a similar degree of discrimination between the dose groups as provided by the conventional method. Despite the reduction in the number of ions detected, a similar pattern in the distribution between the control, low dose (100 mg/kg) and high dose (200 mg/kg) and the QC samples. The OPLS-DA loadings S-plots indicating the similar ions responsible for the group separations were detected by both analytical platforms. These were identified as the conjugated sulfate and glucuronide metabolites and the N-acetylcysteinlyl (mercapturate) conjugate. The major advantage accruing from this methodology was the analysis of all of the samples as a single analytical run thus eliminating the need for batch correction.
The infusion MS PCA differed from the rapid UHPLC results giving less group discrimination. An inspection of the infusion spectra showed the presence of several intense peaks, possibly a result of a high salt content in the rat urine causing ion suppression.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Waters Corporation |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |