High-throughput UHPLC/MS/MS-based metabolic profiling using a vacuum jacketed column

In UHPLC, frictional heating from the eluent flowing through the column at pressures of ca. 10–15 Kpsi causes radial diffusion via temperature differences between the center of the column and its walls. Longitudinal dispersion also occurs due to temperature gradients between the inlet and outlet. These effects cause band broadening but can be mitigated via a combination of vacuum jacketed stainless steel tubing, reduced column end nut mass, and a constant temperature in the column from heating the inlet fitting. Here, vacuum jacketed column (VJC) technology, employing a novel column housing located on the source of the mass spectrometer and minimized tubing from the column outlet to the electrospray probe, was applied to profiling metabolites in urine. For a 75 s reversed-phase gradient separation, the average peak widths for endogenous compounds in urine were 1.2 and 0.6 s for conventional LC/MS and VJC systems, respectively. The peak tailing factor was reduced from 1.25 to 1.13 when using the VJC system compared to conventional UHPLC, and the peak capacity increased from 65 to 120, with a 25% increase in features detected in urine. The increased resolving power of the VJC system reduced co-elution, simplifying MS and MS/MS spectra, providing a more confident metabolite identification. The increased LC performance also gave more intense MS peaks, with a 10–120% increase in response, improving the quality of the MS data and detection limits. Reducing the LC gradient duration to 37 s gave peak widths of ca. 0.4 s and a peak capacity of 84

Advances in high throughput LC/MS based metabolomics: A review

Properly implemented, metabolic and lipidomic profiling can provide a deeper understanding of mammalian, plant and bacterial biology. These omics-tools have developed and matured over the last 40-years and are now being deployed to provide valuable information in epidemiological studies, drug toxicology and pharmacology, disease biology and progression and patient stratification. LC/MS has become the technology of choice for both metabolic and lipid profiling, due to its speed, sensitivity and structural elucidation capabilities. In the preceding two decades there have been many technological and methodological advances in LC/MS that have facilitated the evolution of the technology into a rugged, reliable, and easily deployed tool. These advances include, but are not limited to, improvements in chromatography (phases, columns, and delivery system), instruments for mass spectrometry, optimization of sample preparation, the introduction of ion mobility, data analysis tools, metabolite databases, harmonized protocols, and the more widespread use of quality control methods and reference standards/matrices. Here, recent developments and advances in high throughput liquid chromatography/high resolution mass spectrometry for metabolic phenotyping are described. These advances which may provide improved feature detection, increased laboratory efficiency and data quality, as well as “biomarker” identification, are discussed in relation to their potential application to the analysis of large clinical studies, or biobank collections

High throughput UHPLC-MS-based lipidomics using vacuum jacketed columns

Reversed-phase UHPLC-MS is extensively employed for both the profiling of biological fluids and tissues to characterize lipid dysregulation in disease and toxicological studies. With conventional LC-MS systems the chromatographic performance and throughput are limited due to dispersion from the fluidic connections as well as radial and longitudinal thermal gradients in the LC column. In this study vacuum jacketed columns (VJC), positioned at the source of the mass spectrometer, were applied to the lipidomic analysis of plasma extracts. Compared to conventional UHPLC, the VJC-based methods offered greater resolution, faster analysis, and improved peak intensity. For a 5 min VJC analysis, the peak capacity increased by 66%, peak tailing reduced by up to 34%, and the number of lipids detected increased by 30% compared to conventional UHPLC. The narrower peaks, and thus increased resolution, compared to the conventional system resulted in a 2-fold increase in peak intensity as well a significant improvement in MS and MS/MS spectral quality resulting in a 22% increase in the number of lipids identified. When applied to mouse plasma samples, reproducibility of the lipid intensities in the pooled QC ranged from 1.8–12%, with no related drift in tR observed

Development of a rapid profiling method for the analysis of polar analytes in urine using HILIC-MS and ion mobility enabled HILIC-MS

Introduction As large scale metabolic phenotyping is increasingly employed in preclinical studies and in the investigation of human health and disease the current LC–MS/MS profiling methodologies adopted for large sample sets can result in lengthy analysis times, putting strain on available resources. As a result of these pressures rapid methods of untargeted analysis may have value where large numbers of samples require screening. Objectives To develop, characterise and evaluate a rapid UHP-HILIC-MS-based method for the analysis of polar metabolites in rat urine and then extend the capabilities of this approach by the addition of IMS to the system. Methods A rapid untargeted HILIC LC–MS/MS profiling method for the analysis of small polar molecules has been developed. The 3.3 min separation used a Waters BEH amide (1 mm ID) analytical column on a Waters Synapt G2-Si Q-Tof enabled with ion mobility spectrometry (IMS). The methodology, was applied to the metabolic profiling of a series of rodent urine samples from vehicle-treated control rats and animals administered tienilic acid. The same separation was subsequently linked to IMS and MS to evaluate the benefits that IMS might provide for metabolome characterisation. Results The rapid HILIC–MS method was successfully applied to rapid analysis of rat urine and found, based on the data generated from the data acquired for the pooled quality control samples analysed at regular intervals throughout the analysis, to be robust. Peak area and retention times for the compounds detected in these samples showed good reproducibility across the batch. When used to profile the urine samples obtained from vehicle-dosed control and those administered tienilic acid the HILIC-MS method detected 3007 mass/retention time features. Analysis of the same samples using HILIC–IMS–MS enabled the detection of 6711 features. Provisional metabolite identification for a number of compounds was performed using the high collision energy MS/MS information compared against the Metlin MS/MS database and, in addition, both calculated and measured CCS values from an experimentally derived CCS database. Conclusion A rapid metabolic profiling method for the analysis of polar metabolites has been developed. The method has the advantages of speed and both reducing sample and solvent consumption compared to conventional profiling methods. The addition of IMS added an additional dimension for feature detection and the identification of metabolites

Capillary ultra performance liquid chromatography-tandem mass spectrometry analysis of tienilic acid metabolites in urine following intravenous administration to the rat

Capillary scale (100 mm × 150 μm id) UPLC/MS/MS, performed using reversed-phase gradient chromatography on sub 2 μm particles, has been successfully employed for the characterization of the metabolites of the drug tienilic acid (TA) excreted via the urine following oral administration to the rat. The capillary LC system provided a significant increase (range ca. 11-33-fold) in sensitivity compared with a conventional 150 mm × 2.1 mm id UPLC system. An investigation of the effect of the injection volume and sample mass loading on the capillary column on the results obtained for both endogenous metabolites and TA was performed. This demonstrated that the injection of up to 2 μL of rat urine onto the system was permitted whilst still providing excellent chromatographic results and robustness. Qualitative analysis of the urine revealed the presence of TA itself and a total of 15 metabolites of the drug, including those resulting from biotransformations such as hydroxylation or conjugation. The capillary chromatography system was shown to be robust, and capable of providing comprehensive drug metabolite profiles from small format urine samples such as those obtained from preclinical studies in rodents

Application of a novel mass spectral data acquisition approach to lipidomic analysis of liver extracts from sitaxentan-treated liver-humanized PXB mice.

The application of a data-independent acquisition (DIA) method ("SONAR") that employs a rapidly scanning quadrupole is described for the lipidomic analysis of complex biological extracts. Using this approach, the MS acquisition window can be varied between 1 and 25 Da, enabling the isolation of ions prior to their entering the collision cell. By rapidly scanning the resolving quadrupole window over a specified mass range, co-eluting precursor ions are transmitted sequentially into the collision cell, where collision energies are cycled between low and elevated levels to induce fragmentation. This method of data generation provides both precursor and fragment ion information at high specificity, allowing for greater accuracy of compound identification, whether using a database, spectral libraries, or comparison to authentic standards. The value of the approach in simplifying and "de-cluttering" the spectra of co-eluting lipids is shown with examples from lipidomic profiles obtained in investigations of the composition of organic extracts of livers obtained from SCID and chimeric liver-humanized mice administered under various experimental conditions

Ion Mobility Spectrometry Combined With Ultra Performance Liquid Chromatography/Mass Spectrometry For Metabolic Phenotyping of Urine: Effects of Column Length, Gradient Duration and Ion Mobility Spectrometry on Metabolite Detection.

The need for rapid and efficient high throughput metabolic phenotyping (metabotyping) in metabolomic/metabonomic studies often requires compromises to be made between analytical speed and metabolome coverage. Here the effect of column length (150, 75 and 30 mm) and gradient duration (15, 7.5 and 3 min respectively) on the number of features detected when untargeted metabolic profiling of human urine using reversed-phase gradient ultra performance chromatography with, and without, ion mobility spectrometry, has been examined. As would be expected, reducing column length from 150 to 30 mm, and gradient duration, from 15 to 3 min, resulted in a reduction in peak capacity from 311 to 63 and a similar reduction in the number of features detected from over ca. 16,000 to ca. 6500. Under the same chromatographic conditions employing UPLC/IMS/MS to provide an additional orthogonal separation resulted in an increase in the number of MS features detected to nearly 20,000 and ca. 7500 for the 150 mm and the 30 mm columns respectively. Based on this limited study the potential of LC/IMS/MS as a tool for improving throughput and increasing metabolome coverage clearly merits further in depth study

Rapid profiling method for the analysis of lipids in human plasma using ion mobility enabled-reversed phase-ultra high performance liquid chromatography/mass spectrometry

The incorporation of ion mobility (IM) into LC–MS analysis has been demonstrated to result in the generation of superior quality MS and MS/MS spectral data as well as providing enhanced resolution in the IM dimension based on lipid class. Here a sub 4 min microbore LC-ion mobility-accurate mass MS (LC-IM-MS) method has been developed for the rapid, profiling of lipids in biological fluids. The method was scaled directly from a conventional, 12  min, LC-MS analysis maintaining the chromatographic performance and lipid separation observed in the longer methodology giving a 75% saving in mobile phase consumption and analysis time. Because of the additional dimension of separation provided by IM, improvements in mass spectral quality from the increased resolution of co-eluting species were also seen when compared to the same separation without IM, thus aiding the identification of target lipids. When applied to human plasma samples some 5037 (positive ESI) and 2020 (negative ESI) mass/retention time features were detected following adduct deconvolution and, of these, 3727 and 800 of those present in the pooled plasma QC samples had a CV of below 30% for positive and negative ESI modes respectively. The method was applied to the analysis of a pilot set of commercially sourced breast cancer plasma samples enabling the differentiation of samples from healthy controls and patients based on their lipid phenotypes. Analysis of the resulting data showed that phosphatidylcholines, triglycerides and diglycerides exhibited lower expression and phosphatidylserine showed increased expression in the breast cancer samples compared to those of healthy subjects. The coefficients of variation, determined by reference to the QC data, for all of the features identified as potential markers of disease, were 6% or less