A targeted reversed-phase gradient UPLC-MS/MS assay has been developed for the quantification /monitoring of 66 amino acids and amino-containing compounds in human plasma and serum using precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQTag Ultra). Derivatization of the target amines required minimal sample preparation and resulted in analytes with excellent chromatographic and mass spectrometric detection properties. The resulting method, which requires only 10 μL of sample, provides the reproducible and robust separation of 66 analytes in 7.5 min, including baseline resolution of isomers such as leucine and isoleucine. The assay has been validated for the quantification of 33 amino compounds (predominantly amino acids) over a concentration range from 2 to 20 and 800 μM. Intra- and interday accuracy of between 0.05 and 15.6 and 0.78-13.7% and precision between 0.91 and 16.9% and 2.12-15.9% were obtained. A further 33 biogenic amines can be monitored in samples for relative changes in concentration rather than quantification. Application of the assay to samples derived from healthy controls and patients suffering from acetaminophen (APAP, paracetamol)-induced acute liver failure (ALF) showed significant differences in the amounts of aromatic and branched chain amino acids between the groups as well as a number of other analytes, including the novel observation of increased concentrations of sarcosine in ALF patients. The properties of the developed assay, including short analysis time, make it suitable for high-throughput targeted UPLC-ESI-MS/MS metabonomic analysis in clinical and epidemiological environments.
IntroductionAs 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.ObjectivesTo 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.MethodsA 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.ResultsThe 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.ConclusionA 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.Electronic supplementary materialThe online version of this article (10.1007/s11306-019-1474-9) contains supplementary material, which is available to authorized users.
Background
Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease characterized by fibrosis and progressive loss of lung function. The pathophysiological pathways involved in IPF are not well understood. Abnormal lipid metabolism has been described in various other chronic lung diseases including asthma and chronic obstructive pulmonary disease (COPD). However, its potential role in IPF pathogenesis remains unclear.
Methods
In this study, we used ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) to characterize lipid changes in plasma derived from IPF patients with stable and progressive disease. We further applied a data-independent acquisition (DIA) technique called SONAR, to improve the specificity of lipid identification.
Results
Statistical modelling showed variable discrimination between the stable and progressive subjects, revealing differences in the detection of triglycerides (TG) and phosphatidylcholines (PC) between progressors and stable IPF groups, which was further confirmed by mass spectrometry imaging (MSI) in IPF tissue.
Conclusion
This is the first study to characterise lipid metabolism between stable and progressive IPF, with results suggesting disparities in the circulating lipidome with disease progression.
The effects of intravenous gefitinib (10 mg/kg), an anilinoquinazoline thymidylate kinase inhibitor (TKI), selective for the epidermal growth factor receptor (EGFR), on the urinary metabotypes of mice were studied. We hypothesized that, in response to the administration of gefitinib, there might be significant changes in the excretion of many endogenous metabolites in the urine, which could be correlated with the plasma pharmacokinetics (PK) of the drug. In order to investigate this conjecture, urine from male C57 BL6 mice was collected before IV dosing (10 mg/kg) and at 0–3, 3–8, and 8–24 h post-dose. The samples were profiled by UPLC/IM/MS and compared with the profiles obtained from undosed control mice with the data analyzed using multivariate statistical analysis (MVA). This process identified changes in endogenous metabolites over time and these were compared with drug and drug metabolite PK and excretion. While the MVA of these UPLC/IM/MS data did indeed reveal time-related changes for endogenous metabolites that appeared to be linked to drug administration, this analysis did not highlight the presence of either the drug or its metabolites in urine. Endogenous metabolites affected by gefitinib administration were identified by comparison of mass spectral, retention time and ion mobility-derived collision cross section data (compared to authentic standards wherever possible). The changes in endogenous metabolites resulting from gefitinib administration showed both increases (e.g., tryptophan, taurocholic acid, and the dipeptide lysyl-arginine) and decreases (e.g., deoxyguanosine, 8-hydroxydeoxyguanosine, and asparaginyl-histidine) relative to the control animals. By 8–24 h, the post-dose concentrations of most metabolites had returned to near control values. From these studies, we conclude that changes in the amounts of endogenous metabolites excreted in the urine mirrored, to some extent, the plasma pharmacokinetics of the drug. This phenomenon is similar to pharmacodynamics, where the pharmacological effects are related to the drug concentrations, and by analogy, we have termed this effect “pharmacometabodynamics”.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.