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1.
J Chromatogr A ; 1714: 464552, 2024 Jan 11.
Article in English | MEDLINE | ID: mdl-38113579

ABSTRACT

The untargeted global profiling of endogenous metabolites and lipids has the potential to increase knowledge and understanding in many areas of biology. LC-MS/MS is a key technology for such analyses however, several different LC methodologies, using different mobile phase compositions, are required to cover the diversity in polarity and analyte structure encountered in biological samples. Most notably many lipid screening methods make use of isopropanol (IPA) as a major component of mobile phases employed for comprehensive lipidomic profiling. In order to increase laboratory efficiency, and minimize opportunities for errors, a suite of methods, based on a single acetonitrile (ACN)-aqueous buffer mobile phase combination, has been developed. This mobile phase can be used for hydrophobic interaction liquid chromatography on an amide stationary phase (for polar analytes), reversed-phase (RP) LC analysis on a C8 stationary phase (for moderately polar-non-polar compounds) and RPLC using a CSH phenyl-hexyl bonded column (for lipids). All of these sub 10 minute separations had good throughput and reproducibility with CV's of analyte response <25 % whilst eliminating the need for complex mobile phase preparation and the use of IPA as an organic modifier for lipidomics. Advantages of removing IPA and replacing it with the ACN-based method were a 58 % increase in peak capacity for lipids, with improved resolution for the di- and triglycerides and cholesterol esters compared to current methods. Compared to the IPA-containing solvent system the ACN-based mobile phase also resulted in a 61 % increase in lipid feature detection. The utility of this "universal" mobile phase approach was demonstrated by its application to a rat toxicology study investigating the consequences of methapyrilene administration through on the endogenous metabolite profiles of plasma and urine. Methapyrilene and its metabolites were also profiled in these samples.


Subject(s)
Chemical and Drug Induced Liver Injury , Methapyrilene , Rats , Animals , Chromatography, Liquid/methods , Lipidomics , Reproducibility of Results , Tandem Mass Spectrometry , Lipids
2.
J Agric Food Chem ; 71(19): 7593-7603, 2023 May 17.
Article in English | MEDLINE | ID: mdl-37139986

ABSTRACT

This work aimed to develop an analytical method for the screening of multiple aminoglycoside residues in foods of animal origin using an ethylene-bridged hybrid (BEH) particle-based sulfoalkylbetaine stationary phase. The effects of chromatographic conditions on the separation of 17 aminoglycosides have been systematically investigated. Sample preparation and mass spectrometry detection have also been investigated and optimized. In contrast to high buffer concentrations in the mobile phase required for silica-based sulfoalkylbetaine stationary phases, a moderate buffer concentration (20 mM) provided the optimal separation of 17 aminoglycosides with the BEH sulfoalkylbetaine stationary phase. The developed method has been evaluated in milk, beef, pork, liver, and honey samples with good performance for retention, selectivity, sensitivity, linearity, precision, and accuracy. The majority of the limit of quantitation estimated with the matrix was less than 25 µg/kg. The overall accuracy across five matrices was in the range from 96 to 111%, with standard deviations of less than 19%.


Subject(s)
Aminoglycosides , Tandem Mass Spectrometry , Animals , Cattle , Aminoglycosides/analysis , Tandem Mass Spectrometry/methods , Chromatography, Liquid/methods , Anti-Bacterial Agents/analysis , Solid Phase Extraction , Ethylenes , Chromatography, High Pressure Liquid/methods
3.
Talanta ; 254: 124089, 2023 Mar 01.
Article in English | MEDLINE | ID: mdl-36459869

ABSTRACT

The use of vacuum jacketed LC columns (VJC) to minimize on- and post-column band broadening to maximize chromatographic performance has been evaluated as a potential route to improved high throughput (HT) analysis. Here the use of the "VJC" approach has been applied to the HT bioanalysis of the antidiabetic GPR40 agonist drug fasiglifam in rat plasma samples obtained following a 5 mg/kg IV dose. The data obtained from a 1 minute VJC/MS-based analysis showed significant improvements compared to that from a conventional 2 minute UHPLC method for the drug. Notably, using VJC/MS with the rapid 1 min analysis provided a ca. 50% reduction in peak width coupled with a 2-5 fold higher peak response whilst doubling analytical throughput when compared to a conventional UHPLC/MS method. In addition, the increased resolution provided by the VJC system also improved the separation of fasiglifam from common matrix interferences such as co-extracted phospholipids thereby reducing the potential for matrix effects. The concatenation of these improvements suggests that the VJC approach may indeed provide a pathway to more sensitive, robust and high throughput drug bioanalysis, with particular advantages for drug discovery applications.


Subject(s)
Tandem Mass Spectrometry , Rats , Animals , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid/methods , Vacuum , Chromatography, Liquid/methods
4.
J AOAC Int ; 106(2): 464-471, 2023 Mar 01.
Article in English | MEDLINE | ID: mdl-36173311

ABSTRACT

BACKGROUND: Baseline separation of nonivamide (NON) and capsaicin (CAP) has not been achieved by using the existing liquid chromatography (LC) methods for the capsaicinoid analysis. This could lead to large errors in the determination of capsaicinoids for capsicum products. OBJECTIVE: The development of an ultrahigh-performance liquid chromatography (UHPLC) method that simultaneously separates NON and CAP as well as other capsaicinoids for the routine analysis of capsaicinoids in capsicum products. METHOD: Capsaicinoids were separated on a Waters CORTECSTM T3 Column (2.1 mm i.d. × 150 mm, 1.6 µm particle size) that was maintained at 45°C on a UHPLC system with a 3-step gradient elution using a binary mobile phase system consisting of water and acetonitrile. Florescence detection was set at 280 nm excitation wavelength and 325 nm emission wavelength. RESULTS: The UHPLC method was able to simultaneously separate NON and CAP, with a minimum resolution of 1.5, as well as other seven capsaicinoids with a total run time of 27 min. Method selectivity, robustness, accuracy, and precision were evaluated, and excellent performance was achieved. CONCLUSIONS: The UHPLC method for NON and CAP and other seven capsaicinoids has been successfully developed and found suitable for the routine analysis of capsaicinoids. HIGHLIGHTS: For the first time, NON and CAP are well separated (Rs >1.5) in a 27 min LC separation. This UHPLC method offers a suitable solution for the determination of nine capsaicinoids in QC labs.


Subject(s)
Capsaicin , Capsicum , Capsaicin/analysis , Capsicum/chemistry , Chromatography, Liquid , Water , Camphor/analysis , Menthol/analysis , Vegetables , Chromatography, High Pressure Liquid/methods
5.
Article in English | MEDLINE | ID: mdl-35709669

ABSTRACT

Recently, a novel hybrid surface technology (HST) has been developed to mitigate metal analyte adsorption in liquid chromatography. The HST provides a hybrid organic-inorganic surface on the metal fluidic path, from injection to detector and including the column frits and wall, to mitigate the interaction between analytes and metals. Here the impact of the HST on the analysis of B group vitamins using liquid chromatography coupled with electrospray tandem mass spectrometry (LC-ESI-MS/MS) has been evaluated. Significant improvements in analyte intensity, limit of quantification (LOQ), carry-over, and peak shape were observed using an LC-ESI-MS/MS system and column that incorporated the HST. The key observed improvements include a 3-10 times increase in sensitivity (providing a lower LOQ) for riboflavin, thiamine, nicotinamide, FMN, PLP, and 5MTHF, no carry-over, and a more symmetrical peak for thiamine. When applied to the analysis of B group vitamins in energy drinks and B vitamin dietary supplement samples, the HST system demonstrated excellent accuracy and repeatability.


Subject(s)
Vitamin B Complex , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Technology , Thiamine/analysis , Vitamin B Complex/analysis
6.
J Chromatogr A ; 1672: 463013, 2022 Jun 07.
Article in English | MEDLINE | ID: mdl-35436684

ABSTRACT

Metabolic phenotyping studies using mouse liver extracts as a model, performed on a novel zwitterionic HILIC UHPLC column, which is based on ethylene-bridged hybrid organic/inorganic particles bonded with sulfobetaine groups and packed into column hardware modified with hybrid surface technology are reported. Initially the chromatographic performance was evaluated under different mobile phase conditions using selected metabolite standards. Following optimization of the chromatographic conditions for 88 hydrophilic metabolites both targeted and untargeted profiling analyses were performed on tissue extracts using LC-MS/MS and LC-TOF/MS, respectively. Chromatographic efficiency parameters such as peak resolution, peak shapes, selectivity and precision in retention and peak areas as well as characteristics that are critical for metabolic profiling analysis such as metabolite coverage and retention time distribution were assessed. The hybrid zwitterionic column exhibited efficient chromatographic separations providing analysis of ca 80 hydrophilic metabolites from different chemical classes and polarities. Utilizing a one-dimensional separation both targeted and untargeted profiling provided comprehensive metabolic signatures that enabled the acquisition of the metabolic phenotypes of the tissue extracts.


Subject(s)
Metabolomics , Tandem Mass Spectrometry , Animals , Chromatography, Liquid/methods , Hydrophobic and Hydrophilic Interactions , Metabolomics/methods , Mice , Tandem Mass Spectrometry/methods , Tissue Extracts
7.
J Proteome Res ; 21(3): 691-701, 2022 Mar 04.
Article in English | MEDLINE | ID: mdl-34968064

ABSTRACT

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.


Subject(s)
Lipidomics , Tandem Mass Spectrometry , Animals , Chromatography, High Pressure Liquid/methods , Lipids , Mice , Reproducibility of Results , Vacuum
8.
Anal Chem ; 93(30): 10644-10652, 2021 08 03.
Article in English | MEDLINE | ID: mdl-34279080

ABSTRACT

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.


Subject(s)
Tandem Mass Spectrometry , Chromatography, High Pressure Liquid , Chromatography, Liquid , Diffusion , Vacuum
9.
Article in English | MEDLINE | ID: mdl-34218093

ABSTRACT

The accurate determination of the pharmacokinetics (PK) of a candidate drug molecule is critical in both drug discovery and development. Over the last 30 years, the sensitivity and selectivity of LC/MS has resulted in it being established as the technology of choice for these studies. However, unwanted chemical interactions between analyte(s) and the metal components in a chromatography system can result in poor peak shape and reduction in signal response, which can adversely affect the analysis of low concentrations of drugs and their metabolites in biological samples. This study evaluated the benefits of employing an inert hybrid surface technology (HST) applied to the metallic components in the LC flow path, column frits and column wall to mitigate these interactions. The results obtained were compared with that of an identical conventional LC for the bioanalysis of two steroid phosphate drugs (dexamethasone phosphate and hydrocortisone phosphate) and an epidermal growth factor receptor (EGFR) inhibitor (gefitinib) in human plasma. The results showed that for the two steroid phosphates, the peak width was reduced by 20%, peak tailing factors reduced by up to 30% and the assay sensitivity improved by factors of 7.5 and 10. This resulted in a significant improvement in the limit of detection. The new LC system also improved the reproducibility of peak integration for gefitinib, thereby reducing assay coefficients of variation (%CV) from greater than 10% to less than 5% at the lower limit of quantification.


Subject(s)
Chromatography, Liquid/instrumentation , Metals/chemistry , Pharmaceutical Preparations , Tandem Mass Spectrometry/methods , Chromatography, Liquid/methods , Chromatography, Liquid/standards , Drug Discovery , Humans , Limit of Detection , Pharmaceutical Preparations/blood , Pharmaceutical Preparations/chemistry , Reproducibility of Results , Surface Properties
10.
Anal Chem ; 93(2): 1009-1015, 2021 01 19.
Article in English | MEDLINE | ID: mdl-33290053

ABSTRACT

We describe a method for the analysis of organic acids, including those of the tricarboxylic acid cycle (TCA cycle), by mixed-mode reversed-phase chromatography, on a CSH Phenyl-Hexyl column, to accomplish mixed-mode anion-exchange separations, which results in increased retention for acids without the need for ion-pairing reagents or other mobile phase additives. The developed method exhibited good retention time reproducibility for over 650 injections or more than 5 days of continuous operation. Additionally, it showed excellent resolution of the critical pairs, isocitric acid and citric acid as well as malic acid and fumaric acid, among others. The use of hybrid organic-inorganic surface technology incorporated into the hardware of the column not only improved the mass spectral quality and subsequent database match scoring but also increased the recovery of the analytes, showing particular benefit for low concentrations of phosphorylated species. The method was applied to the comparative metabolomic analysis of urine samples from healthy controls and breast cancer positive subjects. Unsupervised PCA analysis showed distinct grouping of samples from healthy and diseased subjects, with excellent reproducibility of respective injection clusters. Finally, abundance plots of selected analytes from the tricarboxylic acid cycle revealed differences between healthy control and disease groups.


Subject(s)
Body Fluids/metabolism , Citric Acid Cycle , Citric Acid/metabolism , Fumarates/metabolism , Isocitrates/metabolism , Malates/metabolism , Body Fluids/chemistry , Chromatography, High Pressure Liquid , Citric Acid/chemistry , Citric Acid/urine , Fumarates/chemistry , Fumarates/urine , Humans , Isocitrates/chemistry , Isocitrates/urine , Malates/chemistry , Malates/urine , Mass Spectrometry , Molecular Structure
11.
J Pharm Biomed Anal ; 193: 113729, 2021 Jan 30.
Article in English | MEDLINE | ID: mdl-33171338

ABSTRACT

The application of the Quality by Design (QbD) principles in developing a new ultra high performance liquid chromatography method for the analysis of formoterol/budesonide and related substances using Fusion QbD® software is explored. The effect of various chromatographic parameters including, column stationary phase, pH, temperature, flow rate, and gradient time on separations were systematically investigated. Results show that optimal separations of these compounds in a standard solution can be achieved using a BEH C18 column (2.1 × 1.7 µm × 10 cm) applying a pH of 8.2, a temperature of 35 °C, a flow rate of 0.35 mL min-1 and a gradient time of 25 min. Furthermore, the results show that the main parameters affecting the performance of the method were the mobile phase pH, gradient time, and the temperature. For example, the most important factor for peak tailing was the pH of the mobile phase and the critical factors affecting resolution of the analytes were the gradient time and the temperature. As an application, the method was further used to analyze budesonide and formoterol in a sample obtained from a Symbicort® metered dose inhaler and it was found to provide similar separations to those obtained with the standard solution. These findings indicate that applying the QbD principles in analytical method development can be very advantageous not only in obtaining deep understanding of the effect of input parameters but also potential regulatory flexibility.


Subject(s)
Budesonide , Metered Dose Inhalers , Administration, Inhalation , Chromatography, High Pressure Liquid , Ethanolamines , Formoterol Fumarate
12.
J Chromatogr A ; 1611: 460597, 2020 Jan 25.
Article in English | MEDLINE | ID: mdl-31619360

ABSTRACT

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.


Subject(s)
Chromatography, High Pressure Liquid/methods , Lipids/blood , Tandem Mass Spectrometry/methods , Case-Control Studies , Discriminant Analysis , Female , Humans , Ion Mobility Spectrometry , Least-Squares Analysis , Metabolome , Phosphatidylcholines , Principal Component Analysis
13.
J Proteome Res ; 18(11): 4055-4064, 2019 11 01.
Article in English | MEDLINE | ID: mdl-31550900

ABSTRACT

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.


Subject(s)
Isoxazoles/pharmacology , Lipidomics/methods , Lipids/analysis , Liver Extracts/metabolism , Liver/drug effects , Mass Spectrometry/methods , Thiophenes/pharmacology , Animals , Chromatography, Liquid/methods , Endothelin Receptor Antagonists/pharmacology , Ions/analysis , Liver/metabolism , Male , Mice, SCID , Reproducibility of Results
14.
Metabolomics ; 15(2): 17, 2019 01 22.
Article in English | MEDLINE | ID: mdl-30830424

ABSTRACT

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.


Subject(s)
High-Throughput Screening Assays/methods , Metabolomics/methods , Urine/chemistry , Animals , Body Fluids , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid/methods , Humans , Male , Metabolome , Quality Control , Rats/urine , Rats, Sprague-Dawley/urine , Reproducibility of Results , Tandem Mass Spectrometry/methods
15.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1087-1088: 142-148, 2018 Jun 15.
Article in English | MEDLINE | ID: mdl-29738964

ABSTRACT

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.


Subject(s)
Chromatography, High Pressure Liquid/methods , Tandem Mass Spectrometry/methods , Ticrynafen/urine , Administration, Intravenous , Animals , Male , Rats , Rats, Sprague-Dawley , Ticrynafen/administration & dosage , Ticrynafen/metabolism
16.
Anal Chim Acta ; 982: 1-8, 2017 Aug 22.
Article in English | MEDLINE | ID: mdl-28734348

ABSTRACT

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.


Subject(s)
Chromatography, High Pressure Liquid , Ion Mobility Spectrometry , Metabolome , Urine/chemistry , Humans
17.
PLoS One ; 10(8): e0134206, 2015.
Article in English | MEDLINE | ID: mdl-26244785

ABSTRACT

Prostate cancer (PCa) is the most prevalent cancer amongst men and the second most common cause of cancer related-deaths in the USA. Prostate cancer is a heterogeneous disease ranging from indolent asymptomatic cases to very aggressive life threatening forms. The goal of this study was to identify differentially expressed metabolites and lipids in prostate cells with different tumorigenic phenotypes. We have used mass spectrometry metabolomic profiling, lipidomic profiling, bioinformatic and statistical methods to identify, quantify and characterize differentially regulated molecules in five prostate derived cell lines. We have identified potentially interesting species of different lipid subclasses including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), glycerophosphoinositols (PIs) and other metabolites that are significantly upregulated in prostate cancer cells derived from distant metastatic sites. Transcriptomic and biochemical analysis of key enzymes that are involved in lipid metabolism demonstrate the significant upregulation of choline kinase alpha in the metastatic cells compared to the non-malignant and non-metastatic cells. This suggests that different de novo lipogenesis and other specific signal transduction pathways are activated in aggressive metastatic cells as compared to normal and non-metastatic cells.


Subject(s)
Lipids/analysis , Metabolome , Metabolomics/methods , Blotting, Western , Cell Line, Tumor , Choline Kinase/genetics , Choline Kinase/metabolism , Chromatography, Liquid , Gene Expression Regulation, Neoplastic , Humans , Inositol Phosphates/analysis , Lipid Metabolism , Male , Middle Aged , Phenotype , Phosphatidylcholines/analysis , Phosphatidylethanolamines/analysis , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Reverse Transcriptase Polymerase Chain Reaction , Tandem Mass Spectrometry , Transcriptome/genetics
18.
Analyst ; 140(16): 5546-56, 2015 Aug 21.
Article in English | MEDLINE | ID: mdl-26146891

ABSTRACT

An integrated capillary scale (300 µm id) ceramic microfluidic LC system combined with MS/MS has been successfully employed for the quantitative analysis of pharmaceutical compounds in human plasma. The capillary ceramic microfluidic LC/MS/MS system showed an approximate 20-fold (range 11-38-fold) increase in sensitivity compared with a standard 2.1 mm scale UPLC/MS/MS system for a broad range of analytes. The loading capacity of the devices capillary separations channel allowed injection of 2 µL of an aqueous solution, and up to 1.2 µL of a typical protein-precipitated plasma sample, onto the reversed-phase chromatography system. The system also showed excellent chromatographic performance and robustness, with no deleterious effects on the chromatography observed over the course of 1000 injections of protein-precipitated plasma. The ability of the ceramic microfluidic LC/MS/MS system to deliver this level of sensitivity and performance enables the routine quantification of pharmaceutical compounds from small format samples, such as those obtained by dried blood spot or other blood microsampling approaches, to be performed.


Subject(s)
Ceramics/chemistry , Chromatography, Liquid/methods , Dried Blood Spot Testing/instrumentation , Lab-On-A-Chip Devices , Pharmaceutical Preparations/blood , Plasma/chemistry , Tandem Mass Spectrometry/methods , Humans , Pharmaceutical Preparations/chemistry
19.
Bioanalysis ; 7(11): 1397-411, 2015.
Article in English | MEDLINE | ID: mdl-26110713

ABSTRACT

Capillary LC (cLC) coupled to MS has the potential to improve detection limits, address limited sample volumes and allow multiple analyses from one sample. This is particularly attractive in areas where ultrahigh assay sensitivity, low limits of detection and small sample volumes are becoming commonplace. However, implementation of cLC-MS in the bioanalytical-drug metabolism area had been hampered by the lack of commercial instrumentation and the need for experts to operate the system. Recent advances in microfabricated devices such as chip-cube and ion-key technologies offer the potential for true implementation of cLC in the modern laboratory including the benefits of the combination of this type of separation with high-resolution MS.


Subject(s)
Chromatography, Liquid/instrumentation , Metabolomics/instrumentation , Microfluidic Analytical Techniques/instrumentation , Pharmaceutical Preparations/metabolism , Pharmacokinetics , Animals , Body Fluids/chemistry , Body Fluids/metabolism , Chromatography, Liquid/methods , Equipment Design , Humans , Metabolomics/methods , Mice , Microfluidic Analytical Techniques/methods , Pharmaceutical Preparations/blood
20.
Bioanalysis ; 7(7): 857-67, 2015.
Article in English | MEDLINE | ID: mdl-25932520

ABSTRACT

BACKGROUND: Increased pressure to obtain more, higher sensitivity data from less sample is especially critical for large peptides, whose already optimized LC-MS methods are heavily challenged by traditional ligand-binding assays. RESULTS: Critical bioanalytical assays were adapted to integrated microscale LC to reduce sample volumes while increasing sensitivity. Assays for teriparatide, glucagon and human insulin and five analogs were transferred from 2.1 mm analytical scale LC to a 150 µm scale system. This resulted in a 15-30 fold overall improvement in sensitivity derived from increased signal to noise, three to six fold reduction in injection volumes, and a two to five fold reduction in sample consumption. CONCLUSION: Integrated microscale LC reduces sample consumption while enabling single picomolar quantification for therapeutic and endogenous peptides.


Subject(s)
Blood Chemical Analysis/methods , Lab-On-A-Chip Devices , Peptides/blood , Systems Integration , Blood Chemical Analysis/instrumentation , Chromatography, Liquid , Humans , Injections , Linear Models , Mass Spectrometry , Time Factors
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