LC-MS Response Research Articles

Development and application of a dual isotopic labeling method for enhanced detection and quantification of stimulants in urine samples using high-resolution mass spectrometry.

Given the critical nature of anti-doping efforts, the detection of stimulant substances is shifting from accurate qualitative analysis to precise quantitative analysis. Additionally, the use of liquid chromatography-high-resolution mass spectrometry (LC-HRMS) in detecting stimulants is becoming more widespread. However, the lack of isotope-labeled internal standards is causing increasing issues with quantitative accuracy. Furthermore, challenges such as the mass spectrometric response of small molecules and the separation of isomers present additional difficulties. We have developed a quantitative method for stimulant substances containing amine or phenol hydroxyl groups, using a dual-label derivatization system. This method offers a new perspective for analyzing and detecting low molecular weight substances, isomers, or those with poor LC-MS response, and proposes a solution to the problem of missing isotope-labeled internal standards. Methodological validation has shown that this approach has promising application potential.

Tracking Extensive Portfolio of Cyanotoxins in Five-Year Lake Survey and Identifying Indicator Metabolites of Cyanobacterial Taxa.

Cyanobacterial blooms require monitoring, as they pose a threat to ecosystems and human health, especially by the release of toxins. Along with widely reported microcystins, cyanobacteria coproduce other bioactive metabolites; however, information about their dynamics in surface waters is sparse. We investigated dynamics across full bloom successions throughout a five-year lake monitoring campaign (Greifensee, Switzerland) spanning 150 sampling dates. We conducted extensive suspect screening of cyanobacterial metabolites using the database CyanoMetDB. Across all 850 samples, 35 metabolites regularly co-occurred. Microcystins were present in 70% of samples, with [d-Asp3,(E)-Dhb7]MC-RR reaching concentrations of 70 ng/L. Anabaenopeptins, meanwhile, were detected in 95% of all samples with concentrations of Oscillamide Y up to 100-fold higher than microcystins. Based on LC-MS response and frequency, we identified indicator metabolites exclusively produced by one of three cyanobacteria isolated from the lake, these being [d-Asp3,(E)-Dhb7]MC-RR from Planktothrix sp. G2020, Microginin 761B from Microcystis sp. G2011, and Ferintoic acid B from Microcystis sp. G2020. These indicators showed distinct temporal trends and peaking seasons that reflect the variance in either the abundance of the producing cyanobacteria or their toxin production dynamics. Our approach demonstrates that selecting high LC-MS response and frequent and species-specific indicator metabolites can be advantageous for cyanobacterial monitoring.

Rapid LC–MS assay for targeted metabolite quantification by serial injection into isocratic gradients

Liquid chromatography mass spectrometry (LC–MS) has emerged as a mainstream strategy for metabolomics analyses. One advantage of LC–MS is that it can serve both as a biomarker discovery tool and as a platform for clinical diagnostics. Consequently, it offers an exciting opportunity to potentially transition research studies into real-world clinical tools. One important distinction between research versus diagnostics-based applications of LC–MS is throughput. Clinical LC–MS must enable quantitative analyses of target molecules in hundreds or thousands of samples each day. Currently, the throughput of these clinical applications is limited by the chromatographic gradient lengths, which—when analyzing complex metabolomics samples—are difficult to conduct in under ~ 3 min per sample without introducing serious quantitative analysis problems. To address this shortcoming, we developed sequential quantification using isotope dilution (SQUID), an analytical strategy that combines serial sample injections into a continuous isocratic mobile phase to maximize throughput. SQUID uses internal isotope-labelled standards to correct for changes in LC–MS response factors over time. We show that SQUID can detect microbial polyamines in human urine specimens (lower limit of quantification; LLOQ = 106 nM) with less than 0.019 normalized root mean square error. Moreover, we show that samples can be analyzed in as little as 57 s. We propose SQUID as a new, high-throughput LC–MS tool for quantifying small sets of target biomarkers across large cohorts.Graphical

Oligomers in polyethylene furanoate - identification and quantification approach via LC-UV LC-MS response ratio

ABSTRACTPolyethylene furanoate polymer is intended to be used as a food contact material. A PEF polymer sample was investigated for its oligomer composition by solvent extraction and using HRLC-MS. The 20 oligomers found were divided into four groups: group I contains cyclic oligomers consisting of furandicarboxylic acid and monoethylene glycol units, group II comprises cyclic oligomers consisting of furandicarboxylic acid, monoethylene glycol units and one diethylene glycol unit, group III are cyclic oligomers were two monoethylene glycol units are substituted by diethylene glycol units and group IV are linear oligomers consisting of furandicarboxylic acid and monoethylene glycol units. Oligomers of group I account for around 87% of the total oligomer content, group II oligomers 12% and group III oligomers 1%. The contribution of group IV oligomers is very small: less than 0.05%. MS-MS experiments showed similar fragmentation patterns for all oligomers. The results of this study demonstrate that oligomers are abundant in the PEF material and are potential migrants to foods that are in contact with the polymer. Oligomers of group I and group II have the same absorption maxima in UV detection which was used to develop a quantification approach for these oligomers using dimethyl 2,5-furandicarboxylate as external standard.

Optimised method to estimate octanol water distribution coefficient (logD) in a high throughput format

Lipophilicity is one of the molecular properties assessed in early drug discovery. Direct measurement of the octanol-water distribution coefficient (logD) requires an analytical method with a large dynamic range or multistep dilutions, as the analyte's concentrations span across several orders of magnitude. In addition, water/buffer and octanol phases which have very different polarity could lead to matrix effects and affect the LC-MS response, leading to erroneous logD values. Most compound libraries use DMSO stocks as it greatly reduces the sample requirement but the presence of DMSO has been shown to underestimate the lipophilicity of the analyte. The present work describes the development of an optimised shake flask logD method using deepwell 96 well plate that addresses the issues related to matrix effects, DMSO concentration and incubation conditions and is also amenable to high throughput. Our results indicate that the equilibrium can be achieved within 30min by flipping the plate on its side while even 0.5% of DMSO is not tolerated in the assay. This study uses the matched matrix concept to minimise the errors in analysing the two phases namely buffer and octanol in LC-MS.

Epimers of Azaspiracids: Isolation, Structural Elucidation, Relative LC-MS Response, and in Vitro Toxicity of 37-epi-Azaspiracid-1

Since azaspiracid-1 (AZA1) was identified in 1998, the number of AZA analogues has increased to over 30. The development of an LC-MS method using a neutral mobile phase led to the discovery of isomers of AZA1, AZA2, and AZA3, present at ~2-16% of the parent analogues in phytoplankton and shellfish samples. Under acidic mobile phase conditions, isomers and their parents are not separated. Stability studies showed that these isomers were spontaneous epimerization products whose formation is accelerated with the application of heat. The AZA1 isomer was isolated from contaminated shellfish and identified as 37-epi-AZA1 by nuclear magnetic resonance (NMR) spectroscopy and chemical analyses. Similar analysis indicated that the isomers of AZA2 and AZA3 corresponded to 37-epi-AZA2 and 37-epi-AZA3, respectively. The 37-epimers were found to exist in equilibrium with the parent compounds in solution. 37-epi-AZA1 was quantitated by NMR, and relative molar response studies were performed to determine the potential differences in LC-MS response of AZA1 and 37-epi-AZA1. Toxicological effects were determined using Jurkat T lymphocyte cells as an in vitro cell model. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that 37-epi-AZA1 elicited a lethal response that was both concentration- and time-dependent, with EC50 values in the subnanomolar range. On the basis of EC50 comparisons, 37-epi-AZA1 was 5.1-fold more potent than AZA1. This data suggests that the presence of these epimers in seafood products should be considered in the analysis of AZAs for regulatory purposes.

UHPLC-ESI-HRMS Quantitation of Metabolites without Using Reference Standards: Impact of LC Flow Rate and Mobile Phase Composition on MS Responses

During LC-MS quantitation of drugs for pharmacokinetic assessment, usually metabolites are not quantified due to the unavailability of reference standards. If a metabolite is quantified without a reference standard, then it is assumed that the LC-MS response to a drug is similar to that of its metabolite and the standard curve, of the parent compound, is used to quantitate the metabolite. This approach could result in an over or underestimation of the metabolite. To evaluate the impact of mobile phase composition on LC-MS response, the mobile phases were interchanged between methanol, acetonitrile and a 50/50 mixture of methanol/acetonitrile. UHPLC flow rates were varied from 200-500 μL/min, with and without the addition of reverse composition of mobile phases, at the parent drug retention time. This change was necessary to achieve uniform MS responses for drugs and their metabolites. In this study, HRMS data, obtained using orbi-trap, resulted in a linear response over a wider dynamic range than that obtained using the linear ion trap. Overall, the parameters, required for achieving standard free quantitation, are dependent upon the mobile phase composition and LC flow rates.

Implication of Free Cholesterol in Lc–Ms Response Enhancement

In the context of matrix effects in bioanalytical LC-MS/MS, a speculative link between free cholesterol, the recoveries of the compound from three common extraction procedures, and response enhancement was qualitatively investigated. Injections on-column of cholesterol both in solution and extracts, in conjunction with post-column infusion of three representative drugs, reveal a direct role in pronounced response enhancement for two out of three analytes, under one set of LC-MS/MS conditions, for the majority of typical plasma extraction procedures, where electrospray-based gaseous ion generation is used. Cholesterol has been shown to have a strong association with LC-MS/MS response enhancement and ideally should be monitored during method development, reinforcing the reasoning behind minimizing SPE elution volumes and avoiding less selective means of sample preparation.

Microscale Phosphoproteome Analysis of 10 000 Cells from Human Cancer Cell Lines

We developed a miniaturized LC-MS system with a high-recovery phosphopeptide enrichment protocol that allows phosphoproteome analysis of 10(4) cells. In the enrichment protocol, the key step is to add sodium deoxycholate and sodium lauroyl sarcosinate to the buffer solution for protein extraction and digestion and to omit any subsequent desalt/desurfactant step before phosphopeptide enrichment. The phosphopeptides enriched by hydroxy acid-modified metal oxide chromatography (HAMMOC) are directly injected onto a miniaturized LC column using a nitrogen-pressure-driven cell, instead of switching valve-type injectors. The miniaturized analytical column of 25 μm diameter provided a 3.6-fold improvement in sensitivity over the conventional 100 μm diameter column. Overall, our analytical system provided approximately 80-fold improvement on average in the LC-MS response, and we identified 1011 unique phosphorylated sites based on 995 unique phosphopeptides from a single analysis of 10(4) HeLa cells (approximately 1 μg of proteins). This is the most sensitive phosphoproteomics system that has so far been reported for proteome-wide analysis of in vivo phosphorylation in mammalian cells.

Towards the quantification of residual milk allergens in caseinate-fined white wines using HPLC coupled with single-stage Orbitrap mass spectrometry

A method based on LC-ESI-high-resolution (HR)-MS analysis, using a single-stage Orbitrap mass spectrometer, was developed for the quantification of casein allergens potentially present in white wines as a result of fining by caseinate. The method consists of (1) extraction from the matrix by ultrafiltration, (2) digestion with trypsin and (3) detection/quantification of residual caseins, obtained by monitoring the LC-MS response of representative tryptic peptides (peak areas in extracted-ion chromatograms). Method linearity was assessed first on caseinate solutions prepared either in water or in wine matrix (the ultrafiltration residue of a protein-free white wine). Limits of detection (LOD) ranged from 0.1 to 0.3 µg ml−1 (S/N = 3) in water, and between 0.15 and 0.7 µg ml−1 in wine matrix, depending on the selected peptide. Method repeatability and reproducibility, measured as response variability (standard deviation) due to LC-MS analysis alone and to both enzymatic digestion and LC-MS analysis, were assessed on caseinate standard solutions in water and ranged from 5 to 12% and from 8 to 20%, respectively. A higher variability was usually observed for the peptide marker response in the case of matrix-matched samples, the only exception being peptide GPFPIIV from β-casein, the marker also providing the highest sensitivity. The method was finally applied to a casein-free white wine (‘Greco di Tufo’) fined with caseinate at different concentrations, after discarding the precipitate due to casein–wine components aggregation. Minimum detectable added caseinate concentrations (i.e. those corresponding to responses with S/N = 3) were estimated between 39 and 51 µg ml−1, according to the peptide marker chosen. These limits are compatible with caseinate concentrations typically adopted for wine-fining purposes. Moreover, a cross-check with the calibration performed in wine matrix led to an estimation of the concentration of dissolved caseinate to be in the low ng ml−1 range.

A low flow ionization technique to integrate quantitative and qualitative small molecule bioanalysis

Mass spectrometry-based assays are used in drug discovery and development to detect, characterize and quantify drugs, metabolites, impurities and degradants. Recently, high resolution-based mass spectrometers have begun to emerge as a platform with potential for performing integrated qualitative and quantitative assays in order to streamline the drug discovery and development process. However, the widely different LC–MS response observed for a drug and its metabolites limit the direct use of LC–MS responses for relative quantitative determination of metabolites. This in turn limits the use of conventional LC–ESI-MS methods, in the absence of reference standards, as an integrated technique for detection, characterization and quantification of drugs and metabolites. The goal of this study was to explore the use of LC–captive spray ionization (CSI)-mass spectrometry for detection, characterization and quantification of drugs and metabolites. CSI allows the use of conventional HPLC or uHPLC columns and flow rates of 0.35–0.6 mL/min (before post-column flow splitting) and can be considered as a technique which can function as a nanospray or microspray. Also, in comparison to conventional nanospray ionization (NSI) techniques, setup and maintenance of CSI do not require: (1) X, Y, and Z positioning or cameras to guide the spray positioning, (2) difficult to control splitters to deliver nano-flow ratios and difficult to maintain nanospray nozzles. Evaluations using equimolar mixture of buspirone and four monoxy metabolites present in human plasma show that LC–CSI-MS is a highly sensitive technique that gives a near equimolar response for the compounds used in this example. Comparisons of LC–ESI-MS data with that obtained using LC–CSI-MS show that reasonable quantification of metabolites may be achievable without using reference standards or administration of radiolabeled drugs.

Novel MS Solutions Inspired by MIST

To improve patient safety and to help avoid costly late-stage failures, the pharmaceutical industry, along with the US FDA and International Committee on Harmonization (ICH), recommends the identification of differences in drug metabolism between animals used in nonclinical safety assessments and humans as early as possible during the drug-development process. LC-MS is the technique of choice for detection and characterization of metabolites, however, the widely different LC-MS response observed for a new chemical entity (NCE) and its structurally related metabolites limits the direct use of LC-MS responses for quantitative determination of NCEs and metabolites. While no method provides completely accurate universal response, UV, corona charged aerosol detection (CAD), radioactivity, NMR and low-flow (< 20 µl/min) nanospray approaches provide opportunities to quantify metabolites in the absence of reference standards or radiolabeled material with enough precision to meet the needs of early clinical development.

Analysis of tricyclic antidepressant drugs in plasma by means of solid‐phase microextraction‐liquid chromatography‐mass spectrometry

Solid-phase microextraction coupled to liquid chromatography and mass spectrometry (SPME-LC-MS) was used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. SPME was performed by direct extraction on a PDMS/DVB (60 microm) coated fiber, employing a stirring rate of 1200 rpm for 30 min, pH 11.0, and temperature of 30 degrees C. Drug desorption was carried out by exposing the fiber to the liquid chromatography mobile phase for 20 min, using a labmade SPME-LC interface at 50 degrees C. The main variables experimentally influencing LC-MS response were evaluated and mathematically modeled. A rational optimization with fewer experiments was achieved using a factorial design approach. The constructed empirical models were adjusted with 96-98% of explained deviation allowing an adequate data set comprehension. The chromatographic separation was realized using an RP-18 column (150 mm x 2.1 mm, 5 microm particles) and ammonium acetate buffer (0.01 mol/l, pH 5.50) : acetonitrile (50 : 50 v/v) as mobile phase. Low detection levels were achieved with electrospray interface (0.1 ng/ml). The developed method showed specificity, linearity, precision, and limit of quantification adequate to assay tricyclic antidepressant drugs in plasma.

Response Normalized Liquid Chromatography Nanospray Ionization Mass Spectrometry

The widely different LC-MS response observed for many structurally different compounds limits the use of LC-MS in full scan detection mode for quantitative determination of drugs and metabolites without using reference standard. The recently introduced nanospray ionization (NSI) technique shows comparable MS response for some compounds under non-LC-MS conditions. However, in the presence of numerous endogenous compounds commonly associated with biological samples such as urine, plasma, and bile, LC-MS is required to separate, detect, identify, and measure individual analytes. An LC-NSI-MS system was devised and the MS response obtained in this system for a variety of pharmaceutical drugs and their metabolites. The set-up involves two high-performance liquid chromatography (HPLC) systems, a chip-based NSI source and a quadrupole-time-of-flight (Q-TOF) mass spectrometer. Herein this is referred to as the response normalized-liquid chromatography NSI-MS (RNLC-NSI-MS) system. One HPLC unit performs the analytical separation, while the other unit adds solvent post-column with an exact reverse of the mobile phase composition such that the final composition entering the NSI source is isocratic throughout the entire HPLC run. The data obtained from four different structural classes of compounds [vicriviroc (VCV), desloratadine (DL), tolbutamide, and cocaine] and their metabolites indicate that by maintaining the solvent composition unchanged across the HPLC run, the influence of the solvent environment on the ionization efficiency is minimized. In comparison to responses obtained from radiochromatograms, responses from conventional LC-ESI-MS overestimated the VCV and DL responses, respectively, by 6- and 20-fold. Although VCV and DL responses obtained using LC-NSI-MS are within 2- to 6-fold from the respective radiochromatographic responses, the response normalization modification results in nearly uniform LC-NSI-MS response for all compounds evaluated.