SpheriCal® -ESI: A dendrimer-based nine-point calibration solution ranging from m/z 273 to 1716 for electrospray ionization mass spectrometry peptide analysis.

RATIONALE: A calibration solution for mass spectrometry needs to cover the range of interest with intense and sufficiently narrowly spaced peaks. Limited options fulfilling this may lead to compromises between performance and ease of use. SpheriCal® -ESI was designed to combine high calibration performance for electrospray ionization (ESI) mass spectrometric analysis of peptides in positive mode with quick and easy use. METHODS: The developed calibration solution was tested using three mass spectrometers: two ion traps and one tandem quadrupole. The m/z errors of SpheriCal® -ESI itself and of a tryptic digest of cytochrome C were measured after calibration. The results were compared with those achieved with ESI Tuning Mix. The memory effects of the dendrimers, and contamination from Na+ in the calibration solution, were evaluated. RESULTS: SpheriCal® -ESI showed good shelf life as powder and was quickly reconstituted for use. Achieving intense and stable signals was straightforward. The accuracies and precisions were as expected for the instruments. SpheriCal® -ESI was more precise and at least as accurate as ESI Tuning Mix. The memory effects and Na+ contamination were found to be negligible in typical peptide solvents. In addition, the dendrimers showed predictable dissociations with product ions common to collision-induced dissociation in both ion trap and tandem quadrupole mass spectrometers. CONCLUSIONS: SpheriCal® -ESI provided easily accessible calibration by showing intense signals at low infusion rates and at source settings equal or similar to those used in peptide analysis. Nine calibration points in the range of interest gave precise and accurate results. Memory effects and contamination were negligible even without rinsing.

Concurrent identification of 11 major primary active compounds in Huangqin Qingfei Decoction by liquid chromatography tandem mass spectrometry via liquid chromatography tandem mass spectrometry.

Huangqin Qinfei Decoction (HQD) is a traditional Chinese medicine that is administered for acute pneumonia, bronchial inflammation, acute bronchitis and acute lung infection. In this study, we used liquid chromatography linked with tandem mass spectrometry (LC-MS/MS) for the concurrent identification of 11 bioactive compounds; namely, baicalin, baicalein, wogonoside, scutellarin, wogonin, oroxylin A, geniposide, genipin, geniposidic acid, chlorogenic acid, and crocin-I, for the quality control of HQD. The evaluation was conducted on an Agilent Poroshell 120 EC-C18 (2.1mm×100mm, 2.7µm) with gradient elution in the mobile phase with 0.1% formic acid and 1mM/L ammonium acetate in water as solvent A and methanol as solvent B at a flow rate of 0.3mL/min in under 12 min. Mass spectrometric detection was conducted in the selected reaction monitoring mode utilizing electro spray ionization in the positive and negative modes. Every one of the calibration curves had good linearity with R2 >0.9992. Intra-day and inter-day accuracies for every one of the evaluated components were expressed as the relative standard deviation (RSD) from 1.72%-5.02% and 0.63%-5.99%, respectively. The recuperation of the 11 compounds that were measured at the three concentrations was within 94.05%-105.18%, with the RSD ≤ 6.26%. The use of this method was determined through the effective evaluation of 11 compounds in 5 batches of HQD. The confirmed method is precise, sensitive, and effective for identifying the contents of the chosen compounds in HQD for quality control.

DMS as an orthogonal separation to LC/ESI/MS/MS for quantifying isomeric cerebrosides in plasma and cerebrospinal fluid.

Cerebrosides, including glucosylceramides (GlcCers) and galactosylceramides (GalCers), are important membrane components of animal cells with deficiencies resulting in devastating lysosomal storage disorders. Their quantification is essential for disease diagnosis and a better understanding of disease mechanisms. The simultaneous quantification of GlcCer and GalCer isomers is, however, particularly challenging due to their virtually identical structures. To address this challenge, we developed a new LC/MS-based method using differential ion mobility spectrometry (DMS) capable of rapidly and reproducibly separating and quantifying isomeric cerebrosides in a single run. We show that this LC/ESI/DMS/MS/MS method exhibits robust quantitative performance within an analyte concentration range of 2.8-355 nM. We further report the simultaneous quantification of nine GlcCers (16:0, 18:0, 20:0, 22:0, 23:0, 24:1, 24:0, 25:0, and 26:0) and five GalCers (16:0, 22:0, 23:0, 24:1, and 24:0) molecular species in human plasma, as well as six GalCers (18:0, 22:0, 23:0, 24:1, 24:0 and 25:0) and two GlcCers (24:1 and 24:0) in human cerebrospinal fluid. Our method expands the potential of DMS technology in the field of glycosphingolipid analysis for both biomarker discovery and drug screening by enabling the unambiguous assignment and quantification of cerebroside lipid species in biological samples.

Improved Dried Blood Spot-Based Metabolomics Analysis by a Postcolumn Infused-Internal Standard Assisted Liquid Chromatography-Electrospray Ionization Mass Spectrometry Method.

Dried blood spots (DBSs) have gained increasing attention recently with their growing importance in precision medicine. DBS-based metabolomics analysis provides a powerful tool for investigating new biomarkers. Until now, very few studies have discussed measures for improving analytical accuracy with the consideration of the special characteristics of DBSs. The present study proposed a postcolumn infused-internal standard (PCI-IS) assisted strategy to improve data quality for DBS-based metabolomics studies. An efficient sample preparation protocol with 80% acetonitrile as the extraction solvent was first established to improve the metabolite recovery. The PCI-IS assisted liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was used to simultaneously estimate the blood volume and correct the signal change caused by ion source contamination and the matrix effect to evaluate the spot volume effect and hematocrit (Hct) variation effect on target metabolites. Phenylalanine-d8 was selected as the single PCI-IS to correct the matrix effect. For calibration of errors caused by the blood volume difference, 75% of the test metabolites showed good correlation (R2 ≥ 0.9) between the spot volume and the signal intensity after PCI-IS correction compared to less than 50% metabolites with good correlation before calibration. The spot volume was further calibrated by the same PCI-IS. Investigation of the Hct variation effect on target metabolites revealed that it affected the concentrations of metabolites in the DBS samples depending on their abundance in the red blood cell (RBC) or plasma; it is essential to preinvestigate the distribution of metabolites in blood to minimize the comparison bias in metabolomics studies. Finally, the PCI-IS assisted method was applied to study acetaminophen-induced liver toxicity. The results indicated that the proposed PCI-IS strategy could effectively remove analytical errors and improve the data quality, which would make the DBS-based metabolomics more feasible in real-world applications.

Combining untargeted and targeted metabolomics approaches for the standardization of polyherbal formulations through UPLC-MS/MS.

INTRODUCTION: Polyherbal formulations are an integral part of various indigenous medicinal systems such as Traditional Chinese Medicine (TCM) and Ayurveda. The presence of a very large number of compounds makes the quality control of polyherbal formulations very difficult. OBJECTIVES: To overcome this problem, we have developed a comprehensive strategy for the dereplication of natural products in polyherbal formulations by using Adhatoda vasica as a case study. METHODS: The strategy is based on five major steps: the collection of plant samples from different locations to observe the effects of environmental variables; LC-ESI-MS/MS-based untargeted metabolite profiling of the plant samples to identify marker compounds using extensive chemometric analysis of the obtained data; the identification of marker compounds in polyherbal products; the isolation, purification and characterization of the marker compounds; and MRM-based quantitative analysis of the isolated marker compounds using LC-ESI-MS/MS. RESULTS: Using this strategy, we identified a total of 51 compounds in the methanolic extract of A. vasica plants from 14 accessions. Chemical fingerprinting of the plant led to the identification of characteristic peaks that were used to confirm the presence of A. vasica in complex polyherbal formulations. Four quinazoline alkaloids (marker compounds) were isolated, purified and quantified in various herbal formulations containing A. vasica. CONCLUSION: This method demonstrates a comprehensive strategy based on untargeted and targeted metabolite analysis that can be used for the standardization of complex polyherbal formulations.

Metabolomics by UHPLC-MS: benefits provided by complementary use of Q-TOF and QQQ for pathway profiling.

INTRODUCTION: Non-targeted metabolic profiling using high-resolution mass spectrometry (HRMS) is a standard approach for pathway identification despite technical limitations. OBJECTIVES: To assess the performance of combining targeted quadrupole (QQQ) analysis with HRMS for in-depth pathway profiling. METHODS: Serum of exercising patients with type 1 diabetes (T1D) was profiled using targeted and non-targeted assays. RESULTS: Non-targeted analysis yielded a broad unbiased metabolic profile, targeted analysis increased coverage of purine metabolism (twofold) and TCA cycle (three metabolites). CONCLUSION: Our screening strategy combined the benefits of the unbiased full-scan HRMS acquisition with the deeper insight into specific pathways by large-scale QQQ analysis.

Validation of a multiplexed LC-MS/MS clinical assay to quantify insulin-like growth factor-binding proteins in human serum and its application in a clinical study.

Circulating insulin-like growth factor-binding proteins (IGFBPs) continue to gain attention as biomarkers of drug activities on insulin like growth factor (IGF)/IGF receptor signaling pathways. A multiplexed LC-MS/MS method was validated for the absolute quantitation of IGFBPs in human serum. The method was used to measure screening concentrations of IGFBPs in spinal and bulbar muscular atrophy (SBMA) patients in a phase 2 clinical trial. Concentrations of IGFBP 1, 2, 3, and 5 were simultaneously determined based on representative signature peptides derived from an optimized trypsin digestion procedure. Signature peptide levels were absolutely quantitated using a sensitive/specific targeted LC-MS/MS method. Corresponding mass-shifted, stable isotope-labeled peptides were employed as internal standards. A true blank matrix for the quantitation of IGFBPs was not available since they are endogenous proteins in human serum. In this method, calibration standards/curves were prepared using authentic synthetic peptides spiked into a surrogate matrix. The surrogate matrix was generated from human serum treated in the same way as the study samples, but using iodoacetic acid instead of iodoacetamide as the alkylation reagent. This surrogate matrix approach allowed for the direct and sensitive/specific quantification of IGFBP 1, 2, 3, and 5 due to the lack of any endogenous background. Equivalent matrix effect and recovery of analytes was achieved for the authentic and surrogate matrices. The fully validated LC-MS/MS assay will allow further evaluation of the utility of IGFBP biomarkers in clinical trials.

Evaluation of the High-Field Orbitrap Fusion for Compound Annotation in Metabolomics.

Annotation of signals of interest represents a key point in mass spectrometry-based metabolomics studies. The first level of investigation is the elemental composition, which can be deduced from accurately measured masses and isotope patterns. However, accuracy of these two parameters remains to be evaluated on last generation mass spectrometers to determine the level of confidence that can be used during the annotation process. In this context, we evaluated the performance of the Orbitrap Fusion mass spectrometer for the first time and demonstrated huge potential for metabolite annotation via elemental composition determination. This work was performed using a set of 50 standard compounds analyzed under LC/MS conditions in solvent and biological media. Accurate control of the number of trapped ions proved mandatory to avoid space charge effects, ensure sub-ppm mass accuracy (using external calibration), and reliable measurement of isotopic patterns at 500,000 resolution. On the basis of the results, we propose standard optimized experimental conditions for performing robust and accurate untargeted metabolomics on the Orbitrap Fusion at high mass measurement and mass spectral accuracy.

Internal Standard Quantification Using Tandem Mass Spectrometry of a Tryptic Peptide in the Presence of an Isobaric Interference.

Model mixtures of isobaric peptides were studied to evaluate the possibility, using tandem mass spectrometry experiments, for internal standard quantification of a tryptic peptide in the presence of an isobaric interference. To this end, direct injection electrospray ionization-tandem mass spectrometry (ESI-MS/MS) experiments were performed on an ion trap instrument using a large mass-selection window (15 m/ z) encompassing the isobaric mixture and the internal standard; MS/MS experiments were carried out to remove completely the interference from the mixture by fragmenting it. This allowed for the correct intensity assignment for the protonated peptide peak and, thus, for the analyte to be quantified through the relative intensity estimate of this peak with respect to the internal standard. This was done by monitoring the 15 m/ z mass-selection window only and without the necessity for careful inspection of any fragment ions peaks. The interference removal was assessed by determining an excitation voltage large enough for the analyte/internal standard ratio to remain constant ensuring correct quantification despite isobaric contamination. A calibration curve was obtained to predict reference samples and compared to reference samples purposely spiked with the interference using the proposed methodology; internal standard quantification of the analyte was made possible with ∼1% deviation despite the isobaric contamination.

Creating a Mass Spectral Reference Library for Oligosaccharides in Human Milk.

We report the development and availability of a mass spectral reference library for oligosaccharides in human milk. This represents a new variety of spectral library that includes consensus spectra of compounds annotated through various data analysis methods, a concept that can be extended to other varieties of biological fluids. Oligosaccharides from the NIST Standard Reference Material (SRM) 1953, composed of human milk pooled from 100 breastfeeding mothers, were identified and characterized using hydrophilic interaction liquid chromatography electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS) and the NIST 17 Tandem MS Library. Consensus reference spectra were generated, incorporated into a searchable library, and matched using the newly developed hybrid search algorithm to elucidate unknown oligosaccharides. The NIST hybrid search program facilitates the structural assignment of complex oligosaccharides especially when reference standards are not commercially available. High accuracy mass measurement for precursor and product ions, as well as the relatively high MS/MS signal intensities of various oligosaccharide precursors with Fourier transform ion trap (FT-IT) and higher energy dissociation (HCD) fragmentation techniques, enabled the assignment of multiple free and underivatized fucosyllacto- and sialyllacto-oligosaccharide spectra. Neutral and sialylated isomeric oligosaccharides have distinct retention times, allowing the identification of 74 oligosaccharides in the reference material. This collection of newly characterized spectra based on a searchable, reference MS library of annotated oligosaccharides can be applied to analyze similar compounds in other types of milk or any biological fluid containing milk oligosaccharides.

Paper spray ionization mass spectrometry: recent advances and clinical applications.

INTRODUCTION: Paper spray mass spectrometry has provided a rapid, quantitative ambient ionization method for xenobiotic and biomolecule analysis. As an alternative to traditional sample preparation and chromatography, paper spray demonstrates the sampling ionization of a wide range of molecules and significant sensitivity from complex biofluids. The amenability of paper spray with dried blood spots and other sampling types shows strong potential for rapid, point-of-care (POC) analysis without time-consuming separation procedures. Areas covered: This special report summarizes the current state and advances in paper spray mass spectrometry that relate to its applicability for clinical analysis. It also provides our perspectives on the future development of paper spray mass spectrometry and its potential roles in clinical settings. Expert commentary: Paper spray has provided the fundamental aspects of ambient ionization needed for implementation at the POC. With further clinical management and standardization, paper spray has the potential to replace traditional complex analysis procedure for rapid quantitative detection of illicit drugs, therapeutic drugs and metabolites. Surface and substrate modifications also offer significant improvement in desorption and ionization efficiencies, resulting in enhanced sensitivity. Comprehensive analysis of metabolites and lipids will further extend the implementation of paper spray ionization mass spectrometry into clinical applications.

Selection of internal standards for accurate quantification of complex lipid species in biological extracts by electrospray ionization mass spectrometry-What, how and why?

Lipidomics is rapidly expanding because of the great facilitation of recent advances in, and novel applications of, electrospray ionization mass spectrometry techniques. The greatest demands have been for successful quantification of lipid classes, subclasses, and individual molecular species in biological samples at acceptable accuracy. This review addresses the selection of internal standards in different methods for accurate quantification of individual lipid species. The principles of quantification with electrospray ionization mass spectrometry are first discussed to recognize the essentials for quantification. The basics of different lipidomics approaches are overviewed to understand the variables that need to be considered for accurate quantification. The factors that affect accurate quantification are extensively discussed, and the solutions to resolve these factors are proposed-largely through addition of internal standards. Finally, selection of internal standards for different methods is discussed in detail to address the issues of what, how, and why related to internal standards. We believe that thorough discussion of the topics related to internal standards should aid in quantitative analysis of lipid classes, subclasses, and individual molecular species and should have big impacts on advances in lipidomics. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:693-714, 2017.

Effects of solvent parameters on the electrospray ionization tandem mass spectrometry response of glucose.

RATIONALE: The importance of saccharides, the most abundant biomolecules on Earth, extends beyond their biological roles and to consumer products and industrial processes. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) is an attractive tool for the analysis of underivatized saccharides (US), but they tend to have relatively low sensitivities due to their low surface activities and lack of easily protonable or deprotonable chemical groups. An understanding of the influences that solvent parameters have on their signal intensities would enhance the usefulness of ESI-MS/MS for their analysis. METHODS: Solutions of glucose, a model analyte for US, in various combinations of solvent, additive, additive concentration, and pH were analyzed by flow injection analysis ESI-MS/MS in both the positive and negative ionization mode. The blank-corrected signal intensities of the solvent parameter combinations were then compared. RESULTS: The addition of acetonitrile led to severe ionization suppression in the positive ionization mode through its competition with glucose for cation adduction. High signal intensity was achieved under wide pH and concentration ranges for methanol: water solutions containing ammonium trifluoroacetate in the positive ionization mode. The highest signal intensities for acetonitrile: water solutions were those containing ammonium formate or lithium fluoride in the negative ionization mode. CONCLUSIONS: An understanding of the influence of solvent parameters on the signal intensity of a given analyte is useful for guiding the selection process of mobile phases/flow solvents that lead to low limits of detection or the minimization of matrix effects by allowing its detection at high dilution factors.

Neutral Loss Is a Very Common Occurrence in Phosphotyrosine-Containing Peptides Labeled with Isobaric Tags.

While developing a multiplexed phosphotyrosine peptide quantification assay, an unexpected observation was made: significant neutral loss from phosphotyrosine (pY) containing peptides. Using a 2000-member peptide library, we sought to systematically investigate this observation by comparing unlabeled peptides with the two highest-plex isobaric tags (iTRAQ8 and TMT10) across CID, HCD, and ETD fragmentation using high resolution high mass accuracy Orbitrap instrumentation. We found pY peptide neutral loss behavior was consistent with reduced proton mobility, and does not occur during ETD. The site of protonation at the peptide N-terminus changes from a primary to a tertiary amine as a result of TMT labeling which would increase the gas phase basicity and reduce proton mobility at this site. This change in fragmentation behavior has implications during instrument method development and interpretation of MS/MS spectra, and therefore ensuing follow-up studies. We show how sites not localized to tyrosine by search and site localization algorithms can be confidently reassigned to tyrosine using neutral loss and phosphotyrosine immonium ions. We believe these findings will be of general interest to those studying pY signal transduction using isobaric tags.

Mass Spectrometry of Intact Proteins Reveals +98 u Chemical Artifacts Following Precipitation in Acetone.

Protein precipitation in acetone is frequently employed ahead of mass spectrometry for sample preconcentration and purification. Unfortunately, acetone is not chemically inert; mass artifacts have previously been observed on glycine-containing peptides when exposed to acetone under acidic conditions. We herein report a distinct chemical modification occurring at the level of intact proteins when incubated in acetone. This artifact manifests as one or more satellite peaks in the MS spectrum of intact protein, spaced 98 u above the mass of the unmodified protein. Other artifacts (+84, +112 u) also appear upon incubation of proteins or peptides in acetone. The reaction is pH-sensitive, being suppressed when proteins are exposed to acetone under acidic conditions. The +98 u artifact is speculated to originate through an intermediate product of aldol condensation of acetone to form diacetone alcohol and mesityl oxide. A +98 u product could originate from nucleophilic attack on mesityl oxide or through condensation with diacetone alcohol. Given the extent of modification possible upon exposure of proteins to acetone, particularly following overnight solvent exposure or incubation at room temperature, an awareness of the variables influencing this novel modification is valued by proteomics researchers who employ acetone precipitation for protein purification.

Development of a Postcolumn Infused-Internal Standard Liquid Chromatography Mass Spectrometry Method for Quantitative Metabolomics Studies.

Quantitative metabolomics has become much more important in clinical research in recent years. Individual differences in matrix effects (MEs) and the injection order effect are two major factors that reduce the quantification accuracy in liquid chromatography-electrospray ionization-mass spectrometry-based (LC-ESI-MS) metabolomics studies. This study proposed a postcolumn infused-internal standard (PCI-IS) combined with a matrix normalization factor (MNF) strategy to improve the analytical accuracy of quantitative metabolomics. The PCI-IS combined with the MNF method was applied for a targeted metabolomics study of amino acids (AAs). D8-Phenylalanine was used as the PCI-IS, and it was postcolumn-infused into the ESI interface for calibration purposes. The MNF was used to bridge the AA response in a standard solution with the plasma samples. The MEs caused signal changes that were corrected by dividing the AA signal intensities by the PCI-IS intensities after adjustment with the MNF. After the method validation, we evaluated the method applicability for breast cancer research using 100 plasma samples. The quantification results revealed that the 11 tested AAs exhibit an accuracy between 88.2 and 110.7%. The principal component analysis score plot revealed that the injection order effect can be successfully removed, and most of the within-group variation of the tested AAs decreased after the PCI-IS correction. Finally, targeted metabolomics studies on the AAs showed that tryptophan was expressed more in malignant patients than in the benign group. We anticipate that a similar approach can be applied to other endogenous metabolites to facilitate quantitative metabolomics studies.

Temporal Signal Pattern Recognition in Mass Spectrometry: A Method for Rapid Identification and Accurate Quantification of Biomarkers for Inborn Errors of Metabolism with Quality Assurance.

Mass spectrometry (MS)-based metabolomic initiatives that use conventional separation techniques are limited by low sample throughput and complicated data processing that contribute to false discoveries. Herein, we introduce a new strategy for unambiguous identification and accurate quantification of biomarkers for inborn errors of metabolism (IEM) from dried blood spots (DBS) with quality assurance. A multiplexed separation platform based on multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) was developed to provide comparable sample throughput to flow injection analysis-tandem MS (FIA-MS/MS) but with greater selectivity as required for confirmatory testing and discovery-based metabolite profiling of volume-restricted biospecimens. Mass spectral information is encoded temporally within a separation by serial injection of three or more sample pairs, each having a unique dilution pattern, alongside a quality control (QC) that serves as a reference in every run to facilitate between-sample comparisons and/or batch correction due to system drift. Optimization of whole blood extraction conditions on DBS filter paper cut-outs was first achieved to maximize recovery of a wide range of polar metabolites from DBS extracts. An interlaboratory comparison study was also conducted using a proficiency test and retrospective neonatal DBS that demonstrated good agreement between MSI-CE-MS and validated FIA-MS/MS methods within an accredited facility. Our work demonstrated accurate identification of various IEM based on reliable measurement of a panel of primary or secondary biomarkers above an upper cutoff concentration limit for presumptive screen-positive cases without stable isotope-labeled reagents. Additionally, nontargeted metabolite profiling by MSI-CE-MS with temporal signal pattern recognition revealed new biomarkers for early detection of galactosemia, such as N-galactated amino acids, that are a novel class of pathognomonic marker due to galactose stress in affected neonates.

Implications for Metabolite Quantification by Mass Spectrometry in the Absence of Authentic Standards.

Quantification of metabolites by mass spectrometry in the absence of authentic reference standards or without a radiolabel is often called "semiquantitative," which acknowledges that mass spectrometric responses are not truly quantitative. For many researchers, it is tempting to pursue this practice of semiquantification in early drug discovery and even preclinical development, when radiolabeled absorption, distribution, metabolism, and excretion studies are being deferred to later stages of drug development. The caveats of quantifying metabolites based on parent drug response are explored in this investigation. A set of 71 clinically relevant drugs/metabolites encompassing common biotransformation pathways was subjected to flow injection analysis coupled with electrospray ionization (ESI) mass spectrometry. The results revealed a large variation in ESI response even for structurally similar parent drug/metabolite pairs. The ESI response of each metabolite was normalized to that of the parent drug to generate an ESI relative response factor. Overall, relative response factors ranged from 0.014 (>70-fold lower response than parent) to 8.6 (8.6-fold higher response than parent). Various two-dimensional molecular descriptors were calculated that describe physicochemical, topological, and structural properties for each drug/metabolite. The molecular descriptors, along with the ESI response factors, were used in univariate analyses as well as a principal components analysis to ascertain which molecular descriptors best account for the observed discrepancies in drug/metabolite ESI response. This investigation has shown that the practice of using parent drug response to quantify metabolites should be used with caution.

Relative molar response of lipophilic marine algal toxins in liquid chromatography/electrospray ionization mass spectrometry.

RATIONALE: Accurate quantitative analysis of lipophilic toxins by liquid chromatography/mass spectrometry (LC/MS) requires calibration solution reference materials (RMs) for individual toxin analogs. Untargeted analysis is aimed at identifying a vast number of compounds and thus validation of fully quantitative untargeted methods is not feasible. However, a semi-quantitative approach allowing for profiling is still required and will be strengthened by knowledge of the relative molar response (RMR) of analogs in LC/MS with electrospray ionization (ESI). METHODS: RMR factors were evaluated for toxins from the okadaic acid (OA/DTXs), yessotoxin (YTX), pectenotoxin (PTX), azaspiracid (AZA) and cyclic imine (CI) toxin groups, in both solvent standards and environmental sample extracts. Since compound ionization and fragmentation influences the MS response of toxins, RMRs were assessed under different chromatographic conditions (gradient, isocratic) and MS acquisition modes (SIM, SRM, All-ion, target MS/MS) on low- and high-resolution mass spectrometers. RESULTS: In general, RMRs were not significantly impacted by chromatographic conditions (isocratic vs gradient), with the exception of DTX1. MS acquisition modes had a more significant impact, with PnTX-G and SPX differing notably. For a given toxin group, response factors were generally in the range of 0.5 to 2. The cyclic imines were an exception. CONCLUSIONS: Differences in RMRs between toxins of a same chemical base structure were not significant enough to indicate major issues for non-targeted semi-quantitative analysis, where there is limited or no availability of standards for many compounds, and where high degrees of accuracy are not required. Differences in RMRs should be considered when developing methods that use a standard of a single analogue to quantitate other toxins from the same group.

Identification and quantification of thymol metabolites in plasma, liver and duodenal wall of broiler chickens using UHPLC-ESI-QTOF-MS.

In the present study, we aimed to develop a method for thymol sulfate and thymol glucuronide determination in plasma, liver and duodenal wall of broiler chickens after feeding with a Thymus vulgaris essential oil at the different concentrations (0.01, 0.05 and 0.1% w/w). UHPLC coupled with accurate-mass QTOF-MS was used for identification and quantification of thymol metabolites. Novel Waters Oasis Prime HLB solid-phase extraction cartridges were applied to sample clean-up with extraction recoveries ranged from 85 to 92%. The presence of thymol glucuronide was confirmed by MS software according to molecular formula, score, mass error and double bond equivalent. In terms of validation, calibration curves of thymol sulfate were constructed in matrix samples with linearity from 3.91 to 250.0 ng/mL and correlation coefficients were within the range of 0.9979-0.9995. Limits of detection were 0.97, 0.29 and 0.63 ng/mL and limits of quantification were 3.23, 0.97 and 2.09 ng/mL for plasma, liver and duodenal wall, respectively. Intra-day and inter-day precision expressed as relative standard deviation were <4.35%. To highlight, thymol metabolites were directly detected for the first time in liver and duodenal wall and this method was shown to be successfully applicable for investigation of thymol metabolism in chickens after thyme essential oil ingestion.