MS/MS Research Articles

(S)-1-(5-(4-Methylpiperazin-1-yl)-2,4-dinitrophenyl)pyrrolidine-2-carboxylic acid as a derivatization reagent for ultrasensitive detection of amine enantiomers by HPLC-MS/MS and its application to the chiral metabolite analysis of (R)-1-aminoindan in saliva

(S)-1-(5-(4-Methylpiperazin-1-yl)-2,4-dinitrophenyl)pyrrolidine-2-carboxylic acid (Pro-PPZ) was employed as a chiral derivatization reagent (CDR) for the efficient enantioseparation and ultrasensitive mass spectrometric detection of chiral amines. Pro-PPZ was prepared from the one-step reaction of 1-(5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine (PPZ) and l-proline. Two amines and two amino acid methyl esters were selected as model chiral amines, which were easily labeled with Pro-PPZ under mild reaction conditions (35 °C for 10 min) generating Pro-PPZ-amine derivatives. The resulting diastereomers were completely separated by reversed-phase liquid chromatography (RP-LC) using an ODS column (Rs = 3.4–17.0 for amines). Ultrasensitive detection limits on femtomolar level were obtained for the tested amines using multiple reaction monitoring (MRM) chromatograms at a single monitoring ion, m/z 289 (0.1–5.0 fmol for amines). The practical metabolite analysis of (R)-1-aminoindan (R-AI) in saliva samples was performed by LC–MS/MS using the Pro-PPZ derivatization method. The method was validated in terms of precision, accuracy, and linearity. Using this method, R-AI concentrations in saliva were determined after a single oral administration of the drug rasagiline to healthy male and female subjects, but no (S)-1-aminoindan (S-AI) was detected, which suggesting that R-AI was not converted into S-enantiomer in the metabolic process. R-AI concentrations in four healthy volunteers ranged from 32.85 nM to 49.45 nM, with an average value of 43.76 nM. To date, there is no LC–MS (or MS/MS) method reported for the enantioselective determination of R-AI in human saliva samples.

Evaluation of Different Tandem MS Acquisition Modes to Support Metabolite Annotation in Human Plasma Using Ultra High-Performance Liquid Chromatography High-Resolution Mass Spectrometry for Untargeted Metabolomics.

Ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) is a powerful and essential technique for metabolite annotation in untargeted metabolomic applications. The aim of this study was to evaluate the performance of diverse tandem MS (MS/MS) acquisition modes, i.e., all ion fragmentation (AIF) and data-dependent analysis (DDA), with and without ion mobility spectrometry (IM), to annotate metabolites in human plasma. The influence of the LC separation was also evaluated by comparing the performance of MS/MS acquisition in combination with three complementary chromatographic separation modes: reversed-phase chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) with either an amide (aHILIC) or a zwitterionic (zHILIC) stationary phase. RPLC conditions were first chosen to investigate all the tandem MS modes, and we found out that DDA did not provide a significant additional amount of chemical coverage and that cleaner MS/MS spectra can be obtained by performing AIF acquisitions in combination with IM. Finally, we were able to annotate 338 unique metabolites and demonstrated that zHILIC was a powerful complementary approach to both the RPLC and aHILIC chromatographic modes. Moreover, a better analytical throughput was reached for an almost negligible loss of metabolite coverage when IM-AIF and AIF using ramped instead of fixed collision energies were used.

Exploration of ovine milk whey proteome during postnatal development using an iTRAQ approach.

BackgroundOvine milk is a rich source of bioactive proteins that supports the early growth and development of the newborn lambs. A large number of researches had targeted to the identification of ovine milk fat globule membrane proteins (MFGMPs), caseins (CNs), mastitis milk proteins in past years, but the dynamic change tendency of milk whey proteins during postnatal development has received limited attention. This research aimed to investigate the dynamic changes of ovine milk whey proteins after delivery, and explore the functions of whey proteins on early development of the newborns.MethodsIn this research, Hu sheep milk samples were collected from six individuals by manual milking manner, at 0 d, 3 d, 7 d, 14 d, 28 d and 56 d after delivery, respectively. The milk whey proteins were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) coupled with liquid chromatography (LC)-electrospray ionization (ESI) tandem MS (MS/MS) methods. In addition, biological functions of differentially expressed proteins (DEPs) were annotated by Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.ResultsA total of 310 proteins were identified , of which 121 were differentially expressed. In detail, 30 (10 up-regulated and 20 down-regulated), 22 (11 up-regulated and 11 down-regulated), 11 (four up-regulated and seven down-regulated), 11 (eight up-regulated and three down-regulated), 10 (six up-regulated and four down-regulated) DEPs were identified in 3 d vs. 0 d, 7 d vs. 3 d, 14 d vs. 7 d, 28 d vs. 14 d, 56 d vs. 28 d comparison groups, respectively. The GO annotation analysis revealed that biological process principally involved metabolic and biological regulation, the major cellular location were organelle, cell and extracellular region, and the mainly molecular function were binding and catalytic activity. Circadian rhythm, fatty acid biosynthesis and African trypanosomiasis were enriched by KEGG annotation analysis.ConclusionThe study reveals a comprehensive understanding of Hu sheep milk proteome, suggesting whey proteins change dramatically in early development of newborn lambs, which provide a potential guidance for early weaning of lambs.

Multifunctional Reactive MALDI Matrix Enabling High-Lateral Resolution Dual Polarity MS Imaging and Lipid C═C Position-Resolved MS2 Imaging.

Local lipid variations in tissues are readily revealed with mass spectrometry imaging (MSI) methods, and the resulting lipid distributions serve as bioanalytical signatures to reveal cell- or tissue-specific lipids. Comprehensive MSI lipid mapping requires measurements in both ion polarities. Additionally, structural lipid characterization is necessary to link the lipid structure to lipid function. Whereas some structural elements of lipids are readily derived from high-resolution mass spectrometry (MS) and tandem-MS (MSn), the localization of C═C double bonds (DBs) requires specialized fragmentation and/or functionalization methods. In this work, we identify a multifunctional matrix-assisted laser desorption/ionization (MALDI) matrix for spatially resolved lipidomics investigations that reacts with lipids in Paternò-Büchi (PB) reactions during laser irradiation facilitating DB-position assignment and allows dual-polarity high-resolution MALDI-MSI and MALDI MS2I studies. By screening 12 compounds for improved ionization efficiency in positive-/negative-ion mode and the functionalization yield compared to the previously introduced reactive MALDI matrix benzophenone, 2-benzoylpyridine (BzPy) is identified as the best candidate. The new matrix enables DB localization of authentic standards belonging to 12 lipid classes and helps to assign 133/58 lipid features in positive-/negative-ion mode from mouse cerebellum tissue. The analytical capabilities of BzPy as a multifunctional MALDI-MSI matrix are demonstrated by imaging endogenous and PB-functionalized lipids in mouse kidney sections with 7 μm lateral resolution in both ion modes. Tracking diagnostic lipid DB-position fragment ions in mouse pancreatic tissue with down to 10 μm pixel size allows us to identify the islets of Langerhans associated with lipid isomer upregulation and depletion.

Analysis of oligomeric complexes of the amyloid-forming FYLLYY peptide by collision-induced dissociation with electrospray ionization mass spectrometry.

The monomeric and oligomeric structures of the "FYLLYY" β2 microglobulin (β2m) active sequence, formed in (DMSO/CH3CN) solution, were investigated using electrospray ionization (ESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Dissociation of dimer and trimer ions was investigated by tandem mass spectrometry using collision induced dissociation (CID). The covalent bond fragmentation patterns were observed in the 21+ and 32+ MS/MS spectra (21+ = [dimer+H]1+ and 32+ = [trimer + 2H]2+). A π-π stacking geometry for the FYLLYY 21+ complex and partial parallel β-sheet geometry for the 32+ complex are proposed to be stable structures. The observed covalent bond fragment ions in the MS/MS spectra of the 32+ complex are considered to have originated from the partial parallel β-sheet moiety. The FYLLYY → AALLGY (or FYLLAA) substituted sequence was also investigated by CID-MS/MS. Our MS/MS analysis suggests that the π-π stacking interaction structures are important in dimer binding rather than the structures of a complete parallel or anti-parallel β-sheet 21+ complex.

Escherichia coli and Sf9 Contaminant Databases to Increase Efficiency of Tandem Mass Spectrometry Peptide Identification in Structural Mass Spectrometry Experiments.

Filtering of nonspecifically binding contaminant proteins from affinity purification mass spectrometry (AP-MS) data is a well-established strategy to improve statistical confidence in identified proteins. The CRAPome (contaminant repository for affinity purification) describes the contaminating background content present in many purification strategies. However, full contaminant lists for nickel-nitrilotriacetic acid (NiNTA) and glutathione S-transferase (GST) affinity matrices are lacking. Similarly, no Spodoptera frugiperda (Sf9) contaminants are available, and only the FLAG-purified contaminants are described for Escherichia coli. For MS experiments that use recombinant protein, such as structural mass spectrometry experiments (hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking, and radical foot-printing), failing to include these contaminants in the search database during the initial tandem MS (MS/MS) identification stage can result in complications in peptide identification. We have created contaminant FASTA databases for Sf9 and E. coli NiNTA or GST purification strategies and show that the use of these databases can effectively improve HDX-MS protein coverage, fragment count, and confidence in peptide identification. This approach provides a robust strategy toward the design of contaminant databases for any purification approach that will expand the complexity of systems able to be interrogated by HDX-MS.

HPTLC and UPLC-MS/MS Methods for Quality Control Analysis of Itrifal Formulations of Unani System of Medicine.

Ultra-performance LC (UPLC)-tandem MS (MS/MS) and high-performance TLC (HPTLC) assay methods were developed for chemical fingerprinting and quantitative analysis of bioactive constituents of a certain "Itrifal formulation," a traditional kind of Unani medicine. In the present investigation, HPTLC and UPLC-MS/MS methods were developed and validated for the detection and quantification of major metabolites present in itrifal formulation. The metabolites present in the formulation were separated using modern chromatographic techniques, and a quantitative analysis was performed. Analytical performance of the proposed HPTLC and UPLC-MS/MS methods was validated as per the defined guidelines with respect to linearity, accuracy, precision, robustness, and specificity. The developed UPLC-MS/MS and HPTLC methods were used for quantification of gallic acid, tannic acid, catechin, and quercetin. All four constituents were quantified by UPLC-MS/MS, while two constituents were quantified by HPTLC in the commercial itrifal formulation. The calibration plot was found to be linear, accurate, precise, robust, and specific for both HPTLC and UPLC-MS/MS. The present methods were successfully applied for analysis of the given markers in itrifal formulations. The same can be used for QC and stability testing of itrifal formulations.

GC\u2009\xd7\u2009GC\u2013HRMS nontarget fingerprinting of organic micropollutants in urban freshwater sediments

BackgroundSediments are sinks for organic micropollutants, which are traditionally analysed by gas chromatography–mass spectrometry (GC–MS). Although GC–MS and GC–tandem MS (MS/MS) are preferred for target screening, they provide only limited chromatographic resolution for nontarget screening. In this study, a comprehensive two-dimensional GC–high-resolution MS method (GC × GC–HRMS) was developed for nontarget screening and source identification of organic micropollutants in sediments from an urban channel and adjacent lake in Copenhagen, Denmark. The GC × GC–HRMS data were processed by pixel-based chemometric analysis using baseline subtraction, alignment, normalisation, and scaling before principal component analysis (PCA) of the pre-processed GC × GC–HRMS base peak ion chromatograms (BPCs). The analysis was performed to identify organic micropollutants of high abundance and relevance in the urban sediments and to identify pollution sources. Tentative identifications were based on match factors and retention indices and tagged according to the level of identification confidence.ResultsThe channel contained both a significantly higher abundance of micropollutants and a higher diversity of compounds compared to the lake. The PCA models were able to isolate distinct sources of chemicals such as a natural input (viz., a high relative abundance of mono-, di- and sesquiterpenes) and a weathered oil fingerprint (viz., alkanes, naphthenes and alkylated polycyclic aromatic hydrocarbons). A dilution effect of the weathered oil fingerprint was observed in lake samples that were close to the channel. Several benzothiazole-like structures were identified in lake samples close to a high-traffic road which could indicate a significant input from asphalt or tire wear particles. In total, 104 compounds and compound groups were identified.ConclusionsSeveral chemical fingerprints of different sources were described in urban freshwater sediments in Copenhagen using a pixel-based chemometric approach of GC × GC–HRMS BPCs. Various micropollutants of anthropogenic origin were identified. Tailored pre-processing and careful interpretation of the identification results is inevitable and still requires further research for an automated workflow.

Copper(II) partially protects three histidine residues and the N‐terminus of amyloid‐β peptide from diethyl pyrocarbonate (DEPC) modification

Diethyl pyrocarbonate (DEPC) has been primarily used as a residue‐specific modifying agent to study the role of His residues in peptide/protein and enzyme function; however, its action is not specific, and several other residues can also be modified. In the current study, we monitored the reaction of DEPC with amyloid‐beta (Aβ) peptides and insulin by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and determined the modification sites by electrospray ionization quadrupole time‐of‐flight tandem MS (ESI Q‐TOF MS/MS). Our results indicate that five residues in Aβ1–42 are modified in the presence of 30‐fold molar excess of DEPC. After hydroxylamine treatment (which removes modifications from three His residues), two labels remain bound at the peptide N terminus and Lys16. DEPC treatment of Aβ1–42 promotes peptide aggregation, as monitored through the loss of soluble Aβ42 in a semi‐quantitative MALDI‐TOF MS assay. It has been previously proposed that Cu(II) ions protect Aβ1–16 from DEPC modification through binding to His6. We confirmed that Cu(II) ions decrease the stoichiometry of Aβ1–16 modification with the excess of DEPC being lower as compared to the control, which indicates that Cu(II) protects Aβ from DEPC modification. Sequencing of obtained Cu(II)‐protected Aβ1–16 samples showed that Cu(II) does not protect any residues completely, but partially protects all three His residues and the N terminus. Thus, the protection by Cu(II) ions is not related to specific metal binding to a particular residue (e.g. His6), but rather all His residues and the N terminus are involved in binding of Cu(II) ions. These results allow us to elucidate the effect of DEPC modification on amyloidogenity of human Aβ and to speculate about the role of His residues in these processes.

Ultrahigh-Performance Hydrophilic Interaction Liquid Chromatography with Tandem Mass Spectrometry Method for the Determination of Paralytic Shellfish Toxins and Tetrodotoxin in Mussels, Oysters, Clams, Cockles, and Scallops: Collaborative Study.

An ultrahigh-performance LC (UHPLC)-tandem MS (MS/MS) method for determination of paralytic shellfish poisoning toxins and tetrodotoxin (TTX) in bivalve molluscs was developed. To be used for regulatory testing, it needed to be validated through collaborative study. The aim was to conduct a collaborative study with 21 laboratories, using results to assess method performance. Study materials incorporated shellfish species mussels, oysters, cockles, scallops, and clams and were assessed to demonstrate stability and homogeneity. Mean concentrations determined by participants for blind duplicate samples were used to assess reproducibility, repeatability, and trueness. Method performance characteristics were excellent following statistical assessment of participant data, with method trueness showing excellent method accuracy against expected values. No significant difference was found in the trueness results determined by different chromatographic column types. Acceptability of the between-laboratory reproducibility for individual analytes was evidenced by >99% of valid Horwitz ratio values being less than the 2.0 limit of acceptability. With excellent linearity and sensitivity fit-for-purpose over a range of mass spectrometer instruments, the UHPLC-MS/MS method compared well against other detection methods. It includes additional paralytic shellfish toxin (PST) analogues as well as TTX, which, to date, have not been incorporated into any other hydrophilic marine toxin official method of analysis. The results from this study demonstrate that the method is suitable for the analysis of PST analogues and TTX in shellfish tissues and is recommended as an official alternative method of analysis for regulatory control. A new mass spectrometric method for PST and TTX has been validated successfully through collaborative study.

Collision-Induced Dissociation Studies of Protonated Ions of Alkylated Thymidine and 2′-Deoxyguanosine

Mass spectrometry and tandem MS (MS/MS) have been widely employed for the identification and quantification of damaged nucleosides in DNA, including those induced by alkylating agents. Upon collisional activation, protonated ions of alkylated nucleosides frequently undergo facile neutral loss of a 2-deoxyribose in MS/MS, and further cleavage of the resulting protonated nucleobases in MS3 can sometimes be employed for differentiating regioisomeric alkylated DNA lesions. Herein, we investigated systematically the collision-induced dissociation (CID) of the protonated ions of O4-alkylthymidine (O4-alkyldT), O2-alkyldT, O6-alkyl-2'-deoxyguanosine (O6-alkyldG), and N2-alkyldG through MS3 analysis. The MS3 of O2- and O4-MedT exhibit different fragmentation patterns from each other and from other O2- and O4-alkyldT adducts carrying larger alkyl groups. Meanwhile, elimination of alkene via a six-membered ring transition state is the dominant fragmentation pathway for O2-alkyldT, O4-alkyldT, and O6-alkyldG adducts carrying larger alkyl groups, whereas O6-MedG mainly undergoes elimination of ammonia. The breakdown of N2-alkyldG is substantially influenced by the structure of the alkyl group, where the relative ease in eliminating ammonia and alkene is modulated by the chain length and branching of the alkyl groups. We also rationalize our observations with density functional theory (DFT) calculations.

Unique Distribution of Diacyl-, Alkylacyl-, and Alkenylacyl-Phosphatidylcholine Species Visualized in Pork Chop Tissues by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging.

Phosphatidylcholine (PC) is the major phospholipid in meat and influences meat qualities, such as healthiness. PC is classified into three groups based on the bond at the sn-1 position: Diacyl, alkylacyl, and alkenylacyl. To investigate their composition and distribution in pork tissues, including longissimus thoracis et lumborum (loin) spinalis muscles, intermuscular fat, and transparent tissues, we performed matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI–MSI). Eleven diacyl-, seven alkylacyl-, and six alkenylacyl-PCs were identified using liquid chromatography (LC)-tandem MS (MS/MS) analysis. Despite many alkylacyl- and alkenylacyl-PC species sharing identical m/z values, we were able to visualize these PC species using MALDI–MSI. Diacyl- and alkylacyl- and/or alkenylacyl-PC species showed unique distribution patterns in the tissues, suggesting that their distribution patterns were dependent on their fatty acid compositions. PCs are a major dietary source of choline in meat, and the amount was significantly higher in the muscle tissues. Consumption of choline mitigates age-related memory decline and neurodegenerative diseases; therefore, the consumption of pork muscle tissues could help to mitigate these diseases. These results support the use of MALDI–MSI analysis for assessing the association between PC species and the quality parameters of meat.

Binding ability of arsenate towards Cu2+ and Zn2+: thermodynamic behavior and simulation under natural water conditions.

A study on the sequestering ability between arsenate, AsO43-, and Cu2+ and Zn2+ in aqueous solution is reported. The results of the elaboration of potentiometric data include only species with 1 : 1 metal to ligand ratio for Cu2+-arsenate system, namely CuLH2, CuLH, CuL, and CuLOH (L = AsO43-). For the Zn2+-arsenate system, a speciation model with only two species with both 1 : 1 and 1 : 2 metal to ligand ratios was obtained, namely ML and ML2. Spectrophotometric titrations were also employed in the study of the Cu2+-AsO43- system, and the results of the analysis of experimental data fully confirmed potentiometric ones. The potentiometric titrations were performed under different conditions of temperature (288.15 ≤ T/K ≤ 310.15, at I = 0.15 mol L-1) and ionic strength (0.15 ≤ I/mol L-1 ≤ 1 in NaCl). The dependence of formation constants of the complex species on ionic strength and temperature was also evaluated, as well as the enthalpy and entropy change values were obtained. Laser desorption mass spectrometry (LD MS) and tandem mass spectrometry (MS/MS) were exploited to confirm Cu2+-AsO43- and Zn2+-AsO43- complex formation and to determine both their composition and structural characteristics. Simulation of speciation profiles under natural water conditions was performed. The sequestering ability of arsenate towards Cu2+ and Zn2+ was quantified under different conditions of pH, temperature and ionic strength, typical of several natural waters. Examples of arsenate distribution under seawater and freshwater conditions were reported.

Simultaneous Determination of 12 Bioactive Constituents in Bupi Yiqi No. 1 Recipe by Ultrahigh-Performance Liquid Chromatography Tandem Mass Spectrometry.

Bupi Yiqi No. 1 Recipe (BPYQ), a traditional Chinese medicine formula, has been widely used as a treatment of multidrug resistance during chemotherapy in colorectal cancer and for the prevention of gastrointestinal cancer recurrence for decades. In this study, a rapid, reliable, and accurate ultrahigh-performance LC (UHPLC) coupled with electrospray ionization (ESI)-tandem MS (MS/MS) method was developed for the simultaneous determination of 12 major bioactive components, including protocatechuic acid, astilbin, rutin, calycosin-7-O-β-D-glucoside, ginsenoside Re, ononin, isoliquiritigenin, calycosin, apigenin, ginsenoside Rb1, formononetin, and glycyrrhizic acid in BPYQ. The chromatographic separation of the analytes was achieved on a Poroshell 120 SB-Aq column (50 × 2.1 mm, 1.7 µm) with a mobile phase of acetonitrile and 0.1% (v/v) formic acid aqueous solution. The flow rate and column temperature were set at 0.4 mL/min and 30°C, respectively. Mass spectrometric detection of the analyses was performed on multiple reaction monitoring mode in positive and negative ESI mode. The established UHPLC-ESI-MS/MS method was validated in terms of the linearity, precision, repeatability, stability, and accuracy. All calibration curves of the 12 compounds showed good linearity, with correlation coefficients (r) greater than 0.9980 within the test ranges. The LODs and LOQs for the 12 compounds were in the ranges of 0.08-1.32 and 0.27-5.28 ng/mL, respectively. The average recoveries of all the standard compounds were between 98.4 and 102.9%, and their relative SD values ranged from 1.24 to 3.78%. The proposed method can provide a meaningful basis for the QC of BPYQ. The established UHPLC-ESI-MS/MS method was demonstrated to be a powerful tool for quantifying the 12 compounds in BPYQ.

Cerebrospinal fluid: Profiling and fragmentation of gangliosides by ion mobility mass spectrometry

The proximity of cerebrospinal fluid (CSF) with the brain, its permanent renewal and better availability for research than tissue biopsies, as well as ganglioside (GG) shedding from brain to CSF, impelled lately the development of protocols for the characterization of these glycoconjugates and discovery of central nervous system biomarkers expressed in CSF. Currently, the investigation of CSF gangliosides is focused on concentration measurements of the predominant classes and much less on their profiling and structural analysis.Since we have demonstrated recently the high performance of ion mobility separation mass spectrometry (IMS MS) for compositional and structural determination of human brain GGs, in the present study we have implemented for the first time IMS MS for the exploration of human CSF gangliosidome, in order to generate the first robust mass spectral database of CSF gangliosides. IMS MS separation and screening revealed 113 distinct GG species in CSF, having similar compositions to the species detected in human brain. In comparison with the brain tissue, we have discovered in CSF several components containing fatty acids with odd number of carbon atoms and/or short glycan chains. By tandem MS (MS/MS) we have further analyzed the structure of GD3(d18:1/18:0) and GD2(d18:1/18:0), two glycoforms exhibiting short carbohydrate chains found in CSF, but discovered and characterized previously in brain as well. According to the present results, human CSF and brain show a similar ganglioside pattern, a finding that might be useful in clinical research focused on discovery of ganglioside species associated to neurodegenerative diseases and brain tumors.

Mycotoxin Testing Paradigm: Challenges and Opportunities for the Future

Abstract Mycotoxins are one of the great global challenges to agri-food and feed safety. Industry requires fast, reliable, and economical testing methods for the most important regulated mycotoxins to manage this problem. Climate change and changes in agricultural practice are complicating this situation, triggering the movement of some mycotoxins into new regions, which are unprepared for their management. Modern LC–tandem MS (LC–MS/MS) instruments have addressed this analytical challenge, but such instruments are expensive and require highly qualified personnel and dedicated facilities. As a result of these limitations, traditional LC–MS/MS is not amenable for use on farms or at small to midsized processing facilities, such as a grain elevator. To address the need for on-site rapid testing, the mycotoxin community has focused on antibody-based and spectrophotometric approaches. The development of innovative technologies such as miniaturized MS would allow for the acquisition of more information on mixtures of toxins present in a sample at costs comparable to those of the existing rapid methods such as ELISA. The capital costs are higher, but it would reduce per-sample testing costs and time requirements and provide better value for money while maintaining the accuracy and selectivity achieved in a laboratory setting. In this article, we review the available techniques and contrast them in the context of three main criteria: method performance, speed of analysis, and cost. We define the integration of these three parameters as the “mycotoxin testing paradigm.”

FaPEx® Multipesticide Residues Extraction Kit for Minimizing Sample Preparation Time in Agricultural Produce

Abstract Background: The quick, easy, cheap, effective, rugged, and safe method, generally applied to the determination of pesticide residues in food, has been recently modified and adopted for the analysis of pesticide residues worldwide, including Taiwan. Objective: The method still needs to be improved, particularly in efficiency and normalization, because the time costs 20–30 min for extraction and cleanup of multiple pesticides in every food. Methods: In this study, we present a highly efficient and simple sample preparation method that was developed for the determination of 380 pesticide residues in food, including mangoes, scallions, bok choy, apples, carrots, and pea seedlings. The homogenized 1 g samples mixed with 5 mL 1.0% acetic acid in acetonitrile were pushed through the FaPEx® (fast pesticide extraction) kits in a dropwise manner to obtain sample extracts. The total processing time was less than 15 min. The extracts were subject to chromatographic separation followed by GC–tandem MS (MS/MS) and LC–MS/MS analysis. Results: For more than 270 pesticide residues in these foods, the test gave acceptable mean recoveries, ranging between 70 and 120%, and relative SDs below 20%. The LOQ was at least 0.01 mg/kg for 380 pesticides. Conclusions: The developed method can greatly reduce the time needed for multiple pesticide residues analysis. Highlights: FaPEx is used for the extraction of pesticide residues that relies on single-use pre-filled sealed cartridges. FaPEx diminishes operation time, glassware demand, and laboratory space requirement. It also significantly decreases the amount of chemical solvents.

Analytical Method of Multi-Mycotoxins in Table-Ready Foods for a Total Diet Study Using Stable Isotope Dilution Liquid Chromatography–Tandem Mass Spectrometry

Abstract Background: Determining the multi-mycotoxins present in table-ready foods is necessary for a total diet study. However, so far, most methods of analyzing multi-mycotoxins apply to raw foods. Therefore, a reliable method for analyzing multi-mycotoxins in table-ready foods is needed. Objective: We developed and validated methods of multi-mycotoxin analysis that employed stable isotope dilution with LC–tandem MS (LC–MS/MS) using two representative matrices. Methods: Samples were fortified with [13C]-labeled mycotoxins as internal standards and extracted with 50% acetonitrile in water for high-carbohydrate foods and 3% formic acid in acetonitrile for high-protein and/or high-fat foods, cleaned up with n-hexane and the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, and followed up by LC–MS/MS. Results: Validation of the methods was performed, and the results were as follows: the correlation coefficient, R2, was 0.99; method detection limit, 0.01–2.4 μg/kg; recoveries, 83.6–114%; precision, 0.8–10 (intraday) and 3.1–22% (interday); interlaboratory reproducibility, ≤15%; and uncertainty, 3.5–19%error. The applicability of the methods was evaluated by analyzing table-ready foods spiked with standards. Conclusions: These methods were successfully evaluated and deemed appropriate for determining the multi-mycotoxins in table-ready foods. Highlights: This work demonstrates that stable isotope dilution with LC–MS/MS can be effectively used to analyze multi-mycotoxins simultaneously in a total diet study.

Identifying Challenges and Risks Associated with the Analysis of Major Mycotoxins in Feed and Botanicals

Abstract Changing weather conditions have heightened the risk of growth of mycotoxigenic molds on crops and various agricultural commodities. Mycotoxins, which are linked to carcinogenic and nephrotoxic effects in animals and humans, have been traditionally analyzed by immunoassays, gas, and LC techniques with spectrophotometric detectors. This review discusses the current techniques and challenges in commercial settings associated with the analysis of mycotoxins in unique matrices such as animal feeds, herbal products, and dietary supplements containing botanicals. Because of the advantages and growing acceptance of LC-tandem MS (MS/MS) over traditional approaches, discussion is mainly based on LC-MS/MS-based approaches. Considering the impact of sample preparation on accuracy of quantitative results, discussion about pros and cons of recently introduced sample preparation techniques is integrated with analytical methods. A section of the review explains the importance and availability of reference materials for mycotoxins. The present discussion provides good insight into the current challenges and developments during mycotoxin analysis of feed and botanicals and addresses the need for researchers in terms of an official MS-based method.

Exploiting the Semidestructive Nature of Gas Cluster Ion Beam Time-of-Flight Secondary Ion Mass Spectrometry Imaging for Simultaneous Localization and Confident Lipid Annotations.

Lipids have been recognized as key players in cell signaling and disease. Information on their location and distribution within a biological system, under varying conditions, is necessary to understand the contributions of different lipid species to an altered phenotype. Imaging mass spectrometry techniques, such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) and matrix-assisted laser desorption/ionization (MALDI), are capable of revealing global lipid distributions in tissues in an untargeted fashion. However, to confidently identify the species present in a sample, orthogonal analyses like tandem MS (MS/MS) are often required. This can be accomplished by bulk sample analysis with liquid chromatography (LC)-MS/MS, which can provide confident lipid identifications, at the expense of losing location-specific information. Here, using planarian flatworms as a model system, we demonstrate that imaging gas cluster ion beam (GCIB)-ToF-SIMS has the unique capability to simultaneously detect, identify, and image lipid species with subcellular resolution in tissue sections. The parallel detection of both, intact lipids and their respective fragments, allows for unique identification of some species without the need of performing an additional orthogonal MS/MS analysis. This was accomplished by correlating intact lipid and associated fragment SIMS images. The lipid assignments, respective fragment identities, and locations gathered from ToF-SIMS data were confirmed via LC-MS/MS on lipid extracts and ultrahigh mass resolution MALDI-MS imaging. Together, these data show that the semidestructive nature of ToF-SIMS can be utilized advantageously to enable both confident molecular annotations and to determine the locations of species within a biological sample.