Variable Data Independent Acquisition Research Articles

Using Variable Data-Independent Acquisition for Capillary Electrophoresis-Based Untargeted Metabolomics.

Capillary electrophoresis coupled with tandem mass spectrometry (CE-MS/MS) offers advantages in peak capacity and sensitivity for metabolic profiling owing to the electroosmotic flow-based separation. However, the utilization of data-independent MS/MS acquisition (DIA) is restricted due to the absence of an optimal procedure for analytical chemistry and its related informatics framework. We assessed the mass spectral quality using two DIA techniques, namely, all-ion fragmentation (AIF) and variable DIA (vDIA), to isolate 60-800 Da precursor ions with respect to annotation rates. Our findings indicate that vDIA, coupled with the updated MS-DIAL chromatogram deconvolution algorithm, yields higher spectral matching scores and annotation rates compared to AIF. Additionally, we evaluated a linear migration time (MT) correction method using internal standards to accurately align chromatographic peaks in a data set. Postcorrection, the data set exhibited less than 0.1 min MT drifts, a difference mostly equivalent to that of conventional reverse-phase liquid chromatography techniques. Moreover, we conducted MT prediction for metabolites recorded in mass spectral libraries and metabolite structure databases containing a total of 469,870 compounds, achieving an accuracy of less than 1.5 min root mean squares. Our platform provides a peak annotation platform utilizing MT information, accurate precursor m/z, and the MS/MS spectrum recommended by the metabolomics standards initiative. Applying this procedure, we investigated metabolic alterations in lipopolysaccharide (LPS)-induced macrophages, characterizing 170 metabolites. Furthermore, we assigned metabolite information to unannotated peaks using an in silico structure elucidation tool, MS-FINDER. The results were integrated into the nodes in the molecular spectrum network based on the MS/MS similarity score. Consequently, we identified significantly altered metabolites in the LPS-administration group, where glycinamide ribonucleotide, not present in any spectral libraries, was newly characterized. Additionally, we retrieved metabolites of false-negative hits during the initial spectral annotation procedure. Overall, our study underscores the potential of CE-MS/MS with DIA and computational mass spectrometry techniques for metabolic profiling.

Authenticity and traceability of goat milk: Molecular mechanism of β-carotene biotransformation and accessibility

The efficiently extraction and accurately quantify of β-carotene and its metabolites are crucial for authenticity and traceability in goat milk. Nevertheless, its reliability can be largely improved. In this study, meticulously designed native ESI-MS, fluorescence spectroscopy and molecular docking in combination with cold-induced acetonitrile aqueous two-phase separation system weaken the interaction between β-lactoglobulin and β-carotene metabolites and realized the efficiently extraction. Furthermore, established non-targeted quantitative metabolomics with optimal ion source and variable data-independent acquisition minimized the matrix effects and potential ion suppression. Validated atmospheric pressure chemical ionization-ultra high performance liquid chromatography-Orbitrap method showed that β-carotene as distinctive biomarker in cow milk, and retinol, retinaldehyde, retinoic acid and abscisic acid in goat milk. Collectively, the proposed method is a powerful tool to detect cow adulteration risks in goat milk samples and provides valuable information for availability on authenticity of goat milk.

Variable Data Independent Acquisition and Data Mining Exploring Feature-Based Molecular Networking Analysis for Untargeted Screening of Synthetic Cannabinoids in Oral Fluid.

Novel psychoactive substances (NPS) are constantly emerging in the drug market, and synthetic cannabinoids (SCs) are included in this NPS family. Forensic laboratories often struggle with these continually emerging SCs, forcing them to develop an untargeted workflow to incorporate these psychoactive drugs in their procedures. Usually, forensic laboratories select analytical methods based on targeted mass spectrometry (MS) technologies for strictly tracking already known NPS. The appropriate way to tackle unknown substances is to develop pipelines for untargeted analysis that include LC-HRMS analytical methods and data analysis. Once established, this strategy would allow drug testing laboratories to be always one step ahead of the new trends concerning the "designer drugs" market. To address this challenge an untargeted workflow based on mass spectrometry data acquisition and data analysis was developed to detect SCs in oral fluid (OF) samples at a low concentration range. The samples were extracted by mixed-mode solid-phase extraction and analyzed by Liquid Chromatography - High-Resolution Mass Spectrometry (LC-HRMS). Tandem mass spectra (MS2) were recorded performing a variable isolation width across a mass range of all theoretical precursor ions (vDIA) after the chromatographic separation. After raw data processing with the MSDial software, the deconvoluted features were sent to GNPS for Feature-Based Molecular Networking (FBMN) construction for nontargeted data mining. The FBMN analysis created a unique integrated network for most of the SCs assessed in the OF at a low level (20 ng/mL). These results demonstrate the potential of an untargeted approach to detect different derivatives of SCs at trace levels for forensic applications.

Development and validation of qualitative screening, quantitative determination and post-targeted pesticide analysis in tropical fruits and vegetables by LC-HRMS

The following qualitative screening and quantitative determination of 201 pesticides present a detailed optimization making use of design of experiments (DoE). The post-targeted screening took advantage of an upgradeable database to extend the survey further to perform a standard-free novel quantitation by liquid chromatography high resolution mass spectrometry (LC-HRMS). The fine tuning of instrumental parameters allowed the best method selectivity, sensitivity and reproducibility for the compounds under study. Among the factors considered, the heated electrospray source (HESI), the sample composition and the MS/MS acquisition modes were assessed, including the all-ion fragmentation (AIF) mode and different versions of the variable data independent acquisition (vDIA). The results of the optimization showed that selected HESI conditions, a methanol/water (1:1) sample composition and a vDIA acquisition method increased the signal for most compounds with a remarkable benefit on signal shape and intensity. The method showed adequate analytical performance when assessed in local produce.

Multiclass pesticide residue analysis in tobacco (Nicotiana tabacum) using high performance liquid chromatography-high resolution (Orbitrap) mass spectrometry: A simultaneous screening and quantitative method

Public exposure to pesticides through tobacco has attracted serious attention. Here we report a simultaneous screening and quantitation method for the non-target multiresidue analysis of pesticides in different tobacco types. The method involved extraction of a homogenate (20 g, containing 2 g tobacco) in ethyl acetate (10 mL), cleanup of 2 mL extract by dispersive solid phase extraction with PSA (50 mg)+C18 (50 mg)+GCB (25 mg)+MgSO4 (100 mg), followed by reconstitution in 1 mL acetonitrile:water (3:7) and analysis using HPLC with Quadrupole-Orbitrap mass spectrometry. The high resolution accurate mass analysis was performed through sequential full-scan (resolution=35000) and variable data independent acquisition (resolution=17500) events. When the method was evaluated in a mixture of 181 pesticides, it effectively minimised matrix interferences and false negatives. The target compounds included 5 pairs of isomers and 27 pairs of isobars, which were distinguished based on chromatographic separation, mass resolving power and/or unique product ions. The screening detection limit (SDL) for 86.4% of the test pesticides was set at 5 ng/g, while the remainder had the SDLs at 10 ng/g (9.3%) and 40 ng/g (4.3%). Nearly, 75% of the compounds showed recoveries of 70–120% at 10 ng/g. The rest of the compounds showed satisfactory recoveries at 40 and 100 ng/g. In all cases, precision-RSDs were < 20%. The established method demonstrated a successful performance in four different types of tobacco matrices while aligning with the guidelines of SANTE and US-FDA. Owing to its efficiency, the method is recommended for screening and quantitation of multiclass pesticides in tobacco.

High-Coverage Screening of Sulfonamide Metabolites in Goat Milk by Magnetic Doped S Graphene Combined with Ultrahigh-Performance Liquid Chromatography-High-Resolution Mass Spectrometry.

Currently, there are more than 1000 varieties of synthetic sulfonamides universally used as antibiotics causing severe results of potential carcinogenicity and drug resistance for human health due to excessive residue of animal-derived food. A facile and novel approach for untargeted screening of sulfonamides (SAs) and metabolites was proposed based on magnetic solid-phase extraction-ultrahigh-performance liquid chromatography-tandem high-resolution mass spectrometry (MSPE-UHPLC-HRMS). Compared with QuEChERS without the clean-up procedure and SPE in terms of matrix effect and absolute recovery, magnetic doped S graphene (S-doping level: 2.82%) synthesized via a solid-state microwave approach and the aggregation wrap mechanism was used as a novel adsorbent for nonspecific extraction of desired analytes by the noncovalent interaction between electron-deficient thiophene sulfur and electron donors such as amino or amide as well as π-π stacking interactions. Combined with variable data-independent acquisition, characteristic fragment-ion filtering (m/z 156.01138 or m/z 108.04439) and assignment of extracted-ion chromatograms of marked fragment ions were successfully utilized to screen the desired analytes and subsequently confirmed with the availability of reference standards. The optimized and validated approach for spiked 26 SAs and 9 metabolites in control goat milk demonstrated satisfactory accuracy (80.1-112.6%) and precision (RSDs < 6.4%) for matrix-matched standard addition. After applying suspect goat milk samples, untargeted SA analytes including sulfanilamide or an N4-acetylsulfamethazine metabolite with concentration ranging from 66.3 to 398.5 ng L-1 were determined in 5 of 45 goat milk samples.

High-throughput planar solid-phase extraction coupled to orbitrap high-resolution mass spectrometry via the autoTLC-MS interface for screening of 66 multi-class antibiotic residues in food of animal origin

Antibiotic residues in food pose a major threat to the health of humans and animals worldwide. Their trace-level analysis is still too time- and cost-intensive to be adequately covered in routine analysis. Thus, a new high-throughput planar solid-phase extraction method has been developed for rapid screening of 66 antibiotics. Via simple clicks on the image, the autoTLC-MS interface automatically eluted the target analyte zones directly into an orbitrap high-resolution mass spectrometer operated in the variable data-independent acquisition mode. Muscle tissue, cow milk and chicken eggs were analyzed regarding nine different antibiotic classes, including sulfonamides, diaminopyrimidines, lincosamides, pleuromutilins, macrolides, cephalosporins, penicillins, amphenicols and nitroimidazoles. The planar clean-up took 7 min per sample, which is 5-fold faster than the routine state-of-the-art. The screening method has been validated for one representative of each class according to the European Commission Decision 2002/657/EC. Most analytes were successfully detected at half of their required maximum residue limit.

Pyrrolizidine alkaloid profiling of four Boraginaceae species from Northern Germany and implications for the analytical scope proposed for monitoring of maximum levels

ABSTRACT Pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs) have been determined in food and feed at levels relevant for consumer health. More than 660 different PAs have been detected, but few are available as reference substances for analytical demands. In the context of the European legislation on maximum levels of PAs in food products, a defined analytical scope of 21 PAs for determination has been suggested. An expansion of the scope from 21 to 35 PAs, including 14 structural isomers, is currently under discussion. In the present study, a target screening method was established for a comprehensive characterisation of PA profiles of the species Echium vulgare, Heliotropium europaeum, Cynoglossum officinale and Symphytum spp. to assess whether an expansion of the analytical scope is required to quantitatively cover the total PA contents of Boraginaceae species. The scope of the method comprised known and unknown PAs previously screened and confirmed in the respective plant extracts. A total of 176 PAs and PANOs were detected. The toxic 1,2-unsaturated PAs represent the predominant PA type with about 98% of the mean total content. This PA profiling demonstrates that an expansion of the scope from 21 to 35 PAs is required to adequately cover the mean total PA contents of Cynoglossum officinale and H. europaeum, whereas in the case of Symphytum spp. and Echium vulgare an expansion would not be necessary. Abbreviations Pyrrolizidine alkaloid: PA, Pyrrolizidine alkaloid N-oxide: PANO, European Food Safety Authority: EFSA, German Federal Institute for Risk Assessment: BfR, ultra-high performance liquid chromatography: UHPLC, high-resolution: HR, tandem mass spectrometer: MS/MS, multiple reaction monitoring: MRM, data-independent MS2: ddms2, electrospray ionisation: ESI, limit of detection: LOD, limit of quantification: LOQ, gas chromatography coupled with mass spectrometry: GC-MS, fragmentation (data): MS2 (data), full scan: MS1, variable data-independent acquisition: vDIA, monoester esterified with a necic acid at position C9 of the necine base: O9-monoester, relative proportion: rel. prop., intraperitoneal: i.p., intravenous: i.v., higher-energy C-trap dissociation: HCD, all-ion fragmentation: AIF, parallel reaction monitoring: PRM

Effects of spices on the formation of biogenic amines during the fermentation of dry fermented mutton sausage

A precise and unambiguous quantitative strategy for six biogenic amines in dry fermented mutton sausage with liquid chromatography tandem quadrupole Orbitrap mass spectrometry was developed to investigate the inhibitory effects of star anise, amomum tsao-ko, clove, cassia, fennel, bay leaf, and nutmeg on the accumulation of biogenic amines. Compared the data-dependent MS2 fragmentation (dd-MS2), variable data-independent acquisition (vDIA), and multiplex data-independent acquisition (mDIA), and the mDIA acquisition mode was selected for further analysis. The LODs and LOQs were 0.9–1.5 μg kg−1 and 2.9–5.0 μg kg−1. The maximum inhibition of spice on tryptamine, putrescine, spermidine, 2-phenylethylamine, tyrosamine, and histamine were 21.8%, 19.3%, 27.5%, 24.6%, 18.7% and 24.4%, which revealed that the accumulation of BAs was significantly inhibited by spice extracts, while cassia and fennel extracts showed the best effect. Thus, the established method can provide a better reference for dry fermented mutton sausage quality, safety and trade analysis.

High-throughput mass spectrometry scheme for screening and quantification of flavonoids in antioxidant nutraceuticals

Antioxidant nutraceuticals functional characteristic science is a challenging field for combining sensitivity and comprehensiveness. A untargeted screening and quantification method based on ultra-high performance liquid chromatography coupled to Quadrupole-Orbitrap high resolution mass spectrometry has been developed for determination of multiple classes of flavonoids in eight-three nutraceuticals samples. The data acquisition is based on a non-target approach of sequential full scan and variable data independent acquisition of twenty consecutive fragmentation events. The flavonoids include flavanols, flavones, flavanones, anthocyanidins, flavonols and isoflavones. A processing strategy is introduced to implementing filtering methods based on data feature extraction, common ion selection, shoulder peak removal, response threshold adjustment, mass shift and characteristic structural fragments evaluation. Confirmation is based on both accurate mass and isotopic assignment of standards, and further quantification is achieved by fragmentation. This scheme allows in depth characterization of flavonoids with the entire fragments.

Non-target data acquisition for target analysis (nDATA) of 845 pesticide residues in fruits and vegetables using UHPLC/ESI Q-Orbitrap.

A non-target data acquisition for target analysis (nDATA) workflow based on accurate mass measurements using UHPLC/ESI Q-Orbitrap full MS-data-independent acquisition and a compound database was developed to screen pesticide residues in fruit and vegetable samples. The compound database of 845 pesticides was built from dd-MS2 (data-dependent acquisition) product ion spectral data and LC retention times of individual pesticide standards. MS2 spectra of samples were acquired using multiplexing data-independent acquisition (mDIA) and variable data-independent acquisition (vDIA). Screening of pesticides in samples was based on either the retention time (± 0.5min) and the mass accuracy (± 5ppm) of a precursor (RTP by full MS) or the retention time (± 0.5min) and the mass accuracy (± 5ppm) of a precursor and its fragment ion (RTFI by full MS/DIA). In validation studies involving mDIA and vDIA analysis of 10 fruits and vegetables spiked with pesticides prior to QuEChERS sample preparation, RTP correctly found up to 765 and 796 pesticides at 10 and 100μg/kg, respectively, whereas RTFI correctly identified up to 729 and 764 pesticides at the same respective concentrations. UHPLC/ESI Q-Orbitrap full MS/mDIA or vDIA proved to be a comprehensive detection technique and has potential for pesticide residue screening in fruits and vegetables. Graphical Abstract.

A rapid non-target screening method for determining prohibited substances in human urine using liquid chromatography/high-resolution tandem mass spectrometry.

Target analysis using liquid chromatography-tandem mass spectrometry is applied for rapidly detecting various prohibited doping substances. Frequent modification is required as additional substances are prohibited. We developed and validated a non-target screening method requiring no further modification because it analyzes the full spectrum of data in fixed m/z ranges. Urine samples were extracted using solid-phase extraction and analyzed by employing a method that combines full scan and variable data independent acquisition using high-resolution mass spectrometry; and all prohibited substances in the urine samples were successfully detected using our screening method. The method was validated in terms of specificity (no interferences), recoveries (29%-131%), matrix effects (35%-237%), limites of detection (0.0002-100ng/mL), and intra- and inter-day precisions (coefficients of variation lower than 25%). The applicability of this method to doping tests was evaluated by analyzing 14 urine samples. As a result, the non-target screening method is efficient for conducting anti-doping tests because it can be applied without any further modification to prohibited drugs as well as to unknown targets that can be prohibited in the future.

In-house validation and small-scale collaborative study to evaluate analytical performances of multimycotoxin screening methods based on liquid chromatography-high-resolution mass spectrometry: Case study on Fusarium toxins in wheat.

A strong trend toward using highly selective mass spectrometry technologies for screening of multiple mycotoxins has been observed in recent years. In the present study, the process of validation of a multimycotoxin screening method based on liquid chromatography-high-resolution mass spectrometry method is presented. The method was intended for the simultaneous screening of the major Fusarium toxins (deoxynivalenol, 3- and 15-acetyl deoxynivalenol, T-2 and HT-2 toxins, zearalenone, enniatins A, A1, B, and B1, and beauvericin) in wheat. The sample preparation protocol was based on a double extraction (methanol followed by acetonitrile/water mixture) and purification through solid-phase extraction C18 column. To provide insights for full exploitation of the potential of the double-stage high-resolution mass spectrometry detection, a full-scan acquisition event followed by a sequence of 5 fragmentation events (variable data-independent acquisition) was set for mycotoxin detection, the latter to be exploited for confirmatory purposes. Method analytical performances were evaluated through in-house validation and small-scale interlaboratory study, designed according to Commission Regulation 519/2014/EU, setting performance requirements for screening methods for mycotoxins. Screening target concentrations were close to European Union maximum permitted or indicative levels. The in-house validation provided the precision of the response under repeatability conditions and the intermediate precision (both resulting lower than 30%), the cutoff value, and the rate of false suspect results for negative (free of the mycotoxin of interest) samples, which resulted lower than 0.1% in all cases. The collaborative study provided reproducibility and laboratory independent cutoff values. Analysis of reference materials proved method trueness and suitability for screening of the major Fusarium mycotoxins in wheat. Finally, the applicability of the full-scan/variable data-independent acquisition detection approach was successfully tested on a set of naturally contaminated wheat samples, where 2 characteristic product ions could be detected for all identified mycotoxins even at levels in the low μg/kg range.

A simultaneous screening and quantitative method for the multiresidue analysis of pesticides in spices using ultra-high performance liquid chromatography-high resolution (Orbitrap) mass spectrometry

A novel screening and quantitation method is reported for non-target multiresidue analysis of pesticides using ultra-HPLC-quadrupole-Orbitrap mass spectrometry in spice matrices, including black pepper, cardamom, chili, coriander, cumin, and turmeric. The method involved sequential full-scan (resolution = 70,000), and variable data independent acquisition (vDIA) with nine consecutive fragmentation events (resolution = 17,500). Samples were extracted by the QuEChERS method. The introduction of an SPE-based clean-up step through hydrophilic-lipophilic-balance (HLB) cartridges proved advantageous in minimizing the false negatives. For coriander, cumin, chili, and cardamom, the screening detection limit was largely at 2 ng/g, while it was 5 ng/g for black pepper, and turmeric. When the method was quantitatively validated for 199 pesticides, the limit of quantification (LOQ) was mostly at 10 ng/g (excluding black pepper, and turmeric with LOQ = 20 ng/g) with recoveries within 70–120%, and precision-RSDs <20%. Furthermore, the method allowed the identification of suspected non-target analytes through retrospective search of the accurate mass of the compound-specific precursor and product ions. Compared to LC–MS/MS, the quantitative performance of this Orbitrap-MS method had agreements in residue values between 78–100%.

Simultaneous quantitative determination, identification and qualitative screening of pesticides in fruits and vegetables using LC-Q-Orbitrap™-MS

A method based on QuEChERS extraction and LC-quadrupole-Orbitrap™ MS detection was established utilising an improved fully non-targeted way of data acquisition with and without fragmentation. A full-scan acquisition event without fragmentation (resolving power 70 000) was followed by five consecutive fragmentation events (variable data independent acquisition – vDIA; resolving power 35 000) where all ions from the full-scan range are fragmented. Compared with fragmentation in a single event (all-ion fragmentation – AIF), this improves both selectivity and sensitivity for the fragment ions, which is beneficial for screening performance and identification capability. The method was validated, using the data from the same measurements, for two types of analysis: quantitation/identification and qualitative screening. The quantitative validation, performed according to the guidelines in SANCO/12571/2013, tested the performance of the method for 184 compounds in lettuce and orange at two spiking levels: 10 and 50 ng g−1. The validation showed that the vast majority of the compounds met the criteria for trueness and precision set in the SANCO guidance document. In the qualitative validation the same 184 compounds were used to test the untargeted screening capabilities of the method. In this validation the compounds were spiked at three levels into 11 different fruit and vegetable matrices, which were measured twice on separate days. Taking all data from the qualitative validation together, an overall detection rate of 92% was achieved at the 10 ng g−1 level, increasing to 98% at 200 ng g−1. A screening detection limit (as defined in the SANCO guidelines) of 10 ng g−1 could be achieved for 134 compounds. For 39 and two pesticides the SDL was 50 and 200 ng g−1, respectively. For the other nine compounds no SDL could be established. The identification (ion ratio) criteria as recommended in the SANCO document could be met for 93% of the detected pesticide/matrix/concentration combinations. The outcome of both validations shows that the described method can be used to combine quantitative analysis and the identification of frequently detected pesticides (so far typically done using triple quadrupole MS/MS) with a qualitative screening to be used for a wide range of less frequent detected compounds in one measurement.