Scan MS Research Articles

Toxicokinetics and Tissue Distribution of the Hepatotoxic Triterpenoid Saponin Pterocephin A in Rats Using the Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) Method.

Pterocephin A is a natural triterpenoid saponin isolated from Pterocephalus hookeri, a traditional Tibetan medicine with slight toxicity, which can induce liver injury in rats. This study aimed to establish a sensitive and reliable UPLC-MS/MS method for exploring the toxicokinetics and tissue distribution of pterocephin A following single intravenous and intragastric administration. Pterocephin A and prosapogenin 1C (internal standard, IS) were extracted using a simple protein precipitation technique with methanol as the precipitant for plasma samples and methanol/acetonitrile = 1:1 (v/v) for tissue samples. UPLC separation was achieved by gradient elution with 0.3 mL/min and a mobile phase consisting of 5 mM ammonium formate (A) and acetonitrile (B) (0-2 min 30% B; 2-4 min: 30-80% B; 4-5 min: 80-98% B; 5-6.5 min: 98% B; 6.5-7 min: 98-30% B; and 7-8 min: 30% B, v/v) with a column temperature of 35 °C. MS spectrometry adopted negative ion scanning mode, primary MS spectrometry adopted full scan monitoring mode, and secondary MS spectrometry adopted targeted MS2 scan monitoring mode. The assay exhibited a linear dynamic range of 0.02-15 μg/mL for pterocephin A in biological samples, with the low limit of quantification set at 0.02 μg/mL. Non-compartmental toxicokinetic parameters indicated that pterocephin A was well absorbed into the systemic circulation and had a long residual time after intravenous (10 mg/kg) and intragastric (60 mg/kg) administration, as it could still be detected after 72 h. Tissue distribution analysis revealed detectable levels of pterocephin A in various tissues, and a high concentration was maintained in the liver after intravenous (10 mg/kg) administration, with the highest concentration being 610.95 ± 25.73 ng/mL and a specific distribution pattern of liver > lung > kidney > intestine > spleen > testes > heart > stomach. The toxicokinetic process and tissue distribution characteristics of pterocephin A were expounded in this study, which can provide relevant data support for further research and clinical application of pterocephin A with its slight toxicity.

PM10 Organic Aerosol Fingerprints by Using Liquid Chromatography Orbitrap Mass Spectrometry: Urban vs. Suburban in an Eastern Mediterranean Medium-Sized Coastal City

This study compares the PM10 (particulate matter of diameter smaller than 10 μm) organic aerosol composition between urban and suburban stations in Heraklion, Crete, during winter 2024 in order to highlight the impact of local anthropogenic activities on urban atmospheric particulate matter pollution. Using an HPLC-ESI-MS Orbitrap analyzer (High Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry) in full MS scan mode at a resolution of 140,000, 48 daily aerosol filter extracts were analyzed in both positive and negative modes, resulting in the detection of 2809 and 3823 features, respectively. Features with at least five times higher intensity in the urban environment compared to the suburban, and p < 0.05, were deemed significant. A correlation with black carbon (r > 0.6) was observed for 71% of significant urban features in positive mode. These features showed a predominance of low O:C ratios (<0.2) and the majority were classified as intermediate volatility organic compounds (IVOCs), indicating fresh primary emissions. A clear urban–suburban distinction was shown by PCA of positive mode features, unlike the negative mode features. Regarding the total intensity of the features, urban samples were on average 55% higher than suburban samples in positive mode and 39% higher in negative mode. This study reveals the molecular profile of locally emitted combustion related organics observed in positive mode in an urban environment.

Forensic application of sandpaper spray ionization mass spectrometry (SPS-MS): Direct analysis of solid pharmaceutical formulations and edible cannabis products.

In this work, we employed a new ambient ionization mass spectrometry technique, sandpaper spray mass spectrometry (SPS-MS), as an efficient tool to analyze pills and tablets of pharmaceutical formulations. The following samples were analyzed: regulators of blood pressure, cholesterol, and diabetes, and drugs for the treatment of erectile dysfunction (ED). Additionally, a hard candy of Cannabis sativa containing Δ9-tetrahydrocannabinol (Δ9-THC) and its related isomer cannabidiol (CBD) was also evaluated. The surfaces of the samples, without any prior treatment, were sanded onto triangular-cut sandpaper, and full MS scans (and MS/MS) were acquired in both positive and negative ionization modes. SPS-MS (and MS/MS) allowed for prompt detection of the active pharmaceutical ingredients (APIs) in each formulation. Other components of the formulations, added as excipients, were also tentatively identified. The results described herein indicate that the SPS-MS technique can be applied to fast screening of pills and tablets being potentially used as an efficient tool to detect counterfeit pharmaceutical and illicit products, a current issue of increasing concern.

Comprehensive Identification of the Chemical Substances in Effective Component Groups of Polygalae Radix Based on Targeted Tandem Mass Spectrometry Analysis Strategy

ABSTRACTPolygalae Radix is a commonly used traditional Chinese medicine with multiple pharmacological activities. Modern research have revealed that saponins, xanthones, and oligosaccharide esters are its effective component groups. However, up to date the chemical substances in these effective component groups are still unclear, which has become a fundamental scientific issue needing to be addressed. In our study, an analysis strategy based on targeted tandem mass spectrometry (MS/MS) analysis was proposed to comprehensively identify the chemical substances in effective component groups. First, the extract of the Polygalae Radix was detected by ultra‐performance liquid chromatography coupled with electrospray ionization–orbitrap mass spectrometry in a full MS scan mode to minimize the losses of signals; second, the precursor ions of all compounds were extracted from the obtained full MS scan data using Compound Discoverer software; third, the interested precursor ions were locked based on their molecular features and constructed in a precursor ion list; finally, each precursor ion in the list was subjected to targeted MS/MS analysis for structure characterization. Using our analysis strategy, a total of 565 components were successfully identified.

Elucidating important factors and corresponding method optimization for sensitive detection of small peptide drugs in human urine by solid-phase extraction and UPLC-HRMS: The influence of MS scan modes, protein precipitants, and ammonium formate.

Small peptide hormones are widely used in sports as performance-enhancing substances, making it crucial to develop sensitive analytical methods for their detection in doping control analysis. Various factors significantly affect analytical sensitivity, such as the selection of ultra-performance liquid chromatography (UPLC) mobile phase, high-resolution mass spectrometry (HRMS) scanning modes, and extraction solvents for pretreatment. Herein, comparative study approach was utilized to investigate the sensitivity of each peptide analyte under both full scan and parallel reaction monitoring (PRM) modes of HRMS and assess the effects of some protein precipitants as a part of extraction solvents on solid-phase extraction (SPE). The results showed that full scan should be selected as the primary scan mode of HRMS, and the combination with PRM mode could effectively compensate for the limitations of full scan, and the addition of protein precipitants would adversely affect the detection of certain small peptide analytes. Meanwhile, influences of ammonium formate in reverse UPLC mobile phase on the charge state distribution of small peptides were investigated and elucidated. Based on these findings, a sensitive and reliable UPLC-HRMS analytical method combining full scan and PRM mode was validated for screening and confirmation of 63 small peptide analytes after SPE, with limits of detection (LODs) ranging between 0.010 and 0.473 ng/ml and limits of identification (LOIs) ranging between 0.015 and 1.512 ng/ml. Additionally, suggestions were provided for the detection of [Arg8]-vasopressin, dermorphin, and its analogues.

Comprehensive identifying flavonoids in Citri Reticulatae Pericarpium using a novel strategy based on precursor ions locked and targeted MS/MS analysis

Citri Reticulatae Pericarpium is a traditional Chinese medicine with extremely high health benefits as well as clinical value. In vivo and in vitro tests have proved that its main active secondary metabolites are flavonoids. However, they have not been comprehensively analyzed up to now mainly due to lack of suitable analysis method. To solve this problem, a novel strategy based on precursor ions locked and targeted MS/MS analysis was proposed. Firstly, the database of the flavonoids previously isolated from Citri Reticulatae Pericarpium was established to obtain the characteristics of their precursor ions. Secondly, after performing the full MS scan of the extract, all compounds in the total ion chromatogram were extracted by Compound Discoverer software. Thirdly, the precursor ions of the flavonoids were locked from the extracted compounds according to their characteristics, forming a precursor ions list. Finally, the precursor ions in the constructed list were performed targeted MS/MS analysis for structures characterization. As a result, total 187 flavonoids were successfully identified, and except for flavones, flavonols as well as dihydroflavones, some chalcones were also characterized from Citri Reticulatae Pericarpium for the first time.

A LC-MS-based serum pharmacochemistry approach to reveal the compatibility features of mutual promotion/assistance herb pairs in Xijiao Dihuang decoction

Xijiao Dihuang decoction (XDT), a famous formula, was usually used to improve the prognosis of patients with blood-heat and blood-stasis syndrome-related diseases. There were some mutual promotion and mutual assistance herb pairs in XDT. However, the exact functions of these herb pairs in the compatibility of XDT were not elucidated due to the lack of appropriate methodologies. Based on the theory of serum pharmacochemistry, a systematic method was established for the qualitative and quantitative analysis of characteristic components in the extracts and drug-containing plasma samples of XDT and its relational mutual promotion/assistance herb pairs. For qualitative analysis, 85 characteristic components were identified using the liquid chromatography with triple time-of-flight mass/mass spectrometry (LC-Triple QTOF-MS/MS) based on the mass defect filtering, product ion filtering, neutral loss filtering and isotope pattern filtering techniques. For quantitative detection, a relative quantitation assay using an extract ion chromatogram (EIC) of the full scan MS experiment was validated and employed to assess the quantity of the 85 identified compounds in the test samples of single herb, herb pairs and XDT. The results of multivariate statistical analyses indicated that both the assistant and guide herbs could improve the solubilization of active compounds from the sovereign and minister herbs in XDT in vitro, might change the trans-membrane transportation, and regulate metabolism in vivo. The methods used in present study might be also valuable for the investigation of multiple components from other classic TCM formulas for the purpose of compatibility feature study.

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2 O. We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

Ultrafast protein digestion using an immobilized enzyme reactor following high-resolution mass spectrometry analysis for rapid identification of abrin toxin.

Abrin toxin, highly dangerous with an estimated human lethal dose of 0.1-1 μg per kg body weight, has attracted much attention regarding criminal and terroristic misuse over the past decade. Therefore, developing a rapid detection method for abrin toxin is of great significance in the field of biosecurity. In this study, based on the specific dissociation method of an immobilized enzyme reactor, the trypsin immobilized reactor Fe3O4@CTS-GA-Try was prepared to replace free trypsin, and the immobilized enzyme digestion process was systematically investigated and optimized by using bovine serum albumin as the simulant of abrin. After 5 min one-step denaturation and reduction, a satisfactory peptide number and coverage were yielded with only 15 s assisted by an ultrasound probe to identify model proteins. Subsequently, abrin was rapidly digested using the established method, resulting in a stable and highly reproducible characteristic peptide number of 39, which can be analyzed by nanoelectrospray ionization coupled with high-resolution mass spectrometry. With the acquisition mode of full MS scan coupled with PRM, not only MS spectroscopy of total abrin peptides but also the corresponding MS/MS spectroscopy of specific abrin peptides can achieve the characteristic detection of abrin toxin and its different isoforms in less than 10 minutes, with high repeatability. This assay provides a universal platform and has great potential for the development of on-site detection and rapid mass spectrometric analysis techniques for macromolecular protein toxins and can further be applied to the integrated detection of chemical and biological agents.

A sensitive high-resolution mass spectrometry method for quantifying intact M-protein light chains in patients with multiple myeloma

To determine the disease status and the response to treatment for patients with multiple myeloma, measuring serum M-protein levels is a widely used alternative to invasive punctures to count malignant plasma cells in the bone marrow. However, the quantification of this monoclonal antibody, which varies from patient to patient, poses significant analytical challenges. This paper describes a sensitive and specific mass spectrometry assay that addresses two objectives: to overcome the potential interference of biotherapeutics in the measurement of M-proteins, and to determine the depth of response to treatment by assessing minimal residual disease. After immunocapture of immunoglobulins and free light chains in serum, heavy and light chains were dissociated by chemical reduction and separated by liquid chromatography. M-proteins were analyzed by high-resolution mass spectrometry using a method combining a full MS scan for isotyping and identification and a targeted single ion monitoring scan for quantification. This method was able to discriminate M-protein from the therapeutic antibody in all patient samples analyzed and allowed quantification of M-protein with a LLOQ of 2.0 to 3.5 µg/ml in 5 out of 6 patients. This methodology appears to be promising for assessing minimal residual disease with sufficient sensitivity, specificity, and throughput.

A Combined Gas-Phase Separation Strategy for ADP-ribosylated Peptides.

ADP-ribosylation (ADPr) is a post-translational modification that is best studied using mass spectrometry. Method developments that are permissive with low inputs or baseline levels of protein ribosylation represent the next frontier in the field. High-field asymmetric waveform ion mobility spectrometry (FAIMS) reduces peptide complexity in the gas phase, providing a means to achieve maximal ADPr peptide sequencing depth. We therefore investigated the extent to which FAIMS with or without traditional gas-phase fractionation-separation (GPS) can increase the number of ADPr peptides. We examined ADPr peptides enriched from mouse spleens. We gleaned additional insight by also reporting findings from the corresponding non-ADPr peptide contaminants and the peptide inputs for ADPr peptide enrichment. At increasingly higher negative compensation voltages, ADPr peptides were more stable, whereas the non-ADPr peptides were filtered out. A combination of 3 GPS survey scans, each with 8 compensation voltages, resulted in 790 high-confidence ADPr peptides, compared to 90 with GPS alone. A simplified acquisition strategy requiring only two injections corresponding to two MS1 scan ranges coupled to optimized compensation voltage settings provided 402 ADPr peptides corresponding to 234 ADPr proteins. We conclude that our combined GPS strategy is a valuable addition to any ADP-ribosylome workflow. The data are available via ProteomeXchange with identifier PXD040898.

High-Field Asymmetric Waveform Ion Mobility Spectrometry Analysis of Carcinogenic Aromatic Amines in Tobacco Smoke with an Orbitrap Tribrid Mass Spectrometer.

Smoking is a risk factor for bladder cancer (BC), although the specific chemicals responsible for BC remain uncertain. Considerable research has focused on aromatic amines (AAs), including o-toluidine (o-tol), o-anisidine (o-anis), 2-naphthylamine (2-NA), and 4-aminobiphenyl (4-ABP), which are linked to human BC based on elevated BC incidence in occupationally exposed factory workers. These AAs arise at nanogram levels per combusted cigarette. The unambiguous identification of AAs, particularly low-molecular-weight monocyclic AAs in tobacco smoke extracts, by liquid chromatography-mass spectrometry (LC-MS) is challenging due to their poor performance on reversed-phase columns and co-elution with isobaric interferences from the complex tobacco smoke matrix. We employed a tandem liquid-liquid and solid-phase extraction method to isolate AAs from the basic fraction of tobacco smoke condensate (TSC) and utilized high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to high-resolution accurate mass (HRAM) Orbitrap LC-MS2 to assay AAs in TSC. The employment of FAIMS greatly reduced sample complexity by removing precursor co-isolation interfering species at the MS1 scan stage, resulting in dramatically improved signal-to-noise of the precursor ions and cleaner, high-quality MS2 spectra for unambiguous identification and quantification of AAs in TSC. We demonstrate the power of LC/FAIMS/MS2 by characterizing and quantifying two low-molecular-weight carcinogenic AAs, o-tol and o-anis, in TSC, using stable isotopically labeled internal standards. These results demonstrate the power of FAIMS in trace-level analyses of AA carcinogens in the complex tobacco smoke matrix.

Study of metal and organic contaminants transported by microplastics in the Lebanese coastal environment using ICP MS, GC-MS, and LC-MS

An untargeted study of multiclass contaminants associated with microplastics (MPs) in the East Mediterranean was carried out. Samples were collected in 2020–2021 from the shoreline at 14 different locations, along the Lebanese coast. Attenuated Total Reflectance (ATR) FTIR spectroscopy showed the predominant presence of polyethylene and polypropylene among plastic debris. The non-polar and polar organic compounds sorbed on the MPs were identified and quantified by GC - TOF MS and LC – electrospray MS/MS, respectively. Deconvolution of accurate GC–MS scan data allowed the identification of >130 organic pollutants, 64 of which could be confirmed by matching with the authentic standards, among which a number of previously unreported in targeted GC–MS(MS) methods. In addition to highly toxic, legacy chlorinated pollutants, high levels (average values from 0.8 to 4.0 μg g−1) of some musks, UV filters and UV absorbers were detected. Untargeted LC-MS demonstrated the persistence of several pesticides (i.e., chlorpyrifos) and pharmaceuticals, such as phenacetin and minoxidil, which were quantified. In addition, a study of metals associated with microplastics using ICP-MS confirmed the high potential of microplastics to act as a vector of, among others, toxic metals, such as Cd, Pb, Bi or Hg.

A mass defect filtering combined background subtraction strategy for rapid screening and identification of metabolites in rat plasma after oral administration of Yindan Xinnaotong soft capsule

The absorbed prototypes and metabolites of traditional Chinese medicines (TCMs) serves an important part in pharmacological action and clinical effects. However, the comprehensive characterization of which is facing actual or possible rigorous challenges due to the lack of data mining methods and the complexity of metabolite samples. Yindan Xinnaotong soft capsule (YDXNT), a typical traditional Chinese medicine prescription consisting of extracts from 8 herbal medicines, is widely used for the treatment of angina pectoris and ischemic stroke in the clinic. This study established a systematic data mining strategy based on ultra-high performance liquid chromatography tandem quadrupole-time-of-fight mass spectrometry (UHPLC-Q-TOF MS) for comprehensive metabolite profiling of YDXNT in rat plasma after oral administration. The multi-level feature ion filtration strategy was primarily conducted through the full scan MS data of plasma samples. All potential metabolites were rapidly fileted out from the endogenous background interference based on the background subtract and the chemical type specifically mass defect filter (MDF) windows including flavonoids, ginkgolides, phenolic acids, saponins, and tanshinones. As the MDF windows of certain types were overlapped, the screened-out potential metabolites were deeply characterized and identified according to their retention times (RT), integrating neutral loss filtering (NLF), diagnostic fragment ions filtering (DFIF), and further confirmed by reference standards. Thus, a total of 122 compounds, consisting of 29 prototype components (16 confirmed with reference standards) and 93 metabolites had been identified. This study provides a rapid and robust metabolite profiling method for researching complicated traditional Chinese medicine prescriptions.

MNeuCode Empowers Targeted Proteome Analysis of Arginine Dimethylation.

Characterization of protein arginine dimethylation presents significant challenges due to its occurrence at the substoichiometric level. To enable a targeted MS/MS analysis of these dimethylation sites, we developed the mNeuCode (methyl-neutron-coding) tag by metabolically labeling methylarginine with stable isotopes during cell culture, which generated a diagnostic peak containing the NeuCode isotopologue signature in a high-resolution MS scan. A software tool, termed NeuCodeFinder, was developed for screening the NeuCode signatures in mass spectra. Therefore, a targeted MS/MS workflow was established for proteome-wide discovery of arginine dimethylation. The efficacy and utility were demonstrated by identifying 176 arginine dimethylation sites residing on 70 proteins in HeLa cells. Among them, 38% of the sites and 29% of the dimethylated proteins are novel, including five novel arginine dimethylation sites on the protein FAM98A, which is a substrate of protein arginine methyltransferase 1 (PRMT1). Our results show that deletion of FAM98A in HeLa cells suppressed cell migration, and importantly, dimethylation-deficient mutation suppressed this process as well. Therefore, the PRMT1-FAM98A pathway mediates cell migration possibly through dimethylation of these newly identified sites of FAM98A. Our study might drive the methodological shift from shotgun-based to targeted proteome analysis for interrogation of the substoichiometric biomolecules by using NeuCode-enabled techniques.

Rapid identification of pyoverdines of fluorescent Pseudomonas spp. by UHPLC-IM-MS

Siderophores are iron-chelating molecules produced by bacteria and other microbes. They are involved with virulence in infections and play key roles in bacterial community assembly and as plant protectants due to their pathogen control properties. Although assays exist to screen whether newly isolated bacteria can produce siderophores, the chemical structures of many of these bio-active molecules remain unidentified due to the lack of rapid analytical procedures. An important group of siderophores are pyoverdines. They consist of a structurally diverse group of chromopeptides, whose amino acid sequence is characteristic for the fluorescent Pseudomonas species that secrets them. Although over 60 pyoverdine structures have been described so far, their characterization is cumbersome and several methods (isoelectrofocusing, iron uptake measurement, mass determination) are typically combined as ambiguous results are often achieved by a single method. Those additional experiments consume valuable time and resources and prevent high-throughput analysis. In this work, we present a new pyoverdine characterisation option by recording their collision cross sections (CCS) using trapped ion mobility spectrometry. This can be done simultaneously in combination with UHPLC and high-resolution MS resulting in a rapid identification of pyoverdines. The high specificity of CCS values is presented for 17 pyoverdines secreted by different Pseudomonas strains. The pyoverdine mass determination by full scan MS was supported by fragments obtained from broadband collision induced dissociation (bbCID). As iron contaminations in laboratories are not uncommon, CCS values of ferripyoverdines were also evaluated. Thereby, unusual and highly characteristic ion mobility patterns were obtained that are suitable as an alternative identification marker.

Identification of Oxindoleacetic Acid Conjugates in Quinoa (Chenopodium quinoa Willd.) Seeds by High-Resolution UHPLC-MS/MS.

Quinoa (Chenopodium quinoa Willd.) has a high nutritional value and it contains a high number and high amounts of specialized metabolites. These metabolites include, for example, phenolic acids, flavonoids, terpenoids and steroids. In addition, it is known to contain N-containing metabolites, such as betalains. Here, we report the presence and identification of 14 new oxindoleacetate conjugates in quinoa by high-resolution ultrahigh-performance liquid chromatography quadrupole-time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) and ultrahigh-resolution UHPLC-QOrbitrap-MS/MS. The oxindoleacetate conjugates were extracted from dried and ground quinoa seeds using either methanol/water or acetone/water (4:1, v/v) and were further concentrated into aqueous phase and analyzed by UHPLC with reverse-phase chromatography using acetonitrile and 0.1% aqueous formic acid as eluents. High-resolution hybrid LC-MS/MS techniques, including full scan MS with in-source collision, induced dissociation, and data dependent-MS2(TopN) with stepped normalized collision energies using N2 as collision gas and data-independent acquisition (MSE) using ramped collision energies and argon as collision gas enabled their analysis directly from the crude quinoa seed extract. The oxindoleacetate conjugates were found to be present in both conventional and organic farmed seeds. According to our best knowledge, this is the first time hydroxy-oxindoles have been reported in quinoa.

Supercritical fluid chromatography coupled to high-resolution tandem mass spectrometry: an innovative one-run method for the comprehensive assessment of chocolate quality and authenticity.

To assess chocolate quality and authenticity comprehensively, a combination of various analytical procedures is involved, thereby making the process time-consuming and costly. Thus, we investigated the potential of ultra-high performance supercritical fluid chromatography coupled to quadrupole-time of flight mass spectrometry (UHPSFC-QTOF-MS) as an alternative to "classic" methods. By combining hexane and aqueous extracts from sequential extraction, a single 8-min analytical run enabled us (i) to determine cocoa butter equivalents (CBEs) and milk fat content based on the detection of selected triacylglycerols, (ii) to calculate dry non-fat cocoa solids based on determined theobromine and caffeine content, and (iii) to profile contained sugars. To obtain the most comprehensive information about sample composition, the MS method comprised a full MS scan for non-target screening and several time-scheduled targeted MS/MS functions ("parallel reaction monitoring") optimized according to the possible concentration ranges of the analytes. For 40 different chocolate samples, our results and those obtained by using standard methods (LC-UV for non-fat cocoa solids, and GC-FID for CBEs) were in good agreement. Compared to the conventional approach for chocolate quality and authenticity control, the presented SFC-MS method is a fast, cost-effective, and efficient alternative, and only samples suspicious for the presence of CBE should be referred to the standard GC-FID method for exact CBE quantification. In the study, also some challenges offered by SFC-MS have been addressed.

Highly sensitive MS/MS detection for confident identification of potent novel synthetic opioids and their metabolites

The aim of this presentation is to demonstrate the use of high-resolution mass spectrometry as a unique tool for the untargeted detection and identification of new emerging substances, with little or no method optimization necessary. The presentation will demonstrate how accurate mass instruments are providing extra levels of confidence in analyte identification by reliably obtaining comprehensive MS/MS spectral fragment information that can be used for identification, confirmation, and/or library matching of potent drugs and metabolites present in discarded authentic case samples. NSO compounds were extracted from human whole blood using a liquid-liquid extraction (LLE) procedure. HPLC separation was performed on an ExionLC system using a Phenomenex Kinetex C18 column Mobile phases were ammonium formate and formic acid in methanol and acetonitrile. The flow rate was 0.4 mL/min with a total LC runtime of 15.5 minutes. The injection volume was 10 μL. MS and MS/MS data were collected for each sample using Zeno IDA for optimal sensitivity on the ZenoTOF 7600 system. Data acquisition consisted of a TOF MS scan to collect accurate mass precursor ions from 100 to 700 Da, followed by a TOF MS/MS full scan ranging from 25 to 700 Da to ensure all fragments were captured for identification using a maximum of 16 candidate ions. Data was acquired using SCIEX OS software 2.1. A series of 9 calibrator solutions were prepared by spiking control human whole blood samples with the 6 targeted analytes at final concentrations ranging from 10 pg/mL to 100 ng/mL. The series of calibrator solutions were injected to evaluate the quantitative performance of the system and its ability to accurately measure low level analytes with a high level of precision and accuracy in TOF MS mode. Each calibrator was injected in triplicate. LLOQs of between 10 and 50 pg/ml were observed for all compounds analysed. Accuracies at LLOQ ranged from 86–111% and imprecision was < 15% for all compounds analysed. In addition, the use of specific technologies afforded by the new SCIEX ZenoTOF 7600 system generated an average 9x sensitivity improvement on spectral data providing significantly improved library matching. A comprehensive and highly sensitive method for the screening and identification of potent NSO in human whole blood is described. The significant gains in MS/MS sensitivity on the SCIEX ZenoTOF 7600 system yielded an improvement in confident identifications of low-level analytes through spectral library matching. The observed sensitivity gains resulted in a 9x improvement, on average, in TOF MS/MS sensitivity across the drugs positively identified in the authentic case samples analyzed. This improvement enabled confident identification of key drugs and metabolites at trace levels that were not previously achievable. The MS/MS sensitivity levels afforded by ZenoTOF 7600 system provide a means to monitor low levels of ultra-potent NSO in poly-drug intake scenarios.

Detection of multiple prohibited ingredients in nutritional supplements in a case of doping

We present a case of a mixed martial art athlete found positive to methasterone metabolites, ligandrol and cardarine metabolites after an anti-doping testing. The athlete denied any drugs consumption but provided the list of 23 nutritional supplements under use during competition. The aim of the present study was to analyse the provided material to support evidence of unintentional doping. Cumulatively, products were commercialised either as pre-workout, intra-workout and post-workout adjuvants containing amino acids, creatine and proteins or as cofactors and vitamins to recover after training. Four products were taken to aid sleeping and one was a “testosterone boost”. All samples were submitted to analysis by liquid-chromatography/high resolution mass spectrometry (LC-QTOF). One hundred milligrams of material (powders, pills, oily pills, capsules) were sampled and dissolved with 10 mL of methanol, vortexed for 30 and sonicated for 30 min. The extracts were centrifuged at 3000 rpm for 5 min at 25 °C. The supernatants were appropriately diluted with deionised water then 5 μL injected into the LC-MS. Separations were carried out in a Zorbax C18 (2.1 × 100, 1.8 μm) column in 5 mM ammonium formate (mobile phase A) and methanol (mobile phase B), both added of 0.1% formic acid, flow at 0.2 mL/min. Acquisitions were carried out in positive polarity MS scan mode and MS/MS for fragment confirmation. Sensitivity expressed as method detection limit (MDL), linearity, carry-over, dilution integrity, accuracy and precision were tested under the described conditions for cardarine, methasterone and ligandrol. The analysis of the samples showed 2 out the 23 nutritional products containing the illicit substances. Methasterone, cardarine and ligandrol were alongside present in one sample (S1) at the respective concentrations of 5 μg/mg, 11.8 μg/mg and 1.4 μg/mg. Cardarine and ligandrol were confirmed in a second sample (S2) at the concentration of 0.6 and 0.2 μg/mg, respectively. Two products resulted contaminated with methasterone, cardarine and ligandrol. The calculated concentration for all substances > 1 μg/mg in S1 product may presume an intentional manipulation with pharmacologically active ingredients that better fitted the promise of enhanced recovery and improved endurance. In S2 ligandrol and cardarine amounts are compatible with a cross-contamination scenario. Conclusion: methasterone, ligandrol and cardarine were complessively detected in 2 samples. Since no labelling declared the retrieved substances, this might have represented a case of unintentional doping.