MS Methodology Research Articles

METHOD DEVELOPMENT AND VALIDATION OF ERYTHROMYCIN AND OLAPARIB IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY

The liquid chromatography–tandem mass spectrometry (LC-MS)/MS methodology was used to develop and validate a method for detecting erythromycin and olaparib in human plasma. Antibiotics such as erythromycin and olaparib fall into this category. Liquid chromatography is used to separate stationary and mobile phases based on differences in their affinities as well as to remove unwanted contaminants. It improves repeatability, sensitivity, resilience, and low-level protein detection. A C18 (C18, 5 m, 100×4.6 mm) column is utilized for high resolution and peak area. The calibration curve is created using linear regression. Internally, telmisartan is utilized as a benchmark. The flow rate of the mobile phase is 0.5 mL/min. Erythromycin and olaparib have mass-to-charge ratios of 735.43–115.97 and 435.08–102.04, respectively. Erythromycin in combination with olaparib resulted in a 98% recovery rate. The precision and accuracy of the results determined by interday QC samples are within acceptable limits. There was no evidence of instability.

Juniper communis L. Essential Oils from Western Romanian Carpathians: Bio-Structure and Effective Antibacterial Activity

The antibacterial activity of four bacterial standard strains that are naturally encountered in humans and animals was investigated by using bioactive compounds from commercial essential oils of Juniperus communis that were collected from the Western Romanian Carpathians. The Juniper communis essential oils, volatile compounds, were recognized through the GC–MS methodology by comparing identified spectra with those held in the NIST 02, Wiley 275 library. The ratio of each component was calculated based on the peak areas of the GC, without utilizing correction factors. The CLSI standardized micro-dilution was used to determine antimicrobial activity, employing 10−3 dilutions of fresh culture, with inoculums equivalent to a standard of 0.5 McFarland being prepared for testing. Four bacterial strains, Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Streptococcus pyogenes (ATCC 19615), were investigated, using 96-well micro-dilution plates. Over each micro-dilution well, the essential oils were poured, introducing gradually 2, 4, 8, and 10 µL/well, respectively. The results were expressed as ±SEM and analyzed by one-way ANOVA with Bonferroni’s multiple comparison test, considering the differences statistically provided when p < 0.05 or lower. The juniper essential oil originating from the Western Romanian Carpathians is rich in four main volatile compounds: β-pinene (34.02%), 1α-pinene (30.43%), p-cymol (20.25%), and β-myrcene (10.20%). The juniper communis essential oil reduced bacterial density for all four strains tested, but compared to Gram-negative bacteria, in our case; a considerably higher antibacterial effectiveness was detected for Gram-positives, with peak reduction of Staphylococcus aureus, recommending the Romanian essential oil as a beneficial antibacterial resource.

Novel hydrophilic-phase extraction, HILIC and high-resolution MS quantification of an RNA oligonucleotide in plasma.

Aim: In the theme of quantitative LC-MS bioanalysis of oligonucleotides free of ion-pairing, a 22-mer RNA oligonucleotide took center stage. The focus was on a unique polar-based retention scheme to produce a high-recovery extraction presenting a high-performance alternative extraction means, also there was the opportunity to involve hydrophilic-interaction liquid chromatography and contemporary high-resolution MS as the end point. Results: Original LC-MS methodology was developed for the oligonucleotide and the performance was robust for both nominal and accurate mass detection, the latter affording 10× improvement in sensitivity and 4000-fold linear dynamic range, 500pM to 2000nM. Conclusion: A novel means of solid-phase extraction is exhibited within a robust pair-free methodology, reaching pM sensitivity with the demonstrably beneficial accurate mass platform.

Phytoprostanes from Date Palm Fruit and Byproducts: Five Different Varieties Grown in Two Different Locations As Potential sources.

Date palm fruit has been considered for centuries as an ancient nutritional constituent in the human diet. Recently, global trade in dates increased at an average that, simultaneously, will be accompanied by an increase in date palm byproducts. Supported by date phytochemicals and their health benefits, the aim of this work is to evaluate for the first time the presence of special metabolites of plant called phytoprostanes (PhytoPs) in five different varieties of the Phoenix dactylifera L. pulps and pits using a microLC-ESI-QTrap-MS/MS methodology. Results obtained showed the interest of using these matrices as potential sources of several PhytoPs (ent-16-B1-PhytoP; ent-9-L1-PhytoP; and epimers of ent-16-F1t-PhytoP and of 9-F1t-PhytoP). The variation in concentration between different varieties and different DPF parts was also evaluated. Results obtained will help to unravel the biological activities associated with DPF consumption that could be related to these bioactive metabolites.

Rapid Structure Determination of Bioactive 4″-Tetrahydrofurfuryl Macrozone Reaction Mixture Components by LC-SPE/Cryo NMR and MS.

LC-SPE/cryo NMR and MS methodologies have been developed and employed for a rapid structure determination of 4″-tetrahydrofurfuryl macrozone reaction mixture components. Macrozones, novel conjugates of azithromycin, and thiosemicarbazones have shown very good in vitro antibacterial activities against susceptible and some resistant bacterial strains and are promising agents for further development. The post-column multiple trapping of the chromatographically separated reaction mixture components on the SPE cartridges increased the sensitivity and together with cryogenically cooled NMR probe made it possible to identify and structurally characterize main 4″-tetrahydrofurfuryl macrozone reaction mixture compounds including those present at very low concentration level. This approach has several advantages over a classical off-line procedure, efficiency and low solvent consumption being the two most important ones. All identified components were process-related. It has been demonstrated that two different kinds of compounds with respect to structure were identified, i.e., macrolide-related and thiosemicarbazone-related ones. This methodology can serve as a platform for reliable and effective macrolides reaction components structure profiling, serving as both isolation and identification tools.

In-depth correlation analysis demonstrates that 4-hydroxyproline at the Yaa position of Gly-Xaa-Yaa repeats dominantly stabilizes collagen triple helix

There is a general consensus that collagen stability is largely maintained by Pro and its major hydroxylated form, 4-hydroxyproline (4Hyp). However, positional difference in their stabilizing effect at the Xaa or Yaa position of collagenous Gly-Xaa-Yaa sequences has remained inconclusive. Here, we position-specifically evaluated the correlation of imino acid contents to denaturation temperature (Td) of collagen among various vertebrate and invertebrate species, using a recently developed LC–MS methodology. 4Hyp at the Yaa position showed the highest positive correlation with Td, followed by Pro at the Xaa position, which was even further increased by excluding invertebrates. We confirmed that Gly-Pro-4Hyp liberated after bacterial collagenase digestion was highly positively correlated with Td. Furthermore, other tripeptides with Yaa position 4Hyp also had comparable positive correlation, excepting negative correlation of Gly-Gly-4Hyp, while tripeptides with Xaa position Pro did not. These data provide evidence that 4Hyp dominantly contributes to thermal stability of collagen depending on its sequence position, especially in vertebrates.

The Enzymatic Activities of Nucleoside Triphosphate Diphosphohydrolases Towards Tenofovir Diphosphate and Their Spatial Distributions in Human Colorectal Tissue

Tenofovir (TFV; prescribed as the tenofovir disoproxil fumarate prodrug) must be phosphorylated to form a competitive inhibitor of HIV, TFV-diphosphate (TFV-DP). Previously, we reported heterogeneity in TFV-DP distribution in colorectal tissue (a putative site of HIV infection) sections collected from human participants receiving a TFV-containing enema. This observed heterogeneity is likely multifactorial. TFV-DP is structurally similar to ATP. It is known that nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases) dephosphorylate ATP. Therefore, we hypothesized that NTPDase-mediated dephosphorylation plays a role in regulating TFV-DP levels in colorectal tissue. To test this, we incubated recombinant NTPDase proteins (NTPDase 1, 3, 4, 5, and 8), individually, with TFV-DP to determine the abilities of these enzymes to dephosphorylate TFV-DP. Following incubation, TFV-DP levels were measured using ultra-high performance liquid chromatography tandem mass spectrometry. We observed TFV-DP dephosphorylation in the presence of NTPDase 1 or 3 enzymes. Further, a malachite green phosphate assay that measures the release of inorganic phosphate was employed to further confirm the activities of NTPDase 1 and 3 towards TFV-DP. Immunoblotting revealed that NTPDase 1 and 3 proteins are indeed expressed in human colorectal tissue lysates. To visualize the distributions of NTPDases across human colorectal tissue, we employed Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI). Initially, trypsin digestions on a range of recombinant NTPDases including 1 and 3 were carried out to detect and identify their tryptic peptides that could be targeted for detection in MALDI MSI. From these digestions, NTPDase 1 yielded peptides, SQHQETPVYLGATAGMR, SLSNYPFDFQGAR, and IITGQEEGAYGWITINYLLGK whereas NTPDase 3 exhibited the following peptides: YGIVLDAGSSR, GSGISSYGNNPQDVPR, and MFTVLTR. Five tryptic peptides corresponding to NTPDase 5 (which did not exhibit enzymatic activity towards TFV-DP) were identified: LYTHSYLGFGLK, LHQPEEVQR, GSFYAFSYYYDR, QGAETVQGLLEVAK, and LATLGALETEGTDGHTFR. Next, the abovementioned MALDI MS methodology was applied to directly visualize tryptic peptides of NTPDase 1, 3 and 5 in human colorectal tissue sections. In these studies, human colorectal biopsies were collected and sectioned to 20-micron thickness. Tissue sections were subjected to serial washing steps using ethanol. Trypsin and matrix (α-cyano-4-hydroxycinnamic acid) were deposited onto the tissue using a robotic sprayer. We detected tryptic peptides, SQHQETPVYLGATAGMR, and LATLGALETEGTDGHTFR corresponding to NTPDase 1 and 5, respectively. The distribution profiles of NTPDase 1 and 5 were generated using the m/z values of above peptides. The identities of the detected peptides were validated using mass spectrometry-based proteomics analyses. Thus, in this work, we established a MALDI MSI methodology for simultaneous visualization of tryptic peptides corresponding to NTPDases 1 and 5. Results from our in vitro studies suggest that NTPDase 1 and 3 may contribute to the regulation of TFV-DP levels.

Quantifying Capsaicinoids from Chili Pepper and Motor Oil Mixtures Used in Elephant Deterrent Fences

Human–elephant conflicts continue to escalate as African elephants (Loxodonta africana) attempt to forage on cultivated crops. Chili pepper fencing is used as a deterrent but has varying success rates. These fences are constructed with crushed chili peppers, typically mixed in motor oil, placed on cloths, and strung around crop fields. This mixture is made using different formulas involving oil and sometimes water, but each takes advantage of the “heat” and discomfort produced from capsaicinoids, the active ingredients in chili peppers. Using LC–MS technology, we examined some of these formulas and preparation methods. The development of this methodology is to our knowledge the first time this type of analysis has been conducted with chili and motor oil mixtures. By examining the differences in Scoville Heat Units (SHU), we confirmed that mixing with oil played an important role and that the SHU was no different by boiling crushed chilis in water before combining with oil compared to directly combining the crushed peppers with oil. Allowing the crushed peppers to remain in the mixture rather than filtering before mixing showed only a slight benefit. This LC–MS methodology provides a new tool for conservationists to compare the efficacy of chili fence formulas, which may allow for improvement of this deterrent method via further field testing.

Valuable products from the flowers of lemon (Citrus limon (L.) Osbeck) and grapefruit (Citrus paradisi Macfad.) Italian trees

Citrus products are items of fundamental importance in the global market. The fruit of lemon (Citrus limon (L.) Osbeck) are mainly processed for the production of the juice, and of the peel essential oil, as a by-product. The same can be said for grapefruit (Citrus paradisi Macfad.), even if its production quantities are considerably lower. In the present study, the absolutes obtained from the flowers of Italian lemon and grapefruit plants were evaluated for their chemical composition and sensory properties. The GC analysis unveiled a volatile composition different from that of peel oils, being present compounds such as indole (0.04–0.22%) and methyl anthranilate (0.31–3.63%). The GC findings were correlated with sensory evaluation, carried out by a panel of trained assessors, that highlighted a pronounced floral character in grapefruit, while a predominant citrusy note was described in lemon. By means of an MDGC system, equipped with a chiral selector, a number of optical isomers were separated, allowing to define the enantiomeric ratios in these matrices for the first time. The nonvolatile fraction of the absolutes was investigated about its coumarin content by exploiting LC–UV and LC–MS methodologies. The predominant coumarins in grapefruit were epoxyaurapten (20,977.0±300.1mgL−1) and aurapten (3259.0±68.4mgL−1). A total amount of 9587.0±210.6mgL−1 was determined in lemon absolute, the highest content being that of bergamottin (2776.0±65.5mgL−1). The findings obtained in this study highlight the valuable properties of the absolutes of lemon and grapefruit flowers, as unique raw material for perfume and cosmetic industries.

Nucleoside Triphosphate Diphosphohydrolases Dephosphorylate the Pharmacologically Active Metabolite of Tenofovir

Tenofovir (TFV; prescribed as the tenofovir disoproxil fumarate prodrug), an acyclic nucleotide analog, inhibits human immunodeficiency virus (HIV) replication and is currently used for both HIV treatment and prevention (as pre‐exposure prophylaxis). The pharmacologically active form of TFV is TFV‐diphosphate (TFV‐DP). Previously, we reported the heterogeneous distribution of TFV and TFV‐DP in colorectal tissue (a putative site of HIV infection) sections collected from research participants receiving a TFV‐containing enema. Although the observed heterogeneity in TFV‐DP distribution in colorectal tissue is multifactorial, it could be envisioned that dephosphorylation plays a role in regulating TFV‐DP levels since this molecule is structurally similar to ATP, which is a substrate for nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases). Therefore, in order to gain increased understanding of TFV‐DP disposition, the ability of recombinant human NTPDase 1, 3, 4, 5, and 8 to dephosphorylate TFV‐DP was investigated by incubating these enzymes with TFV‐DP, individually. TFV‐DP levels were measured using an ultra‐high‐performance liquid chromatography tandem mass spectrometry approach. From these incubations, TFV‐DP substrate depletion, a measure of dephosphorylation, was observed in the presence of NTPDase 1 or 3 enzymes. The activities of NTPDase 1 and 3 towards TFV‐DP were further confirmed using a malachite green phosphate assay that measures the release of inorganic phosphate. Further, using immunoblotting, we tested whether NTPDase 1 and 3 are expressed in colorectal tissue. These studies revealed the presence of both proteins in colorectal tissue lysate. In order to gain an understanding of the spatial distribution patterns of NTPDase 1 and 3 in colorectal tissue, Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) was employed. As a first step, we developed a MALDI MSI method to directly detect and identify peptides of NTPDase 1 and 3 following trypsin digestion. Trypsin digestions on NTPDases were carried out using a concentration of 0.05 μg/μl, and α‐cyano‐4‐hydroxycinnamic acid was used as the MALDI matrix. Following trypsin digestions and MALDI MS analysis, peptides corresponding to NTPDase 1 and 3 were detected and identified. From these, NTPDase 1 exhibited peptides, SLSNYPFDFQGAR, SQHQETPVYLGATAGMR, and IITGQEEGAYGWITINYLLGK whereas NTPDase 3 yielded following peptides: GSGISSYGNNPQDVPR, YGIVLDAGSSR, FTEETWPQIHFEK, and MFTVLTR. Thus, in this study, we established a MALDI MS methodology for detection and identification of peptides corresponding to NTPDase 1 and 3. Furthermore, the above developed method can be used to generate spatial distribution profiles (including cell‐type and region‐specific localization patterns) of NTPDase 1 and 3 in colorectal and other tissues of interest that are susceptible to HIV infection. Data from in vitro incubations indicate that NTPDase 1 and 3 may contribute to the regulation of TFV‐DP levels.Support or Funding InformationThis work was funded by NIH grants U19AI113127, UM1 AI068613 and R01AI128781.

Optimisation of extraction procedure and development of LC–DAD–MS methodology for anthocyanin analysis in anthocyanin-pigmented corn kernels

An ultra-high-performance liquid chromatography–diode array detector–mass spectrometry method was developed for characterisation and quantification of anthocyanin components in complex corn-kernel matrices. The anthocyanin profiles and total anthocyanin content (TAC) of mature seeds of five types of anthocyanin-pigmented corn were reported. Internal standard was used to validate the efficiency of extraction and optimise the liquid extraction procedure for anthocyanins. A total of eighteen anthocyanins were identified and quantified. Cyanidin-based glucosides were the major pigments of purple-pericarp sweetcorn (75.5% of TAC) and blue-aleurone maize (91.6%), while pelargonidin-based glucosides composed the main anthocyanins of reddish-purple-pericarp sweetcorn (61.1%) and cherry-aleurone maize (74.6%). Importantly, previous studies reported the presence of acetylated and succinylated anthocyanins in corn kernels; these compounds were found to be artefact pigments, generated during the extraction process. These crucial findings provide the correct anthocyanin profiles of pigmented corns, and emphasise the importance of using acidified solutions for the extraction of corn-based anthocyanins.

Rhodnius spp. are differentiated based on the peptide/protein profile by matrix-assisted laser desorption/ionization mass spectrometry and chemometric tools.

Triatominae are hematophagous insects involved in the transmission of Chagas disease. Among the 19 genera of the subfamily, those with the highest epidemiological importance regarding the dissemination of Trypanosoma cruzi are Panstrongylus, Rhodnius, and Triatoma. Of these three genera, Rhodnius presents the greatest difficulties for specific identification. Thus, there is a need to overcome the difficulties in identifying phenotypes of similar species of this genus. In the present study, the MALDI-TOF MS methodology was used to identify 12 Rhodnius species, among the 21 admitted. The MALDI-TOF MS methodology allowed specific characterization through the identification of peptides and proteins, starting from four different methods of extraction: (A) acetonitrile/formic acid (ACN/AF), (B) acetonitrile/trifluoroacetic acid (ACN/TFA), (C) isopropyl/formic acid (IPA/AF), and (D) methanol/formic acid (MeOH/AF), and four types of MALDI-TOF matrices: α-cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (SA), 6-aza-2-thiothymine (ATT), and 2,6-dihydroxyacetophenone (DHAP). The experiments were performed by combining the four solvents and four matrices to select the best MALDI extraction/matrix. The application of the MALDI-TOF MS technique, through the digital mass spectrometry approach combined with chemometric tools, such as partial least squares-discriminant analysis (PLS-DA), was able to discriminate 12 species of Rhodnius genus, which are difficult to identify using morphological characteristics. Thus, in view of the results obtained, the methodology described in the present article can be applied with speed and efficiency for the discrimination of Triatominae species. Graphical Abstract.

Exploitation of artichoke byproducts to obtain bioactive extracts enriched in inositols and caffeoylquinic acids by Microwave Assisted Extraction

Byproducts from artichoke represent the majority of the mass collected from the plant and constitute an interesting source of bioactive compounds such as inositols and caffeoylquinic acids. In this work, a microwave assisted extraction (MAE) methodology was developed for the simultaneous extraction of these compounds from artichoke stalks, leaves, receptacles and external bracts. Optimal MAE conditions to maximize the extraction of these bioactives and the antioxidant activity were 97 °C, 3 min, ethanol:water (50:50, v/v). Moreover, a GC–MS methodology was also developed for the simultaneous determination of these compounds in a single run; optimal derivatization conditions were achieved using hexamethyldisilazane and N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. Artichoke receptacle extracts were the richest in caffeoylquinic acids (28–35 mg g−1 dry sample), followed by the bracts (9–18 mg g−1 dry sample), while those from leaves showed the highest concentrations of inositols (up to 15 mg g−1 dry sample). Receptacle extracts also had the highest antioxidant activity (123 mg TE g−1 dry sample) and the greatest concentration of total phenolic compounds (47 mg GAE g−1 dry sample). Therefore, the developed methodology could be considered as a valuable procedure to obtain and characterize bioactive ingredients with industrial interest from artichoke byproducts, opening new routes of revalorization of artichoke agro-industrial residues.

Biodistribution and metabolic profile of 3,4-dimethylmethcathinone (3,4-DMMC) in Wistar rats through gas chromatography–mass spectrometry (GC–MS) analysis

3,4-Dimethylmethcathinone (3,4-DMMC) is a new psychoactive substance whose recreational use and trade have recently increased. Given the absence of information on the toxicokinetics of 3,4-DMMC, the present work aimed at validating a GC–MS methodology for the drug quantification in biological matrices, and further characterizing its biodistribution in Wistar rats.The method was validated based on the evaluation of the drug stability, limit of detection and quantification, linearity, selectivity, precision, accuracy and recovery. To characterize biodistribution, Wistar rats were administered with 20 or 40 mg/Kg of 3,4-DMMC i.p.. After 1 h or 24 h, rats were anaesthetized, euthanized and blood, brain, liver, heart, kidneys, lungs, spleen, urine (only at 24 h), and a portion of gut, muscle and adipose tissue were collected for analysis.After 1 h, 3,4-DMMC was present in all analysed matrices, and the presence of two metabolites was further detected in all of them. The drug accumulation was higher in kidneys, lungs, spleen and brain. After 24 h, 3,4-DMMC was only present in urine, along with five metabolites. All metabolites were tentatively identified.Through elucidation of the most appropriate analytical matrices and the metabolites that may have the largest detection windows, these data are expected to assist in future clinical and forensic investigations.

Advances in LC–MS/MS Methods for Allergen Testing, Meat Speciation, and Gelatin Speciation

Abstract Background: Food authenticity is demanded by the consumer at all times. The consumer places trust in the manufacturer that the food product is genuine in terms of what is recorded on the packaging label. Objective: Recent advancements in LC–tandem MS methodology in the detection of allergens, meat, and gelatin speciation in raw food products and processed foods are detailed in this paper. Method: For each of the three methods, initial proteome analysis and the screening leading to the determination of unique tryptic peptides were conducted using a high-resolution, accurate tandem mass spectrometer. Having identified the unique markers, the method was transferred to a tandem quadrupole mass spectrometer for a higher-sensitivity quantitative study, multiple reaction monitoring transition analysis. Results: For the allergens method a detection limit of at least 10 ppm was attained across the 12 allergen peptides in this workflow. In the gluten workflow the resulting chromatograms show good detection down to 5 ppm, with no interference from the food matrices. The meat speciation method details that signature peptides could be readily identified at 1% w/w with no matrix interference. Conclusions: These single-injection workflows with cycle-time optimization enable wide coverage of analytes to identify multiple species within challenging matrix samples.

Identification and quantification of the native carotenoid composition in fruits from the Brazilian Amazon by HPLC–DAD–APCI/MS

In this study the native carotenoid composition of previously uninvestigated fruits from the Brazilian Amazon was determined; marirana (Couepia subcordata Benth.), inajá (Maximiliana maripa) and caranã (Mauritiella armata) were analyzed by an HPLC–DAD–APCI–MS methodology. Three carotenoids were identified in marirana pulp, all-trans-α-carotene (6407 ± 380 μg 100 g−1, fresh weight) and all-trans-β-carotene (6331 ± 410 μg 100 g−1, fresh weight) being the two major carotenoids, followed by zeaxanthin dilaurate (1061 ± 80 μg 100 g−1, fresh weight). Only β-carotene was found in inajá pulp (1371 ± 370 μg 100 g−1, fresh weight). In caranã pulp, four carotenoids were identified, all-trans-β-carotene being the most predominant (373 ± 80 μg 100 g−1, fresh matter), followed by all-trans-α-carotene (230 ± 60 μg 100 g−1, fresh weight), lutein (198 ± 40 μg 100 g−1, fresh weight) and 9-cis-β-carotene (111 ± 30 μg 100 g−1, fresh weight). The cultivation and consumption of these fruits should be encouraged, since they could contribute to the intake of important carotenoids that could have beneficial effects on human health.

Advances in LC-MS/MS Methods for Allergen Testing, Meat Speciation, and Gelatin Speciation.

Background: Food authenticity is demanded by the consumer at all times. The consumer places trust in the manufacturer that the food product is genuine in terms of what is recorded on the packaging label. Objective: Recent advancements in LC-tandem MS methodology in the detection of allergens, meat, and gelatin speciation in raw food products and processed foods are detailed in this paper. Method: For each of the three methods, initial proteome analysis and the screening leading to the determination of unique tryptic peptides were conducted using a high-resolution, accurate tandem mass spectrometer. Having identified the unique markers, the method was transferred to a tandem quadrupole mass spectrometer for a higher-sensitivity quantitative study, multiple reaction monitoring transition analysis. Results: For the allergens method a detection limit of at least 10 ppm was attained across the 12 allergen peptides in this workflow. In the gluten workflow the resulting chromatograms show good detection down to 5 ppm, with no interference from the food matrices. The meat speciation method details that signature peptides could be readily identified at 1% w/w with no matrix interference. Conclusions: These single-injection workflows with cycle-time optimization enable wide coverage of analytes to identify multiple species within challenging matrix samples.

A MALDI-TOF-based Method for Studying the Transport of BBB Shuttles\u2014Enhancing Sensitivity and Versatility of Cell-Based In Vitro Transport Models

In recent decades, peptide blood-brain barrier shuttles have emerged as a promising solution for brain drugs that are not able to enter this organ. The research and development of these compounds involve the use of in vitro cell-based models of the BBB. Nevertheless, peptide transport quantification implies the use of large amounts of peptide (upper micromolar range for RP-HPLC-PDA) or of derivatives (e.g. fluorophore or quantum-dot attachment, radiolabeling) in the donor compartment in order to enhance the detection of these molecules in the acceptor well, although their structure is highly modified. Therefore, these methodologies either hamper the use of low peptide concentrations, thus hindering mechanistic studies, or do not allow the use of the unmodified peptide. Here we successfully applied a MALDI-TOF MS methodology for transport quantification in an in vitro BBB cell-based model. A light version of the acetylated peptide was evaluated, and the transport was subsequently quantified using a heavy internal standard (isotopically acetylated). We propose that this MALDI-TOF MS approach could also be applied to study the transport across other biological barriers using the appropriate in vitro transport models (e.g. Caco-2, PAMPA).

MALDI-TOF MS based procedure to detect KPC-2 directly from positive blood culture bottles and colonies

In this study, we identified specific carbapenemase-producing isolates applying an easy and rapid protocol for the detection of mature KPC-2 β-lactamase by MALDI-TOF MS from colony and positive blood culture bottles. In addition, we evaluated the correlation of the ~11,109 Da signal as a biomarker associated with KPC-2 production. A collection of 126 well-characterized clinical isolates were evaluated (including 60 KPC-2-producing strains). Presence of KPC-2 was assessed by MALDI-TOF MS on protein extracts. Samples were prepared using the double layer sinapinic acid technique. In order to identify mature KPC-2, raw spectra were analyzed focusing on the range between m/z 25,000–30,000 Da. A single distinctive peak, at approximately m/z 28,544 Da was found in all clinical and control KPC-2-producing strains, and consistently absent in the control groups (ESBL producers and susceptible strains). This peak was detected in all species independently of where the gene blaKPC-2 was embedded. Statistical results showed 100% sensitivity, CI95%: [94.0%; 100%] and 100% specificity, CI95%: [94.6%; 100%], indicating a promising test with a high discriminative power. KPC-2 β-lactamase could be directly detected from both colonies and blood culture bottles. On the other hand, the m/z 11,109 Da signal determinant was only associated with 32% of Klebsiella pneumoniae and Escherichia coli KPC positive isolates. This MALDI-TOF MS methodology has the potential to detect directly the widespread and clinically relevant carbapenemase, KPC-2, in Enterobacterales with a straightforward, low cost process, assuming MALDI-TOF MS is already adopted as the main identification tool, with clear clinical implications on antibiotic stewardship for early infection treatment.

Comparative evaluation of the identification of rapidly growing non-tuberculous mycobacteria by mass spectrometry (MALDI-TOF MS), GenoType Mycobacterium CM/AS assay and partial sequencing of the rpoβ gene with phylogenetic analysis as a reference method

IntroductionThe American Thoracic Society and the Infectious Diseases Society of America recommend that clinically significant non-tuberculous mycobacteria (NTM) should be identified to the species level in order to determine their clinical significance. The aim of this study was to evaluate identification of rapidly growing NTM (RGM) isolated from clinical samples by using MALDI-TOF MS and a commercial molecular system. The results were compared with identification using a reference method. MethodsWe included 46 clinical isolates of RGM and identified them using the commercial molecular system GenoType® CM/AS (Hain, Lifescience, Germany), MALDI-TOF MS (Bruker) and, as reference method, partial rpoβ gene sequencing followed by BLAST and phylogenetic analysis with the 1093 sequences available in the GeneBank. ResultsThe degree of agreement between GenoType® and MALDI-TOF MS and the reference method, partial rpoβ sequencing, was 27/43 (62.8%) and 38/43 cases (88.3%) respectively. For all the samples correctly classified by GenoType®, we obtained the same result with MALDI-TOF MS (27/27). However, MALDI-TOF MS also correctly identified 68.75% (11/16) of the samples that GenoType® had misclassified (p=0.005). ConclusionsMALDI-TOF MS classified significantly better than GenoType®. When a MALDI-TOF MS score >1.85 was achieved, MALDI-TOF MS and partial rpoβ gene sequencing were equivalent. GenoType® was not able to distinguish between species belonging to the M. fortuitum complex. MALDI-TOF MS methodology is simple, rapid and associated with lower consumable costs than GenoType®. The partial rpoβ sequencing methods with BLAST and phylogenetic analysis were not able to identify some RGM unequivocally. Therefore, sequencing of additional regions would be indicated in these cases.