UHPLC-tandem Mass Spectrometry Research Articles

Quantification and enantiomeric distribution of clenbuterol in several bovine tissues using UHPLC-tandem mass spectrometry: Evaluation of a risk factor associated with meat contamination.

Clenbuterol (Clb) (4-amino-α-[(tert-butylamine) methyl]-3,5-dichlorobenzyl alcohol) is a sympathomimetic agent that exhibits β2-agonist activity. It is applied as a bronchodilatory, tocolytic, and mucolytic agent and is authorized for clinical management in both human and veterinary therapeutics as a racemic mixture. However, its use is strictly prohibited in animals destined for food production in countries in the European Union and in the United States and Mexico, among many others. The R-(-) enantiomer in clenbuterol stimulates β2-receptors, whereas the S-(+) enantiomer blocks the effect of β1-receptors. The aims of this study were to develop a method for detecting and quantifying Clb and its enantiomeric distribution in several bovine tissues. The UHPLC-MS/MS method developed to quantify the target compound at trace levels in these tissues combines high sensitivity with good selectivity and short chromatographic run time. The tissue samples tested were found to contain racemic Clb in concentrations of 5-447 pg g-1 . The enantiomeric analysis of Clb showed that R-(-)-Clb is present at higher concentrations in some tissues, whereas S-(+)-Clb was detected in a ratio of 55/45 in the liver and heart tissues.

Branched medium-chain fatty acid profiling and enantiomer separation of anteiso-forms of teicoplanin fatty acyl side chain RS3 using UHPLC-MS/MS with polysaccharide columns

This work reports on targeted UHPLC-tandem mass spectrometry methods for the chiral separation of anteiso-methyl branched fatty acids (aiFAs). The methods involve precolumn derivatization with 1-naphthylamine and chiral separation on Chiralpak IG-U. anteiso-Methyl branched fatty acids with up to eight carbons can be separated. A method was used for the assignment of the absolute configuration of an aiFA present as fatty acyl residue of the teicoplanin mixture, namely teicoplanin RS3. Furthermore, the excellent methylene selectivity and improved selectivity for constitutional isomers of the polysaccharide columns was exploited for the elucidation and structural confirmation of previously unknown fatty acyl residues in teicoplanin. This shows the versatility and practical applicability of polysaccharide columns as orthogonal stationary phases to reversed-phase for structural elucidation of natural compounds. The developed methods are useful tools for related subdisciplines such as targeted metabolomics and lipidomics.

Characterization of the Metabolome of Breast Tissues from Non-Hispanic Black and Non-Hispanic White Women Reveals Correlations between Microbial Dysbiosis and Enhanced Lipid Metabolism Pathways in Triple-Negative Breast Tumors.

Simple SummaryWe previously showed that breast tumor tissues from women display an imbalance in abundance and composition of microbiota compared to normal healthy breast tissues. It is unknown whether these differences in breast tumor microbiota may be driven by alterations in microbial metabolites, leading to potentially protective or pathogenic consequences. The aim of our study was to conduct global metabolic profiling on normal and breast tumor tissues to identify differences in metabolite profiles and to determine whether breast microbial dysbiosis may be associated with enrichment of microbial metabolites in triple-negative breast cancer (TNBC) which disproportionately affects women of African ancestry. We observed significant correlations between elevated lipid metabolism pathways and microbial dysbiosis in TNBC tissues from both non-Hispanic black and white women. This is the first study to report an association between breast microbial dysbiosis and alterations in host metabolic pathways in breast tumors, including TNBC, of non-Hispanic black and non-Hispanic white women.Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that is non-responsive to hormonal therapies and disproportionately impact women of African ancestry. We previously showed that TN breast tumors have a distinct microbial signature that differs from less aggressive breast tumor subtypes and normal breast tissues. However, it is unknown whether these differences in breast tumor microbiota may be driven by alterations in microbial metabolites, leading to potentially protective or pathogenic consequences. The goal of this global metabolomic profiling study was to investigate alterations in microbial metabolism pathways in normal and breast tumor tissues, including TNBC, of non-Hispanic black (NHB) and non-Hispanic white (NHW) women. In this study, we profiled the microbiome (16S rRNA) from breast tumor tissues and analyzed 984 metabolites from a total of 51 NHB and NHW women. Breast tumor tissues were collected from 15 patients with TNBC, 12 patients with less aggressive luminal A-type (Luminal) breast cancer, and 24 healthy controls for comparison using UHPLC-tandem mass spectrometry. Principal component analysis and hierarchical clustering of the global metabolomic profiling data revealed separation between metabolic signatures of normal and breast tumor tissues. Random forest analysis revealed a unique biochemical signature associated with elevated lipid metabolites and lower levels of microbial-derived metabolites important in controlling inflammation and immune responses in breast tumor tissues. Significant relationships between the breast microbiome and the metabolome, particularly lipid metabolism, were observed in TNBC tissues. Further investigations to determine whether alterations in sphingolipid, phospholipid, ceramide, amino acid, and energy metabolism pathways modulate Fusobacterium and Tenericutes abundance and composition to alter host metabolism in TNBC are necessary to help us understand the risk and underlying mechanisms and to identify potential microbial-based targets.

Spatial quantitation of antibiotics in bone tissue compartments by laser-capture microdissection coupled with UHPLC-tandem mass spectrometry

Bones are the site of multiple diseases requiring chemotherapy, including cancer, arthritis, osteoporosis and infections. Yet limited methodologies are available to investigate the spatial distribution and quantitation of small molecule drugs in bone compartments, due to the difficulty of sectioning undecalcified bones and the interference of decalcification methods with spatially resolved drug quantitation. To measure drug concentrations in distinct anatomical bone regions, we have developed a workflow that enables spatial quantitation of thin undecalcified bone sections by laser-capture microdissection coupled to HPLC/tandem mass spectrometry, and spatial mapping on adjacent sections by mass spectrometry imaging. The adhesive film and staining methods were optimized to facilitate histology staining on the same sections used for mass spectrometry image acquisition, revealing drug accumulation in the underlying bone tissue architecture, for the first time. Absolute spatial concentrations of rifampicin, bedaquiline, doxycycline, vancomycin and several of their active metabolites are shown for both small rodent bones and larger rabbit bones that more closely resemble human bone density. Overlaid MALDI mass spectrometry images of drugs and histology staining enabled the generation of semi-quantitative data from regions of interest within anatomical bone compartments. These data correlated with absolute drug concentrations determined by HPLC–MS/MS in laser-capture microdissection samples. Collectively, these techniques enable semi- and fully quantitative drug distribution investigations within bone tissue compartments for the first time. Our workflow can be translated to image and quantify not only drugs but also biomarkers of disease to investigate drug penetration as well as mechanisms underlying bone disorders.Graphical abstract

The Influence of Increasing Lecithin Concentrations on Carotenoid Bioaccessibility and Cell Uptake

ObjectivesWe sought to determine if lecithin would produce a dose-response effect, increasing carotenoid bioaccessibility and Caco-2 cell uptake. MethodsA mixture of β-carotene, lycopene, lutein and zeaxanthin was digested with increasing concentrations of lecithin (0–10 mg lecithin/mL test meal) and ∼4% vegetable oil (n = 6 per condition). In vitro gastric, duodenal, and jejunal digestion was mimicked for 1 h each, and the micellar fraction isolated for analysis. Additionally, Caco-2 cells producing a monolayer reflecting a small intestinal phenotype were incubated with of the micellular fraction for 4 h, and cells harvested for extraction. The micellular fraction and cell were extracted with hexane and dichloromethane, and the organic phase dried under argon. Micellular carotenoids were quantitated using high performance liquid chromatography-diode-array detection (HPLC-DAD), while cellular carotenoids were quantitated using UHPLC-tandem mass spectrometry (UHPLC-MS/MS). One-way ANOVA followed by Tukey’s post-hoc test was used to assess statistically significant differences between lecithin doses on carotenoid bioaccessibility and cellular uptake. Fractions of digesta were also visualized using confocal microscopy, to better elucidate the lecithin-micelle interaction. ResultsLecithin significantly increased β-carotene and zeaxanthin bioaccessibility only when comparing the most extreme doses tested. No significant differences were observed for lycopene or lutein bioaccessibility. Likewise, lecithin did not produce a significant increase in the cell uptake in the range tested. ConclusionsOverall, lecithin had minimal impact on carotenoid bioaccessibility, and Caco-2 cell uptake of carotenoids. Funding SourcesThis work was supported by a research grant from Nu Skin Enterprises, Inc.

A New Strategy for Efficient Retrospective Data Analyses for Designer Benzodiazepines in Large LC-HRMS Datasets.

The expanding and dynamic market of new psychoactive substances (NPSs) poses challenges for laboratories worldwide. The retrospective data analysis (RDA) of previously analyzed samples for new targets can be used to investigate analytes missed in the first data analysis. However, RDA has historically been unsuitable for routine evaluation because reprocessing and reevaluating large numbers of forensic samples are highly work- and time-consuming. In this project, we developed an efficient and scalable retrospective data analysis workflow that can easily be tailored and optimized for groups of NPSs. The objectives of the study were to establish a retrospective data analysis workflow for benzodiazepines in whole blood samples and apply it on previously analyzed driving-under-the-influence-of-drugs (DUID) cases. The RDA workflow was based on a training set of hits in ultrahigh-performance liquid chromatography–quadrupole time-of-flight–mass spectrometry (UHPLC-QTOF-MS) data files, corresponding to common benzodiazepines that also had been analyzed with a complementary UHPLC–tandem mass spectrometry (MS/MS) method. Quantitative results in the training set were used as the true condition to evaluate whether a hit in the UHPLC-QTOF-MS data file was true or false positive. The training set was used to evaluate and set filters. The RDA was used to extract information from 47 DBZDs in 13,514 UHPLC-QTOF-MS data files from DUID cases analyzed from 2014 to 2020, with filters on the retention time window, count level, and mass error. Sixteen designer and uncommon benzodiazepines (DBZDs) were detected, where 47 identifications had been confirmed by using complementary methods when the case was open (confirmed positive finding), and 43 targets were not reported when the case was open (tentative positive finding). The most common tentative and confirmed findings were etizolam (n = 26), phenazepam (n = 13), lorazepam (n = 9), and flualprazolam (n = 8). This method efficiently found DBZDs in previously acquired UHPLC-QTOF-MS data files, with only nine false-positive hits. When the standard of an emerging DBZD becomes available, all previously acquired DUID data files can be screened in less than 1 min. Being able to perform a fast and accurate retrospective data analysis across previously acquired data files is a major technological advancement in monitoring NPS abuse.

A newborn screening approach to diagnose 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

3‐Hydroxy‐3‐methylglutaryl‐coenzyme A lyase deficiency (HMGCLD) is a rare autosomal recessively inherited metabolic disorder. Patients suffer from avoidable neurologically devastating metabolic decompensations and thus would benefit from newborn screening (NBS). The diagnosis is currently made by measuring dry blood spot acylcarnitines (C5OH and C6DC) followed by urinary organic acid profiling for the differential diagnosis from several other disorders. Using untargeted metabolomics (reversed‐phase UHPLC coupled to an Orbitrap Elite hybrid mass spectrometer) of plasma samples from 5 HMGCLD patients and 19 age‐matched controls, we found 3‐methylglutaconic acid and 3‐hydroxy‐3‐methylglutaric acid, together with 3‐hydroxyisovalerylcarnitine as the most discriminating metabolites between the groups. In order to evaluate the NBS potential of these metabolites we quantified the most discriminating metabolites from untargeted metabolomics in 23 blood spots from 4 HMGCLD patients and 55 controls by UHPLC tandem mass spectrometry. The results provide a tool for expanded NBS of HMGCLD using tandem mass spectrometry. Selected reaction monitoring transition 262/85 could be used in a first‐tier NBS analysis to screen for elevated 3‐hydroxyisovalerylcarnitine. In a positive case, a second‐tier analysis of 3‐hydroxy‐3‐methylglutaric acid and 3‐methylglutaconic acid in a dry blood spot using UHPLC tandem mass spectrometry instruments confirms the diagnosis. In conclusion, we describe the identification of new diagnostic biomarkers for HMGCLD and their application in NBS in dry blood spots. By using second‐tier testing, all patients with HMGCLD were unequivocally and correctly diagnosed.

Development of a High Coverage Pseudotargeted Lipidomics Method Based on Ultra-High Performance Liquid Chromatography-Mass Spectrometry.

Lipid coverage is crucial in comprehensive lipidomics studies challenged by high diversity in lipid structures and wide dynamic range in lipid levels. Current state-of-the-art lipidomics technologies are mostly based on mass spectrometry (MS), including direct-infusion MS, chromatography-MS, and matrix-assisted laser desorption ionization (MALDI) imaging MS, each with its pros and cons. Due to the need or favorability for measurement of isomers and isobars, chromatography-MS is preferable for lipid profiling. The ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based nontargeted lipidomics approach and UHPLC-tandem MS (UHPLC-MS/MS)-based targeted approach are two representative methodological platforms for chromatography-MS. In the present study, we developed a high coverage pseudotargeted lipidomics method combining the advantages of nontargeted and targeted lipidomics approaches. The high coverage of lipids was achieved by integration of the detected lipids derived from nontargeted UHPLC-HRMS lipidomics analysis of multiple matrices (e.g., plasma, cell, and tissue) and the predicted lipids speculated on the basis of the structure and chromatographic retention behavior of the known lipids. A total of 3377 targeted lipid ion pairs with over 7000 lipid molecular structures were defined. The pseudotargeted lipidomics method was well validated with satisfactory analytical characteristics in terms of linearity, precision, reproducibility, and recovery for lipidomics profiling. Importantly, it showed better repeatability and higher coverage of lipids than the nontargeted lipidomics method. The applicability of the developed pseudotargeted lipidomics method was testified in defining differential lipids related to diabetes. We believe that comprehensive lipidomics studies will benefit from the developed high coverage pseudotargeted lipidomics approach.

Yinchenhao Decoction Ameliorates Alpha-Naphthylisothiocyanate Induced Intrahepatic Cholestasis in Rats by Regulating Phase II Metabolic Enzymes and Transporters.

Yinchenhao Decoction (YCHD), a famous traditional Chinese formula, has been used for treating cholestasis for 1000s of years. The cholagogic effect of YCHD has been widely reported, but its pharmacodynamic material and underlying therapeutic mechanism remain unclear. By using ultra-high-performance liquid chromatography (UHPLC)-quadrupole time-of-flight mass spectrometry, 11 original active components and eight phase II metabolites were detected in rats after oral administration of YCHD, including three new phase II metabolites. And it indicated that phase II metabolism was one of the major metabolic pathway for most active components in YCHD, which was similar to the metabolism process of bilirubin. It arouses our curiosity that whether the metabolism process of YCHD has any relationship with its cholagogic effects. So, a new method for simultaneous quantitation of eight active components and four phase II metabolites of rhein, emodin, genipin, and capillarisin has been developed and applied for their pharmacokinetic study in both normal and alpha-naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis rats. The results indicated the pharmacokinetic behaviors of most components of YCHD were inhibited, which was hypothesized to be related to different levels of metabolic enzymes and transporters in rat liver. So dynamic changes of intrahepatic enzyme expression in cholestasis and YCHD treated rats have been monitored by an UHPLC-tandem mass spectrometry method. The results showed expression levels of UDP-glucuronosyltransferase 1-1 (UGT1A1), organic anion-transporting polypeptide 1A4 (OATP1A4), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1, sodium-dependent taurocholate cotransporter, and organic anion-transporting polypeptide 1A2 were significantly inhibited in cholestasis rats, which would account for reducing the drug absorption and the metabolic process of YCHD in cholestatic rats. A high dose (12 g/kg) of YCHD remarkably increased the expression of UGT1A1, bile salt export pump, MRP2, OATP1A4 in cholestasis rats presented it exhibited the greatest ameliorative effect on cholestasis, also particularly in histopathological examination and reducing levels of alanine transaminase, aspartate transaminase, total bilirubin, direct bilirubin, and total bile acid. Considering the metabolic process of bilirubin in vivo, the choleretic effect of YCHD is proven to be related to its regulatory action on expression of metabolic enzymes and transporters in cholestatic liver.

Occurrence of aflatoxin M1 in milk samples from Italy analysed by online-SPE UHPLC-MS/MS

The occurrence of aflatoxin M1 in 69 milk samples collected in a south region of Italy in 2016 was evaluated. The samples were analysed using an automated method based on online SPE coupled with UHPLC tandem mass spectrometry. After a salt induced liquid–liquid extraction with acetonitrile to remove protein from milk, the extract was diluted with water and analysed using an automated online SPE MS/MS method. Among the analysed samples no one had AFM1 higher than the legally allowable limits whereas 71.4% of the other analysed samples were above the LOD of the method. The highest contamination level of AFM1 was found in pasteurised milk (44.39 ng kg−1). The results show the worrying and widespread of AFM1 contamination, highlighting the necessity of monitoring studies in order to evaluate the reduction of the maximum legal limit.

Unique Alterations in the Immunopeptidome of Colorectal Cancer Reflect Specific Transformations in Cancer-Associated Signaling Pathways and Reveal Tumor-Specific HLA-Ligand Modulations

Background: Immunotherapy with checkpoint-inhibitors has shown spectacular results in the treatment of certain cancer types including microsatellite instable colorectal cancer (CRC). Applicability is believed to be dependent on the number of potential neo-epitopes derived from genetic mutations that are presented on cancer cells. In case of most microsatellite-stable CRC however, clinical responses to immune checkpoint blockade are so far disappointing. Therefore, we analyzed the non-mutant HLA immunopeptidome of CRC in order to provide an extensive dataset for the development of immunotherapeutic strategies in this very common malignancy.Methods: Tissue specimens from 35 primary CRC and corresponding non-malignant colon were analyzed after HLA immunoprecipitation by uHPLC tandem mass spectrometry. Maximally attainable quantities of source proteins (MAQS) expectable in HLA-ligandomes were estimated by regression analyses. Identified peptides and source proteins were annotated for their pathway association using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the protein analysis through evolutionary relationships (PANTHER). HLA-ligands and source proteins were further analyzed semi-quantitatively assessing significant modulations on CRC tissue compared to adjacent benign colon, with particular focus on specific alterations observed exclusively in CRC tissue. Immunogenicity of the identified HLA-ligands (prioritized ligands for the 8 most frequent HLA-alleles HLA A*01, A*02, A*03, A*24, B*07, B*08, B*44, C*07) was evaluated in peripheral blood mononuclear cells (PBMC) from 50 additional CRC patients and 120 healthy controls (15 each for the 8 most frequent HLA-types) using ELISpot and flow cytometry.Results: For MHC class I, peptides from 7684 source proteins (81% MAQS) were identified on CRC, as well as peptides from 6312 source proteins on (non-malignant) colon tissue (79% MAQS). For MHC class II, peptides from 1602 source proteins (63% MAQS) were identified, as well as peptides from 3835 source proteins on non-malignant colon tissue (75% MAQS). HLA-ligands and their respective source proteins were compared on tissue level (CRC vs. adjacent benign tissue), as well as against a database of 100 non-malignant human tissues of different origin in order to identify ligands and source proteins exclusively presented on CRC tissue. Implementing KEGG and PANTHER pathway analysis, overrepresented source proteins within MHC class I and II restricted ligands could be assigned to classical tumorigenesis pathways like WNT- and integrin signaling, as well as to the p53 signaling pathway. HLA ligands were further semi-quantitatively analyzed comparing tumor and autologous adjacent colon tissue leading to the exclusive identification of 1364 up-modulated and 1070 down-modulated source proteins in CRC tissue. Notably, 3 source proteins (represented by 10 HLA-ligands) showing significant up-modulation and frequent tumor-exclusive detection of derived HLA-ligands (in ≥3 CRC) were identified (LAMC-2, SLC52A2, SULF-1). For MHC class II, a single up-modulated source protein with exclusive detection in ≥2 CRC tissues (IL6R) was identified. Further analyses of HLA class I restricted peptides (n=359) derived from simultaneously up- and down-modulated source proteins revealed that the respective modulation was mainly a peptide sequence specific feature (31/359 (8.6%) peptides with up- and down-modulation). Preexisting T cell responses were observed against one tumor-exclusive up-modulated peptide (SULF-1) in CRC patients and 3 HLA restricted peptides established as immunogenic epitopes in CRC patients (TACC2, TNS4, IGHG2), as well as 2 additional HLA-restricted peptides confirmed as epitopes in healthy controls (GLA, ESRRA).Conclusions: We provide the first comprehensive analysis of the HLA immunopeptidome in a solid cancer (CRC). We observed that the presented ligandome can reflect tumor-specific alterations in protein metabolism. Moreover, tumor-exclusive up- and down-modulation of HLA-peptides was mainly sequence-specific, suggesting a differential posttranslational regulation of HLA-restricted peptides in CRC. The described approach for identification of relevant antigens might also enable patient-specific immunotherapeutic approaches in CRC patients. DisclosuresKowalewski:Immatics Biotechnologies GmbH: Employment. Bernhardt:DECODON: Employment.

UHPLC–MS/MS method with protein precipitation extraction for the simultaneous quantification of ten antihypertensive drugs in human plasma from resistant hypertensive patients

Today the management of resistant hypertension is a critical health problem: the main difficulty on this field is the discrimination of cases of poor therapeutic adherence from cases of real resistance. This gives rise to the need of high throughput and reliable quantification methods for the Therapeutic Drug Monitoring (TDM) of antihypertensive drugs. The aim of this work was the development and validation of a UHPLC-Tandem mass spectrometry assay for this application and its use in plasma from patients with resistant hypertension. The novelty of this method resides in the ability to simultaneously quantify a wide panel of antihypertensive drugs: amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan, ramipril and telmisartan. Moreover, this method stands out for its simplicity and cheapness, resulting feasible for clinical routine. Both standards and quality controls were prepared in human plasma. After the addition of internal standard, each sample underwent protein precipitation with acetonitrile and was then dried. Extracts were resuspended in water:acetonitrile 90:10 (0.05% formic acid) and then injected into the chromatographic system. Chromatographic separation was performed on an Acquity® UPLC HSS T3 1.8μm 2.1×150mm column, with a gradient of water and acetonitrile, both added with 0.05% formic acid.Accuracy, intra-day and inter-day precision fitted FDA guidelines for all analytes, while matrix effects and recoveries resulted stable between samples for each analyte. Finally, we tested this method by monitoring plasma concentrations in 22 hypertensive patients with good results. This simple analytical method could represent a useful tool for the management of antihypertensive therapy.

A validated UHPLC-tandem mass spectrometry method for quantitative analysis of flavonolignans in milk thistle (Silybum marianum) extracts

Validated methods are needed for the analysis of natural product secondary metabolites. These methods are particularly important to translate in vitro observations to in vivo studies. Herein, a method is reported for the analysis of the key secondary metabolites, a series of flavonolignans and a flavonoid, from an extract prepared from the seeds of milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)]. This report represents the first UHPLC MS-MS method validated for quantitative analysis of these compounds. The method takes advantage of the excellent resolution achievable with UHPLC to provide a complete analysis in less than 7min. The method is validated using both UV and MS detectors, making it applicable in laboratories with different types of analytical instrumentation available. Lower limits of quantitation achieved with this method range from 0.0400μM to 0.160μM with UV and from 0.0800μM to 0.160μM with MS. The new method is employed to evaluate variability in constituent composition in various commercial S. marianum extracts, and to show that storage of the milk thistle compounds in DMSO leads to degradation.

Interplay Between the Immune System and Colorectal Carcinoma - Towards Tumor-Specific Peptide-Based Vaccination for Any HLA-Type

Introduction: Peptide-basedcancer vaccines targeting tumor-associated antigens comprised of synthetic peptides have shown promising clinical results and have progressed to Phase III clinical testing in several tumor entities, including colorectal carcinoma (CRC). As of yet, these vaccines have typically been limited to HLA-A*02 positive patients, which means that more than half of the patient pool will not be eligible for those treatment strategies. The development of novel strategies for vaccine design, which are applicable to a larger fraction of potential patients, is therefore required. CRC seems to be particularly suitable for antigen-specific immunotherapy as it is among those cancers with highest rates of genetic mutations leading to alterations in protein metabolism. In this study, we aimed to identify HLA class I and II tumor-associated antigens from CRC covering the most frequent HLA-allotypes, in order to develop a peptide warehouse containing HLA-specific peptide panels that can be compiled in a patient-individualized manner, according to the respective patient's HLA-typing.Methods: The HLA presented immunopeptidome of 30 primary CRC samples and matched autologous non-malignant colon tissue was analyzed after HLA-immunoprecipitation by uHPLC tandem mass spectrometry. Identified source proteins and peptides were cross-evaluated with an in-house database of ligandome data derived from different benign tissues to preclude off-tumor presentation of HLA-ligands in order to to prevent any possible induction of autoimmunity by antigen-specific T cells against these ligands. Furthermore, we evaluated pre-existing antigen-specific T cell responses against these novel CRC-associated peptides in a cohort of 25 CRC patients.Results: About 17,000 MHC class I presented peptides could be identified on CRC and were cross-evaluated with our in-house database containing 39,000 MHC class I peptides presented on non-malignant tissue. The ligands identified were derived from 7,500 unique tumor-associated proteins and from 11,500 tumor-exclusive source proteins, respectively. Moreover, about 2,000 different MHC class II presented peptides from more than 1,600 source proteins were identified on CRC tissue. Interestingly, MHC class II peptides could contain shorter amino-acid sequences which - after further proteasomal degradation - might also bind to MHC class I molecules. For clinical applicability, we prioritized the identified ligands according to their frequencies of presentation and detectability on CRC. For representation in the HLA-specific peptide sets, a cutoff expression on at least 25% of allotype-matched tumors was required. This guided the assembly of a peptide vaccine warehouse consisting of 40 highly specific CRC-associated HLA ligands. Therefore, this selected set of HLA-specific peptides allows vaccination against CRC for about 95% of Caucasian patients. So far, we detected specific T cell reactivity against two peptides (detection of 10-fold and 17-fold increase of IFNγ-producing T cells representing spots as compared to background in enzyme linked immunospot assays - ELISPOT) in CRC patients.Conclusions: We here provide for the first time a comprehensive evaluation of HLA-ligandomes in CRC across the most frequent Caucasian HLA-alleles. We developed a HLA-allotype specific warehouse of newly identified CRC associated HLA-ligands, which can be individually assembled according to patient specific HLA-alleles. This approach expands the applicability of peptide-based cancer vaccines to literally every CRC patient. DisclosuresNo relevant conflicts of interest to declare.

Simultaneous quantitative analysis of tamsulosin and finasteride in pharmaceutical dosage form by U-HPLC Tandem mass spectrometry

A sensitive, rapid, selective and accurate liquid chromatography coupled to quadrupole tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous identification and quantification of tamsulosin and finasteride in bulk and in their combined dosage form. Chromatography was performed on a Hypersil gold 50 mm × 2 mm (1.9 μm) column, using acetonitrile:ammonium acetate (90:10, v : v ) pH = 3.5 as the mobile phase. Protonated ions formed by a turbo ion-spray in positive mode were used to detect the analytes as well as the internal standard (IS). MS/MS detection was carried out by monitoring the fragmentation of 408.74 → 227.29 ( m/z ), 373.11 → 304.96 ( m/z ) and 255.75 → 166.15 ( m/z ) for tamsulosin, finasteride and diphenhydramine (IS), respectively, on a triple quadrupole mass spectrometer. The linearity was obtained over the concentration range of 1.6-40.0 ng/mL for tamsulosin and 20.0-500.0 ng/mL for finasteride with a lower limit of detection of 0.5 ng/mL and 5.0 ng/mL for the two drugs, respectively. The proposed method was successfully applied to tamsulosin and finasteride determination in pharmaceutical dosage form. The results obtained were statistically analyzed and compared with those of reference ones; in addition, the method was validated according to USP 34 recommendations. The simplicity and sensitivity of this method allows its use in the quality control of the cited drugs and can be extended for routine analysis of the drugs in their pharmaceutical preparations.

Quantitative determination of β-hydroxymethylbutyrate and leucine in culture media and microdialysates from rat brain by UHPLC-tandem mass spectrometry

The main objective of the present work was to develop a method to determine β-hydroxymethylbutyrate (HMB) and leucine (Leu) in culture media and brain microdialysates. An accurate, selective, and cost-effective method, based on the use of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), was developed for the identification and quantification of both compounds. The method consisted of sample dilution, direct injection onto the chromatographic equipment, and quantification with a triple quadrupole mass spectrometer using an electrospray ionization interface in positive mode. The procedure and the UHPLC-MS/MS parameters were accurately optimized to achieve the highest recoveries and to enhance the analytical characteristics of the method. For chromatographic separation, an Acquity UPLC BEH Hilic column using acetonitrile-water gradient with formic acid as additive was employed. The total run time was 4 min. The limits of detection (LODs) obtained ranged from 0.01 to 0.04 μg mL(-1), and the limits of quantification (LOQs) ranged from 0.04 to 0.12 μg mL(-1). Precision (expressed as relative standard deviation) was lower than 15 %, and the determination coefficient (R (2)) was higher than 99.0 % with a residual deviation for each calibration point lower than ±25 %. Mean recoveries were between 85 and 115 %. The method was successfully applied to the analysis of both compounds, HMB and Leu, in samples obtained from an experiment of blood-brain barrier (BBB) passage in vitro and to an experiment of brain microdialysis in rats in vivo after an oral challenge with HMB to detect its appearance in the brain.

Association of cannabinoid receptor expression with anandamide concentrations in endometrial cancer

BackgroundThe incidence of endometrial cancer is increasing worldwide. Despite improved overall survival rates, its aetiopathogenesis remains complicated, although oestrogens are known causal factors. Among other factors thought to be implicated are the endocannabinoids. Anandamide (AEA), the prototypical endocannabinoid, has emerged as a possible factor in the pathogenesis of endometrial cancer because oestrogen regulates AEA concentrations and the expression of the cannabinoid receptors in other tissues. Furthermore, the endocannabinoid system is protective against the growth and spread of different malignancies. We aimed to investigate and quantify anandamide and its receptors in endometrial cancer. MethodsIn this single-centre study conducted at a UK teaching hospital, 31 patients with endometrial carcinomas (27 type 1, four type 2) and seven healthy age-matched controls with atrophic endometria were recruited. Biopsy samples were taken and divided into two—one for this study and the other for histological confirmation of diagnosis (graded according to the FIGO system). The biopsy sample for this study was further divided into two—one for AEA measurement and the other for quantitative RT-PCR. CB1 and CB2 transcript levels were measured in triplicate and normalised against the housekeeping genes PPIA, MRPL19, and IPO8. 15 of 31 cancer patients (12 type 1, three type 2) and all seven of the control group, volunteered blood for plasma collection. At hysterectomy, 31 endometrial cancer samples (27 type 1, four type 2) and seven age-matched atrophic control endometrial biopsy samples were obtained for tissue AEA measurement. Extracts from the plasma and endometrial homogenates were assessed for the presence of AEA with UHPLC-tandem mass spectrometry. FindingsIn the endometrial cancer tissues, both CB1 and CB2 transcript levels were significantly decreased by 90% (p<0·0004) and 80% (p< 0·0001), respectively, compared with atrophic tissues. Plasma AEA concentrations were significantly higher in patients with endometrial cancer than in the control group (mean 1·95 nM [SE 0·59] vs 1·06 [0·08], p=0·037). Plasma AEA concentrations in patients with type 1 disease were significantly higher than in control patients (2·17 [0·72] vs 1·06 [0·08], p<0·05), but in patients with type 2 disease there was no difference (1·08 [0·05]). Similarly, tissue AEA in patients with endometrial cancer was significantly higher than in the control group (5·45 nmol/g [SE 0·91] vs 1·77 [0·66], p=0·046). Tissue AEA was significantly higher in type 1 endometrial cancer than in atrophic samples (6·12 [0·98] vs 1·77 [0·66], p<0·05), but type 2 samples did not differ (0·90 [0·16]). Furthermore, in samples available for comparison (15 endometrial cancer, seven atrophic) there was a statistically significant correlation between plasma and tissue AEA concentrations (r=0·55, p=0·008). InterpretationThe results from this study suggest that downregulation of CB1 and CB2 receptor transcript levels and raised tissue and plasma AEA concentrations are linked in some way. We speculate that the endocannabinoid system is implicated in the aetiopathogenesis of endometrial cancer, but further work is warranted. The correlation between plasma and tissue AEA concentrations suggests that plasma AEA could be used as a biomarker for some forms of endometrial malignant disease, especially type 1 endometrioid adenocarcinoma. The endocannabinoid system thus presents an attractive novel target for pharmacological intervention in some endometrial malignancies. FundingUniversity Hospitals of Leicester NHS Trust.

Quantitative determination of neurotransmitters, metabolites and derivates in microdialysates by UHPLC–tandem mass spectrometry

The main objective of the present work is to study the time-course of rat brain neurotransmitters in vivo after an oral challenge with a nutritional ingredient or an external stimulus, such as a chemical agent. An ultrahigh performance liquid chromatography–tandem mass spectrometry method for the identification and quantification of neurotransmitters, metabolites and derivates in microdialysates from rat brain was previously developed. Betaine, glutamine, glutamic acid, gamma-aminobutyric acid, phosphocholine, glycerophosphocholine, cytidine 5'-diphosphocholine, choline, acetylcholine, dopamine, norepinephrine, serotonin, tyrosine, epinephrine, tryptophan and 5-hydroxyindoleacetic acid were selected as analytes. The method involves direct injection of samples of microdialysates from rat brain onto the chromatographic equipment and quantification with a triple quadrupole mass spectrometer detector using an electrospray ionization interface in positive mode. The limits of detection ranged from 0.1 to 50ngmL−1 and the limits of quantification from 0.3 to 200ngmL−1. The inter- and intra-day variability were lower than 15%. Recovery rates ranged from 85% to 115%.

Analysis of Agricultural Residues on Tea Using d-SPE Sample Preparation with GC-NCI-MS and UHPLC-MS/MS

This study presents new sample preparation and analytical procedures for the quantification of pesticides on processed tea leaves. The new method includes tea extraction and dispersive solid phase extraction (d-SPE) to prepare gas chromatography (GC) and ultrahigh-performance liquid chromatography (UHPLC)-ready samples, providing a fast and cost-effective solution for time-sensitive industrial analysis to fulfill regulatory requirements. Both GC-negative chemical ionization mass spectrometry (GC-NCI-MS) and UHPLC-tandem mass spectrometry (UHPLC-MS/MS) were employed to produce highly sensitive and reproducible data. Excellent limits of detection (typically below 1 μg/kg for GC and 10 μg/kg for UHPLC), wide linearity ranges, and good recoveries (mostly >70%) were achieved on the selected pesticides. Twenty-seven tea samples purchased from local grocery stores were analyzed using the newly developed methods. Among the pesticides analyzed, endosulfan sulfate and kelthane were the most frequently detected by GC-NCI-MS and imidacloprid and acetamiprid by UHPLC-MS/MS in these teas. The samples were found to be relatively clean, with <1 mg/kg of total pesticide residues. The organic-labeled teas were significantly cleaner than nonorganic ones. The cost per gram of tea did not correlate with pesticide residue levels detected.