The gene expression and proteomic profiling of Acanthamoeba isolates.

INTRODUCTION: The free-living protozoan Acanthamoeba can cause severe keratitis known as Acanthamoeba Keratitis (AK) and granulomatous amoebic encephalitis (GAE). The pathogenesis of Acanthamoeba includes intricate interactions between the organism and the host's immune system. The downstream analysis of a well-annotated genome assembly along with proteomic analysis can unravel several biological processes and aid in the identification of potential genes involved in pathogenicity. METHODS: Based on the next-generation sequencing data analysis, genes including lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein were selected as probable pathogenic targets that were validated by conventional PCR in a total of 30 Acanthamoeba isolates. This was followed by real-time PCR for the evaluation of relative gene expression in the keratitis and amoebic encephalitis animal model induced using keratitis (CHA5), encephalitis (CHA24) and non-pathogenic environmental isolate (CHA36). In addition, liquid chromatography-mass spectrometry (LC-MS/MS) was performed for keratitis, encephalitis, and non-pathogenic environmental isolate before and after treatment with polyhexamethylene biguanide (PHMB). RESULTS: The conventional PCR demonstrated the successful amplification of lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein genes in clinical and environmental isolates. The expression analysis revealed phospholipase, lysophospholipase, and mannose-binding genes to be significantly upregulated in the keratitis isolate (CHA 5) during AK in the animal model. In the case of the amoebic encephalitis model, phospholipase, lysophospholipase, S8/S53 peptidase, and carboxylesterase were significantly upregulated in the encephalitis isolate compared to the keratitis isolate. The proteomic data revealed differential protein expression in pathogenic versus non-pathogenic isolates in the pre and post-treatment with PHMB. CONCLUSION: The gene expression data suggests that lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein (MBP) could play a role in the contact-dependent and independent mechanisms of Acanthamoeba pathogenesis. In addition, the proteomic profiling of the 3 isolates revealed differential protein expression crucial for parasite growth, survival, and virulence. Our results provide baseline data for selecting possible pathogenic targets that could be utilized for designing knockout experiments in the future.

Comparison of two LC-MS/MS methods for the quantification of 24,25-dihydroxyvitamin D3 in patients and external quality assurance samples.

OBJECTIVES: In-house developed liquid-chromatography mass spectrometry (LC-MS/MS) methods are used more and more frequently for the simultaneous quantification of vitamin D metabolites. Among these, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) is of clinical interest. This study assessed the agreement of this metabolite in two validated in-house LC-MS/MS methods. METHODS: 24,25(OH)2D3 was measured in 20 samples from the vitamin D external quality assurance (DEQAS) program and in a mixed cohort of hospital patients samples (n=195) with the LC-MS/MS method at the Medical University of Graz (LC-MS/MS 1) and at the University of Liège (LC-MS/MS 2). RESULTS: In DEQAS samples, 24,25(OH)2D3 results with LC-MS/MS 1 had a proportional bias of 1.0% and a negative systemic difference of -0.05%. LC-MS/MS 2 also showed a proportional bias of 1.0% and the negative systemic bias was -0.22%. Comparing the EQA samples with both methods, no systemic bias was found (0.0%) and the slope was 1%. The mean difference of 195 serum sample measurements between the two LC-MS/MS methods was minimal (-0.2%). Both LC-MS/MS methods showed a constant bias of 0.31 nmol/L and a positive proportional bias of 0.90%, respectively. CONCLUSIONS: This study is the first to assess the comparability of 24,25(OH)2D3 concentrations in a mixed cohort of hospitalized patients with two fully validated in-house LC-MS/MS methods. Despite different sample preparation, chromatographic separation and ionization, both methods showed high precision measurements of 24,25(OH)2D3. Furthermore, we demonstrate the improvement of accuracy and precision measurements of 24,25(OH)2D3 in serum samples and in the DEQAS program.

Structure-function analyses of alkylhydroperoxidase D from Streptococcus pneumoniae reveal an unusual three-cysteine active site architecture.

During aerobic growth, the Gram-positive facultative anaerobe and opportunistic human pathogen Streptococcus pneumoniae generates large amounts of hydrogen peroxide that can accumulate to millimolar concentrations. The mechanism by which this catalase-negative bacterium can withstand endogenous hydrogen peroxide is incompletely understood. The enzyme alkylhydroperoxidase D (AhpD) has been shown to contribute to pneumococcal virulence and oxidative stress responses in vivo We demonstrate here that SpAhpD exhibits weak thiol-dependent peroxidase activity and, unlike the previously reported Mycobacterium tuberculosis AhpC/D system, SpAhpD does not mediate electron transfer to SpAhpC. A 2.3-Å resolution crystal structure revealed several unusual structural features, including a three-cysteine active site architecture that is buried in a deep pocket, in contrast to the two-cysteine active site found in other AhpD enzymes. All single-cysteine SpAhpD variants remained partially active, and LC-MS/MS analyses revealed that the third cysteine, Cys-163, formed disulfide bonds with either of two cysteines in the canonical Cys-78-X-X-Cys-81 motif. We observed that SpAhpD formed a dimeric quaternary structure both in the crystal and in solution, and that the highly conserved Asn-76 of the AhpD core motif is important for SpAhpD folding. In summary, SpAhpD is a weak peroxidase and does not transfer electrons to AhpC, and therefore does not fit existing models of bacterial AhpD antioxidant defense mechanisms. We propose that it is unlikely that SpAhpD removes peroxides either directly or via AhpC, and that SpAhpD cysteine oxidation may act as a redox switch or mediate electron transfer with other thiol proteins.

Evaluation of the 25-hydroxy vitamin D assay on a fully automated liquid chromatography mass spectrometry system, the Thermo Scientific Cascadion SM Clinical Analyzer with the Cascadion 25-hydroxy vitamin D assay in a routine clinical laboratory.

Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers advantages over immunoassay due to its increased specificity and ability to multiplex metabolites within a single run. Wide scale adoption of LC-MS/MS in routine clinical laboratories is restricted in part due to the high level of technical expertise required. The Thermo Scientific™ Cascadion™ SM Clinical Analyzer is the first fully automated, random access clinical analyser that utilises LC-MS/MS technology. We report an analytical validation of the 25-hydroxy vitamin D2 and D3 assays on the Cascadion Analyzer and an assessment of its performance within a routine clinical laboratory. Methods Analyser usability was assessed by staff with no previous experience of LC-MS/MS. An analytical validation included analysis of 154 patient samples on two different Cascadion Analyzers and a four-way method comparison of 146 patient samples on Roche and Siemens immunoassays and an in-house LC-MS/MS method. Accuracy was assessed using external quality assurance and reference materials. Seven third party IQC materials were tested on Cascadion. Results Cascadion proved easy to use by scientific and non-scientific staff. The assay passed all validation criteria. Excellent agreement was demonstrated between two different Cascadions (y = 0.97x + 3.9 nmol/L, r2 > 0.99). A method comparison demonstrated no significant difference (p > 0.05) between the Cascadion and the Roche immunoassay. A significant difference (p < 0.0001) was observed between the Cascadion and an LC-MS/MS and Siemens methods. Results obtained from EQA and reference material showed a mean bias of +3.09% and all samples were within ±10% of assigned concentrations. All third party IQC samples tested were compatible for use with Cascadion. Conclusions The Cascadion Analyzer is a fully automated LC-MS/MS system that requires no prior LC-MS/MS expertise. The vitamin D assays demonstrated excellent performance with high levels of accuracy.

Dissipation of neonicotinoid insecticides imidacloprid, indoxacarb and thiamethoxam on pomegranate (Punica granatum L.).

Neonicotinoid insecticides such as imidacloprid, indoxacarb and thiamethoxam are widely used for control of a large number of insect pests of pomegranate crop. Their residue levels were evaluated on pomegranate fruits over 2 years during the same cropping season. The QuEChERS analytical method in conjunction with LC-MS/MS was validated to analyse the insecticides on pomegranate fruits with peel (whole fruit), without peel (aril) and in the field soil. The method performance was satisfactory with the limit of quantification (LOQ) of 0.005 mg/kg which was below the maximum residue limits (MRLs) in pomegranate for the 3 compounds. A first order reaction kinetics was observed for the three insecticides with the half -life of degradation of 8-11.1 days for imidacloprid; 7.4-8.4 days for indoxacarb and 9.8-14.2 days for thiamethoxam. Though the insecticides are systemic in nature, the residues in the edible pomegranate aril were always < LOQ. The maximum residue levels of imidacloprid on pomegranate was less than its MRL of 1 mg/kg, so the pre-harvest interval (PHI) required was 1 day only. For indoxacarb, 31-42 days PHI was needed for the residues to reduce to its MRL of 0.02 mg/kg. The PHI of thiamethoxam was 46-77 days, the time required for its residues to reduce to its MRL of 0.01 mg/kg. Higher rainfall possibly facilitated faster dissipation of imidacloprid residues from pomegranate whereas indoxacarb and thiamethoxam remained unaffected. The results of the study can be utilized to incorporate these three chemicals in the plant protection program of pomegranate and fixation of MRL in India.

Peptidome analysis of lung tissues from a hyperoxia-induced bronchopulmonary dysplasia mouse model: Insights into the pathophysiological process of bronchopulmonary dysplasia.

The aim of this study was to identify and compare the peptidomic profiles of lung tissues from neonatal mice with and without bronchopulmonary dysplasia (BPD). Hyperoxia was used to establish the BPD mouse model. Lung tissues obtained on postnatal day (PND) 9 were processed for analysis via histological staining and label-free liquid chromatography-mass spectrometry (LC-MS/MS). Histological analysis of the lung sections from the BPD group showed significant alveolar simplification and aberrant pulmonary vascularization. We identified 3,704 total peptides, of which 63 were differentially expressed in the lung tissues from the BPD group compared with those from the control group. Within this subset, 31 peptides were downregulated, and 32 peptides were upregulated. Bioinformatics analysis suggested several potential roles of the differentially expressed peptides in the pathophysiological process of BPD. In summary, this study highlights novel peptide candidates, and provides new insights for further understanding the molecular mechanism of BPD development.

Activity of the liver enzyme ornithine carbamoyltransferase (OTC) in blood: LC-MS/MS assay for non-invasive diagnosis of ornithine carbamoyltransferase deficiency.

BACKGROUND: Liver enzymes are released from hepatocytes into circulation and their activity can be measured in the blood. We examined whether the plasma activity of the liver enzyme ornithine carbamoyltransferase, determined by a novel liquid chromatography-mass spectrometry (LC-MS/MS) assay, could be utilized for the detection of OTC deficiency (OTCD), an X-linked inborn error of the urea cycle. METHODS: The plasma ornithine carbamoyltransferase (OTC) activity was assayed in the reverse reaction using isotopically labeled citrulline-d4 as a substrate and by determination of the product, ornithine-d4, by LC-MS/MS analysis. RESULTS: The plasma OTC activity in the controls was in the range of 111-658 pkat/L (n=49, median 272 pkat/L), and the activity increased linearly with serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in patients with hepatopathy. The OTC activity was subsequently determined in 32 individuals carrying mutations in the OTC gene, and OTC/ALT and OTC/AST ratios were calculated to account for the degree of hepatopathy, which is a common finding in OTCD. The OTC/ALT ratio enabled clear differentiation of OTCD hemizygotes (n=11, range 0-69×10-6) from controls (504-3440×10-6). This ratio also enabled the detection of 11 of 12 symptomatic heterozygotes (range 38-794×10-6), while this marker did not allow for reliable differentiation of asymptomatic heterozygotes (n=9) from controls. CONCLUSIONS: LC-MS/MS assay of plasma OTC activity enabled the detection of all hemizygous and the majority of symptomatic heterozygous OTCD patients in the tested cohort. This study demonstrates that non-invasive assay of enzymes expressed predominantly in the liver could be used as an alternative approach for diagnosing inborn errors of metabolism.

Proteomics reveals that cell density could affect the efficacy of drug treatment.

In vitro cell biology study plays a fundamental role in biological and drug development research, but the repeatability and accuracy of cell studies remain to be low. Various uncertainties during the cell culture process could introduce bias into drug research. In this study, we evaluate the potential effects and underlying mechanisms induced by cell number differences in the cell seeding process. Normally, drug experiments are initiated 24 h after cell seeding, and the difference in the cell number at the time of inoculation leads to the difference in cell confluence (cell density) when drug research is conducted. While cell confluence is closely related to intercellular communication, surface protein interaction, cell autocrine as well as paracrine protein expression of cells, it might have a potential impact on the effect of biological studies such as drug treatment. This study used proteomics technology to comprehensively explore the different protein expression patterns between cells with different confluences. Due to the high sensitivity and high throughput of liquid chromatography-mass spectrometry (LC-MS/MS) detection, it was hired to evaluate the protein expression differences of Hep3B cells with 3 different confluences (30%, 50%, and 70%). The differential expressed proteins were analyzed by the Reactome pathway and the Gene Ontology (GO) pathway. Significant differences were identified across three confluences in terms of the number of proteins identified, the protein expression pattern, and the expression level of certain KEGG pathways. We found that those proteins involved in the cell cycle pathway were differently expressed: the higher the cell confluence, the higher these proteins expressed. A cell cycle inhibitor palbociclib was selected to further verify this observation. Palbociclib in the same dose was applied to cells with different confluence, the results indicated that the growth inhibition effect of palbociclib increases along with the increasing trend of cell cycle protein expression. The result indicated that cell density did influence the effect of drug treatment. Furthermore, three other drugs, cisplatin, paclitaxel, and imatinib, were used to treat the three liver cancer cell lines Hep3B, SUN387, and MHCC97, and a similar observation was obtained that drug effect would be different when the cell confluences were different. Therefore, selecting an appropriate number of cells for plating is vitally important at the beginning of a drug study.

Potential Health Benefits of Banana Phenolic Content during Ripening by Implementing Analytical and In Silico Techniques.

Banana ranks as the fifth most cultivated agricultural crop globally, highlighting its crucial socio-economic role. The banana's health-promoting benefits are correlated with its composition in bioactive compounds, such as phenolic compounds. Thus, the present study attempts to evaluate the potential health benefits of banana phenolic content by combing analytical and in silico techniques. Particularly, the total phenolic content and antioxidant/antiradical activity of banana samples during ripening were determined spectrophotometrically. In parallel, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was implemented to unravel the variations in the phenolic profile of banana samples during ripening. Chlorogenic acid emerged as a ripening marker of banana, while apigenin and naringenin were abundant in the unripe fruit. In a further step, the binding potential of the elucidated phytochemicals was examined by utilizing molecular target prediction tools. Human carbonic anhydrase II (hCA-II) and XII (hCA-XII) enzymes were identified as the most promising targets and the inhibitory affinity of phenolic compounds was predicted through molecular docking studies. This class of enzymes is linked to a variety of pathological conditions, such as edema, obesity, hypertension, cancer, etc. The results assessment indicated that all assigned phenolic compounds constitute great candidates with potential inhibitory activity against CA enzymes.

Quality Assessment of Ground Coffee Samples from Greek Market Using Various Instrumental Analytical Methods, In Silico Studies and Chemometrics.

Coffee is one of the most widely consumed beverages worldwide due to its sensory and potential health-related properties. In the present comparative study, a preparation known as Greek or Turkish coffee, made with different types/varieties of coffee, has been investigated for its physicochemical attributes (i.e., color), antioxidant/antiradical properties, phytochemical profile, and potential biological activities by combining high-throughput analytical techniques, such as infrared spectroscopy (ATR-FTIR), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and in silico methodologies. The results of the current study revealed that roasting degree emerged as the most critical factor affecting these parameters. In particular, the L* color parameter and total phenolic content were higher in light-roasted coffees, while decaffeinated coffees contained more phenolics. The ATR-FTIR pinpointed caffeine, chlorogenic acid, diterpenes, and quinic esters as characteristic compounds in the studied coffees, while the LC-MS/MS analysis elucidated various tentative phytochemicals (i.e., phenolic acids, diterpenes, hydroxycinnamate, and fatty acids derivatives). Among them, chlorogenic and coumaric acids showed promising activity against human acetylcholinesterase and alpha-glucosidase enzymes based on molecular docking studies. Therefore, the outcomes of the current study provide a comprehensive overview of this kind of coffee preparation in terms of color parameters, antioxidant, antiradical and phytochemical profiling, as well as its putative bioactivity.

Effect of a Carotenoid Extract from Citrus reticulata By-Products on the Immune-Oxidative Status of Broilers.

Although carotenoids generally possess antimicrobial and antioxidant properties, the in vivo synergistic action of carotenoid blends derived from plant-based by-products has not been thoroughly studied. Therefore, the carotenoid characterization and antimicrobial potential of Citrus reticulata extract as well as the impact of this carotenoid-rich extract (CCE) dietary supplementation on the performance, meat quality, and immune-oxidative status of broiler chickens were determined. One hundred and twenty one-day-old hatched chicks (Ross 308) were allocated to two dietary groups, with four replicate pens of 15 birds each. Birds were fed either a basal diet (CON) or the basal diet supplemented with 0.1% CCE (25 mg carotenoid extract included in 1 g of soluble starch) for 42 d. ß-Cryptoxanthin, ß-Carotene, Zeaxanthin, and Lutein were the prevailing carotenoid compounds in the Citrus reticulata extract. The CCE feed additive exerted inhibitory properties against both Gram-positive (Staphylococcus aureus) and negative (Klebsiella oxytoca, Escherichia coli, and Salmonella typhimurium) bacteria. Both the broiler performance and meat quality did not substantially differ, while the breast muscle malondialdehyde (MDA) concentration tended to decrease (p = 0.070) in the CCE-fed broilers. The inclusion of CCE decreased the alanine aminotransferase and MDA concentration, and the activity of glutathione peroxidase, while the activity of superoxide dismutase was increased in the blood. Catalase and NADPH oxidase 2 relative transcript levels were significantly downregulated in the livers of the CCE-fed broilers. Additionally, Interleukin 1ß and tumor necrosis factor (TNF) relative transcript levels were downregulated in the livers of the CCE- fed broilers, while TNF and interferon γ (IFNG) tended to decrease in the spleens and bursa of Fabricius, respectively. The present study provided new insights regarding the beneficial properties of carotenoids contained in Citrus reticulata in broilers' immune-oxidative status. These promising outcomes could be the basis for further research under field conditions.

Characterization of glyphosate and AMPA concentrations in the urine of Australian and New Zealand populations.

Glyphosate is the most used herbicide globally, but our understanding of human exposure and how different uses affect exposure is not well understood. The aim of this study was to obtain the first data on glyphosate and its primary degradation product aminomethylphosphonic acid (AMPA) concentrations in pooled and individual urine from the Australia and New Zealand region using a sensitive direct injection method and compare results with studies from elsewhere. Pooled urine samples from the Australian general population (n = 125 pools representing >1875 individuals) and individual urine samples (n = 27) from occupationally exposed New Zealand farmers were analysed by LC-MS/MS. Glyphosate was detected above the LOD (0.20-1.25 µg/L) in 8 % of the Australian population pooled urine samples with most detections in the 45-60 years age group. Furthermore, glyphosate (0.85 to 153 µg/L) and AMPA (0.50 to 3.35 µg/L) were detected in 96 % and 33 % of farmers, respectively. The maximum glyphosate urine concentration was 1.7 times above the recommended acceptable daily intake (ADI), when assuming a urinary excretion rate of 1 %. The pooled sampling and analysis approach proved effective for rapid large-scale screening of populations and could be used to determine where targeted and more specific individual sampling may be required.

Phytochemical Analysis and Immunomodulatory Potential on Diabetic-Infected Tuberculosis by Fruit Etlingera rubroloba A.D. Poulsen.

<b>Background and Objective:</b> <i> Etlingera rubroloba</i> (<i>E. rubroloba</i>) A.D. Poulsen is an endemic plant in South-East Sulawesi and is a newly discovered species. This plant is expected to have the potential as an immunomodulator in patients with diabetes mellitus (DM), which can prevent tuberculosis infection by increasing the phagocytic function of macrophage cells and interleukin-12 (IL-12) levels. <b>Materials and Methods:</b> Phytochemical analysis of the ethanolic extract of the fruit of <i>E. rubroloba</i> A.D. Poulsen using Liquid Chromatography-Mass Spectrometry (LC-MS/MS) was carried out. The immunomodulatory potential <i>in vivo</i> on BALB/c mice model DM was carried out by oral induction of TB antigen with extract dose, control positive, negative and normal groups. Furthermore, the phagocytic activity of macrophage cells can be seen with a microscope and the levels of IL-12 with the Elisa kit. <b>Results:</b> The results showed the ethanol extract of the fruit of <i>E. rubroloba</i> contained eight chemical compounds and had potential as immunomodulators in BALB/c DM mice induced by TB antigen by increasing the phagocytic activity of macrophage cells and levels of IL-12, which were significantly different from the negative control (p<0.05). <b>Conclusion:</b> The chemical composition of the ethanol extract of the fruit of <i>E. rubroloba</i> has the potential as an immunomodulator in TB antigen-induced DM <i>in vivo</i>.

Development and applicability of a multi-residue method for dyes, including new residue markers, to detect drug misuse in aquaculture.

A qualitative and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the sensitive and exhaustive analysis of residues from triarylmethane dyes, triarylmethane-derivative dyes, phenothiazines, phenoxazines and xanthenes in aquaculture samples. For a wider and more robust detection of dye misuse on farms, other residue markers were also included the leuco forms of brilliant green, crystal violet and malachite green; one direct metabolite of Victoria pure blue BO and methylene blue and three bile acids, which are endogenous markers of the effects of dye contamination in fish. We optimised the extraction method by comparing several extraction solvents and sample solvents reported in the literature to have the best extraction efficiency. The residues were determined using a positive electrospray ionisation source. We assessed the parameters of this LC-MS/MS method by evaluating the matrix effects, identification and quantitative parameters according to the criteria stipulated in the European Commission Decision No. 2002/657/EC. A study on the applicability of the method was conducted on various aquaculture species and on a positive catfish.

Reaction dynamics and residue identification of haemoglobin modification by acrolein, a lipid-peroxidation by-product.

BACKGROUND: Lipid hydroperoxides decompose to reactive aldehydes, such as acrolein. Measurement of oxidative stress markers in the clinic could improve risk stratification for patients. METHODS: To aid the development of diagnostic oxidative stress markers, we defined the acrolein modifications of haemoglobin using mass spectrometry. RESULTS: Acrolein modifications have little effect on the secondary structure of haemoglobin. They do not disrupt the quaternary structure, but instead promote crosslinked octamers. For acrolein modified haemoglobin the response to O2 binding is altered such that cooperativity is lost. Mass spectrometry experiments at a 1:1 acrolein:haemoglobin molar ratio demonstrate that the α-chain quickly forms an aza-Michael adduct (+56 Da), which then forms a more stable adduct, Nε-(3-methylpyridinium)lysine (MP-lysine, +76 Da) over 7 days. The ß-chain remains relatively unchanged over the duration of the 7 days and the aza-Michael adduct is dominant. At 2:1 and 5:1 molar ratios the α-chain was consistently modified at K7, H20, H50, and the ß-chain at C93 and H97 with the aza-Michael adduct. Beyond 5 h, an MP-adduct (+76 Da) was located predominantly at K7 of the α-chain, while an FDP-adduct (+94 Da) was observed at K95 of the ß-chain. CONCLUSIONS: We have generated qualitative evidence identifying the acrolein target sites on haemoglobin, a potential oxidative stress marker that is easily measured in circulation. GENERAL SIGNIFICANCE: We provide data for the community to develop targeted mass spectrometric or immunometric assays for acrolein modified haemoglobin to further validate the potential of haemoglobin as an oxidative stress marker in patients .

Confirmation of five nitrofuran metabolites including nifursol metabolite in meat and aquaculture products by liquid chromatography-tandem mass spectrometry: Validation according to European Union Decision 2002/657/EC.

LC-MS/MS method for confirmation of nitrofuran metabolites in meat and aquaculture products, including the nifursol metabolite (DNSH), was developed. The nitrofuran metabolites investigated were as follows: 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoine (AHD), semicarbazide (SEM) and 3,5-dinitrosalicylic acid hydrazide (DNSH). The sample preparation includes a washing step, allowing to analyze only the fraction of protein-bound residues. The final optimized recovery solvent for injection was found to be a mixture of ammonium acetate 2 mM/acetonitrile (60/40 ; v/v). Matrix effects and stability in biological matrix and standard solution for DNSH have been also carried out. Method performances were assessed using criteria of the Decision (EC) No 2002/657 and considering the proposed-for-adoption reference point for action (RPA) of 0.50 µg.kg-1 under Reg 1871/2019. Trueness ranged 86.5%-103.7% and precision ranged 2.0%-6.5% on intra-laboratory reproducibility conditions. Decision limit (CCα) ranged 0.028-0.182 µg.kg-1 and capability of detection limit (CCß) ranged 0.032-0.233 µg.kg-1.

Comparison of the efficacy of different androgens measured by LC-MS/MS in representing hyperandrogenemia and an evaluation of adrenal-origin androgens with a dexamethasone suppression test in patients with PCOS.

BACKGROUND: The aims of this study were to compare the efficacy of different androgens measured by liquid chromatography-mass spectrometry (LC-MS/MS) in representing hyperandrogenemia and to evaluate adrenal-origin androgens with a dexamethasone suppression test in patients with polycystic ovary syndrome (PCOS). METHODS: One hundred and two patients with PCOS and 41 healthy volunteers were recruited and total serum testosterone (TT), androstenedione (AD), dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) were measured by LC-MS/MS. ROC analysis was performed to compare the efficacy of different androgens in representing hyperandrogenemia. Dexamethasone suppression test was performed in 51 patients with PCOS and above indicators were measured after dexamethasone administration. The prediction efficacy of DHEA and DHEA-S at baseline in the dexamethasone suppression test was evaluated with ROC analysis. RESULTS: The AUCs of TT, AD, free androgen index (FAI) and DHEA-S in ROC analysis for representing hyperandrogenemia were 0.816, 0.842, 0.937 and 0.678, respectively. The optimal cutoff value of TT was 0.337 ng/ml, with a sensitivity of 72.0% and specificity of 82.93%. The optimal cutoff value for AD was 1.309 ng/ml, with a sensitivity of 81.0% and specificity of 73.17%. The optimal cutoff value of the FAI was 2.50, with a sensitivity of 87.0% and specificity of 92.68%. Alternatively, AD or FAI more than the optimal cutoff values as evidence of hyperandrogenemia had the highest sensitivity of 91.18%. The levels of cortisol, DHEA and DHEA-S were all suppressed to narrow ranges after dexamethasone administration. Nine and 8 of 51 patients with PCOS had significant decreases in TT and AD, respectively. DHEA can be used as a indicator for predicting significant decrease of TT in dexamethasone suppression test with cutoff value of 13.28 ng/ml. A total of 27.5% (14/51) of patients had DHEA-S excess, but only 1 of 9 patients who had a significant decrease in TT had elevated level of DHEA-S at baseline. CONCLUSIONS: AD measured by LC-MS/MS can represent hyperandrogenemia in PCOS patients and, combined with TT or FAI, can improve the screening efficiency of hyperandrogenemia. Seventeen percent of PCOS patients had adrenal-origin androgen dominance, with TT significantly decreasing after 2 days of dexamethasone administration. Adrenal-origin androgen dominance was not parallel with DHEA-S excess in patients with PCOS.

How to Study the Proteomes and Phosphoproteomes of Anther and Pollen.

Proteomics analysis was a powerful technology for characterizing proteins and protein posttranslational modification (PTMs). Recently, many anther and pollen-related proteomic analyses have been reported, which have expanded our understanding of anther and pollen development and regulation. In this chapter, we describe a detailed, optimized protocol for the separation, digestion, tagging, and subsequent mass spectrometry-based identification and quantification of proteins and phosphoproteins from anther and pollen.

Solid phase-extraction procedure for the determination of amitraz degradation products in honey.

Solid-phase extraction (SPE) was carefully optimised for preconcentration of 2-4-dimethylaniline (2,4-DMA) and 2,4-dimethylformanilide (DMF) from honey samples. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used for the separation and quantification of these analytes. By applying the SPE procedure with 200 mg of OASIS HLB sorbent, the limits of detection achieved in honey samples can be lowered to 0.41 µg·kg-1 for 2,4-DMA and 0.69 µg L-1 for DMF. The proposed method achieves good recoveries (81.1-114%) and precision (RSD 1.07-4.05%, n = 3) for analysed honey sample spiked at two concentration levels 1 µg L-1 and 2 µg L-1. The results demonstrated our method can be applied as a simple way for sample preparation of honey samples for determination of amitraz degradation products. In 1 out of 5 of the analysed Polish honey samples, the DMF residues exceed the maximum residual limits (0.2 mg·kg-1 for total amitraz residues).

LC-MS/MS Analysis of the Emerging Toxin Pinnatoxin-G and High Levels of Esterified OA Group Toxins in Galician Commercial Mussels.

The occurrence of marine harmful algae is increasing worldwide and, therefore, the accumulation of lipophilic marine toxins from harmful phytoplankton represents a food safety threat in the shellfish industry. Galicia, which is a commercially important EU producer of edible bivalve mollusk have been subjected to recurring cases of mussel farm closures, in the last decades. This work aimed to study the toxic profile of commercial mussels (Mytilus galloprovincialis) in order to establish a potential risk when ingested. For this, a total of 41 samples of mussels farmed in 3 Rías (Ares-Sada, Arousa, and Pontevedra) and purchased in 5 local markets were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Chromatograms showed the presence of okadaic acid (OA), dinophysistoxin-2 (DTX-2), pectenotoxin-2 (PTX-2), azaspiracid-2 (AZA-2), and the emerging toxins 13-desmethyl spirolide C (SPX-13), and pinnatoxin-G (PnTX-G). Quantification of each toxin was determined using their own standard calibration in the range 0.1%-50 ng/mL (R2 > 0.99) and by considering the toxin recovery (62-110%) and the matrix correction (33-211%). Data showed that OA and DTX-2 (especially in the form of esters) are the main risk in Galician mollusks, which was detected in 38 samples (93%) and 3 of them exceeded the legal limit (160 µg/kg), followed by SPX-13 that was detected in 19 samples (46%) in quantities of up to 28.9 µg/kg. Analysis from PTX-2, AZA-2, and PnTX-G showed smaller amounts. Fifteen samples (37%) were positive for PTX-2 (0.7-2.9 µg/kg), 12 samples (29%) for AZA-2 (0.1-1.8 µg/kg), and PnTX-G was detected in 5 mussel samples (12%) (0.4 µg/kg-0.9 µg/kg). This is the first time Galician mollusk was contaminated with PnTX-G. Despite results indicating that this toxin was not a potential risk through the mussel ingestion, it should be considered in the shellfish safety monitoring programs through the LC-MS/MS methods.