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Validation of an HPLC-fluorescence method for the simultaneous free and glycosylated pyridinium crosslinks determination in urine
No full textBACKGROUND: Pyridinium crosslinks, released during bone resorption, are excreted in urine as free pyridinoline (Pyr) and deoxypyridinoline (DPyr), or bound to peptide or to sugars, as galactosyl-pyridinoline (Gal-Pyr) and glucosyl-galactosyl pyridinoline (GluGal-Pyr). Commonly, only total Pyr and D-Pyr urinary amounts (free + bound forms) are evaluated.
METHOD: We developed and validated an analytical method based on HPLC-fluorescence for the evaluation of the collagen crosslinks Pyr and DPyr (free and total), GluGal-Pyr and Gal-Pyr in the urine of healthy women (n = 20; aged 27-41) and girls (n = 20; aged 5-10). Urine, spiked with an unnatural D-Pyr homologue, as IS, was solid-phase extracted prior to HPLC analysis. The use of this IS and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr, synthesized to be used as primary calibrators, guarantees the specificity of the method and the correct crosslinks quantification. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis.
RESULTS: The method demonstrates good selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Pyr and D-Pyr, both free and total, and GluGal-Pyr amounts were significantly higher in girls than in women (p < 0.0001). Gal-Pyr, evaluated in girls for the first time, was under its lower quantification limit (< 21.20 pmol/mL) in women.
CONCLUSIONS: The quantification of free and glycosylated pyridinium crosslinks might provide more information on the degradation of various types of collagen, respect to the measurement of total Pyr and D-Pyr alone. Moreover, this validated method could be a useful non-invasive technique for studying pathological conditions characterized by modified glycosylation enzyme activity and for other clinical investigations on bone fragility
Development of a multi-analyte method for the determination of anabolic hormones in bovine urine by isotope-labelled GC-MS/MS
No full textThe use of natural and synthetic hormones for growth promotion purposes in meat producing animals is prohibited in the European Community since 1986 to protect consumers from possible developmental, neurobiological, genotoxic and carcinogenic effects due to the intake of hormone residues and their metabolites. Consequently, every EU Member State has to monitor a set proportion of the total annual production of different animal food commodities for their anabolic residues, determining them either in biological fluids (blood or urine) or muscle tissue and edible organs.
Screening analysis of anabolic hormones using immunoassays suffer from cross-reactivity with structurally related hormones. In order to reduce analytical interferences and improve accuracy, we developed a method using gas chromatography coupled to ion trap tandem mass spectrometry (GC-MS/MS) for the simultaneous determination of 11 anabolics in bovine urine (hexestrol, diethylstilbestrol, dienestrol, 17α-estradiol, 17β-estradiol, 17α-ethynylestradiol, 19-nortestosterone, 17α-methyltestosterone, β-testosterone, α-zeranol and β-zeranol). The assay consists of a simple sample preparation procedure, which utilizes only one solid-phase extraction step and one liquid-liquid extraction step for sample clean-up prior to derivatization for positive electron impact GC-MS/MS analysis. Two types of derivatization agents were assayed, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane and heptafluorobutyric anhydride (HFBA). The three possible acquisition modes of the GC-MS system, Full Scan, selective ion monitoring (SIM) and tandem mass spectrometry (MS/MS), were compared and best results were obtained in the MS/MS mode in the narrow concentration range of 0.25 to 8.0 ng/mL. Better results and good linearity were obtained using BSTFA as a derivatization agent by which no matrix effects were observed. The limits of detection were in the range of 0.005 ng/mL (diethylstilbestrol) to 0.38 ng/mL (17α-methyltestosterone and 17α-ethynylestradiol). Recoveries varied from 81 ± 4% (α-zeranol) to 149 ± 24% (17α-methyltestosterone) using the MS/MS mode. Repeatability values were obtained from 4 replicate analysis of spiked urine samples at 1 ng/mL and ranged from 2.1 to 22.7%
Development of a multianalyte method for the determination of anabolic hormones in bovine urine by isotope-dilution GC-MS/MS
No full textA sensitive, specific and selective multianalyte GC–MS/MS method has been developed for the determination of 11 anabolic hormones in bovine urine. After adjusting the urine pH to 4.8, the samples were spiked with deuterated internal standards and submitted to enzymatic hydrolysis with β-glucuronidase/arylsulfatase. Hormones were eluted with methanol through a C18 solid phase cartridge and submitted to a liquid–liquid extraction. Analytes were derivatized by adding N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane and GC–MS data were obtained in the positive electron impact tandem mass mode. Under these conditions, no matrix effects were observed and limit of detection values were in the range of 0.005 ng/mL (diethylstilbestrol) to 0.38 ng/mL (17α-methyltestosterone and 17α-ethynylestradiol). Recoveries from 81% (α-zeranol) to 149% (17α-methyltestosterone) were found under the selected conditions. These results were better than those found using heptafluorobutyric anhydride (HFBA) as derivative reagent and those measured in full scan and selective ion monitoring modes
Microwave-assisted extraction of pyrethroid insecticides from soil
No full textAn analytical method has been developed for the microwave-assisted extraction of synthetic pyrethroid insecticide residues in soils and their determination by gas chromatography with electron capture detector (ECD) and mass spectrometry (MS) detector. Clean soil samples were spiked with nine pyrethroids: tetramethrin, cyfluthrin, flucythrinate, deltamethrin, bifenthrin, permethrin, cypermethrin and fluvalinate were used for this study. Concentration levels were 50 ng g−1 for all the compounds studied except to sumitrin for which a 10 μg g−1 was evaluated. Two grams of sample were treated in a closed PTFE reactor with 10 mL toluene and 1 mL water and irradiated at 700 W during 9 min. The toluene extract was evaporated and reconstituted in 2 mL hexane and copper wires added in order to remove sulphur interferences. Clean-up with 2 g of florisil and elution with 20 mL ethyl acetate:hexane 33% (v/v) was performed. Best chromatographic conditions were established for GC-ECD analysis for routine analysis and GC-NCI-MS for confirmation. Recovery yields of spiked soils were from 97 to 106% for eight of the pyrethroid and 86% for the tetramethrin, with relative standard deviations from 1 to 7%. Results were comparable with those found by ultrasonic extraction in the analysis of real samples and limit of detection values obtained were in the range from 1 to 200 ng/g for ECD and from 0.3 to 2 ng/g for NCI-MS
Quantification of bone turnover markers in control human urine by HPLC-fluorescence using a new proposed internal standard
No full textThe aim of this study is to validate an HPLC-fluorescence method for the quantification of free pyridinoline (Pyd), free deoxypyridinoline (D-Pyd), indices of bone degradation, together with galactosyl (Gal-Pyd) and glucosyl-galactosyl (Glu-Gal-Pyd) pyridinolines in non-hydrolyzed healthy women urine (n=7, age=34±7). For the first time, a synthesized D-Pyd superior homologue is proposed as internal standard.
For all these compounds, the method is linear in the range 0-170 pmol injected and no matrix effect is found. The coefficients of variation for intra- and inter-day repeatability are between 3.1 and 10.4%. The limit of detection is around 2.3 pmol injected.
Free D-Pyd, free Pyd and Glu-Gal-Pyd evaluated in urine account for 40.5±4.7, 215.1±25.0 and 96.4±20.1 pmol/mL (mean±SD), respectively. Gal-Pyd is absent in control urines or under our detection limit.
The addition of the new proposed internal standard to urine before any pre-analytical step, allows the unequivocal quantification of the selected bone resorption indices. Moreover, the correct HPLC identification of all selected analytes is guaranteed by the availability of pure corresponding synthesized standards
Dimethylarginines in complicated type 1 diabetes: roles of insulin, glucose, and oxidative stress
No full textTo investigate the roles of insulin, glucose, and oxidative stress on plasma asymmetric and symmetric dimethylarginine (ADMA, SDMA) levels in complicated diabetes, we studied patients with type 1 diabetes (T1D; n = 20), T1D + end-stage renal disease under hemodialysis (T1D + ESRD; n = 12), T1D + ESRD who received kidney transplant (KD; n = 16), and T1D + ESRD who received kidney-pancreas transplant (KP; n = 20) and healthy controls (n = 50). Levels of ADMA, SDMA, and free and total malondialdehyde (MDA) were increased in all patients, with the highest rises for SDMA and free MDA in T1D+ESRD. In KP, the normalized glycemia contributes to the recovery of ADMA, SDMA, and MDA levels toward normal values. From the covariance analyses, both glucose and insulin relate significantly to ADMA in T1D + ESRD (beta = +0.004, beta = -0.038, respectively) and in KP (beta = +0.032, beta = +0.032, respectively). Creatinine clearance and insulin relate to SDMA in all patient groups (beta = -0.006). Our results provide evidence for the effect of kidney-pancreas transplant on the recovery of ADMA, SDMA, and indexes of oxidative stress toward normal values. Only free MDA allows one to discriminate the magnitude of the oxidative status, as increased total MDA could also be attributable to a reduced renal function
Evaluation of oxidative status in human serum: comparison of different methodological approaches
No full textDifferent biomarker assays have been developed for assessing oxidative stress in human serum. In this retrospective study the analytical performance of two different methodological approaches was evaluated in subjects with known increased oxidative stress to measure serum peroxidation indices: mild (n=35) and heavy (n=61) smokers, chronic renal failure (n=19) and kidney transplanted patients (n=59) compared to healthy controls (n=56).
Serum oxidative stress, assessing Reactive Oxygen Metabolite derivatives (d-ROMs, Diacron International, Italy) levels and Total Antioxidant Capacity (TAC, Diacron International, Italy) by commercial spectrophotometric assays, was compared with malondialdehyde (MDA) concentrations, quantified both in free (FMDA) and total (T-MDA) forms, by isotope-dilution gas chromatography-mass spectrometry (GC-MS) technique (“gold standard” reference method), generally unsuitable for routine use.
Sensitivity, specificity and cut-off points of T-MDA and F-MDA, d-ROMs, TAC assays were evaluated by receiver-operator characteristic (ROC) analyses together with the area under ROC curve (AUC).
ROC analyses accuracy: the best for T-MDA (AUC: 1; sensitivity and specificity: 100%), good for d-ROMs (AUC: 0.87; sensitivity 72.8%, specificity: 100%) and F-MDA (AUC: 0.82; sensitivity 74.7%, specificity: 100%), and not as good as the previous for TAC (AUC: 0.66; sensitivity: 52% and specificity: 92.9%). The increased peroxidative damage was best proved only by T-MDA levels. The assessment of d-ROMS concentrations and TAC by reliable assays is useful for routine clinical purposes; even if less sensitive, it determines the balance of oxidative status. The comparison between “gold standard” and routine methods allows biochemists and clinicians to evaluate data more thorough
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Oxidative status in different settings and with different methodological approaches compared by Receiver Operating Characteristic curve analysis
Full text linkObjectives: To test the performance of different analytical approaches in highlighting the occurrence of deregulated redox status in various physio-pathological situations.
Design and methods: 35 light and 61 heavy smokers, 19 chronic renal failure, 59 kidney transplanted patients, and 87 healthy controls were retrospectively considered for the study. Serum oxidative stress and antioxidant status, assessed by spectrophotometric Reactive Oxygen Metabolites (d-ROMs) and Total Antioxidant Capacity (TAC) tests, respectively, were compared with plasma free (F-MDA)
and total (T-MDA)malondialdehyde, both quantified by isotope-dilution-gas chromatography–mass spectrometry (ID-GC–MS). Sensitivity, specificity and cut-off points of T-MDA, F-MDA, d-ROMs and TAC were evaluated by both Receiver Operating Characteristic (ROC) analyses and area under the ROC curve (AUC).
Results: Only T-MDA assay showed a clear absence of oxidative stress in controls and significant increase in all patients (AUC 1.00, sensitivity and specificity 100%). Accuracy was good for d-ROMs (AUC 0.87, sensitivity 72.8%, specificity 100%) and F-MDA (AUC 0.82, sensitivity 74.7%, specificity 83.9%), but not high enough for TAC to show in patients impaired antioxidant defense (AUC 0.66, sensitivity 52.0%, specificity 92.9%).
Conclusions: This study reveals T-MDA as the best marker to detect oxidative stress, shows the ability of d-ROMs to identify modified oxidative status particularly in the presence of high damages, and evidences the poor TAC performance. d-ROMs and TAC assays could be useful for routine purposes; however, for an accurate clinical data evaluation, their comparison versus a “gold standard method” is required
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