Individual Calibration Curves Research Articles

Determination of dose-response calibration curves for gamma radiation using gamma-H2AX immunofluorescence based biodosimetry.

Gamma-H2AX immunofluorescence assay has gained popularity as a DNA double strand break marker. In this work, we have investigated the potential use of gamma H2AX immunofluorescence assay as a biological dosimeter for estimation of dose in our institution. Seven healthy individuals were selected for the study and the blood samples collected from the first five individuals were irradiated to low doses (0-10 cGy) and high doses (50-500 cGy) in a telecobalt unit. All the samples were processed for gamma-H2AX immunofluorescence assay and the dose-response calibration curves for low and high doses were determined. In order to validate the determined dose-response calibration curves, the blood samples obtained from the sixth and seventh subjects were delivered a test dose of 7.5 cGy and 250 cGy. In addition, time and cost required to complete the assay were also reported. The goodness of fit (R2) values was found to be 0.9829 and 0.9766 for low and high dose-response calibration curves. The time required to perform the gamma-H2AX immunofluorescence assay was found to be 7 hours and 30 minutes and the estimated cost per sample was 5000 rupees (~ 60 USD). Based on this study we conclude that the individual dose-response calibration curves determined with gamma-H2AX immunofluorescence assay for both low and high dose ranges of gamma radiation can be used for biological dosimetry. Further, the gamma-H2AX immunofluorescence assay can be used as a rapid cost-effective biodosimetric tool for institutions with an existing confocal microscope facility.

Sub-ppb detection limit of Cr, Pb, and Cu in water by surface-enhanced LIBS with femtosecond laser

Water pollution caused by heavy metal contamination significantly threatens human health. Detecting trace levels of heavy metals in water is of utmost importance. Surface-enhanced laser-induced breakdown spectroscopy (SE-LIBS) has become a reliable detection method. In this study, we improved the analytical sensitivity of trace heavy metals in water by introducing femtosecond lasers into the SE-LIBS technique, replacing the traditional nanosecond lasers. An aluminum plate was utilized as a substrate to transform the liquid into a solid, overcoming the inadequacy in sensitivity and precision of LIBS when applied to water samples. We developed individual calibration curves for Cr, Pb, and Cu to determine their respective limit of detection (LOD). The LODs obtained were 0.3, 2.5, and 0.08 ng/mL for Cr, Pb, and Cu, respectively. These LODs were lower than traditional nanosecond SE-LIBS, and even lower than the LODs obtained by combining other LIBS enhancement techniques and SE-LIBS. We also determined good accuracy through the coefficient of determination R2, which was 0.97, 0.99, and 0.97, respectively, for Cr, Pb, and Cu. These results provide valuable and insightful information for the sensitive analysis of trace heavy metals in water using SE-LIBS with a femtosecond laser.

Real-time measurement of the moisture contents of dewatered sewage sludge during thermal drying using a low-cost ECH2O EC-5 soil moisture sensor

Real-time measurement of sewage sludge moisture content renders it possible to optimize the consumption of auxiliary fuel and the thermal treatment. Thus, we explored whether a dielectric soil moisture sensor could be used to measure real-time changes in sludge moisture content. This was possible with the aid of a unified calibration curve. The explanatory variable was the third-order sensor output voltage. The calibration curve well-fitted the data; the mean measurement error was 6.14 wt.%. The use of individual calibration curves for each sludge batch reduced the mean measurement error to 3.09 wt.%. The mean measurement error improves when multiple sensors are simultaneously used. The sensor operates very well when the moisture content is low; the results are minimally affected by the bulk density and Fe level. Considering feed-back to the process, it is ideal to measure sludge moisture content near the outlet of the thermal treatment.

Low-Cost Nitric Oxide Sensors: Assessment of Temperature and Humidity Effects.

High equipment cost is a significant entry barrier to research for small organizations in developing solutions to air pollution problems. Low-cost electrochemical sensors show sensitivity at parts-per-billion by volume mixing ratios but are subject to variation due to changing environmental conditions, in particular temperature. In this study, we demonstrate a low-cost Internet of Things (IoT)-based sensor system for nitric oxide analysis. The sensor system used a four-electrode electrochemical sensor exposed to a series of isothermal/isohume conditions. When deployed under these conditions, stable baseline responses were achieved, in contrast to ambient air conditions where temperature and humidity conditions may be variable. The interrelationship between working and auxiliary electrodes was linear within an environmental envelope of 20-40 °C and 30-80% relative humidity, with correlation coefficients from 0.9980 to 0.9999 when measured under isothermal/isohume conditions. These data enabled the determination of surface functions that describe the working to auxiliary electrode offsets and calibration curve gradients and intercepts. The linear and reproducible nature of individual calibration curves for stepwise nitric oxide (NO) additions under isothermal/isohume environments suggests the suitability of these sensors for applications aside from their role in air quality monitoring. Such applications would include nitric oxide kinetic studies for atmospheric applications or measurement of the potential biocatalytic activity of nitric oxide consuming enzymes in biocatalytic coatings, both of which currently employ high-capital-cost chemiluminescence detectors.

A novel amperometric biosensor for multi-enzymatic biosensing of triglycerides

This work has been focused on developing a novel biosensor assisted by multivariate calibration methods to determine triglycerides (TGs, triacetin, tributyrin, tricaproin, tricaprylin, and tricaprin) in lyophilized serum samples. To achieve this goal, a bare glassy carbon electrode (GCE) was modified and used as the biosensing platform. To increase the sensitivity of the developed method, hydrodynamic methods were used to calibrate the biosensor response. To increase the selectivity of the developed biosensor, it was assisted by partial least squares-1 (PLS-1), radial basis function-PLS (RBF-PLS), and RBF-artificial neural network (RBF-ANN) for exploiting first-order advantage. After characterization of the modifications, the first-order advantage was exploited to increase the selectivity of the method by building a multivariate calibration set in a pre-analyzed lyophilized serum with different TGs concentrations, which were chosen according to the individual calibration curve. Calibration models were then built in the same pre-analyzed lyophilized serum and analyzed by PLS-1, RBF-PLS, and RBF-ANN. Therefore, their performances were examined to predict concentrations of a validation set. The results confirmed the successfulness of the calibration model developed by RBF-ANN. Finally, it was used to analyze two serum samples, and the results demonstrated that the method was successful because its results were compared with a reference method.

Comparison for quantification of eight components in Alpinia officinarum Hance by using high-performance liquid chromatography coupled with diode array detector and charged aerosol detector with individual and substitute reference compound

An efficient HPLC-DAD-CAD method was developed and compared for simultaneous quantification of four flavonoids and four diarylheptanoids in Alpinia officinarum Hance (A. officinarum) using individual and substitute reference compound. All calibration curves for investigated analytes showed good linear regression (R2> 0.9991). The LODs of investigated compounds for DAD and CAD were 0.15–7.92 ng (0.03–1.58 μg/mL) and 2.91–3.95 ng (0.58–0.79 μg/mL), respectively, whereas the LOQs were 0.52–26.39 ng (0.10–5.28 μg/mL) for DAD, and 9.70–13.18 ng (1.94–2.64 μg/mL) for CAD. Recoveries of all analytes, which ranged from 96.58% to 100.06% for DAD, and from 96.29% to 99.61% for CAD, were acceptable. According to the quantitative results, the eight compounds in A. officinarum can be accurately quantified with individual calibration curves by two detectors. In addition, to overcome the bottleneck of shortage of reference standards, diphenylheptane A and galangin, respectively, were selected for direct or calibrated quantitative determination of other diarylheptanoids and flavonoids in A. officinarum. The results showed the contents of eight components in A. officinarum determined by these methods were similar, which suggested that substitute reference compound was suitable for quantification of its analogues.

Pulse arrival time as a surrogate of blood pressure

Various models have been proposed for the estimation of blood pressure (BP) from pulse transit time (PTT). PTT is defined as the time delay of the pressure wave, produced by left ventricular contraction, measured between a proximal and a distal site along the arterial tree. Most researchers, when they measure the time difference between the peak of the R-wave in the electrocardiogram signal (corresponding to left ventricular depolarisation) and a fiducial point in the photoplethysmogram waveform (as measured by a pulse oximeter attached to the fingertip), describe this erroneously as the PTT. In fact, this is the pulse arrival time (PAT), which includes not only PTT, but also the time delay between the electrical depolarisation of the heart’s left ventricle and the opening of the aortic valve, known as pre-ejection period (PEP). PEP has been suggested to present a significant limitation to BP estimation using PAT. This work investigates the impact of PEP on PAT, leading to a discussion on the best models for BP estimation using PAT or PTT. We conducted a clinical study involving 30 healthy volunteers (53.3% female, 30.9 ± 9.35 years old, with a body mass index of 22.7 ± 3.2 kg/m^{2}). Each session lasted on average 27.9 ± 0.6 min and BP was varied by an infusion of phenylephrine (a medication that causes venous and arterial vasoconstriction). We introduced new processing steps for the analysis of PAT and PEP signals. Various population-based models (Poon, Gesche and Fung) and a posteriori models (inverse linear, inverse squared and logarithm) for estimation of BP from PTT or PAT were evaluated. Across the cohort, PEP was found to increase by 5.5 ms ± 4.5 ms from its baseline value. Variations in PTT were significantly larger in amplitude, − 16.8 ms ± 7.5 ms. We suggest, therefore, that for infusions of phenylephrine, the contribution of PEP on PAT can be neglected. All population-based models produced large BP estimation errors, suggesting that they are insufficient for modelling the complex pathways relating changes in PTT or PAT to changes in BP. Although PAT is inversely correlated with systolic blood pressure (SBP), the gradient of this relationship varies significantly from individual to individual, from − 2946 to − 470.64 mmHg/s in our dataset. For the a posteriori inverse squared model, the root mean squared errors (RMSE) for systolic and diastolic blood pressure (DBP) estimation from PAT were 5.49 mmHg and 3.82 mmHg, respectively. The RMSEs for SBP and DBP estimation by PTT were 4.51 mmHg and 3.53 mmHg, respectively. These models take into account individual calibration curves required for accurate blood pressure estimation. The best performing population-based model (Poon) reported error values around double that of the a posteriori inverse squared model, and so the use of population-based models is not justified.

Multilabel Per-Pixel Quantitation in Mass Spectrometry Imaging.

In quantitative mass spectrometry imaging (MSI), the gold standard adds a single structural homologue of the target compound at a known concentration to the sample. This internal standard enables to map the detected intensity of the target molecule against an external calibration curve. This approach, however, ignores local noise levels and disproportional ion suppression effects, which might depend on the concentration of the target compound. To overcome these issues, we propose a novel approach that applies several isotopically labeled versions, each at a different concentration, to the sample. This allows creating individual internal calibration curves for every MSI pixel. As proof of principle, we have quantified an endogenous peptide of histone H4 by matrix-assisted laser desorption/ionization-Q-MSI (MALDI-Q-MSI), using a mixture of three isotopically labeled versions. The usage of a fourth label allowed us to compare the gold standard to our multilabel approach. We observed substantial heterogeneity in ion suppression across the tissue, which disclosed itself as varying slopes in the per-pixel regression analyses. These slopes were histology-dependent and differed from each other by up to a factor of 4. The results were validated by liquid chromatography–mass spectrometry (LC-MS), exhibiting a high agreement between LC-MS and MALDI-Q-MSI (Pearson correlation r = 0.87). A comparison between the multilabel and single-label approaches revealed a higher accuracy for the multilabel method when the local target compound concentration differed too much from the concentration of the single label. In conclusion, we show that the multilabel approach provides superior quantitation compared to a single-label approach, in case the target compound is inhomogeneously distributed at a wide concentration range in the tissue.

Direct Catalytic Fuel Cell Device Coupled to Chemometric Methods to Detect Organic Compounds of Pharmaceutical and Biomedical Interest.

Making use of a small direct methanol fuel cell device (DMFC), used as an analytical sensor, chemometric methods, organic compounds very different from one another, can be determined qualitatively and quantitatively. In this research, the following seven different organic compounds of pharmaceutical and biomedical interest, having in common only one –OH group, were considered: chloramphenicol, imipenem, methanol, ethanol, propanol, atropine and cortisone. From a quantitative point of view, the traditional approach, involving the building of individual calibration curves, which allow the quantitative determination of the corresponding organic compounds, even if with different sensitivities, was followed. For the qualitative analysis of each compound, this approach has been much more innovative. In fact, by processing the data from each of the individual response curves, obtained through the fuel cell, using chemometric methods, it is possible to directly identify and recognize each of the seven organic compounds. Since the study is a proof of concept to show the potential of this innovative methodological approach, based on the combination of direct methanol fuel cell with advanced chemometric tools, at this stage, concentration ranges that may not be the ones found in some real situations were investigated. The three methods adopted are all explorative methods with very limited computation costs, which have different characteristics and, therefore, may provide complementary information on the analyzed data. Indeed, while PCA (principal components analysis) provides the most parsimonious summary of the variability observed in the current response matrix, the analysis of the current response behavior was performed by the “slicing” method, in order to transform the current response profiles into numerical matrices, while PARAFAC (Parallel Factor Analysis) allows to obtain a finer deconvolution of the exponential curves. On the other hand, the multiblock nature of “ComDim” (Common Components and Specific Weight Analysis) has been the basis to relate the variability observed in the current response behavior with the parameters of the linear calibrations.

Gas Chromatography-Tandem Mass Spectrometry Verification of Sulfur Mustard Exposure in Humans through the Conversion of Protein Adducts to Free Sulfur Mustard.

Exposures to sulfur mustard (HD; bis(2-chloroethyll) sulfide) are well-known to result in the formation of adducts with free aspartate and glutamate residues of plasma proteins (Lawrence, R. J., Smith, J. R., and Capacio, B. R. 2008 32, (1), 31-36). A modified version of the analytical method reported previously for the verification of HD exposure has been developed (Lawrence, R. J., Smith, J. R., and Capacio, B. R. 2008 32, (1), 31-36). The method reported herein involves the reaction of hydrochloric acid with HD-adducted plasma proteins, resulting in the simultaneous cleavage and conversion of the adduct to free HD. A water scavenger, 2,2-dimethoxypropane, was added to the mixture to increase the reaction yield. Deuterated (d8) thiodiglycol was added as an internal standard and underwent conversion to deuterated sulfur mustard. The analytes were isolated by hexane liquid-liquid extraction and subsequently analyzed by gas chromatography tandem mass spectrometry (GC-MS-MS). An interday and intraday study was performed to evaluate the accuracy and precision of the method. Individual calibration curves with quality control (QC) standards were prepared on 5 days, and a calibration curve with five sets of QCs was prepared on a single day. All results were within the acceptable limits of the validation criteria. Linearity, limit of detection, and limit of quantitation were also verified for each calibration curve. This highly sensitive (pg/mL limit of detection) method can be used for rapid analysis of a definitive marker of sulfur mustard exposure.

A sensitive and reliable RP-HPLC method for the detection of cimetidine – a H2 receptor antagonist in human plasma

Bioanalytical methods for bioequivalence studies require high sensibility and rapidity due to the large number of samples and the low plasma concentration of drugs. The present study aimed to develop and validate a high-performance liquid chromatography method to quantify cimetidine (CMT) in human plasma and to apply it in a bioequivalence study. Spiked plasma of 500 µl (l, m and h concentration) was used for the assay. The HPLC injection volume was 20μl of the reconstitute sample where, 2 ml of ethyl acetate used for extraction purposes. Cimetidine was prepared separately for low (80 ng/ml), medium (2000 ng/ml) and high (3600 ng/ml) concentrations and internal standard (ranitidine) concentration was 3000 ng/ml. Freeze thawing and long terms stability were conducted at -25º c. The individual calibration curve for spiked standards was linear with R2= 0.99. The inaccuracy values for QC samples were within 15% of the actual value and not more than 20% for the LOQ. The limit of quantitation (LOQ) was 40 ng/ml, which was also the lowest concentration of cimetidine that was quantitated with the variability of 5.9%. The within day precision and between day precision for LOQ were 10.8 and 5.9 respectively. The retention time for the analyte was 4.1-4.5 minutes during the within a day and between day results. The mean % inaccuracy values for low, medium and high concentration were 6.8, 5.6 and 7.8 respectively for within day and 2.4, 6.1 and 7.9 respectively for between days. The within day and between day % inaccuracy for LOQ concentration was 12.4 and 5.5 respectively. The mean recoveries for low, medium and high concentration of cimetidine were 80.2, 70.9 and 74.2. The overall mean recovery for cimetidine was 75.1%. The maximum inaccuracy for freeze thaw cycle and long term stability samples for low, medium and high was found with CV less than 15% for all concentrations, indicating that cimetidine is stable. The developed method was precise and accurate and was suitable to be applied for the bioequivalence study of cimetidine.

Quality evaluation of monoenergetic images generated by dual-energy computed tomography for radiotherapy: A phantom study

PurposeQuantification analysis for monoenergetic computed tomography (CT) images obtained from dual-energy CT scanning was performed in the light of their potential use for structures delineation during radiotherapy. MethodsParameters that describe the quality of the images are: linearity, low and high contrast resolution, uniformity, noise and signal to noise ratio (SNR). To evaluate these parameters, a Catphan phantom was scanned using a dual-energy mode at Somatom Definition AS. Based on the polyenergetic CT images, sixteen monoenergetic series (ranged from 40 keV to 190 keV) were created by CT scanner software and automatically analyzed using Artiscan software. ResultsAnalysis of linearity shows that a potential use of any monoenergetic images in radiotherapy planning requires that individual calibration curves are implemented for each of them. While the results of the high contrast resolution analysis were comparable for each energy (5 lp/cm), the results of the analyses for uniformity, low contrast resolution, noise and SNR allowed us to select the best imaging energies. The highest relative uniformity was detected for images reconstructed for energies of 60 keV and 70 keV (98.54% and 98.61%). Similar results were observed for low contrast resolution, where the largest number of disks was detected for these energies, and the noise values (0.42% for 60 keV, 0.44% for 70 keV). The best SNR was observed for images reconstructed for energy of 60 keV. ConclusionsTaking into account these results, the energy of 70 keV was selected as potentially the best for reconstruction of monoenergetic images used for structures delineation during radiotherapy.

A Comparison of GMP Manufactured Ovine Mucosal Enoxaparin and Branded Porcine Enoxaparin

ObjectiveLow Molecular Weight Heparins (LMWH) such as Enoxaparin are currently derived from porcine mucosal heparin (PMH). The same depolymerization methods that retrieve this enoxaparin from unfractionated heparin can be used from other sources, including from sheep, resulting in ovine enoxaparin. Previous studies have compared non‐good manufacturing practice (GMP) batches of ovine enoxaparin and comparing them to porcine mucosal enoxaparin. The purpose of this study is to compare the pharmacokinetics of GMP ovine mucosal enoxaparin (OME) with that of porcine mucosal enoxaparin (PME).Materials and MethodsBranded Enoxaparin (Lovenox, Sanofi‐Aventis, Bridgewater, NJ) and three batches of GMP OME were used. Individual batches of GMP or branded Lovenox were administered subcutaneously (SC) to groups of primates (macaca mulatta) at a dose of 1 mg/kg. Blood samples were collected at baseline and post‐injection times of 2,4, and 6 hours. Plasma samples were frozen for batch analysis. aPTT was measured using clot‐based methods and amidolytic anti‐Xa and anti‐thrombin assays were used to calculate circulating levels of heparin. Individual calibration curves for each the GMP product and branded product were constructed, and the concentrations of drugs were measured at time points in terms of micrograms per milliliter. Drug levels and PK parameters were determined using anti‐Xa and anti‐thrombin assays. The batches of the GMP products were compared among each other and grouped means of all batches were compared with the branded Enoxaparin.ResultsIn the in‐vitro calibration studies, using the aPTT, anti‐Xa and, anti‐IIa assays, despite minor variations, the GMP batches composite values compared well with that of the branded Lovenox. In the PK/PD studies, the three batches of GMP‐produced ovine enoxaparin compared well within the group and the composite mean was similar to that of the branded Lovenox.DiscussionAt a 1 mg/kg SC dosage, three GMP OME preparations produced comparable anti‐Xa and anti‐IIa effects which were similar to the ones observed with PME. The PK parameters calculated based on this data were also comparable for both groups. It is concluded that the SC pharmacokinetics of the three batches of OME were not only comparable between batches but were also similar to the branded Lovenox.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Preparation and Application of Standardized Typical Volatile Components Fraction from Turmeric (Curcuma longa L.) by Supercritical Fluid Extraction and Step Molecular Distillation.

A green and reliable method using supercritical fluid extraction (SFE) and molecular distillation (MD) was optimized for the separation and purification of standardized typical volatile components fraction (STVCF) from turmeric to solve the shortage of reference compounds in quality control (QC) of volatile components. A high quality essential oil with 76.0% typical components of turmeric was extracted by SFE. A sequential distillation strategy was performed by MD. The total recovery and purity of prepared STVCF were 97.3% and 90.3%, respectively. Additionally, a strategy, i.e., STVCF-based qualification and quantitative evaluation of major bioactive analytes by multiple calibrated components, was proposed to easily and effectively control the quality of turmeric. Compared with the individual calibration curve method, the STVCF-based quantification method was demonstrated to be credible and was effectively adapted for solving the shortage of reference volatile compounds and improving the QC of typical volatile components in turmeric, especially its functional products.

Accuracy of Dose Calculation with CBCT Images under Different Scanning Conditions

The treatment plans were designed based on planning CT of Catphan phantom's homogenic and heterogenetical module, respectively. With the OBI system, Catphan phantom was scanned under different scanning conditions. The dose was recalculated by applying treatment plans based on planning CT to the CBCT images with its individual hounsfield unit-electron density calibration curve. The dose distributions were compared with those of the original plan, the results of HI showed a good agreement. CIs in the homogenic module were superior to those in the heterogenetical one. There is a good dose distribution for CBCT images under different scanning conditions. It is helpful for CBCT images directly used for dose re-calculation in adaptive radiation therapy (ART).

Feasibility of a novel multispot nanoarray for antibiotic screening in honey

ABSTRACTPractical solutions for multiple antibiotic determination in food are required by the food industry and regulators for cost-effective screening purposes. This study describes the feasibility in development and preliminary performance of a novel multispot nanoarray for antibiotic screening in honey. Using a multiplex approach, the metabolites of the four main nitrofuran antibiotics, including morpholinomethyl-2-oxazolidone (AMOZ), 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), 1-aminohydantoin (AHD) and chloramphenicol (CAP), were simultaneously detected. Antibodies specific to the five antibiotics were nano-spotted onto microtitre plate wells and a direct competitive assay format was employed. The assay characteristics and performance were evaluated for feasibility as a screening tool for antibiotic determination in honey to replace traditional ELISAs. Optimisation of the spotting and assay parameters was undertaken with both individual and multiplex calibration curves generated in PBS and a honey matrix. The limits of detection as determined by the 20% inhibitory concentrations (IC20) were determined as 0.19, 0.83, 0.09, 15.2 and 35.9 ng ml–1 in PBS, 0.34, 0.87, 0.17, 42.1 and 90.7 ng ml–1 in honey (fortified at the start of the extraction), and 0.23, 0.98, 0.24, 24.8 and 58.9 ng ml–1 in honey (fortified at the end of the extraction) for AMOZ, AOZ, CAP, SEM and AHD respectively. This work has demonstrated the potential of multiplex analysis for antibiotics with results available for 40 samples within a 90-min period for antibiotics sharing a common sample preparation. Although both the SEM and AHD assay do not show the required sensitivity with the antibodies available for use to meet regulatory limits, with further improvements in these particular antibodies this multiplex format has the potential to show a reduction in cost with reduced labour time in combination with the high-throughput screening of samples. This is the first 96-well spotted microtitre plate nanoarray for the semi-quantitative and simultaneous analysis of antibiotics.

Influence of Individual Differences on the Calculation Method for FBG-Type Blood Pressure Sensors.

In this paper, we propose a blood pressure calculation and associated measurement method that by using a fiber Bragg grating (FBG) sensor. There are several points at which the pulse can be measured on the surface of the human body, and when a FBG sensor located at any of these points, the pulse wave signal can be measured. The measured waveform is similar to the acceleration pulse wave. The pulse wave signal changes depending on several factors, including whether or not the individual is healthy and/or elderly. The measured pulse wave signal can be used to calculate the blood pressure using a calibration curve, which is constructed by a partial least squares (PLS) regression analysis using a reference blood pressure and the pulse wave signal. In this paper, we focus on the influence of individual differences from calculated blood pressure based on each calibration curve. In our study, the calculated blood pressure from both the individual and overall calibration curves were compared, and our results show that the calculated blood pressure based on the overall calibration curve had a lower measurement accuracy than that based on an individual calibration curve. We also found that the influence of the individual differences on the calculated blood pressure when using the FBG sensor method were very low. Therefore, the FBG sensor method that we developed for measuring the blood pressure was found to be suitable for use by many people.

Development of a nanoarray capable of the rapid and simultaneous detection of zearalenone, T2-toxin and fumonisin

Fusarium mycotoxins such as trichothecenes, zearalenone and fumonisins occur on a worldwide basis in cereal grains, animal feeds and forages. Practical solutions for multiple mycotoxin determination in samples are required by industry and regulators for cost effective screening purposes. The feasibility of developing a novel multiplex nanoarray for the simultaneous and semi-quantitative detection of three regulated mycotoxins: zearalenone (ZEA), T2-toxin (T2) and fumonisin B1 (FUM) was examined. Additionally, the assay was also able to detect HT2 toxin and fumonisin B2 and B3 due to the cross reactivity profiles of the antibodies used. Individual mycotoxin conjugates specific to the three mycotoxins were nano-spotted onto wells of a microtitre plate. Optimisation of assay parameters and antibodies was undertaken with both individual and multiplex calibration curves generated. A competitive assay format was employed enabling a calibration curve for concentration analysis and duplicate results for up to 40 samples in 70min for the three target mycotoxins. The characteristics and performance of the nanoarray were evaluated including sensitivity and specificity for each target. Additionally, intra and inter spotting precision, cross reactivity, matrix effects and sample analysis in maize and wheat (n=8) was performed. Sensitivity, determined as the concentration causing 50% inhibition, was 70.1, 2.8 and 90.9ppb in PBS, 172.4, 3.2 and 129.3ppb in methanol, 197.4, 0.7 and 216.7ppb in wheat and 43.6, 0.5 and 25.9ppb in maize for ZEA, T2 and FUM respectively. Intra spotting precision was 6%, 11% and 10% for PBS and 5%, 11% and 12% for methanol for ZEA, T2 and FUM respectively. Inter spotting precision was 4%, 14% and 6% for PBS and 3%, 9% and 16% for methanol for ZEA, T2 and FUM respectively. The feasibility of the nanoarray as an easy to use sensitive screening tool in the 96 well format has been demonstrated for the multiplex detection of three regulated mycotoxins. Improvements in automated image and data analysis software for novice end users are required to improve the overall rapidity of analysis.

Complementing the characterization of in vivo generated N-glucuronic acid conjugates of stanozolol by collision cross section computation and analysis.

Detailed structural information on metabolites serving as target analytes in clinical, forensic, and sports drug testing programmes is of paramount importance to ensure unequivocal test results. In the present study, the utility of collision cross section (CCS) analysis by travelling wave ion mobility measurements to support drug metabolite characterization efforts was tested concerning recently identified glucuronic acid conjugates of the anabolic-androgenic steroid stanozolol. Employing travelling-wave ion mobility spectrometry/quadrupole-time-of-flight mass spectrometry, drift times of five synthetically derived and fully characterized steroid glucuronides were measured and subsequently correlated to respective CCSs as obtained in silico to form an analyte-tailored calibration curve. The CCSs were calculated by equilibrium structure minimization (density functional theory) using the programmes ORCA with the data set B3LYP/6-31G and MOBCAL utilizing the trajectory method (TM) with nitrogen as drift gas. Under identical experimental conditions, synthesized and/or urinary stanozolol-N and O-glucuronides were analyzed to provide complementary information on the location of glucuronidation. Finally, the obtained data were compared to CCS results generated by the system's internal algorithm based on a calibration employing a polyalanine analyte mixture. The CCSs ΩN2 calculated for the five steroid glucuronide calibrants were found between 180 and 208 Å(2) , thus largely covering the observed and computed CCSs for stanozolol-N1'-, stanozolol-N2'-, and stanozolol-O-glucuronide found at values between 195.1 and 212.4 Å(2) . The obtained data corroborated the earlier suggested N- and O-glucuronidation of stanozolol, and demonstrate the exploit of ion mobility and CCS computation in structure characterization of phase-II metabolic products; however, despite reproducibly measurable differences in ion mobility of stanozolol-N1'-, N2'-, and O-glucuronides, the discriminatory power of the chosen CCS computation algorithm was found to be not appropriate to allow for accurate assignments of the two N-conjugated structures. Using polyalanine-based calibrations, significantly different absolute values were obtained for all CCSs, but due to a constant offset of approximately 45 Å(2) an excellent correlation (R(2) = 0.9997) between both approaches was observed. This suggests a substantially accelerated protocol when patterns of computed and polyalanine-based experimental data can be used for structure elucidations instead of creating individual analyte-specific calibration curves.

Validation of an assay for quantification of free normetanephrine, metanephrine and methoxytyramine in plasma by high performance liquid chromatography with coulometric detection: Comparison of peak-area vs. peak-height measurements.

Measurements of plasma concentrations of free normetanephrine (NMN), metanephrine (MN) and methoxytyramine (MTY) constitute the most diagnostically accurate screening test for pheochromocytomas and paragangliomas. The aim of this article is to present the results from a validation of an analytical method utilizing high performance liquid chromatography with coulometric detection (HPLC-CD) for quantifying plasma free NMN, MN and MTY. Additionally, peak integration by height and area and the use of one calibration curve for all batches or individual calibration curve for each batch of samples was explored as to determine the optimal approach with regard to accuracy and precision. The method was validated using charcoal stripped plasma spiked with solutions of NMN, MN, MTY and internal standard (4-hydroxy-3-methoxybenzylamine) with the exception of selectivity which was evaluated by analysis of real plasma samples. Calibration curve performance, accuracy, precision and recovery were determined following both peak-area and peak-height measurements and the obtained results were compared. The most accurate and precise method of calibration was evaluated by analyzing quality control samples at three concentration levels in 30 analytical runs. The detector response was linear over the entire tested concentration range from 10 to 2000pg/mL with R(2)≥0.9988. The LLOQ was 10pg/mL for each analyte of interest. To improve accuracy for measurements at low concentrations, a weighted (1/amount) linear regression model was employed, which resulted in inaccuracies of -2.48 to 9.78% and 0.22 to 7.81% following peak-area and peak-height integration, respectively. The imprecisions ranged from 1.07 to 15.45% and from 0.70 to 11.65% for peak-area and peak-height measurements, respectively. The optimal approach to calibration was the one utilizing an individual calibration curve for each batch of samples and peak-height measurements. It was characterized by inaccuracies ranging from -3.39 to +3.27% and imprecisions from 2.17 to 13.57%. The established HPLC-CD method enables accurate and precise measurements of plasma free NMN, MN and MTY with reasonable selectivity. Preparing calibration curve based on peak-height measurements for each batch of samples yields optimal accuracy and precision.