Peak Area Determination Research Articles

Determination of Gamma-Ray Abundance in Photopeaks with Analytic and Numeric Approaches via CERN’s ROOT Analysis Framework

For years in gamma-ray spectrometry, there has been numerious numeric methods implemented and dedicated to integrate the photopeak areas. With the advance of new technology and softwares, determination of peak areas have been digitized. In addition, new statistical approaches have given diverse perspectives in analysing the experimental data. In this case study, Maestro software was used to extract the experimental gamma-ray data from radioactive 60Co source. In general, analysts have been challenged by the calculation of background area under certain gamma-ray peaks when there is especially low counts. For instance, this problem lead them to find different net area values in various accuracies for the same peak. Therefore the precision of determination based on different statistics might affect the results. The systematic error, which is caused by the shapes and models of the background estimation, has been investigated by CERN’s ROOT software. This detailed analysis of digital data from NaI(Tl) scintaillation detector has been tabulated with its comparisons in various modern analytic methods and numeric approaches dated back to 1960s and 1970s.

Box-Behnken design avenue for development and validation of high-performance thin-layer chromatography method for estimation of rutin in Hemidesmus indicus.

The aim of this study was to determine the content of rutin in Hemidesmus indicus and to optimize the high-performance thin-layer chromatography method. The method was validated in compliance with the International Council for Harmonisation guidelines Q2 (R1) for parameters such as linearity, accuracy, precision, robustness, limit of detection, and limit of quantitation. A Box-Behnken design and response surface methodology has been used to investigate the impact of independent variables on the response. Three independent variables, mobile phase composition (% v/v), mobile phase volume (mL), and duration of saturation (min), were studied. Rutin was verified, and its content was determined using a validated high-performance thin-layer chromatography method with good linearity within the range of 200-1000 ng spot-1 with r2 = 0.9998 and correlation coefficient with calibration curve equation y = 0.0297x + 0.0001. The average percentage recovery values varied from 99.03 to 101.15 and 98.88 to 100.12%, respectively, for in-house and marketed mother tincture). The peak area determination at three different concentration levels shows low values of percentage relative standard deviation (<2%) for inter-day (0.04-0.06) and intra-day (0.04-0.05) precision of rutin. The average content of rutin in extract and marketed mother tincture was 229 ± 0.57 and 210 ± 0.57 μg g-1 . The proposed method was simple, precise, and accurate for the determination of rutin with frequent quality control assessment of H. indicus.

Interaction of CO with Pt nanoclusters on a graphene-covered Ru(0001) surface.

The adsorption of CO on Pt nanoclusters on a single layer of graphene epitaxially grown on the Ru(0001) surface [Gr/Ru(0001)] was studied with reflection absorption infrared spectroscopy (RAIRS) and temperature programmed desorption (TPD). The graphene layer was grown through exposure to ethylene using a method that has previously been shown to completely cover the surface. As CO adsorbs on Ru(0001) but not on graphene, the complete coverage of the Ru(0001) surface by graphene was verified with TPD as no CO adsorption was detectable. Previous work has demonstrated that Pt nanoclusters nucleate in the moiré unit cells of the Gr/Ru(0001) surface. Exposure of the Pt/Gr/Ru(0001) surface to CO gives rise to strong RAIRS peaks at 2065-2085 cm-1 assigned to CO at Pt atop sites and at 1848 cm-1 due to CO at Pt bridge sites. The CO TPD peak areas were used to quantify the CO coverage, which allowed for the determination of the RAIRS peak areas per CO molecule. It was found that the RAIRS intensity for CO on Pt/Gr/Ru(0001) is as much as nine times the intensity of CO on Ru(0001) on a per molecule basis. A more modest intensity enhancement was observed compared to CO on Pt islands on the Ru(0001) surface.

Performance assessment of a 500 mm3 CZT and a 2x2 inch LaBr3(Ce) detectors for the determination of the uranium enrichment using the enrichment-meter method and calibration standards for safeguards applications

Determination of the uranium enrichment is an important safeguards verification task, routinely carried out using non-destructive assay methods. The enrichment-meter method is one of the most widely used passive non-destructive X- and gamma-ray based methods used for such tasks. Among its advantages is the highly constrained physical nature of its underlying formalism, allowing it to be used with high-resolution HPGe detectors, as well as with low-resolution NaI detectors. Due to attractive features and spectroscopic performance, CdZnTe and LaBr3(Ce) detectors raised interest in their application to such tasks as well. However, their spectroscopic performance is different to that of the traditional detectors in many ways. Application of the enrichment-meter method requires determination of the net peak areas corresponding to 235U signature photopeaks. The latter requires an adequate algorithm to select the region-of-interest boundaries, which may be sensitive to asymmetrical photopeaks of CZT detectors. In this paper we conduct a performance assessment of a 500 mm3 CZT detector of a quasi-hemispherical design and a 2 × 2 inch LaBr3(Ce) scintillator with the enrichment-meter method using a set of certified uranium standards with enrichment degrees from 0.31% to 4.46% of 235U atomic abundance. We investigate the impact of different methods used for net peak area determination, statistical quality of acquired spectra and size of region-of-interest boundaries on accuracy and uncertainty. We propose an algorithm for symmetrical/asymmetrical region-of-interest boundaries determination and make recommendations on the best combinations of the region-of-interest size and method used for the net peak area determination for each of the detectors. The underlying routines of the algorithm and analysis procedures are described in detail and results are presented.

Silicon Drift Detector response function for PIXE spectra fitting

Abstract The correct determination of the X-ray peak areas in PIXE spectra by fitting with a computer program depends crucially on accurate parameterization of the detector peak response function. In the Guelph PIXE software package, GUPIXWin, one of the most used PIXE spectra analysis code, the response of a semiconductor detector to monochromatic X-ray radiation is described by a linear combination of several analytical functions: a Gaussian profile for the X-ray line itself, and additional tail contributions (exponential tails and step functions) on the low-energy side of the X-ray line to describe incomplete charge collection effects. The literature on the spectral response of silicon X-ray detectors for PIXE applications is rather scarce, in particular data for Silicon Drift Detectors (SDD) and for a large range of X-ray energies are missing. Using a set of analytical functions, the SDD response functions were satisfactorily reproduced for the X-ray energy range 1–15 keV. The behaviour of the parameters involved in the SDD tailing functions with X-ray energy is described by simple polynomial functions, which permit an easy implementation in PIXE spectra fitting codes.

Comparison of multivariate curve resolution strategies in quantitative LCxLC: Application to the quantification of furanocoumarins in apiaceous vegetables

Comprehensive two-dimensional liquid chromatography (LC × LC) has been gaining popularity for the analysis of complex samples in a wide range of fields including metabolomics, environmental analysis, and food analysis. While LC × LC can provide greater chromatographic resolution than one-dimensional LC (1D-LC), overlapping peaks are often still present in separations of complex samples, a problem that can be alleviated by chemometric curve resolution techniques such as multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS has also been previously shown to assist in the quantitative analysis of LC x LC data by isolating pure analyte signals from background signals which are often present at higher levels in LC x LC compared to 1D-LC. In this work we present the analysis of a dataset from the LC × LC analyses of parsley, parsnip and celery samples for the presence and concentrations of 14 furanocoumarins. Several MCR-ALS implementations are compared for the analysis of LC × LC data. These implementations include analyzing the LC x LC chromatogram alone, analyzing the one-dimensional chromatogram alone, as well as two hybrid approaches that make use of both the first and second dimension chromatograms. Furthermore, we compared manual integration of resolved chromatograms versus a simple summation approach, using the resolved chromatographic peaks in both cases. It is found that manual integration of the resolved LC × LC chromatograms provides the best quantification as measured by the consistency between replicate injections. If the summation approach is desired for automation, the choice of MCR-ALS implementation has a large effect on the precision of the analysis. Based on these results, the concentrations of the 14 furanocoumarins are determined in the three apiaceous vegetable types by analyzing the LC × LC chromatograms with MCR-ALS and manual integration for peak area determination. The concentrations of the analytes are found to vary greatly between samples, even within a single vegetable type.

Analysis of N-acetylmuramic acid-6-phosphate (MurNAc-6P) Accumulation by HPLC-MS.

We describe here in detail a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based method to determine N-acetylmuramic acid-6-phosphate (MurNAc-6P) in bacterial cell extracts. The method can be applied to both Gram-negative and Gram-positive bacteria, and as an example we use Escherichia coli cells in this study. Wild type and mutant cells are grown for a defined time in a medium of choice and harvested by centrifugation. Then the cells are disintegrated and soluble cell extracts are generated. After removal of proteins by precipitation with acetone, the extracts are analyzed by HPLC-MS. Base peak chromatograms of wild type and mutant cell extracts are used to determine a differential ion spectrum that reveals differences in the MurNAc-6P content of the two samples. Determination of peak areas of extracted chromatograms of MurNAc-6P ((M-H)- = 372.070 m/z in negative ion mode) allows quantifying MurNAc-6P levels, that are used to calculate recycling rates of the MurNAc-content of peptidoglycan.

Chemometric approach to fatty acid metabolism-distribution networks and methane production in ruminal microbiome

Methane emission has been attracting more and more attention. Unfortunately, a lot of factors influence methane emission (chemical structure of metabolites, time, methane, gas pressure, microbiome composition, diet, etc.). We propose a new chemometric methodology to integrate different laboratory experiments in this field. Firstly, we report (1) new laboratory experiments to measure by separating (1a) methane production (gas phase), (1b) volatile fatty acid (VFA) distribution (liquid phase) and (1c) fatty acid (FA) distribution in rumen microbiome. Next, we also report a new (2) chemometric methodology that integrates all the data in a single theoretical model. The laboratory work includes two experimental sections (a) to measure the methane production, pH, gas pressure, temperature and (b) FA distribution. Section (b) includes two different experimental parts: chromatographic determination of internal peak areas (IPA%) of (b.1) long-chain fatty acids (LCFA) and (b.2) VFA. In all studies, we can use different treatments, distribution phases (media, bacteria, or protozoan microbiome), cis/trans patterns, experimental protocols, etc. Next, we combined perturbation theory (PT), linear free-energy relationships (LFER), linear discriminant analysis (LDA), and artificial neural networks (ANNs) to develop linear and non-linear models of perturbations in methane production–fatty acid distribution network. The best PT-LFER model found presented values of sensitivity, specificity, and accuracy>0.94, and Matthews correlation coefficient (MCC)>0.894 for 545,695 cases of perturbations in experimental data. This methodology may be useful to quantify the effect of perturbations due to the changes in experimental conditions in the study of fatty acid distribution when we need to carry out parallel experiments in different phases.

Uncertainties in gamma-ray spectrometry

High resolution gamma-ray spectrometry is a well-established metrological technique that can be applied to a large number of photon-emitting radionuclides, activity levels and sample shapes and compositions. Three kinds of quantitative information can be derived using this technique: detection efficiency calibration, radionuclide activity and photon emission intensities. In contrast to other radionuclide measurement techniques gamma-ray spectrometry provides unambiguous identification of gamma-ray emitting radionuclides in addition to activity values. This extra information comes at a cost of increased complexity and inherently higher uncertainties when compared with other secondary techniques. The relative combined standard uncertainty associated with any result obtained by gamma-ray spectrometry depends not only on the uncertainties of the main input parameters but also on different correction factors. To reduce the uncertainties, the experimental conditions must be optimized in terms of the signal processing electronics and the physical parameters of the measured sample should be accurately characterized. Measurement results and detailed examination of the associated uncertainties are presented with a specific focus on the efficiency calibration, peak area determination and correction factors. It must be noted that some of the input values used in quantitative analysis calculation can be correlated, which should be taken into account in fitting procedures or calculation of the uncertainties associated with quantitative results. It is shown that relative combined standard uncertainties are rarely lower than 1% in gamma-ray spectrometry measurements.

Development and validation of an LC-MS/MS method for determination of the L-type voltage-gated calcium channel/NMDA receptor antagonist NGP1-01 in mouse serum.

NGP1-01 (8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane) is a heterocyclic cage compound with multifunctional calcium channel blocking activity that has been demonstrated to be neuroprotective in several neurodegenerative models. A sensitive internal standard LC-MS/MS method was developed and validated to quantify NGP1-01 in mouse serum. The internal standard (IS) was 8-(2-phenylethylamino)-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane. Sample preparation involved a protein precipitation procedure by addition of acetonitrile. Chromatographic separation was carried out on a Phenomenex Kinetex phenyl-hexyl column (100 mm×2.1mm, 2.6 μm) employing a gradient (45% isocratic 3 min, 45-95% linear gradient 6 min, 95% isocratic 3 min) of an elution mobile phase of 5mM ammonium acetate in 100% acetonitrile mixing with an application mobile phase of 5mM ammonium acetate in 2% acetonitrile. Detection was achieved by a QTrap 5500 mass spectrometer (AB Sciex) employing electrospray ionization in the positive mode with multiple-reaction-monitoring (MRM) for NGP1-01 (m/z 266→91) and IS (m/z 280→105). The method validation was carried out in accordance with Food and Drug Administration (FDA) guidelines. The method had a linear range of at least 0.5-50 ng/mL with a correlation coefficient 0.999. The intra-assay and inter-assay precisions (%CV) were equal to or within the range of 1.0-4.3% and the accuracies (% relative error) equal to or within -2.5% to 3.4%. The analyte was stable for at least 2 months at -20°C, for at least 8h at room temperature and for at least three freeze-thaw cycles. The extraction recovery was 94.9 to 105.0%, with a %CV ≤ 9.5%. The technique was found to be free of any matrix effects as determined by experiments involving five different lots of mouse serum. Cross-talk interferences were not present. Two different gradient slope chromatography runs were done on dosed mouse serum samples to assess a possible positive error in peak area determination from in-source fragmentation of metabolites generating the same MRM transitions as the parent drug or IS. No such interference was found in the NGP1-01 peak, while a minor interference was identified in the IS peak. The optimized method was applied to the measurement of NGP1-01 in serum of dosed mice.

Comparison of different approaches to estimate uncertainty budget in k 0-INAA measurement

Three CRMs of different matrix composition were analysed, representing an environmental matrix sample (BCR–320R Channel Sediment), a botanical matrix sample (SRM 1547 Peach Leaves) and a zoological matrix sample (SRM 1566b Oyster Tissue). The element mass fractions were obtained using the KayWin program. Analytical measurement uncertainty was determined by two approaches: (1) the routine procedure applying combination of the overall uncertainty u(m) = 3.5 % and statistical uncertainty of the peak area determination and (2) the procedure applying the dedicated ERON program for calculating uncertainty. Performance of altogether 31 certified values was tested by means of calculating En numbers. For the remaining 52 non-certified values, comparison between uncertainties obtained by the two approaches was made. When using the first approach, the En number showed satisfactory performance in 28 cases; by using the second approach, the En number showed satisfactory performance in 27 cases. None of the unsatisfactory performances (En > 1) appeared to be of systematic nature. The uncertainties obtained by applying the two approaches revealed a big extent of consistency. As the present nuclear database lacks lot of data that serve as input to the ERON program, in particular uncertainties of Q0 factors, estimates need to be introduced for the missing values, emphasising the urgent need to upgrade the database with missing data.

Doublet peak area determination in NaI(Tl) scintillation spectrometry using maximum likelihood estimation

A method has been studied for the analysis of the pulse-height spectrum of a mixed 134Cs and 137Cs source obtained with a NaI(Tl) scintillation spectrometer. A peak fitting technique using maximum likelihood estimation was used with a specially developed deconvolution computer program. The activities obtained by this program were compared with the reference value in order to validate the analysis algorithm. Peak-area determination for NaI(Tl) scintillation spectrometry was successfully carried out even for spectra that included peak doublet and were recorded with low statistics. The results obtained by the maximum likelihood estimation agreed with each reference value within their statistical uncertainties.

Uncertainty evaluation in radon concentration measurement using charcoal canister

Active charcoal detectors are used for testing the concentration of radon in dwellings. The method of measurement is based on radon adsorption on coal and measurement of gamma radiation of radon daughters. The contributions to the final measurement uncertainty are identified, based on the equation for radon activity concentration calculation. Different methods for setting the region of interest for gamma spectrometry of canisters were discussed and evaluated. The obtained radon activity concentration and uncertainties do not depend on peak area determination method.

On scattering effects for volume sources in low-energy photon spectrometry

In this study, different aspects of the Compton scattering inside volume sources are illustrated using experimental approach and Monte Carlo simulation. For the low-energy range (below 100keV) scattered events represents around 30% of the whole spectrum. Influence of the source-detector geometry is discussed. The scattering effects induce strong differences in spectrum shape for different geometrical conditions. This should influence efficiency transfer factors. A new approach is proposed, including the scattered events, to avoid complex peak area determination.

Method for Determination of Peak Areas in Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) facilitates determination of both the kinetic constants (k(off)) and the equilibrium constants (K(d)) of complex dissociation from a single experiment. A typical NECEEM electropherogram consists of two peaks and an "exponential bridge" between them, smoothly merging into the peaks. The values of k(off) and K(d) are usually calculated with simple algebraic formulas, by utilizing the areas of the peaks and the bridge. Accurate determination of the two constants requires accurate positioning of the two boundaries separating the bridge from the peaks. Here, we propose a more systematic method for the determination of boundaries between the peaks and the bridge. The method involves a simple geometrical analysis of a NECEEM electropherogram based on an assumption of symmetry in ordinary electrophoretic peaks. To test the method, we (i) constructed a series of computer-simulated NECEEM electropherograms, (ii) determined the two boundaries with our method, and (iii) calculated the values of k(off) and K(d). We found that the deviation of the calculated values from those used to simulate the electropherograms did not exceed 15% for k(off) and 25% for K(d), as long as the peaks and the bridge were visually identifiable. We finally applied the method to the determination of K(d) and k(off) values for the interaction between AlkB protein and its DNA aptamer. The developed method for rational boundary determination in NECEEM will facilitate accurate data analysis in a simple and efficient manner.

On-Line Analysis of a Continuous-Flow Ozonolysis Reaction Using Raman Spectroscopy

We describe the use of an on-line Raman analyzer to quantitatively track the levels of trans-stilbene, benzaldehyde, and alpha-methoxybenzyl hydroperoxide in a continuous flow ozonolysis reactor. Quantitative analysis is carried out using spectral stripping in order to overcome baseline artifacts inherent in simple peak area determinations and to incorporate prior knowledge into the analytical model. The performance of spectral stripping is compared to partial least squares (PLS) analysis. The effect of two process variables, feed flow rate and ozone concentration, are also explored. Finally, the benefit of utilizing an on-line technique is illustrated by demonstrating the bias introduced by sampling for off-line analysis.

Interconversion of Oxazepam Enantiomers During HPLC Separation. Determination of Thermodynamic Parameters

Oxazepam undergoes reversible enantiomerization at ambient temperature. Standard solutions of this compound and extracts from tablets (both with c=0.1 mg/mL) were injected on a chiral HPLC column (Chirobiotic T) at various temperatures (273 K‐313 K, increment 5 K). The mobile phase with the composition MeOH/TEA/Hac (100/0, 1/0, 1) and flow rate 1.0 mL/min were used.1 Both the separation and the interconverson process occurred simultaneously. The various profiles obtained by a UV detector included two peaks (unreacted zone) and a plateau sandwiched between two peaks (reacted zone). These profiles could be explained by on‐column enantiomerization. The unreacted molecule zone on the profile was fused with the reacted molecule zone. The computer assisted peak deconvolution procedure (Origin 7.0 section peak fitting) was used for the determination of peak areas in peak clusters. The deconvolution of experimentally obtained chromatograms enabled the resolved and determined areas of each zones. As follows, the peak areas were used for calculation of thermodynamic parameters of enantiomerization – apparent rate constants (k 1 app and k −1 app ), Gibbs free energy (ΔG 1 app , ΔG −1 app ), enthalpy (ΔH 1 app , ΔH −1 app ), and entropy (ΔS 1 app , ΔS −1 app ).

A simple high-performance liquid chromatography assay for on-line determination of catecholamines in adrenal gland by direct injection on an ISRP column

In the present research, assays were improved for the determination of catecholamines in adrenal gland. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The method involved direct injection of acid extract on to a serum albumin dimethylocadecyl-silane (HSA-C 18) and the utilization of phosphate buffer (pH=3.0): methanol (97:3 v/v), 0.025 g heptanosulfonic acid and 0.0025 g EDTA as mobile phase. The detection was obtained using an electrochemical detector L-ECD-6A-Shimadzu with a potential of the 85 mV. Identification was based on retention time. Quantification was performed by automatic peak-area determination. The detection limit is equal to 0.5 ng ml −1. The HPLC method with electrochemical detection proposed here permits good separation of catecholamines in samples of adrenal gland from rats. The method has various advantages: fast, high precision and good selectivity and do not require sample treatment. The immobilization stress during 5 min did not provoke alteration in catecholamines contains in rat adrenal gland, due to the short time of the stress exposure. This study shows that the catecholamines (norepinephrine and epinephrine) adrenal increased significantly after the acute immobilization stress during 30 min as compared to control group. This increase probably is due to the emotional component of the immobilization stress. In conclusion, our studies suggest an effective participation of the adrenal glands to maintain the homeostasis of organism to the stressful conditions.

Resolution and sensitivity as a function of energy and incident geometry for germanium detectors

The use of modeling programs such as MCNP to predict the response of HPGe detectors is increasing in importance. Accurate simulation of germanium detectors to incident gamma rays relies on knowledge of the performance of the detector in different detector–source geometries. Two important performance parameters are the resolution and sensitivity. The resolution is the FWHM and FW.1M/FWHM ratio. The IEEE 325-1996 standard only specifies the FWHM measurement at one geometry and two energies. Nearly all measurements are made in a different geometry and at other energies. Other investigators [Specifications for Today’s Coaxial HPGe Detectors, 2001 ANS Annual Meeting, Milwaukee, WI; Metzger, private communication, see also: Radionuclide Depth Distribution by Collimated Spectroscopy, 2002 ANS Topical Meeting, Santa Fe, NM], have shown that the sensitivity and resolution change with position of the incident gamma ray on the front of the detector. Such variability has possible implications for the accuracy of peak shape and area determination, since the calibration is potentially a function of angle of incidence. To quantify the sensitivity and resolution variation as a function of energy and point of incidence, measurements have been made on several coaxial detectors of various crystal types and sizes in different source–detector geometries. The full-energy peaks from 59 keV to 2.6 MeV were used. The detectors were placed in a low-background shield to reduce any contribution from external sources. None of the detectors tested was a low-background type. The sources used were an 241Am source, 60Co source and a natural thorium oxide sample. The 241Am 59 keV gamma rays were collimated by a 2 cm thick, 1 mm diameter lead collimator. Several gamma rays from the thorium source were used and collimated by a 10 cm thick and 2 mm diameter tungsten collimator. These collimated sources were used to collect spectra for the incident beam on the front and sides of the detectors. The peak widths were calculated using the methods outlined in IEEE 325-1996. Data are presented to show that the peak shape and sensitivity change with incident beam position and full peak energy.

Quantification of human brain metabolites from in vivo 1H NMR magnitude spectra using automated artificial neural network analysis.

Long echo time (TE=270 ms) in vivo proton NMR spectra resembling human brain metabolite patterns were simulated for lineshape fitting (LF) and quantitative artificial neural network (ANN) analyses. A set of experimental in vivo 1H NMR spectra were first analyzed by the LF method to match the signal-to-noise ratios and linewidths of simulated spectra to those in the experimental data. The performance of constructed ANNs was compared for the peak area determinations of choline-containing compounds (Cho), total creatine (Cr), and N-acetyl aspartate (NAA) signals using both manually phase-corrected and magnitude spectra as inputs. The peak area data from ANN and LF analyses for simulated spectra yielded high correlation coefficients demonstrating that the peak areas quantified with ANN gave similar results as LF analysis. Thus, a fully automated ANN method based on magnitude spectra has demonstrated potential for quantification of in vivo metabolites from long echo time spectroscopic imaging.