Standard Calibration Research Articles

Development and Validation of Polymer-based Porphyrin-Incorporated Reference Materials for Calibration of Quantitative Light-induced Fluorescence device

BackgroundThis study aimed at developing and validating polymer-based reference materials with varying amounts of porphyrin to accurately assess and calibrate quantitative light-induced fluorescence (QLF) device. MethodsReference materials with porphyrin concentrations ranging from 0 to 0.08 wt.% were prepared. The surface properties of the materials were analyzed via gloss and roughness measurements. Color analysis of the specimens was performed on black and white backgrounds with or without filters using a spectrophotometer. This approach revealed the correlations between fluorescence and color. The fluorescence emitted by the specimens was analyzed by measuring ΔR and ΔRmax values using a QLF-D BiluminatorTM. ResultsThe surface gloss and roughness of the reference materials were not affected by the porphyrin content (p > 0.005). Spectrophotometric measurements revealed significant color differences among most specimen groups depending on the background color and the presence of a filter. QLF-D imaging revealed significant differences in fluorescence (ΔR and ΔRmax) among all specimen groups regardless of the background. The fluorescence values observed on the black backgrounds were higher than those observed on the white backgrounds (p < 0.05). ConclusionsThe developed polymer-based porphyrin-incorporated materials serve as reliable reference standards for accurate assessment and calibration of QLF devices. This study demonstrates the importance of background conditions in fluorescence detection and highlights the potential of these materials as standards for QLF device calibration.

Factors influencing the dissolution rate of radon gas in water

Measurements of radon (222Rn) in water are widely utilized across various disciplines, including technology, medicine, exploration, and ecological preservation. For accurate radon measurements in water, the availability of a standardized radon solution is essential. Researchers have predominantly concentrated on the radon concentration in water (RCW) once radon distribution equilibrium is achieved, rather than the rate of radon dissolution prior to equilibrium, in the development of standard radon solutions. This makes the preparation speed of the calibration standards radon solution slow, which seriously restricts the research and development of the standard device. The purpose of this paper is to investigate the correlation between the rate of radon dissolution and both the concentration of gaseous radon and the stirring speed. Four conclusions have been derived through univariate analysis. ➀ The radon concentration in water (RCW) increases with the duration of radon dissolution, eventually reaching a state of equilibrium. ➁ The increase in gas radon concentration correlates with a simultaneous rise in the rate of radon dissolution, while the time required for radon distribution equilibrium remains constant. ➂ The augmentation of the stirring speed enhances the rate of radon dissolution, Concurrently reducing the time needed for RCW to reach equilibrium.➃ When the stirring speed is excessively high, it results in substantial fluctuations in both RCW and the rate of radon dissolution. Consequently, when employing this method for the preparation of radon solutions, it is advisable to regulate the duration of radon dissolution to 30 min at a rate of 380 rad/min and 10 min at a rate of 480 rad/min. This regulation ensures the attainment of precise concentrations. Under conditions of environmental stability, manipulation of gas radon concentration alone proves sufficient for adjusting RCW . These empirical findings furnish a robust foundation for the efficient preparation of standardized radon solutions and the requisite apparatus, essential for the calibration and accuracy of radon measurement instruments.

Smartcard: an integrated approach for contaminant monitoring, from field to laboratory.

Effective food safety monitoring requires a multi-step approach from farm to fork, involving different methods, ranging from convenient screening devices to sophisticated laboratory confirmatory testing. However, sample transportation to routine laboratories is time-consuming and expensive. Simplified on-site sampling followed by laboratory analysis offers a potential solution. Dried blood spot (DBS) cards ensure stability and ease of sample transportation and are used in clinical testing. However, the applicability of such an approach could be broader and include the storage of dried extract from more complex (solid) matrices. Therefore, a simplified approach is presented here, using DBS cards for on-site sampling and subsequent laboratory confirmation for food contaminants. To achieve this, an analytical tool (Smartcard) was designed using 3D-printing technology. As a proof of concept, the approach was applied to detect the pesticide fipronil, which is widely used in ornamental flower production to limit pests and on poultry farms. The Smartcard can securely store the sample extracts on a DBS card (dried extract spot (DExS) card), incorporate the lateral flow immunoassay (LFIA) and immediately provide an estimate of contamination levels. After simplified in-syringe extraction of the sample, the LFIA allows direct screening of fipronil (half maximum inhibitory concentration of 6.5µg/l with calibration standards), and the same sample extract can be directly applied to the DExS card for storage and transport to the laboratory, where analyte re-extraction and instrumental analysis is performed using ultra high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detecting fipronil down to 0.8µg/kg.

Resistance standards with very small calculable AC-DC difference at frequencies up to 2 MHz for the calibration of precision LCR meters

Abstract We have developed novel impedance standards based on thin-film surface mount device (SMD) resistors. Due to the small dimensions of such resistors, the frequency dependence is very small and can be calculated from quantities that can be either measured or numerically calculated. For nominal resistance values in the 10 kΩ range, as for our application, the DC and AC properties of a single thin-film SMD resistor are not sufficient but can be greatly improved by connecting several thin-film SMD resistors in series. Then the calculated frequency dependence at frequencies up to 2 MHz amounts to only a few parts per million of the DC value, which is about four orders of magnitude smaller than for all conventional calculable AC-DC resistors with a similar nominal DC value. A precision inductance-capacitance-resistance (LCR) meter for frequencies up to 2 MHz, which has a resolution and a reproducibility of a few parts per million but a systematic uncertainty of up to 3000 parts per million, is used to measure two very different SMD-based resistance standards. This enables the verification of the model calculation as well as the calibration of the LCR meter and the correction of its systematic effects, both with a relative uncertainty of a few parts per million in the whole frequency range. This boost in precision enables new applications in this frequency range such as the verification of conventional calculable resistance standards, the calibration of impedance standards, and future measurements of the quantum Hall resistance.We have developed novel impedance standards based on thin-film surface mount device&amp;#xD;

Ionizing Radiation Dose to the Skin Assessed after Rapid Detection of 232Th in Consumer Products.

After some consumer products indicated elevated levels of 232Th progeny by gamma-ray spectrometry, a microwave digestion and inductively coupled plasma-mass spectrometry (ICP-MS) procedure was implemented for the direct assay of 232Th content to ensure compliance with Federal regulations and guidelines. Levels of 232Th were determined by ICP-MS based on standard calibration using a 205Tl internal standard. The method had a method detection limit (MDL) of 0.15 Bq g-1 and a lower limit of quantification (LLOQ) of 0.65 Bq g-1 for 232Th, making it a suitable confirmatory method following gamma-ray spectrometry. The 232Th activity concentration calculated from the ICP-MS results ranged from 2.0-3.4 Bq g-1 for the kinesiology tape samples and 20 Bq g-1 for the silicone ion bracelet. The VARSKIN+1.0 software program was used to calculate the shallow dose equivalent of ionizing radiation from 232Th and its progeny from the ICP-MS results. The skin dose to the consumer wearing the kinesiology tape ranged from 0.48-1.6 mSv y-1. The skin dose to the consumer with constant wear of the silicone ion bracelet was estimated to be 17 mSv y-1. Although 232Th may be determined indirectly by assay of high abundance gamma rays produced by its progeny, the US Code of Federal Regulations (CFR) requires the direct assay of 232Th for confirmatory analysis. We found this ICP-MS method to be a rapid 232Th confirmatory technique compared to a chemical separation followed by alpha spectrometry procedure.

Uncertainties of S-Parameter Measurements in Rectangular Waveguides at PTB

Abstract. In this work the determination of measurement uncertainties in scattering parameter measurements for waveguide interfaces ranging from R 100 (WR 90) to R 2.6k (WR 3, WM-864) is presented. For each waveguide band a Thru Reflect Line calibration is performed including uncertainties for calibration standards, cable movement, interface repeatability and the characteristics of the vector network analyzer. For reflection and transmission coefficients, envelopes of uncertainties are determined for magnitude and phase angle respectively. In addition, an experiment on connection (interface) repeatability for R 140 was carried out to systematically investigate the influence of shifting and rotating of waveguide flanges. Translation values in steps of 0.3 mm up to 1.5 mm are examined in simulation as well as measurement. The findings of these investigations can be extended and applied to other waveguide bands.

OPTIMIZATION OF LC-MS/MS METHOD FOR THE SIMULTANEOUS DETERMINATION OF METFORMIN AND ROSIGLITAZONE IN HUMAN PLASMA WITH BOX-BEHNKEN DESIGN

Objective: The objective of this study was to develop a robust Liquid Chromatography – Tandem Mass Spectrometry (LC-MS/MS) methodology for the precise quantification of metformin and rosiglitazone in human plasma. Methods: A Design of Experiments (DOE) framework was utilized, specifically employing a Box-Behnken experimental design, to optimize critical parameters such as Capillary voltage, Cone voltage, Desolvation temperature, and Collision energy. Sample preparation involved protein precipitation using acetonitrile, simplifying the procedure. Chromatography was performed with a mobile phase of 0.1% formic acid and acetonitrile (60:40 V/V) to enhance sensitivity and reproducibility. Quantification was achieved through Multiple Reaction Monitoring (MRM) of the transition’s m/z 130.1 → m/z 60.1 for metformin, m/z 358.2 → m/z 134.9 for rosiglitazone, and m/z 206.3 → m/z 59.9 for phenformin. The methodology was validated according to regulatory guidelines. Results: The developed methodology demonstrated selectivity, linearity, accuracy, precision, recovery, and stability. The calibration curve showed linearity over the concentration range of 5 ng/ml to 1000 ng/ml for metformin and 1.5 ng/ml to 300 ng/ml for rosiglitazone. Accuracy and precision were within acceptable limits across calibration and quality control standards. Assessments of extraction recovery and matrix effects confirmed the robustness of the extraction procedure, with negligible interference from plasma components. Stability studies indicated that the method maintained acceptable limits for metformin and rosiglitazone concentrations under various storage and handling conditions. Conclusion: The validated Liquid Chromatography – Tandem Mass Spectrometry (LC-MS/MS) methodology provides a reliable and accurate platform for the quantification of metformin and rosiglitazone in human plasma. This method shows potential applications in pharmacokinetic studies and clinical research, ensuring consistent performance in routine analysis.

Consideration of spectral interference in total reflection X-ray fluorescence analysis using a limited number of calibration samples: Case study of ocean polymetallic nodules

A novel approach for quantitative analysis in total-reflection X-ray fluorescence (TXRF) which can be employed for the samples with complex matrices inducing overlapping spectral signals in the scenario of limited availability of standard samples is proposed. The approach is based on the least squares (LS) decomposition of real spectra into the weighted sum of simulated individual element subspectra and requires only a single sample of matrix-matched reference material. Oceanic polymetallic nodules and crusts, having complex mineral matrix and containing high concentrations of overlapping elements, were chosen to demonstrate the feasibility of the approach in quantification of 11 elements. The comparison of the results obtained with LS decomposition, internal standard and linear calibration with three standard samples was performed. It was demonstrated that the performance of the proposed approach and of the linear calibration was better than that of internal standard for most of the elements; however, the proposed method requires only a single standard sample.

High performance SERS boosting by Fabry- Pérot cavities of silica-gold-silicon multilayers

Surface-enhanced Raman scattering (SERS), an advanced technique for molecular spectroscopy, relies heavily on the preparation of SERS active materials that can significantly enhance the Raman scattering signals for highly sensitive detection of trace molecules. Traditionally, SERS measurements are performed on silicon or silica substrates, the SERS performance is determined by the structure of SERS materials. Here, we show that the SERS signal can be amplified and modulated using Fabry-Pérot (F-P) cavities made of silica-silicon (SiO2-Si) or silica-gold-silicon (SiO2-Au-Si) multilayers as substrates. Periodic SERS signal variations as SiO2 thickness increases are observed, exhibiting optimal enhancement with the SiO2 thickness of 250 nm due to the optical interference in the cavity. Although the signal enhancement by optical interference is weaker than that by plasmonic resonance, additional signal amplification is essential for highly sensitive SERS materials. Moreover, we applied this strategy to detect thiram in bean sprout extracts, demonstrating that the detection sensitivity is two orders of magnitude higher than that using Si substrates. The utilization of the pseudo-internal standard intensity calibration method facilitates the quantitative analysis of thiram concentrations. Our results provide a promising approach for further amplification of SERS signals with great potential for practical applications.

Utilizing collagen-coated hydrogels with defined stiffness as calibration standards for AFM experiments on soft biological materials: the case of lung cells and tissue

Abstract Atomic Force Microscopy (AFM) is crucial in mechanobiology for high-resolution imaging and nanomechanical measurements of biological samples, providing insights into their mechanical properties. However, AFM faces challenges such as tip damage and cantilever selection errors, impacting measurement accuracy. This study proposes a methodology using collagen-coated hydrogels with predefined stiffness for calibrating AFM measurements on soft biological materials. By facilitating appropriate cantilever selection, assessing systematic errors, and evaluating tip damage, this approach ensures reliable Young’s modulus measurements. The proof of concept with human lung cells and tissue specimens demonstrates improved accuracy and reliability of AFM-based nanomechanical characterizations, essential for understanding cellular mechanics and disease progression.

RainForests: A Machine Learning Approach to Calibrating NWP Precipitation Forecasts

Abstract Probabilistic forecasts derived from ensemble prediction systems (EPSs) have become the standard basis for many products and services produced by modern operational forecasting centers. However, statistical postprocessing is generally required to ensure forecasts have the desired properties expected for probability-based outputs. Precipitation, a core component of any operational forecast, is particularly challenging to calibrate due to its discontinuous nature and the extreme skew in rainfall amounts. A skillful forecasting system must maintain accuracy for low-to-moderate precipitation amounts, but preserve resolvability for high-to-extreme rainfall amounts, which, though rare, are important to forecast accurately in the interest of public safety. Existing statistical and machine learning approaches to rainfall calibration address this problem, but each has drawbacks in design, training approaches, and/or performance. We describe RainForests, a machine learning approach for calibrating ensemble rainfall forecasts using gradient-boosted decision trees. The model is based on the ecPoint system recently developed at ECMWF by Hewson and Pillosu, but uses machine learning models in place of the semisubjective decision trees of ecPoint, along with some other improvements to the model structure. We evaluate RainForests on the Australian domain against some simple benchmarks and show that it outperforms standard calibration approaches both in overall skill and in accurately forecasting high rainfall conditions, while being computationally efficient enough to be used in an operational forecasting system. Significance Statement Weather forecasting typically uses physical models to simulate the atmosphere and produce a set of scenarios, called an ensemble, for each forecast time. Forecast calibration is a set of statistical techniques for using these ensemble forecasts to obtain probability forecasts of weather variables. For example, on a given day, we may predict that there are a 90% chance of at least 1 mm of rain and a 50% chance of at least 3 mm. We introduce RainForests, a new machine learning method for calibrating ensemble forecasts, which uses ensemble forecasts of rainfall and other related variables to produce more accurate probability forecasts of rainfall amounts in operational forecasting settings.

Multi-species cryoEM calibration and workflow verification standard.

Cryogenic electron microscopy (cryoEM) is a rapidly growing structural biology modality that has been successful in revealing molecular details of biological systems. However, unlike established biophysical and analytical techniques with calibration standards, cryoEM has lacked comprehensive biological test samples. Here, a cryoEM calibration sample consisting of a mixture of compatible macromolecules is introduced that can not only be used for resolution optimization, but also provides multiple reference points for evaluating instrument performance, data quality and image-processing workflows in a single experiment. This combined test specimen provides researchers with a reference point for validating their cryoEM pipeline, benchmarking their methodologies and testing new algorithms.

Determination and Quantification of Folpet in Barley using HPLC-DAD Analysis

Although rоutinе pesticide analysеs are mostly performed using GC-MS, GC-MS/MS and/or LC-MS/MS, some pesticides especially those with relatively high MRLs and good detector response could also be precisely quantified with less sensitive equipment which gives the opportunity to some laboratories to expand their scope of work. The study focuses on HPLC-DAD method development for detection of folpet in barley grain. QuEChERS method was performed for extraction after which, additional clean-up was performed using PSA and polypropylene filtration. Pesticide separation was achieved using mobile phase of acidified CAN (pH 2.5) and water. The initial mobile phase was 60:40% (v/v), the elution gradient starts from 80:20% (v/v) to 100:0% (v/v) in 7 min and holds 100:0% (v/v) up to 8.5 min at a flow rate of 1 ml/min after which in the next 6.5 min the column was re-equilibrated to 40% phase-B. Calibration was performed using matrix-matched calibration standards. The obtained limits of detection (LOD) and quantification (LOQ) were 0.18 and 0.55 mg/kg, respectively, and the linear regression coefficient was 0.9973. Recovery, repeatability and reproducibility were investigated at three fortification levels (0.1 mg/kg, 0.6 mg/kg, and 1.6 mg/kg) and were found acceptable with relative standard deviation less than 10%. The method was applied for the analysis of three barley grain samples obtained from different producers and showed that folpet was present in one sample below the LOQ. Overall, the developed method is suitable for the determination of folpet in barley grains at levels below the established MRL of 1 mg/kg.

A Novel PCR-Free Ultrasensitive GQD-Based Label-Free Electrochemical DNA Sensor for Sensitive and Rapid Detection of Francisella tularensis

Biological warfare agents are infectious microorganisms or toxins capable of harming or killing humans. Francisella tularensis is a potential bioterrorism agent that is highly infectious, even at very low doses. Biosensors for biological warfare agents are simple yet reliable point-of-care analytical tools. Developing highly sensitive, reliable, and cost-effective label-free DNA biosensors poses significant challenges, particularly when utilizing traditional techniques such as fluorescence, electrochemical methods, and others. These challenges arise primarily due to the need for labeling, enzymes, or complex modifications, which can complicate the design and implementation of biosensors. In this study, we fabricated Graphene Quantum dot (GQD)-functionalized biosensors for highly sensitive label-free DNA detection. GQDs were immobilized on the surface of screen-printed gold electrodes via mercaptoacetic acid with a thiol group. The single-stranded DNA (ssDNA) probe was also immobilized on GQDs through strong π−π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, leading to a decrease in the effect of GQD but a positive shift associated with the increase in DNA concentration. The specificity of the developed system was observed with different microorganism target DNAs and up to three-base mismatches in the target DNA, effectively distinguishing the target DNA. The response time for the target DNA molecule is approximately 1010 s (17 min). Experimental steps were monitored using UV/Vis spectroscopy, Atomic Force Microscopy (AFM), and electrochemical techniques to confirm the successful fabrication of the biosensor. The detection limit can reach 0.1 nM, which is two–five orders of magnitude lower than previously reported methods. The biosensor also exhibits a good linear range from 105 to 0.01 nM and has good specificity. The biosensor’s detection limit (LOD) was evaluated as 0.1 nM from the standard calibration curve, with a correlation coefficient of R2 = 0.9712, showing a good linear range and specificity. Here, we demonstrate a cost-effective, GQD-based SPGE/F. tularensis DNA test suitable for portable electrochemical devices. This application provides good perspectives for point-of-care portable electrochemical devices that integrate sample processing and detection into a single cartridge without requiring a PCR before detection. Based on these results, it can be concluded that this is the first enzyme-free electrochemical DNA biosensor developed for the rapid and sensitive detection of F. tularensis, leveraging the nanoenzyme and catalytic properties of GQDs.

Towards quantitative microplastic analysis using pyrolysis-gas chromatography coupled with mass spectrometry

Microplastic pollution from everyday plastic items has increased tremendously worldwide. Pyrolysis gas chromatography coupled to mass spectrometry (Py-GC/MS) has been widely investigated for the qualitative and quantitative analysis of microplastics in environmental samples. However, there are several pitfalls to consider when developing an appropriate protocol for their analysis. This study aimed at the development of an in-house database of primary (single) polymers, binary (two) polymers and tertiary (three) polymer mixtures. In this context the potential occurrence of gas phase reactions during pyrolysis of binary and tertiary polymers were investigated. Further, different diluters were tested for the accurate preparation of calibration standards for quantification purposes.Seven different polymers were included in this study, which were chosen due to their prevalence in daily plastic appliances. For each single polymer specific peaks could be identified and recommendation for quantifier analytes given. The analysis of binary polymer mixtures revealed gas phase reactions for PET with PVC, PVC with MDI-PU and PE with PVC. For these binary polymers, several different novel pyrolysis products, specific for the according binary polymer mixture, could be identified. These results confirmed that especially PVC exhibits strong interactions during co-pyrolysis with ester- and ether-based polymers. Similar results were obtained for tertiary polymers.For accurate preparation of calibration standards different diluters (silica, deactivated silica, calcium carbonate, THF and HFIP) were tested. It was observed that deactivated silica had only an influence on the pyrolysis of PET. Whereas, dilution with silica affected PA-6/66, PE, PET and MDI-PU. Only PVC was not influenced by dilution with silica.In conclusion, our results highlight the necessity of an international standard of reference material as well as a standardized analytical protocol for the analysis and quantification of polymers in environmental samples. It is crucial to use diluters suitable for the specific polymer, to exclude potential interactions of diluters with the polymer. The present work has to be seen as a foundation, but future work is needed to adequately address the quantification of polymers in environmental samples.

Incorporating ultrashort-chain compounds into the comprehensive analysis of per- and polyfluorinated substances in potable and non-potable waters by LC-MS/MS

Ultrashort-chain (USC) per- and polyfluoroalkyl substances (PFAS) are small and very polar compounds with carbon chain lengths shorter than C4. Their ubiquitous and high levels of occurrence in environmental aquatic systems are emerging as a significant concern, rivaling the well-established issues associated with long-chain PFAS contamination. Therefore, it is important to analyze both USC and long-chain PFAS together in water samples to comprehensively assess and address the full spectrum of PFAS contamination. The high polarity of USC PFAS poses a challenge for standard chromatographic practices in PFAS analysis, primarily due to insufficient chromatographic retention. In this study, a simple and reliable workflow was developed for the simultaneous analysis of C1 to C14 perfluoroalkyl carboxylic and sulfonic acids, along with other groups of PFAS, in both potable and non-potable waters. The chromatographic separation was conducted using a polar-embedded reversed-phase LC column with an inert coating on the hardware. Three different water matrices (tap water, bottled water, sewage treatment wastewater) were chosen for method evaluation to demonstrate the applicability of the developed workflow for measuring 45 PFAS compounds in diverse water samples. A dilute-and-shoot workflow was evaluated by accuracy and precision analysis at five fortification levels, ranging from 2 to 250 ng/L. Eighteen isotopically labeled PFAS, serving as extracted internal standards, were added to the samples at 100 ng/L to validate the accuracy of the entire workflow. Calibration standards were prepared in reverse osmosis water due to its cleanliness for all analytes. The calibration ranges varied among different analytes, spanning from 1 – 1000 ng/L. All analytes and extracted internal standards exhibited recovery values ranging from 70% to 130% of the nominal concentration across all fortification levels. Satisfactory method precision was demonstrated with %RSD values below 20%. Additional potable and non-potable waters collected from various source waters were tested to further demonstrate that the established workflow is suitable for the accurate quantification of targeted PFAS in a wide range of water matrices.

Zr-MOF-based substrate with inherent internal standards facilitates reliable SERS analysis of sulfonamide antibiotics

A multifunctional structure comprising gold nanoparticles immobilized within a zirconium-based metal-organic framework (AuNPs/Zr-MOF) was systematically designed. With the capabilities of analyte enrichment, Raman signal enhancement and internal standard calibration, the as-prepared AuNPs/Zr-MOF substrates demonstrate the outstanding SERS sensing performance toward the representative sulfonamide antibiotics (SAs), sulfadiazine, sulfamerazine, sulfamethazine and sulfaquinoxaline. For the first time, these four SAs are detected by SERS in real honey samples at levels of 2.8 ∼ 5.2 ng·g−1 within 10 min. The extensive π-bond conjugated system of Zr-MOF significantly enhances the concentration of SAs through π-π interactions. The entrapped SAs are adsorbed perpendicularly onto the surface of Au via coordinated interactions between Au and two oxygen atoms, or between Au and the terminal amino group. To achieve reliable quantitative analysis, the inherent vibration of the Zr-MOF structure was used as an internal standard label. Combined with a simple clean-up protocol, the established SERE sensing method is superior to conventional methods in terms of time, while achieving the impressive sensitivity. This study presents a robust SERS-based approach for the rapid detection and monitoring of multiple analytes.

Long-Term Reproducibility of BMD-Measurements with Clinical QCT Using Simultaneous and Asynchronous Calibration Methods and Different Measurement and Reconstruction Protocols.

Osteoporosis is underdiagnosed and undertreated. To improve timely fracture risk assessment optimized densitometry methods are required such as opportunistic spinal quantitative computed tomography (QCT). However, it is unclear how to best calibrate these scans and correct for potential scanner drift of QCT when used for monitoring bone mineral density (BMD) changes. We compared gold standard simultaneous calibration with asynchronous calibration methods, assessing mid-term (12weeks) and long-term (1.5years) reproducibility of BMD measurements. Cortical and trabecular compartments of the European Spine Phantom were studied with ten different protocols including low dose and high resolution (HR)-modes. Based on weekly phantom data, we compared simultaneous calibration to asynchronous single (termed global) or monthly calibration. The accuracy was better for trabecular measurements than for cortical measurements for all calibration methods. Reproducibility was excellent for all methods and slightly better for asynchronous than for simultaneous calibration both for trabecular and cortical bone. For HR protocols, reproducibility was better than for low dose measurements. In trabecular compartments averaged HR-BMD remained stable for global (- 0.1%/year, ns) but not for simultaneous calibration (- 1.5%/year, p < 0.001). No significant drifts could be detected for averaged low dose BMD (- 0.9 to + 0.8%/year) for either calibration method. Our data suggest that with regard to precision and accuracy measurements with asynchronous calibration are suitable for vertebral BMD assessment (no contrast agents) in clinical practice. Regular (e.g., monthly) stability tests using a calibration phantom could assure long term stability of at least 1year.

Absolute mass spectrometry: A theoretical analytical technique for quantification

Mass spectrometry (MS) is an analytical chemistry technique that requires calibration with standards in order to quantify an analyte in a sample. In many cases the reference standards may be difficult to obtain or unavailable. In fact, when MS is applied to study reactions that yield multiple products, their quantification requires several standards and involves time-consuming and tedious calibration procedures. In other applications like Mass Spectrometry Imaging (MSI), quantification becomes even more tedious when on-tissue standards or mimetic tissue models are required. In this work, we introduce a new measurement technique that we called the Absolute Mass Spectrometry (AbMS), which allows the absolute quantification of an analyte without the need for calibration standards. This technique is based on the “Influence Method” developed in 2015 for nuclear measurements which, when applied to MS, produces this improvement for analyte quantification. Two possible applications of this technique are presented. One is the conceptual development of a mass spectrometer with electronic ionization that does not require calibration to determine the amount of analyte. The other one describes how to apply this technique in a mass spectrometer with other ionization sources like MALDI, DESI, and SIMS, which are used in MSI. AbMS technique uses the conceptual basis of Influence Method and two consecutive measurements to estimate the amount of analyte. Specifically, AbMS can be directly employed as an absolute method for quantification in MSI. Therefore, it constitutes an attractive complement for research applications in pharmaceutical and health sciences.

Toward Absolute Quantification of Soluble Proteins via Proton Nuclear Magnetic Resonance Spectroscopy: Total Protein Concentration in Blood Plasma.

For absolute protein quantification using nuclear magnetic resonance (NMR) spectroscopy, we considered proteins as homopolymers and effective amino acid (AA) residues (AAREff) as monomer units. For diverse classes of proteins, we determined the AAREff molecular weight as 111.5 ± 3.2 Da and the number of hydrogens per AA as 7.8 ± 0.2. Their ratio of 14.3 ± 0.3 (g/LP)/(mol/LH) remains constant across various protein classes and is equivalent to Kjeldahl's nitrogen-to-protein conversion constant of 5.78 ± 0.29 gN/gP. By analogy to the Kjeldahl method, we suggest that the total integral of a 1H NMR solution protein spectrum could be used for total protein quantification. We synthesized low-resolution protein spectra from the weighted sums of individual AA spectra and compared them with experimental spectra. In the methyl region, the ratio of the protein mass to the total number of protons in the synthetic spectra (corrected for the chemical shift mismatch) was ∼1 (mg/mL)/mM, which agrees with an earlier reported experimental ratio for urine (1.05 ± 0.06 (mg/mL)/mM). For human blood plasma, in the methyl region, we found empirical ratios of 1.115 ± 0.006 (mg/mL)/mM (using 96 patient samples) and 1.121 ± 0.011 (mg/mL)/mM for the NIST plasma standard. This numerical agreement points to universal conversion constants, i.e., protein mixtures with unknown compositions could be quantified without the need for calibration standards by measuring the millimolar proton concentration within the methyl region of the NMR spectrum using the same conversion constant.