Stable Reference Materials Research Articles

Repeated lyophilization: A neo-method for urine-based reference materials preparation

In urine drug testing, a cut-off value is often imposed to determine whether the sample is negative or positive. A matrix containing a reference substance helps counteract the adverse effects of the urine matrix across different laboratories to improve the consistency of final results. However, as a biological matrix, urine is prone to corruption and other problems that make it difficult to use as a reference sample. In this study, morphine, nitrazepam, lorazepam, buprenorphine, zolpidem, midazolam, diazepam, and clozapine commonly used in clinical practice were selected as target analytes, and the preparation process was further optimized to repeated lyophilization, in order to obtain more effective, stable, and accurate urine matrix reference materials (mRMs). The appropriate urine density (1.010–1.017 kg/m3) for preparing lyophilized samples was investigated through density determination. Conducting repeated lyophilizations resulted in a denser powder with reduced susceptibility to collapse and improved the quality of lyophilized urine samples. Lyophilized urine mRMs could be stored at room temperature for one month or under refrigeration conditions (4 ℃) for six months.

A comprehensive gas chromatography electron ionization high resolution mass spectrometry study of a new steroidal selective androgen receptor modulator (SARM) compound S42.

The synthetic 20-keto-steroid S42 (1) demonstrated selective androgen receptor modulator (SARM) properties in preclinical studies and, consequently, received growing attention also in the context of sports drug testing programs. Fundamental understanding of the behavior of S42 (1) and of relevant derivatives in gas chromatography-electron ionization MS experiments at high resolution (GC-EI-HRMS) is indispensable to develop a reliable qualitative and quantitative doping control method for S42 (1) and its metabolites in body fluid matrices. We present important fundamental mechanistic data on the EI fragmentation behavior of S42 (1) and of silyl ether derivatives as well as of stable isotope-labelled reference material.

An inter-laboratory comparison between 13 international laboratories for eight components relevant for hydrogen fuel quality assessment

The quality of the hydrogen delivered by refuelling stations is critical for end-users and society. The purity of the hydrogen dispensed at hydrogen refuelling points should comply with the technical specifications included in the ISO 14687:2019 and EN 17124:2022 standards. Once laboratories have set up methods, they need to verify their performances, for example through participation in interlaboratory comparisons. Due to the challenge associated with the production of stable reference materials and transport of these which are produced in hydrogen at high pressure (>10 bar), interlaboratory comparisons have been organized in different steps, with increasing extent.This study describes an inter-laboratory comparison exercise for hydrogen fuel involving a large number of participants (13 laboratories), completed in less than a year and included eight key contaminants of hydrogen fuel at level close to the ISO14687 threshold. These compounds were selected based on their high probability of occurrence or because they have been found in hydrogen fuel samples. For the results of the intercomparison, it appeared that fully complying with ISO 21087:2019 is still challenging for many participants and highlighted the importance of organising these types of exercises. Many laboratories performed corrective actions based on their results, which in turn significantly improved their performances.

Customizable optical tissue phantom platform for characterization of fluorescence imaging device sensitivity.

Optical tissue phantoms serve as inanimate and stable reference materials used to calibrate, characterize, standardize, and test biomedical imaging instruments. Although various types of solid tissue phantoms have been described in the literature, current phantom models are limited in that they do not have a depth feature that can be adjusted in real-time, they cannot be adapted to other applications, and their fabrication can be laborious and costly. Our goal was to develop an optical phantom that could assess the imaging performance of fluorescence imaging devices and be customizable for different applications. We developed a phantom with three distinct components, each of which can be customized. We present a method for fabricating a solid optical tissue that contains (1)an adjustable depth capability using thin film phantoms, (2)a refillable chip loaded with fluorophores of the user's choice in various desired quantities, and (3)phantom materials representative of different tissue types. This article describes the development of phantom models that are customizable, adaptable, and easy to design and fabricate.

A community-led calibration of the Zr isotope reference materials: NIST candidate RM 8299 and SRM 3169

We introduce a zirconium stable isotope reference material (RM) adhering to community-defined best practices: NIST RM 8299. Its calibration provides insights into mass-fractionation laws and accurate determination of absolute ratios using MC-ICP-MS.

Bilateral comparison of primary reference materials (PRMs) containing methanol, ethanol and acetone in nitrogen

A range of oxygenated volatile organic compounds (OVOCs) are present in the atmosphere as a result of direct emissions and as products of atmospheric oxidation. Long-term measurements are important to understand changes to these emission sources and atmospheric oxidation processes. Accurate and stable traceable gaseous primary reference materials are needed to underpin rigorous quality assurance and quality control at monitoring stations such as those organised by the World Meteorological Organization Global Atmosphere Watch (WMO-GAW) programme. The development of a capability for providing traceable primary reference materials (PRMs) of OVOCs is of paramount importance due to the increasing prevalence of these compounds in the urban atmosphere and also because there is currently no Central Calibration Laboratory (CCL) for these components within the WMO-GAW programme. This EURAMET bilateral comparison demonstrates the measurement compatibility (≤ 3%) for three OVOCs (methanol, ethanol and acetone) at nominally 5 µmol mol−1 between two National Metrology Institutes: the National Physical Laboratory (NPL), UK and the Van Swinden Laboratorium (VSL), the Netherlands. The comparison shows that a gravimetric method for value assignment is applicable to acetone but that a more complicated procedure must be employed to value assign methanol and ethanol due to corrections for adsorption effects, which can be as large as 10 % of the nominal value. This work demonstrates the importance of making appropriate corrections to ensure the accuracy of these reference materials. NPL and VSL used different approaches to make these corrections providing confidence and independent verification. This work supports new calibration and measurement capabilities for methanol, ethanol and acetone in the range of 1 μmol mol−1 –10 μmol mol−1 with expanded uncertainties of 6.9 % (3 %), 7.3 % (3 %) and 1.7 % (2 %) for NPL (VSL), respectively, to be realized in the key comparison database and supports the development of the required traceability infrastructure to underpin long-term global measurements of these OVOCs.

Metrology of cellular analysis: problems and solutions

Cellular analysis is one of the perspective developing areas of metrology. The complexity of the measuring object requires novel approaches and correct metrological support for the quantitative methods of cellular analysis. Definition of the quantitative parameters and nominal properties of the cell, standardization of methodology – these are main tasks of the Cellular Analysis Working Group of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology. The results of a number of pilot studies carried out by Working Group can be used as a first step establishing metrological traceability of the cell measurements. Development of the stable reference materials for cell quantification is a challenging problem. One of the promising solutions seems to apply synthetic cell-mimicking particles. Currently we are in the process of developing candidate synthetic reference materials for cell measurements.

Metal acetylacetonates dissolved in UV-curing resin as solid reference material for trace element measurements in organic samples

Vacuum stable and microhomogeneous polymer reference materials with trace amounts of different metals will improve the quality of lateral-resolved quantification significantly.

Preparation of multiplexed control materials for cancer mutation analysis by genome editing in GM12878 cells.

BackgroundQuality control materials are necessary for assay development, test validation, and proficiency testing in cancer mutation analysis. Most of the existing controls for somatic mutations only harbor a single variant and are derived from unstable cell lines. This study aimed to establish a method to create stable multianalyte controls in a defined background by genome editing in GM12878 cells, which also can be applied for the reference of next‐generation sequencing.MethodsGM12878 cells were electroporated with a donor plasmid containing a mutant DNA sequence and a Cas9/sgRNA expressing vector. The genome‐edited GM12878 cell was validated with Sanger sequencing, amplification refractory mutation system (ARMS), and next‐generation sequencing (NGS).ResultsWe have successfully generated a mutant GM12878 cell line harboring the defined variants including single‐nucleotide variants (SNVs), small insertions and deletions (indels), and structural variants (SVs). The introduction of intended mutations in GM12878 cell line was confirmed by both ARMS and sequencing methods.ConclusionsWe developed a method for the preparation of the multiplexed controls for reference mutations in cancer gene by genome editing in GM12878 cells. This methodology can be used to generate other stable cancer reference materials with an unlimited supply.

Synthesis, characterization, method development, and validation of nor-ethylmorphine hydrochloride reference material using established analytical techniques for dope control analysis.

Ethylmorphine is permitted internationally for therapeutic purposes where morphine is not indicated across the globe. Nor-ethylmorphine a major metabolite of ethylmorphine. To differentiate the intake of morphine from ethylmorphine, nor-ethylmorphine stable reference material is desirable. There is no available commercial source and no data for reference material context for this substance. Therefore, nor-ethylmorphine HCl was synthesized and characterized, and purity and potency were assessed using nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HRMS), Fourier transform infrared spectroscopy (FT-IR), thermogravimetry (TGA), and high-performance liquid chromatography (HPLC). Purity and potency were found to be 98.29% and 96.40%, respectively, providing a fit for purpose reference material for doping control analysis in sports.

Long-Term Metabolomics Reference Material.

The use of quality control samples in metabolomics ensures data quality, reproducibility, and comparability between studies, analytical platforms, and laboratories. Long-term, stable, and sustainable reference materials (RMs) are a critical component of the quality assurance/quality control (QA/QC) system; however, the limited selection of currently available matrix-matched RMs reduces their applicability for widespread use. To produce an RM in any context, for any matrix that is robust to changes over the course of time, we developed iterative batch averaging method (IBAT). To illustrate this method, we generated 11 independently grown Escherichia coli batches and made an RM over the course of 10 IBAT iterations. We measured the variance of these materials by nuclear magnetic resonance (NMR) and showed that IBAT produces a stable and sustainable RM over time. This E. coli RM was then used as a food source to produce a Caenorhabditis elegans RM for a metabolomics experiment. The metabolite extraction of this material, alongside 41 independently grown individual C. elegans samples of the same genotype, allowed us to estimate the proportion of sample variation in preanalytical steps. From the NMR data, we found that 40% of the metabolite variance is due to the metabolite extraction process and analysis and 60% is due to sample-to-sample variance. The availability of RMs in untargeted metabolomics is one of the predominant needs of the metabolomics community that reach beyond quality control practices. IBAT addresses this need by facilitating the production of biologically relevant RMs and increasing their widespread use.

Development of a Polystyrene Reference Material for Raman Spectrometer (NMIJ RM 8158-a).

The Raman shift is one of the most important parameters in Raman spectroscopy, and is calculated from the difference in the wavenumbers for excitation and for Raman scattering. Because the observed shifts are strongly dependent on the spectrometer and the measurement conditions, physically and chemically stable reference materials for Raman-shift validation are required. We reliably estimated the Raman shift and evaluated its uncertainty for peaks of a 4-mm-thick, 25-mm-diameter polystyrene disc by using a HeNe laser and Raman-scattered light calibrated with Ne/Ar emission lines. We considered uncertainties originating from the fitting, repeatability, and reproducibility of both the Ne/Ar emission lines and the Rayleigh/Raman-scattered lights, as well as the wavenumber of the unstabilised HeNe laser, and pixel and spectral resolutions. We also considered uncertainty originating from inhomogeneity among and within discs, as well as their long-term stability. The obtained Raman shifts of 11 peaks (reference values) were comparable to those described in ASTM E1840 with expanded uncertainty at 1.1 or 1.2 cm-1 (k = 2, coverage factor).

Method for Reproducible Shipboard Segmented Flow Analysis Ammonium Measurement Using an In-House Reference Material for Quality Control

Ammonium is a fundamental nutrient for phytoplankton growth in seawater and is a key component of the microbial loop. Ammonium measured in parallel with other nutrients is crucial in understanding the small temporal scale changes in oceanographic ecology. Despite the importance of measuring ammonium at sea, owing to its lability, there is no consensus on the best method. The lack of availability of certified reference materials for ammonium in seawater also makes it difficult to assess the accuracy and reproducibility of ammonium measurements. In this study we present a modified segmented flow analysis method using ortho-phthaldialdehyde (OPA) with fluorescence detection to measure ammonium at sea together with four other macro-nutrients (nitrate, nitrite, silicate and phosphate) in near real time. An in-house ammonium quality control (QC) material was produced to improve the accuracy and repeatability of the measurement at sea. The QC was prepared following two different methods and stored in two types of containers. The suitability of the in-house QC’s as a reference material were assessed onboard the RV Investigator in 2018 during two oceanographic voyages, including one on the repeat SR03 CLIVAR transect. This paper describes the production and assessment of the in-house QC for ammonium in seawater, providing groundwork for creating a short-term stable ammonium reference material for sea going voyages. The uncertainty of this method of ammonium measurement was found to be 0.10 μmol/L at ammonium concentration of 1.0 μmol/L. Results show that preparation of the QC inside a laminar flow cabinet and directly into 10 mL polypropylene sample tubes just prior to the commencement of the voyage improved its stability.

Quality Assurance in the molecular diagnostic laboratory

Molecular diagnostics is currently playing an imperative role in the clinical microbiology laboratory. Quality assurance and quality control issues have often remained underdeveloped in our country and are still critical. To relate patient results to these, molecular diagnostics carry values in clinical diagnosis, therapeutic drug monitoring and remission and those results need to be comparable across time and methods. This may be achieved either by producing the identical value across methods and test versions or by using reliable and stable reference materials in the medical laboratories engaged with molecular tests. The establishment of international standards and reference materials, assessment and maintenance of technical competencies, regulatory oversight and best practices considerations are thus of utmost importance. This review focuses on general and specific issues relevant for quality assurance and quality control in the routine molecular diagnostics laboratory.
 Bangladesh J Med Microbiol 2021; 15 (1): 30-34

Construction of Lentivirus-Based Reference Material for RT-PCR Detection of Middle East Respiratory Syndrome Coronavirus and Its Application in External Quality Assessment.

Nucleic acid amplification technology (NAT) has been the most used one for rapid detection of Middle East Respiratory Syndrome coronavirus (MERS-CoV) since MERS-CoV was first detected in 2012. It is important to develop stable and safe reference materials for assessing the quality of NAT kits and performing an external quality assessment (EQA) in different laboratories. In this study, the MERS-CoV RNA fragments including upE, ORF1b, and N were packed within human immunodeficiency virus type 1 (HIV-1)-like particles. The lyophilized virus-like particles (VLPs) were found to be stable at 37 °C or below and safe to be used not only as the control material for PCR detection of MERS-CoV but also as the reference material for EQA. In an EQA organized by Ningbo International Travel Healthcare Center in China, 49 participating institutions achieved 100% agreement in detecting MERS-CoV using various commercial diagnosis kits and different extraction methods. However, different Ct values reported by different sites for the same sample implied that a need exists to standardize the RNA extraction method and/or the PCR detection conditions between the laboratories.

Subnanometer localization accuracy in widefield optical microscopy

The common assumption that precision is the limit of accuracy in localization microscopy and the typical absence of comprehensive calibration of optical microscopes lead to a widespread issue—overconfidence in measurement results with nanoscale statistical uncertainties that can be invalid due to microscale systematic errors. In this article, we report a comprehensive solution to this underappreciated problem. We develop arrays of subresolution apertures into the first reference materials that enable localization errors approaching the atomic scale across a submillimeter field. We present novel methods for calibrating our microscope system using aperture arrays and develop aberration corrections that reach the precision limit of our reference materials. We correct and register localization data from multiple colors and test different sources of light emission with equal accuracy, indicating the general applicability of our reference materials and calibration methods. In a first application of our new measurement capability, we introduce the concept of critical-dimension localization microscopy, facilitating tests of nanofabrication processes and quality control of aperture arrays. In a second application, we apply these stable reference materials to answer open questions about the apparent instability of fluorescent nanoparticles that commonly serve as fiducial markers. Our study establishes a foundation for subnanometer localization accuracy in widefield optical microscopy.

N and O isotope (δ15 Nα , δ15 Nβ , δ18 O, δ17 O) analyses of dissolved NO3- and NO2- by the Cd-azide reduction method and N2 O laser spectrometry.

The nitrogen and oxygen (δ15 N, δ18 O, δ17 O) isotopic compositions of NO3- and NO2- are important tracers of nutrient dynamics in soil, rain, groundwater and oceans. The Cd-azide method was used to convert NO3- or NO2- to N2 O for N and triple-O isotopic analyses by N2 O laser spectrometry. A protocol for laser-based headspace isotope analyses was compared with isotope ratio mass spectrometry. Lasers provide the ability to directly measure 17 O anomalies which can help discern atmospheric N sources. δ15 N, δ18 O and δ17 O values were measured on N/O stable isotopic reference materials (IAEA, USGS) by conversion to N2 O using the Cd-azide method and headspace N2 O laser spectrometry. A 15 N tracer test assessed the position-specific routing of N to the α or β positions in the N2 O molecule. A data processing algorithm was used to correct for isotopic dependencies on N2 O concentration, cavity pressure and water content. NO3- /NO2- nitrogen is routed to the 15 Nα position of N2 O in the azide reaction; hence the δ15 Nα value should be used for N2 O laser spectrometry results. With corrections for cavity pressure, N2 O concentration and water content, the δ15 NαAIR , δ18 OVSMOW and δ17 OVSMOW values (‰) of international reference materials were +4.8 ± 0.1, +25.9 ± 0.3, +12.7 ± 0.2 (IAEA NO3 ), -1.7 ± 0.1, -26.8 ± 0.8, -14.4 ± 1.1 (USGS34) and +2.6 ± 0.1, +57.6 ± 1.2, +51.2 ± 2.0 (USGS35), in agreement with their values and with the isotope ratio mass spectrometry results. The 17 O excess for USGS35 was +21.2 ± 9‰, in good agreement with previous results. The Cd-azide method yielded excellent results for routine determination of δ15 N, δ18 O and δ17 O values (and the 17 O excess) of nitrate or nitrite by laser spectrometry. Disadvantages are the toxicity of Cd-azide chemicals and the lack of automated sampling devices for N2 O laser spectrometers. The 15 N-enriched tracer test revealed potential for position-specific experimentation of aqueous nutrient dynamics at high 15 N enrichments by laser spectrometry, but exposed the need for memory corrections and improved spectral deconvolution of 17 O.

Addressing Analytical Challenges of the Environmental Monitoring for the Water Framework Directive: ERM-CE100, a New Biota Certified Reference Material.

In the context of supporting the EU Member States in the implementation of the EU Water Framework Directive (WFD), a project for the production of a fish reference material (ERM-CE100) certified for its content of the two priority substances hexachlorobenzene (HCB) and hexachlorobutadiene (HCBD) was carried out at the Joint Research Centre (JRC) of the European Commission. The starting material was naturally contaminated Wels catfish (Silurus glanis), caught in the Ebro River (Spain). A novel approach for the processing of the fish was tested that resulted in a homogeneous and stable reference material in the form of a wet paste. The fresh-like texture of the matrix enhances the comparability of this material toward routinely analyzed environmental biota samples and facilitates its use as a quality assurance tool given that the WFD environmental quality standards (EQS) for biota are expressed as wet weight. Certified values for the mass fractions of HCB and HCBD were assigned with 120 ± 8 and 36 ± 4 μg/kg, respectively. The related interlaboratory comparison involved 13 expert laboratories applying a range of analytical methodologies. It is the first biota CRM ever available for HCBD. ERM-CE100 can be used to assess the performance of analytical methods employed in the mandatory monitoring of water bodies under the WFD, thus, providing a benchmark for establishing comparability among measurement results.

A novel cell culture model as a tool for forensic biology experiments and validations

To improve and advance DNA forensic casework investigation outcomes, extensive field and laboratory experiments are carried out in a broad range of relevant branches, such as touch and trace DNA, secondary DNA transfer and contamination confinement. Moreover, the development of new forensic tools, for example new sampling appliances, by commercial companies requires ongoing validation and assessment by forensic scientists. A frequent challenge in these kinds of experiments and validations is the lack of a stable, reproducible and flexible biological reference material. As a possible solution, we present here a cell culture model based on skin-derived human dermal fibroblasts. Cultured cells were harvested, quantified and dried on glass slides. These slides were used in adhesive tape-lifting experiments and tests of DNA crossover confinement by UV irradiation. The use of this model enabled a simple and concise comparison between four adhesive tapes, as well as a straightforward demonstration of the effect of UV irradiation intensities on DNA quantity and degradation. In conclusion, we believe this model has great potential to serve as an efficient research tool in forensic biology.

A revision in hydrogen isotopic composition of USGS42 and USGS43 human-hair stable isotopic reference materials for forensic science

The hydrogen isotopic composition (δ2HVSMOW-SLAP) of USGS42 and USGS43 human hair stable isotopic reference materials, normalized to the VSMOW (Vienna-Standard Mean Ocean Water)–SLAP (Standard Light Antarctic Precipitation) scale, was originally determined with a high temperature conversion technique using an elemental analyzer (TC/EA) with a glassy carbon tube and glassy carbon filling and analysis by isotope-ratio mass spectrometer (IRMS). However, the TC/EA IRMS method can produce inaccurate δ2HVSMOW-SLAP results when analyzing nitrogen-bearing organic substances owing to the formation of hydrogen cyanide (HCN), leading to non-quantitative conversion of a sample into molecular hydrogen (H2) for IRMS analysis. A single-oven, chromium-filled, elemental analyzer (Cr-EA) coupled to an IRMS substantially improves the measurement quality and reliability of hydrogen isotopic analysis of hydrogen- and nitrogen-bearing organic material because hot chromium scavenges all reactive elements except hydrogen. USGS42 and USGS43 human hair isotopic reference materials have been analyzed with the Cr-EA IRMS method, and the δ2HVSMOW-SLAP values of their non-exchangeable hydrogen fractions have been revised:USGS42(Tibetan Hair) δ2HVSMOW−SLAP=−72.9±2.2 mUr (n=6)USGS43(Indian Hair) δ2HVSMOW−SLAP=−44.4±2.0 mUr (n=6)where mUr=0.001=‰. On average, these revised δ2HVSMOW-SLAP values are 5.7mUr more positive than those previously measured. It is critical that readers pay attention to the δ2HVSMOW-SLAP of isotopic reference materials in publications as they may need to adjust the δ2HVSMOW–SLAP measurement results of human hair in previous publications to ensure all results are on the same isotope-delta scale.