Circular Dichroism Spectral Similarity Plots to Extend Validation and Correction to All Measured Wavelengths

Interlaboratory comparisons of circular dichroism (CD) spectra are useful for developing confidence in the measurements associated with optically active molecules. These measurements also help define the higher-order (secondary and tertiary) structure of biopolymers. Unfortunately, the extent of the validity of these measurements has been unclear. In this work, a method is described to extend CD validation over the entire observed wavelength range using what will be called spectral similarity plots. The method involves plotting, wavelength by wavelength, all measured spectral intensities of a sample at one concentration against the intensity values of the same material at a different concentration or pathlength. These spectral similarity plots validate the instrument in terms of spectral shape and whether the shape is shifted in intensity and/or in wavelength. This comparison tests the linearity of instrument’s signal, the balance of its left and right polarizations, its wavelengths, and its spectral intensity scales. When the process is applied to materials with accepted and archived intensity values, the method can be linked to older single-wavelength and double-wavelength calibration techniques. Further, spectral similarity testing of CD spectra from samples with different concentrations run in different labs suggests that improved interlaboratory validation of CD data is possible. Since a database of archival CD measurements is available online, spectral similarity comparisons could possibly provide the ability to compare linearity, polarization balance, wavelength, and spectral intensity between all current CD instruments. If the preliminary results published here prove robust and transferable, then comparisons of full-wavelength range spectra to archived data using spectral similarity plots should become part of the standard process to validate and calibrate the performance of CD instruments.

Interlaboratory Validation of Toxicity Testing Using the Duckweed Lemna minor Root-Regrowth Test

The common duckweed (Lemna minor), a freshwater monocot that floats on the surfaces of slow-moving streams and ponds, is commonly used in toxicity testing. The novel Lemna root- regrowth test is a toxicity test performed in replicate test vessels (24-well plates), each containing 3 mL test solution and a 2–3 frond colony. Prior to exposure, roots are excised from the plant, and newly developed roots are measured after 3 days of regrowth. Compared to the three internationally standardized methods, this bioassay is faster (72 h), simpler, more convenient (requiring only a 3-mL) and cheaper. The sensitivity of root regrowth to 3,5-dichlorophenol was statistically the same as using the conventional ISO test method. The results of interlaboratory comparison tests conducted by 10 international institutes showed 21.3% repeatability and 27.2% reproducibility for CuSO4 and 21.28% repeatability and 18.6% reproducibility for wastewater. These validity criteria are well within the generally accepted levels of <30% to 40%, confirming that this test method is acceptable as a standardized biological test and can be used as a regulatory tool. The Lemna root regrowth test complements the lengthier conventional protocols and is suitable for rapid screening of wastewater and priority substances spikes in natural waters.

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.

DETECTION OF ACID NEUTRALIZERS IN FRAUDULENT MILK: FULL VALIDATION OF A CLASSICAL QUALITATIVE METHOD

Neutralization with alkaline compounds is one of the most common adulterations in milk. The rosolic acid method is a classical test widely used in different countries for detection of neutralizers in milk. The official and a modified version were validated in a single laboratory validation process considering four adulterants: sodium bicarbonate(BI), sodium carbonate(CA), sodium hydroxide(HY) and sodium citrate(CI). The modified version, which presented better performance was selected for interlaboratory validation. In this process, samples of raw milk with acidities of 0.19% were neutralized with different concentrations of BI, CA, HY and C and tested for homogeneity and stability. Eight laboratories, which represented different sectors of the milk production chain, received and analysed these samples. The collaborative trial results confirmed the method performance, although sensitivity and precision were inferior to those obtained in the intralaboratory process, demonstrating its applications and limitations

Critical Review of Analytical and Bioanalytical Verification of the Authenticity of Coffee

Background: The driving factors for the commercial adulteration of coffee are reviewed. Objective: Methods have been assessed for the identification of the most common materials used to adulterate coffee by dilution, to establish the geographic origins, the genotypes of beans, and to assess the authenticity of Kopi Luwak coffee. Method: The literature was surveyed manually and electronically from 1820 to 2018. Results: A flow diagram has been developed to summarize the best approaches to deal with the authentication of coffee. Conclusions: Encouragement is given to the interlaboratory validation of spectroscopic approaches, the exploration of civet cat deoxyribonucleic acid for the identification of Kopi Luwak, and the development of appropriately large and well-curated datasets of authenticity information across multiple techniques. Highlights: The current analytical difficulties in the authentication of coffee are highlighted and suggestions made to improve the situation.

HPLC-UV Estimation of Folic Acid in Fortified Rice and Wheat Flour using Enzymatic Extraction and Immunoaffinity Chromatography Enrichment: An Interlaboratory Validation Study

Background: In India, fortification of cereals with folic acid has been voluntary for many years. However, The Food Safety and Standards Authority of India’s recent Fortification of Foods Regulations (2018) has prompted us to develop and validate a simple analytical method for estimation of folic acid in fortified cereals. Objective: The aim was to develop and validate a simple and rugged HPLC–UV method for quantitative analysis of folic acid in fortified rice and wheat flour. Methods: The enzymatic sample extract was diluted with phosphate buffer, centrifuged, filtered, and then passed slowly through an immunoaffinity cartridge for cleanup. Folic acid in the sample extract was retained by the cartridge and subsequently eluted with 30% acetonitrile [+0.2% trifluoroacetic acid (TFA)]. The elute was collected and analyzed by HPLC–UV at 280 nm. The chromatographic separation of folic acid was achieved on an Agilent Poroshell SB-C18 column (3.0 × 100 mm, 2.7 µm) with 0.1% TFA in methanol as mobile phase. Results: The linearity range of the vitamin was established in the concentration range of 50–800 µg/L, and the regression coefficient was more than 0.999. The LOQ was 5 µg/L. The average spike recovery values of folic acid in rice and wheat flour samples were 90.9 and 80.5%, respectively. The method was subjected to an interlaboratory validation; eight accredited food testing laboratories across India participated in it and resulted in satisfactory z-scores for the reported results. Conclusions: The method will be useful in regulatory compliance testing of folic acid in fortified cereals and processed products. Highlights: A sensitive analysis method is reported for estimation of folic acid in fortified rice and wheat flour. The scope, selectivity, repeatability, and reproducibility of the method establishes it as fit for regulatory compliance check purposes.

Paroxysmal Nocturnal Hemoglobinuria: Grasping the Flow of Diagnostic Dilemmas

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare life-threatening condition due to an acquired somatic mutation of the PIGA gene, leading to nonmalignant clonal expansion of hematopoietic stem cells, which are deficient in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). Fluorescein-labeled proaerolysin (FLAER) and flow cytometry are key tools in the diagnosis of PNH. While clonal detection of PNH in both tests has a sensitive diagnostic threshold of 0.01% in erythrocytes and 0.05% to 1% in leukocytes, one must be cautious in ruling out the possibilities of myelodysplastic syndrome (MDS) or aplastic anemia. We propose guidelines in the differential diagnosis and evaluation of PNH from these and other hematologic disorders that can arise from GPI-AP deficient cells. These guidelines are based on a meta-analysis of five research literature sources, including four case studies. We also compare and contrast our limits of quantification of the in-house PNH assay at University of Kentucky Healthcare with those of an interlaboratory validation of 11 institutions within the United Kingdom. Our report advocates for thorough evaluation of multiple laboratory and clinical variables affecting sensitivity and accuracy of flow cytometry and FLAER in PNH. Furthermore, we recommend lowering of the in-house limit of quantification from the current 1% to 0.01%. This allows for the critical consideration of conditions such as MDS and aplastic anemia and their disease courses, all of which can present with PNH clones as low as 0.01% on flow cytometry and FLAER.