Collaborative Interlaboratory Study Research Articles

Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada

Detection of SARS-CoV-2 RNA in wastewater is a promising tool for informing public health decisions during the COVID-19 pandemic. However, approaches for its analysis by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) are still far from standardized globally. To characterize inter- and intra-laboratory variability among results when using various methods deployed across Canada, aliquots from a real wastewater sample were spiked with surrogates of SARS-CoV-2 (gamma-radiation inactivated SARS-CoV-2 and human coronavirus strain 229E [HCoV-229E]) at low and high levels then provided “blind” to eight laboratories. Concentration estimates reported by individual laboratories were consistently within a 1.0-log10 range for aliquots of the same spiked condition. All laboratories distinguished between low- and high-spikes for both surrogates. As expected, greater variability was observed in the results amongst laboratories than within individual laboratories, but SARS-CoV-2 RNA concentration estimates for each spiked condition remained mostly within 1.0-log10 ranges. The no-spike wastewater aliquots provided yielded non-detects or trace levels (<20 gene copies/mL) of SARS-CoV-2 RNA. Detections appear linked to methods that included or focused on the solids fraction of the wastewater matrix and might represent in-situ SARS-CoV-2 to the wastewater sample. HCoV-229E RNA was not detected in the no-spike aliquots. Overall, all methods yielded comparable results at the conditions tested. Partitioning behavior of SARS-CoV-2 and spiked surrogates in wastewater should be considered to evaluate method effectiveness. A consistent method and laboratory to explore wastewater SARS-CoV-2 temporal trends for a given system, with appropriate quality control protocols and documented in adequate detail should succeed.

Interlaboratory study of the method for direct determination of volatile compounds in alcoholic products using ethanol as internal standard

A collaborative interlaboratory study of the method of direct determination of volatile compounds in spirit drinks and alcoholic products was performed. The proposed method employs ethanol, the major volatile component of an alcoholic product, as an internal standard. For the experiment realization, 9 laboratories from 4 countries – Belarus, Czech Republic, Turkey, Russia – were supplied with standard solutions for gas chromatographic measurements. Five aqueous ethanol 40 % (v/v) standard solutions containing target compounds in concentrations ranging from 10 to 400 mg/L of absolute alcohol were gravimetrically prepared and delivered to the participants. The interlaboratory study was evaluated according to the ISO 5725 standards and the «EuroChem» guide. The repeatability limits (within-laboratory precision) varied between 0.4 and 7.5 % for all samples and compounds, showing a sufficiently high repeatability of the method. The reproducibility limits (between-laboratory precision) were found to vary within a satisfactory range of 0.5–10.0 %. Precision of the method was well within the range predicted by the Horwitz equation for all analytes. In the study experimental estimation of trueness was performed for the first time. Analysis of the obtained results showed that the bias of the method is insignificant at the significance level 5 %. The experimentally determined concentrations of the analytes compared well to the gravimetrical values, thus showing very satisfactory accuracy of the method. The results of the interlaboratory study confirmed that the method «ethanol as internal standard» is robust and reliable and can be used as a standard method for the determination of volatile compounds in water-ethanol samples. Moreover, this method can be validated in any one laboratory without performing additional experimental studies based on the results obtained by validation of the traditional method.

Interlaboratory study of ethanol usage as an internal standard in direct determination of volatile compounds in alcoholic products

A collaborative interlaboratory study on the method of direct quantitation of volatile compounds in spirit drinks and alcoholic products was conducted. The discussed method applies ethanol, the major volatile component of an alcoholic product, as an internal standard. In this study 9 laboratories from 4 different countries were supplied with standard solutions for gas chromatographic measurements. Five aqueous ethanol 40% (v/v) standard solutions containing target compounds in concentrations ranging from 10 mg/L to 400 mg/L of absolute alcohol were prepared and sent to the participants for quantification of acetaldehyde, methyl acetate, ethyl acetate, methanol, 2-propanol, 1-propanol, 2-methyl-1-propanol, 1-butanol and 3-methyl-1-butanol. The interlaboratory study was evaluated according to the ISO 5725 standards and the Eurachem guide. The within-laboratory precision varied between 0.4% and 7.5% for all samples and compounds, showing a sufficiently high repeatability of the method. The between-laboratory precision was found to vary within a satisfactory range of 0.5% ÷ 10.0%. Precision of the method was well within the range predicted by the Horwitz equation for all analytes. The analysis of trueness showed that the bias of the method is insignificant at the significance level α = 5%. The determined concentrations of the analytes compared well to the gravimetrical values thus showing very satisfactory accuracy of the method. The results of the interlaboratory study confirmed that “Ethanol as Internal Standard” method is robust and reliable and can be used as a standard reference method for analysing volatile compounds in water-ethanol samples. The possibilities of method validation according to the previously obtained experimental data were shown.

Collaborative Interlaboratory Studies for the Validation of ELISA Methods for the Detection of Allergenic Fining Agents Used in Wine According to the Criteria of OIV Resolution 427–2010 Modified by OIV–Comex 502–2012

The clarification or fining of wine removes undesired substances (mainly proteins, phenols, and tannins), which would roil the wine and cause bitterness and astringency. A common fining agent, egg white, can be directly added to wine through the inlet of a circulating pump, but more typically egg white comes as commercial preparation in powdered form (commercially named egg albumin). Skimmed milk or more frequently purified caseinates are used to remove bitterness and hardness of white wine and sherry. Both egg white and caseinates are fining agents with optimal enological properties, but their residues could represent a risk for subjects suffering from food allergy. The rules for allergen labeling were detailed in Directives 2003/89/EC, and Directive 2005/26/EC established a list of food ingredients provisionally excluded from labeling, that included wine fining agents. Extended till June 2012, wine labeling exemption can be now maintained only if (1) egg and milk derivatives are not used and cross-contamination is under control; and (2) wine clarified with such products is negative for the presence of residues using techniques with detection and quantification limits of 0.25 and 0.5 ppm, respectively. Analytical requirements were defined in the OIV resolution 427–2010 (OIV 2010) modified by OIV/COMEX 502–2012 (OIV 2012). On the basis of a previous experience, an interlaboratory collaborative trial was organized to validate a commercial ELISA kit designed to measure allergenic residues in red wine fined with egg white proteins. In the meantime, the performance of the commercial caseinate ELISA kit for white wine was rechecked according to the new limit of detection and limit of quantification values, recommended by OIV in 2012. The collaborative interlaboratory studies showed that both ELISA kits had good reproducibility, repeatability, and robustness in detecting residues of allergenic fining agents in wine, in good agreement with the requirements of the OIV resolution 427–2010 modified by OIV/COMEX 502–2012.

Validation by a Collaborative Interlaboratory Study of an ELISA Method for the Detection of Caseinate Used as a Fining Agent in Wine

The clarification or fining of wine removes undesirable substances such as proteins, phenols, and tannin compounds that would cloud the wine and cause bitterness and astringency. Caseinates have useful fining properties, but their residues could present a risk for allergic subjects. A commercial kit that was developed to detect caseinates in food has been examined for its applicability to a wine matrix; it is sensitive to caseinates at concentrations as low as 1 ppm. The general characteristics of the caseinate assay, described below in detail, are as follows. It is a sandwich enzyme-linked immunosorbent assay (ELISA): the microplate is first coated with a specific anti-casein antibody; and after incubation with the wine sample, a secondary anti-casein antibody conjugated with horseradish peroxidase is added to form a sandwich. The antibodies have been tested for their immunoreactivity and the reproducibility of antigen recognition has been measured. An interlaboratory collaborative trial was organized to evaluate the performance of this ELISA method and its ease of use by laboratories routinely dealing with wine/food analyses. The results satisfy the criteria established by the International Organisation of Vine and Wine in the Compendium of International Methods of Analysis MA-EAS1-07-ETCOL.

Laplace random effects models for interlaboratory studies

A model is introduced for measurements obtained in collaborative interlaboratory studies, comprising measurement errors and random laboratory effects that have Laplace distributions, possibly with heterogeneous, laboratory-specific variances. Estimators are suggested for the common median and for its standard deviation. We provide predictors of the laboratory effects, and of their pairwise differences, along with the standard errors of these predictors. Explicit formulas are given for all estimators, whose sampling performance is assessed in a Monte Carlo simulation study.

Estimates of measurement uncertainty from proficiency testing schemes, internal laboratory quality monitoring and during routine enforcement examination of foods

To determine the levels of measurement uncertainty (MU) obtained in proficiency testing and routine microbiological analyses of foods and to compare these with estimates of MU obtained for results of analyses obtained in collaborative interlaboratory studies of microbiological methods. Raw data submitted by participants in the Food Examination Proficiency Assessment Scheme were obtained from the Central Science Laboratory (York). Internal quality monitoring data were obtained from Health Protection Agency (HPA) laboratories, together with data from routine food examinations undertaken in HPA laboratories. The data sets were analysed to determine the relative standard deviations of reproducibility (RSD(R)), based on log(10) colony count values, and thence the relative measures of expanded uncertainty. Analysis of proficiency test data showed extreme values of RSD(R) up to +/-30% depending upon the organism, the laboratory and the method of examination. RSD(R) values on routine samples averaged around +/-12% but ranged up to +/-41% in a few instances. Internal quality assessments for different organisms ranged up to +/-27%, depending upon the particular organism and examination procedure. The results show little difference in uncertainty for counts obtained using different plating systems (e.g. pour plates, spread plates or spiral plating) on the same dilutions of the same food samples. The data are compared with estimates of microbiological uncertainty derived in interlaboratory studies. The estimates of uncertainty ranged widely, both within and between laboratories, and appeared to bear little relationship to the foodstuff under examination. The extent of MU associated with many routine microbiological examinations is generally no worse than those produced in inter-laboratory trials, although notable exceptions were seen. Knowledge of the levels of MU may have wide impact on the establishment of international standard methods for microbiological examination of foods and the ability to set realistic microbiological criteria.

Determination of Phytase Activity in Feed by a Colorimetric Enzymatic Method: Collaborative Interlaboratory Study

Fourteen laboratories participated in a collaborative study (coded fyt9404) and 13 laboratories participated in a study (coded fyt9410) to validate a colorimetric assay for determination of microbial phytase activity in feed. For each study, all laboratories received 6 laboratory samples provided by one commercial supplier (phytase activity levels within the range of 200-400 per kg) to be analyzed in duplicate. Method performance was calculated and statistical calculations were executed according to AOAC guidelines. Results from 3 laboratories for study fyt9404 and from one laboratory for study fyt9410 were excluded from statistical analysis because of invalid data determined during initial review by Youden pair, value versus laboratory. For study fyt9404, repeatability relative standard deviation (RSDr) values ranged from 6.2 to 8.6%, and reproducibility relative standard deviation (RSDR) values ranged from 14.1 to 27.6%. No outliers were identified. For study fyt9410, RSDr values ranged from 3.9 to 7.9%, and RSDR values ranged from 14.0 to 20.5%. With outliers excluded, RSDr values ranged from 2.5 to 7.9%, and RSDR values ranged from 14.0 to 20.5%.

International coordination and validation of analytical methods.

Most experimentation dealing with analytical methodology in the physical and biological sciences has been conducted within a single laboratory. Method validation by other laboratories was considered not only unnecessary but also detrimental because, in the words of one commentator, 'the results are too variable'. Within the last two decades, however, largely as a result of the requirements of international environmental and food standards programmes, it has become increasingly apparent that a collaborative interlaboratory study is the only way to estimate the variability characteristics of methods of analysis as performed by the typical population of laboratories using the methods. To obtain a common basis for measuring the statistical characteristics of analytical methods, representatives of almost two dozen international organizations meeting in Geneva, in May 1987, approved by consensus a protocol for the design and interpretation of data from collaborative studies of chemical methods of analysis.

Validation of ppb/ppt Sulfur Gas Standards by Independent Analytical Methods

Reliable measurements of sulfur-containing gases at ultratrace concentrations have two basic prerequisites: 1) analytical methods with sufficient detectability, specificity, and reproducibility; and 2) accurate standards of these sulfur gases which bracket the expected analyte concentration range in actual air samples. Whereas several potential measurement methods for these sulfur gases have been reported, direct calibration of these methods at low ppb to ppt concentrations has been impossible because of difficulties in preparing dependable, verified standards over this concentration range. A system has been developed in our laboratory for the preparation and validation of sulfur gas standards at concentrations down to 0.01 μg S/m3. The calibration procedure uses commercial G-CAL permeation devices whose emission rates (0.1 to 10 ng S/min) are established via a metal foil collection/flash desorption/flame photometric detection (MFC/FD/FPD) technique that can be standardized with aqueous sulfate standards. The validity of this calibration scheme has been determined by intralaboratory comparisons among four independently calibrated analytical methods and by a collaborative interlaboratory study.

Inorganic chemical analysis of plant tissue: possibilities and limitations.

The variability of analytical results for determination of 23 inorganic components in different plant materials was evaluated, using data obtained over the last 5 years of an extensive (on the average 120 participants) bimonthly collaborative interlaboratory study. In particular, the relation between content level and coefficient of variation (CV) was examined. Usually, a constant CV value was found at high content levels, with a sharp increase in CV at low levels. The precision found for N, P, K, Ca, Cl, Mg, Zn and nitrate was high enough (CV &lt; 20%) to yield reasonably comparable content values. Comparison of analytical results for B, Cu, Fe, Cd, Mn and Na, may be difficult, since about 20% CV was already reached at the levels usually present in plant material. The analytical results for Al, Co, Cr, Mo, Ni, Pb, S, Se and sulphate varied considerably, irrespective of the content level, which means that comparable results are very hard to produce with these components. (Abstract retrieved from CAB Abstracts by CABI’s permission)

Minimum Criteria for Validation of Analytical Methods

Abstract Two aspects must be considered in outlining minimum criteria for validation of analytical methods: management issues and technical issues. Management needs background information and information on issues, options, and implications to identify priorities. The value of the work should be clear to ensure its support and implementation. International adoption of AOAC guidelines for collaborative interlaboratory studies would provide technical criteria for future tests.

Report of the Committee on Collaborative Interlaboratory Studies

Report of the Committee on Collaborative Interlaboratory Studies

Report of the Committee on Collaborative Interlaboratory Studies

Report of the Committee on Collaborative Interlaboratory Studies

The Variability of AOAC Methods of Analysis as Used in Analytical Pharmaceutical Chemistry

Pharmaceutical analytical chemistry, which ordinarily deals with the analysis of formulations containing from 0.1 to 100% of active ingredient, uses methods with a reproducibility (between-laboratory variability) of about 2.5% and a repeatability (within-laboratory variability) of about half that amount. The best between-laboratory precision attainable appears to be about 1.0% and within-laboratory precision, about 0.5%. On the basis of the results available, automated methods do not appear to be any more precise than manual methods, although the studies show fewer outlying data points. Replicates (preferably blind ones) should always be conducted in a collaborative interlaboratory study in order to obtain the important information as to whether efforts should be concentrated on improving the method itself or on the performance of laboratories and analysis in applying it.