Within-laboratory Coefficients Of Variation Research Articles

Development of an all-in-one real-time PCR assay for simultaneous detection of spotted fever group rickettsiae, severe fever with thrombocytopenia syndrome virus and hantaan virus prevalent in central China.

Central China has been reported to be one of the most important endemic areas of zoonotic infection by spotted fever group rickettsiae (SFGR), severe fever with thrombocytopenia syndrome virus (SFTSV) and hantaan virus (HTNV). Due to similar clinical symptoms, it is challenging to make a definite diagnosis rapidly and accurately in the absence of microbiological tests. In the present study, an all-in-one real-time PCR assay was developed for the simultaneous detection of nucleic acids from SFGR, SFTSV and HTNV. Three linear standard curves for determining SFGR-ompA, SFTSV-L and HTNV-L were obtained within the range of 101-106 copies/μL, with the PCR amplification efficiencies ranging from 93.46% to 96.88% and the regression coefficients R2 of >0.99. The detection limit was 1.108 copies/μL for SFGR-ompA, 1.075 copies/μL for SFTSV-L and 1.006 copies/μL for HTNV-L, respectively. Both the within-run and within-laboratory coefficients of variation on the cycle threshold (Ct) values were within the range of 0.53%-2.15%. It was also found there was no statistical difference in the Ct values between single template and multiple templates (PSFGR-ompA = 0.186, PSFTSV-L = 0.612, PHTNV-L = 0.298). The sensitivity, specificity, positive and negative predictive value were all 100% for determining SFGR-ompA and SFTSV-L, 97%, 100%, 100% and 99.6% for HTNV-L, respectively. Therefore, the all-in-one real-time PCR assay appears to be a reliable, sensitive, rapid, high-throughput and low cost-effective method to diagnose the zoonotic infection by SFGR, SFTSV and HTNV.

Evaluation of automated calibration and quality control processes using the Aptio total laboratory automation system

Background: The objective of this study was to determine whether manually performed calibration and quality control (QC) processes could be replaced with an automated laboratory system when installed analyzers fail to provide automated calibration and QC functions.Methods: Alanine aminotransferase (ALT), total cholesterol (TC), creatinine (Cr), direct bilirubin (DB), and lipase (Lip) items were used as analytes. We prepared pooled serum samples at 10 levels for each test item and divided them into two groups; five for the analytical measurement range (AMR) group and five for the medical decision point (MDP) group. Calibration and QC processes were performed for five consecutive days, and ALT, TC, Cr, DB, and Lip levels were measured in the two groups using automated and manual methods. Precision and the mean difference between the calibration and QC methods were evaluated using the reported values of the test items in each group.Results: Repeatability and within-laboratory coefficients of variation (CVs) between the automated system and the conventional manual system in the AMR group were similar. However, the mean reported values for test items were significantly different between the two systems. In the MDP group, repeatability and within-laboratory CVs were better with the automation system. All calibration and QC processes were successfully implemented with the Aptio total laboratory automation system.Conclusion: The Aptio total laboratory automation system could be applied to routine practice to improve precision and efficiency.

Performance of Hair Testing for Cocaine Use-Comparison of Five Laboratories Using Blind Reference Specimens.

The purpose of this study was to compare results from five commercial hair testing laboratories conducting workplace drug testing with regard to bias, precision, selectivity and decontamination efficiency. Nine blind hair specimens, including cocaine-positive drug user specimens (some contaminated with methamphetamine) and negative specimens contaminated with cocaine, were submitted in up to five replicates to five different laboratories. All laboratories correctly identified cocaine in all specimens from drug users. For an undamaged hair specimen from a cocaine user, within-laboratory Coefficients of Variation (CVs) of 5-22% (median 8%) were reported, showing that it is possible to produce a homogenous proficiency testing sample from drug user hair. Larger CVs were reported for specimens composed of blended hair (up to 29%) and curly/damaged hair (19-67%). Quantitative results appeared to be method-dependent, and the reported cocaine concentrations varied up to 5-fold between the laboratories, making interlaboratory comparisons difficult. All laboratories reported at least one positive result in specimens contaminated with cocaine powder, followed by sweat and shampoo treatments. Benzoylecgonine, norcocaine, cocaethylene and hydroxylated cocaine metabolites were all detected in cocaine powder-contaminated specimens. This indicates that current industry standards for analyzing and reporting positive cocaine results are not completely effective at identifying external contamination. Metabolite ratios between meta- or para-hydroxy-cocaine and cocaine were 6- and 10-fold lower in contaminated specimens compared to those observed in cocaine user specimens, supporting their potential use in distinguishing samples positive due to contamination and drug use.

Performance comparison between Elecsys Anti-SARS-CoV-2 and Anti-SARS-CoV-2 S and Atellica IM SARS-CoV-2 Total and SARS-CoV-2 IgG assays

Background: Although serological severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tests from several manufacturers have been introduced in South Korea and some are commercially available, the performance of these test kits has not yet been sufficiently validated. Therefore, we compared the performance of Elecsys Anti-SARS-CoV-2 (ACOV2) and Anti-SARS-CoV-2 S (ACOV2S) and Atellica IM SARS-CoV-2 Total (COV2T) and SARS-CoV-2 IgG (sCOVG) serological tests in this study.Methods: A total of 186 patient samples were used. For each test, we analyzed the positive rate of serological antibody tests, precision, linearity, and agreement among the four assays.Results: The positive rates of COV2T, sCOVG, and ACOV2S were high (81.7%–89.2%) in total, with those for ACOV2S being the highest, while those of ACOV2 were as low as 44.6%. This may be related to the high completion rate of vaccination in Korea. The repeatability and within-laboratory coefficients of variation were within the claimed allowable imprecision; however, further research is needed to establish an allowable imprecision at low concentrations. COV2T showed a linear fit, whereas sCOVG and ACOV2S were appropriately modeled with a nonlinear fit. Good agreement was found among COV2T, sCOVG, and ACOV2S; however, the agreement between ACOV2 and any one of the other methods was poor.Conclusions: Considering the different antigens used in serological SARS-CoV-2 antibody assays, the performance of the tested assays is thought to show no significant difference for the qualitative detection of antibodies to SARS-CoV-2.

Analytical Validation of GFRNMR: A Blood-Based Multiple Biomarker Assay for Accurate Estimation of Glomerular Filtration Rate

Accurate and precise monitoring of kidney function is critical for a timely and reliable diagnosis of chronic kidney disease (CKD). The determination of kidney function usually involves the estimation of the glomerular filtration rate (eGFR). We recently reported the clinical performance of a new eGFR equation (GFRNMR) based on the nuclear magnetic resonance (NMR) measurement of serum myo-inositol, valine, and creatinine, in addition to the immunoturbidometric quantification of serum cystatin C, age and sex. We now describe the analytical performance evaluation of GFRNMR according to the Clinical and Laboratory Standards Institute guidelines. Within-laboratory coefficients of variation (CV%) of the GFRNMR equation did not exceed 4.3%, with a maximum CV% for repeatability of 3.7%. Between-site reproducibility (three sites) demonstrated a maximum CV% of 5.9%. GFRNMR stability was demonstrated for sera stored for up to 8 days at 2–10°C and for NMR samples stored for up to 10 days in the NMR device at 6 ± 2°C. Substance interference was limited to 4/40 (10.0%) of the investigated substances, resulting in an underestimated GFRNMR (for glucose and metformin) or a loss of results (for naproxen and ribavirin) for concentrations twice as high as usual clinical doses. The analytical performances of GFRNMR, combined with its previously reported clinical performance, support the potential integration of this NMR method into clinical practice.

Optimization of a Screening Method for Macroprolactinemia

ObjectiveTo optimize a screening method for macroprolactinemia and improve the accuracy of free prolactin (freePRL) detection. MethodOverall efficiency, calculated as the product of the immunoglobulin G (IgG) precipitation rate and the freePRL recovery rate were employed to determine the concentration of the precipitant polyethylene glycol (PEG). Then, an optimized screening method for macroprolactinemia was established. The concentrations of freePRL, obtained by gel filtration chromatography (GFC), from 66 cases were used as the gold standard, and the sensitivity, specificity, accuracy and precision of the optimized and traditional methods for detecting macroprolactinemia were compared. Results(1) The IgG precipitation rate increased with increasing PEG6000 concentration, and the freePRL recovery rate decreased with increasing PEG6000 concentration; the overall efficiency first increased and then decreased. When the IgG concentrations in the mixture were 10 g/L, 25 g/L and 40 g/L, the concentrations of PEG6000 with the highest overall efficiency were 24%, 20% and 18%, respectively. (2) The effect of high and low IgG on the overall efficiency was 4.7% when using 20% PEG6000, which was lower than the effects when using 18% or 24% PEG6000 (9.2% and 13.2%). (3) In the optimized method established using 20% PEG6000, the macroprolactin (macroPRL) chromatographic peak disappeared, but the freePRL chromatographic peak was retained. The sensitivity of this macroprolactinemia screening method was 96.7%, and the specificity was 100%. (4) The freePRL concentrations obtained by the optimized method for samples from 30 macroprolactinemia cases and 36 true hyperprolactinemia cases were 15.8 (10.2-21.4) ng/mL and 60.2 (51.8-79.9) ng/mL; the concentrations were similar to those obtained using the GFC method (16.3 (11.9-27.2) ng/mL and 68.1 (49.5-92.9) ng/mL, respectively (p > 0.05)) and higher than those obtained using the traditional method (9.1 (6.1-17.6) ng/mL and 51.4 (43.7-71.9) ng/mL), respectively, p < 0.05)). (5) The relative deviation between the optimized and GFC methods was -7.0%, which was significantly lower than the relative deviation between the traditional and GFC methods (-21.4%, p < 0.01). (6) The in-batch coefficients of variation (CVs) for the dual-level quality control materials measured by the optimized method were 1.88% and 1.87%, and the within-laboratory CVs were 2.55% and 2.29%, which were slightly lower than the in-batch CVs (1.93% and 2.81%) and within-laboratory CVs (2.75% and 2.81%) measured by the traditional method. ConclusionThe established optimized method for screening macroprolactinemia using 20% PEG6000 as a precipitant can completely precipitate macroPRL components and effectively retain freePRL components. Compared with traditional methods, the optimized method is simpler, more accurate and more stable for the quantitative detection of freePRL.

A new M23-based ELISA assay for anti-aquaporin 4 autoantibodies: diagnostic accuracy and clinical correlation

PurposeAlthough many assays have been developed to detect anti-aquaporin-4 (AQP4) antibodies, most of these assays require sophisticated techniques and are thus only available at specialized laboratories. The aim of this study was to evaluate the analytical and clinical performance of a new commercial enzyme-linked immunosorbent assay (ELISA RSR, AQP4 Ab Version 2) to detect anti-AQP4 antibodies performed on a fully automated system (SkyLAB 752).MethodsSerum samples from 64 patients with neuromyelitis optica spectrum disorders (NMOSD) (including NMO, longitudinally extensive myelitis-LETM, optical neuritis and myelitis) and 27 controls were tested for anti-AQP4 antibodies. All sera were previously tested using an indirect immunofluorescence (IIF) method on primate tissue, as the reference method. Commercial control sera were used to determine within-run, between-day and within-laboratory precision (CLSI guidelines).ResultsAt a cut-off value of 2.1 U/mL as determined by ROC curves, sensitivity and specificity for NMO were 83.3% and 100%, respectively. The ELISA assay provided 100% concordant results with the reference IIF method. The median concentration of anti-AQP4 antibodies was statistically higher in patients with NMO than in patients with LETM (p = 0.0006) or with other NMOSD and in controls (p < 0.0001). At the concentration of 12.4 and 28.1 U/mL, the within-run, between-day and within-laboratory coefficients of variation (CV) were 3.2% and 3%, 7.6% and 7.4%, and 8.2% and 8.0%, respectively.ConclusionsThis new ELISA method performed on a fully automated system, showed high sensitivity and absolute specificity, good CV in precision tests, and provided observer-independent quantitative results.

A Comparison of Simple Analytical Methods for Determination of Fluoride in Microlitre-Volume Plasma Samples

The aim was to compare potential methods for fluoride analysis in microlitre-volume plasma samples containing nano-gram amounts of fluoride. Methods: A group of 4 laboratories analysed a set of standardised biological samples as well as plasma to determine fluoride concentration using 3 methods. In Phase-1, fluoride analysis was carried out using the established hexamethyldisiloxane (HMDS)-diffusion method (1 mL-aliquot/analysis) to obtain preliminary measurement of agreement between the laboratories. In Phase-2, the laboratories analysed the same samples using a micro-diffusion method and known-addition technique with 200 µL-aliquot/analysis. Coefficients of Variation (CVs) and intra-class correlation coefficients (ICCs) were estimated using analysis of variance to evaluate the amount of variation within- and between-laboratories. Based on the results of the Phase-2 analysis, 20 human plasma samples were analysed and compared using the HMDS-diffusion method and known-addition technique in Phase-3. Results: Comparison of Phase-1 results showed no statistically significant difference among the laboratories for the overall data set. The mean between- and within-laboratory CVs and ICCs were < 0.13 and ≥0.99, respectively, indicating very low variability and excellent reliability. In Phase-2, the overall results for between-laboratory variability showed a poor CV (1.16) and ICC (0.44) for the micro-diffusion method, whereas with the known-addition technique the corresponding values were 0.49 and 0.83. Phase-3 results showed no statistically significant difference in fluoride concentrations of the plasma samples measured with HMDS-diffusion method and known- addition technique, with a mean (SE) difference of 0.002 (0.003) µg/mL. In conclusion, the known-addition technique could be a suitable alternative for the measurement of fluoride in plasma with microlitre-volume samples.

Evaluation of pre-analytical and analytical factors that contribute to variability in CSF biomarker levels

development for tau that shows a marked improvement in precision, linearity and reproducibility when compared to currently available tests. Methods: An in vitro diagnostic test for tau is being developed for the VITROS Immunodiagnostic Systems, and analytical performance was evaluated following CLSI guidelines using two VITROS Tau assay reagent lots, 3 CSF pools, and 3 controls. Linearity was tested with 11 admixtures of endogenous tau in CSF and recombinant tau 441 in buffer. Interference testing included commonly prescribed drugs, recombinant tau, Ab peptides, and endogenous substances. Limit of detection (LoD) and lower limit of quantitation were confirmed with 20 replicates per day on 3 days. Results: The median within-laboratory coefficient of variation (CV) for the 3 CSF pools was 3.3% with a range of 2.4% 4.2%. The controls’ within-laboratory CV was 1.2%. Repeatability CVs were 1.9% for CSF pools and 1.2% for controls. The assay showed good linearity across themeasuring range (08,000pg/mL) with the admixture of endogenous and synthetic peptide. Bias introduced by commonly prescribed drugs at high levels was <610%. The interference from Ab peptides at greater than known physiological concentrations as well as from endogenous substances was <5%. The limit of detection was 9.5pg/mL; the lower limit of quantitation was 40pg/mL. Conclusions: VITROS Immunodiagnostic Products Tau Assay 1 demonstrates excellent analytical performance: it is precise, linear and the tested interferences cause no significant bias. The fully automated format and ability to perform these assays in a clinical laboratory should make testing for these important markers more reliable and routine.

CSF biomarker variability in the Alzheimer's Association quality control program

The cerebrospinal fluid (CSF) biomarkers amyloid-β 1–42 (Aβ42), total-tau (T-tau), and phosphorylated-tau (P-tau) are increasingly used for Alzheimer's disease (AD) research and patient management. However, in addition to significant differences between platform-specific commercial assay kit results, there are still large variations in biomarker measurements between and within laboratories. The main sources of this variability remain undefined. Here data was used from the Alzheimer's Association quality control (QC) program to specifically identify sources of analytical variability. Data from the first nine rounds of the Alzheimer's Association QC program was used. In each round, three CSF samples (aliquots of pooled human CSF) prepared at the Clinical Neurochemistry Laboratory at the University of Gothenburg, Sweden, were analyzed by participating laboratories for tau and Aβ proteins by single analyte enzyme-linked immunosorbent assays (ELISA), a multiplexing xMAP assay or an immunoassay with electrochemiluminescence detection (Meso Scale Discovery; MSD). Moreover, 5 laboratories with extensive experience in performing these tests served as reference laboratories and analyzed all samples in 6 runs. A total of 84 laboratories participated. Coefficients of variation (CV) between the laboratories were around 20–30%, while within-run CV (in each laboratory) was smaller (< 5–10%). Longitudinal within-laboratory CV was 5–19%. Interestingly, longitudinal within-laboratory CVs differed considerably between biomarkers at individual laboratories, suggesting that a component of the variability was assay-dependent. Despite the measurement variability, the overall consistency between laboratories in classification of samples (using common pre-hoc derived cutoffs for AD) was high (>90% between-laboratory consistency in 15/18 samples for ELISA and in 12/18 samples for xMAP). The overall variability of CSF AD biomarker measurements remains too high (target variability may be below 10–15%) to allow assignment of universal cutoff values for a specific intended use, even for laboratories using the same commercially available assay. Thus, each laboratory must ensure longitudinal stability (lot consistency) in their measurements and use internally qualified cutoff levels. Kit lot-dependent effects have a significant influence on variability, especially for Aβ42. Further standardization of laboratory procedures and improvement of kit performance (including adoption of a universal reference standard) will likely increase the usefulness of CSF AD biomarkers for researchers and clinicians.

CSF biomarker variability in the Alzheimer's Association quality control program

BackgroundThe cerebrospinal fluid (CSF) biomarkers amyloid beta 1–42, total tau, and phosphorylated tau are used increasingly for Alzheimer's disease (AD) research and patient management. However, there are large variations in biomarker measurements among and within laboratories. MethodsData from the first nine rounds of the Alzheimer's Association quality control program was used to define the extent and sources of analytical variability. In each round, three CSF samples prepared at the Clinical Neurochemistry Laboratory (Mölndal, Sweden) were analyzed by single-analyte enzyme-linked immunosorbent assay (ELISA), a multiplexing xMAP assay, or an immunoassay with electrochemoluminescence detection. ResultsA total of 84 laboratories participated. Coefficients of variation (CVs) between laboratories were around 20% to 30%; within-run CVs, less than 5% to 10%; and longitudinal within-laboratory CVs, 5% to 19%. Interestingly, longitudinal within-laboratory CV differed between biomarkers at individual laboratories, suggesting that a component of it was assay dependent. Variability between kit lots and between laboratories both had a major influence on amyloid beta 1–42 measurements, but for total tau and phosphorylated tau, between-kit lot effects were much less than between-laboratory effects. Despite the measurement variability, the between-laboratory consistency in classification of samples (using prehoc-derived cutoffs for AD) was high (>90% in 15 of 18 samples for ELISA and in 12 of 18 samples for xMAP). ConclusionsThe overall variability remains too high to allow assignment of universal biomarker cutoff values for a specific intended use. Each laboratory must ensure longitudinal stability in its measurements and use internally qualified cutoff levels. Further standardization of laboratory procedures and improvement of kit performance will likely increase the usefulness of CSF AD biomarkers for researchers and clinicians.

Rapid Bioassay for the Determination of Dioxins and Dioxin-like PCDFs and PCBs in Meat and Animal Feeds

Over the past several years, the numerous contamination incidents have raised concerns over the presence of halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and related chemicals in foods and feeds. Here we applied a sensitive recombinant mouse hepatoma cell (H1L1.1c2) bioassay for the determination of dioxins and dioxin-like polychlorinated dibenzofurans (PCDFs) and biphenyls (PCBs) in meat and animal feeds. These cells responded to TCDD-like chemicals with dose-dependent induction of firefly luciferase activity, and the minimal detection limit of TCDD in the cell was 16 fg. Induction equivalency factors determined for pure TCDD-like polychlorinated dibenzo-p-dioxins (PCDDs), PCDFs, and PCBs in the bioassay were well-correlated with the World Health Organization's toxic equivalency factors. To determine the applicability of the bioassay system to detect those compounds presence in meat and feed samples, cell bioassays for 17 TCDD-like PCDDs and PCDFs congeners-spiked lipid extracted from beef or animal feed were performed. Mean recoveries of TCDD-like chlorinated PCDDs and PCDFs congeners from spiked beef or feed fat ranged from 61.2 to 122.3%. Within-laboratory coefficients of variation for analysis as index of precision were lower than 5.2%, and the calculated limits of detection and quantitation were 0.33 and 1 pg toxicity equivalency quantity (TEQ)/0.5 g fat, respectively. Correlation between bioassay- and high-resolution gas chromatography-mass spectrometry (HR-GC-MS)-determined TEQs for 10 meat samples was 0.85, with 1.2 times higher in bioassay than HR-GC-MS. The correlation between bioassay- and HR-GC-MS-determined TEQs in 10 animal feed products was 0.81, with 2.1 times higher in bioassay than HR-GC-MS. Overall, these results demonstrated that the recombinant cell bioassay can be used for the rapid detection and quantitation of PCDDs and dioxin-like PCDFs and PCBs in meats and animal feeds.

Liquid Chromatographic Determination of Ivermectin in Feed

An analytical method for determining ivermectin in feed at 0.50-3 ppm is presented. The method is based on liquid chromatographic measurement after sample preparation by adsorption chromatography on alumina and solid-phase extraction. Two complete, final, finished medicated feeds and the corresponding control feeds used in their preparation were analyzed. Recoveries from feeds fortified at 50-150% of the 2 ppm ivermectin use concentration also were determined. Mean recoveries from replicate analyses ranged from 90 to 100%, and coefficients of variation (CVs) were less than 4.5%. No significant interferences were found in control feeds. The pooled distribution of individual analytical results (n = 100) gave a mean recovery of 100%, a recovery range of 90-111%, and an overall CV of 5.5%. Resolution of the total variance into its 2 components gave a within-laboratory CV of 4.1% and a between-laboratory CV of 3.4%. There was no significant difference in recoveries among laboratories, days, concentrations, and feed base or between fortified and medicated feeds (P > 0.2).

Screening of Nonfatty Fish for Organochlorine Pesticide Residues by Solid-Phase Extraction Cleanup: Interlaboratory Study

Six U.S. Food and Drug Administration laboratories participated in an Interlaboratory trial of a solid-phase extraction cleanup method for determination of pesticides in nonfatty seafood products. The participants analyzed control and fortified (about 0.050 ppm lindane, heptachlor epoxide, p,p'-DDE, and endrin) croaker and flounder samples and a sea trout sample containing incurred p,p'-DDE residues. Mean recoveries of the fortified residues from the fish ranged from 89.1 to 107.8%. The within-laboratory coefficients of variation (CVs) ranged from 4.2 to 8.5%, and the among-laboratory CVs ranged from 10.9 to 26.5%. The 6 laboratories reported a mean value of 0.040 ppm p,p'-DDE in a fish sample which contained incurred residues. The same value (0.040 ppm) was obtained by using official methodology. The within-laboratory CVs ranged from 3.5 to 18.3%, and the among-laboratory CV was 17.3%.

Liquid Chromatographic Determination of Ivermectin in Feed at the 2-PPM Level: Interlaboratory Study

Abstract An analytical method for the determination of ivermectin in feed at the 2 ppm level was evaluated in an interlaboratory study involving 4 laboratories. The method is based on liquid chromatographic measurement following sample preparation by adsorption chromatography on alumina and solid-phase extraction techniques. Each laboratory analyzed 2 complete, final, finished medicated feeds and the corresponding control feeds used in their preparation. In addition, they also measured recoveries in fortified feeds covering 50–150% of the 2 ppm ivermectin use concentration. The mean recoveries from the replicate analyses reported by the laboratories ranged from 90 to 110% and all coefficients of variation (CVs) were less than 4.5%. No significant interferences were found in the control feeds. The pooled distribution of all the individual analytical results (n = 100) gave a mean recovery of 100%, a recovery range of 90–111%, and an overall CV of 5.5%. Resolution of the total variance into its 2 components gave a withinlaboratory CV of 4.1% and a between-laboratories CV of 3.4%. There was no significant difference in recoveries among laboratories, days, concentrations, and feed base or between fortified and medicated feeds (P &amp;gt; 0.2).

Gas Chromatographic Determination of Trans Unsaturation in Margarine: Collaborative Study

Abstract An international collaborative study of a gas chromatographic (GC) method for the determination of trans unsaturation in margarine was conducted in 1980. Thirteen collaborators analyzed a set of 2 of 3 known mixtures of methyl esters and 4 margarine methyl ester samples. Two of the margarine methyl ester samples were blind duplicates. The experimental data were subjected to statistical analysis to determine within- and between-laboratory variation. The statistical data were in excellent agreement with data from a collaborative study of the AOACIUPAC GC method for the determination of methyl esters of fatty acids. Coefficients of variation (CVs) for components in the range of 4-40% were comparable (CV 2-6% for soybean oil methyl esters (AOAC-IUPAC study) as well as for margarine methyl esters (this study)). Good recoveries of total trans acids were obtained and betweenlaboratory as well as within-laboratory CVs were consistently better for the determination of total trans acids by GC vs infrared analysis. The GC method for determining trans unsaturation in margarine has been adopted official first action.

Performance Characteristics of Methods of Analysis Used for Regulatory Purposes. I. Drug Dosage Forms. B. Gas Chromatographic Methods

Gas chromatographic methods for the analysis of drug dosage forms consist of a simple extraction, dilution with an internal standard solution, and injection, or, even simpler, dilution with the internal standard solution and injection. These methods were used in 7 collaborative studies of the determination of 12 pharmaceuticals, published in the Journal of the AOAC during 1973-1983. A total of 43 individual materials consisting of various dosage forms were each analyzed, usually in duplicate, by an average of 8 laboratories, with a total of 582 reported determinations. The average within-laboratory coefficient of variation (CVo) was 1.25% and the average among-laboratories coefficient of variation (CVx) was 2.41%, for a CVo/CVx ratio of 0.52, at an average outlier rate of 1.4% of the reported values. The line of best fit for CVx plotted against concentration increases with decreasing concentration, extending from a CVx of approximately 1.8% at 100% concentration to a CVx of approximately 3.2% at 1% concentration. The change in CVx for a 10-fold decrease in concentration is approximately 0.7% CVx, independent of analyte and matrix.

Determination and Thin Layer Chromatographic Confirmation of Identity of Aflatoxins B1 and M1 in Artificially Contaminated Beef Livers: Collaborative Study

An international collaborative study involving 13 laboratories was conducted to test methods for the determination and thin layer chromatographic (TLC) confirmation of identity of aflatoxins B1 and M1 in beef liver. For the determination, each collaborator furnished fresh or frozen beef liver. Samples were artificially contaminated by adding solutions containing various concentrations of aflatoxins B1 and M1 (0.032-0.69 ng/g). Two TLC confirmation methods were tested with extracts obtained from the determination. Two measurement methods using 2-dimensional TLC were evaluated. In the first, sample extracts were compared directly with B1 and M1 standards on TLC plates; in the second, internal standards plus sample extracts were compared with B1 and M1 standards on the plates. Average within-laboratory coefficients of variation (CV) for the direct method were 26% for B1 and 26% for M1 compared with 24 and 26%, respectively, for the internal standard method. The average between-laboratory CV values were 39% for B1 and 41% for M1 by the direct method and 36% for B1 and 39% for M1 by the internal method and 36% for B1 and 39% for M1 by the internal standard method. Recoveries ranged from 64 to 90% for B1 and from 72 to 86% for M1. These data indicate that the more convenient direct method was sufficient, and internal standards were unnecessary. An analysis of variance was calculated from combined sample data to determine components of variance. The within-laboratory CV values were 27.0 and 32.3% for B1 and M1, respectively, and the between-laboratory CV values were 47.1 and 53.2%, respectively. Both TLC confirmation methods gave satisfactory results and have been adopted official first action, along with the determination method.

Quantitative Determination of 1-131, Cs-137, and Ba-140 in Milk: Collaborative Studies

The official method for Cs-137 in milk by gamma-ray spectroscopy was extended to include I-131 and Ba-140. A collaborative study was performed on this method applied to I-131 concentration in cow's milk; the original collaborative study of the method including all 3 nuclides was reviewed. In the I-131 study, 1 aliquot of a milk sample containing 82 pCi/L was sent to each of 60 laboratories for triplicate analyses. From 40 responses, the mean of the reported values was 81.6 pCi/L, indicating a method bias below the 5% statistical detectability limit. Within- and between-laboratory coefficients of variation (CVs) were 7 and 8%, respectively. In the 3-nuclide study, 2 samples were sent to 25 laboratories for triplicate analyses; one sample contained 633, 305, and 515 pCi/L, respectively, of I-131, Cs-137, and Ba-140 and the other contained 98, 52, and 72 pCi/L. For the high-activity sample, within-laboratory CVs were 4-5% for the 3 nuclides and between-laboratory CVs were 4-7%. For the low-activity sample, the corresponding results were 6-9% and 8-16%. The method bias was statistically significant at 95% confidence only for Cs-137 in the high-activity sample; reported results were 3% below the known concentration. The extended method was adopted official first action.

Distribution of Aflatoxins B1 and M1 in Contaminated Calf and Pig Livers

The determination of aflatoxins B1 and M1 in multiple sections of livers from 4 calves and 1 pig exposed to high levels of aflatoxins showed a uniform distribution of the aflatoxins in each liver, within the precision of the analytical method used. The thin layer chromatographic method has an expected within-laboratory coefficient of variation of 15%.