Procedures for estimating confidence intervals for selected method performance parameters.

Procedures for estimating confidence intervals (CIs) for the repeatability variance (sigmar2), reproducibility variance (sigmaR2 = sigmaL2 + sigmar2), laboratory component (sigmaL2), and their corresponding standard deviations sigmar, sigmaR, and sigmaL, respectively, are presented. In addition, CIs for the ratio of the repeatability component to the reproducibility variance (sigmar2/sigmaR2) and the ratio of the laboratory component to the reproducibility variance (sigmaL2/sigmaR2) are also presented.

A statistical model to evaluate analyte homogeneity for a material.

An underlying assumption for collaborative studies is that the analyte variation among test samples of the material (i.e., matrix and analyte concentration combination) under study has a negligible influence on the estimates of precision for the method. This assumption is expected to be fulfilled when the material under study is prepared (i.e., thoroughly mixed) such that the analyte is distributed uniformly throughout the matrix. Statistical design and intra-class correlation analysis procedures are proposed to assess the similarity or agreement among analytical results among- and within-containers for single and multiple occasions of use (e.g., collaborative and proficiency studies).

A statistical evaluation of the Youden Matched-Pairs Procedure.

AOAC INTERNATIONAL currently permits investigators to use the Youden Matched-Pairs Procedure to obtain estimates of the method performance indicators repeatability and reproducibility (S(r) and SR, respectively). This report explains the statistical model assumptions upon which the procedure is based, provides validity tests for several of these assumptions, explains conditions under which Youdens' "precision error" is not consistent with precision estimate S(r) or SR as defined by AOAC INTERNATIONAL, and indicates when precision estimates based on the procedure should be interpreted with caution or should not be used.

Repeatability and reproducibility estimates from collaborative studies based on total concentration of trace analytes.

In the process of validating a given analytical method for the total concentration of a trace analyte, the precision indicators, repeatability and reproducibility, are obtained from a collaborative study of the method based on a standard one-way completely randomized model. This report discusses the shortcomings of the statistical models used in such studies, defines the component makeup for estimates of the repeatability and reproducibility variances based on these models, and considers suggestions offered as new policy regarding method performance based on total concentration.

A precollaborative study of weight determination methods for quick frozen shrimp.

A precollaborative study compared the accuracy and precision of official AOAC methods with other selected methods for determining net weight of IQF-glazed shrimp and block-glazed shrimp, assessed the ruggedness of the methods with respect to changes in the levels of the factors under study, and selected candidate methods for use in a collaborative study. Methods tested for determining deglazed (frozen) net weight of IQF-glazed shrimp were (1) AOAC Method 963.18 and (2) the Water Bath Dip Method. Methods tested for determining thawed net weight of IQF-glazed shrimp were (1) AOAC Method 967.13, (2) Modified AOAC Method(mnb) 967.13, (3) Modified AOAC Method(pb) 967.13, (4) the Codex Method, (5) the Air Thaw Method, and (6) Modified AOAC Method 963.18. The same methods except Modified AOAC Method 963.18 were tested for determining thawed net weight of block-glazed shrimp. A total of 864 0.45 kg (1 lb), 0.90 kg (2 lb), and 1.35 kg (3 lb) IQF-glazed shrimp test samples and 234 2.25 kg (5 lb) block-glazed shrimp test samples were collected. During sample preparation, test samples were subjected to either water with or without sodium tripolyphosphate (STP). During deglazing (IQF-glazed shrimp only) and/or thawing, test samples were allocated in a factorial design to assess the effects of STP presence (no STP and STP), sieve mesh sizes (2.83 and 2.38 mm; 0.11 and 0.09 in.), and sieve diameters (20 and 30 cm; 8 and 12 in.). During weighing, test samples were further allocated to a sequence of weighing procedures designed to assess the effects of using sieve weights (dry and wet) in combination with paper towel use (no and yes) and tared pan weights when calculating determined net weights. On the basis of the results of this precollaborative study, Modified AOAC Method(pb) 967.13 and the Air Thaw Method seem to be the best methods to determine net weight of IQF-glazed and block-glazed shrimp. Therefore, to validate method choices in the collaborative study, the authors recommend analysis of IQF-glazed shrimp and block-glazed shrimp test samples, each prepared with or without STP, by Modified AOAC Method(pb) 967.13 and the Air Thaw Method. To fulfill AOAC requirements, IQF-glazed shrimp and block-glazed shrimp test samples, each prepared with or without STP, must be analyzed by official methods: AOAC Method 963.18 (IQF-glazed shrimp only) and AOAC Method 967.13. During testing, sieve mesh size will be either 2.83 or 2.38 mm (0.11 or 0.09 in.), sieve diameter will be limited to 30 cm (12 in.), and weighing procedure will be limited to tared pan.

Determination of fish flesh content in frozen coated fish products (modification of AOAC Official Method 971.13): Collaborative Study.

An intralaboratory collaborative study evaluated a modified version of AOAC Official Method 971.13 for determining the fish flesh content (FFC) in frozen coated fish products by comparing it with the on-line method. Eleven collaborators analyzed 36 products (a total of 6336 test samples). Each product targeted one of 4 percent fish flesh (PFF) levels (35, 50, 65, and 80). Products were manufactured from one of 3 raw materials (fillet blocks, minced blocks, and natural fillets) and processed in one of 4 forms (sticks, portions, formed portions, and fillets) and one of 4 styles (raw breaded, batter-dipped, precooked, and fully cooked). Each "official" test sample was tracked through the processing system and weighed (1) before battering and/or breading and, depending on product style, before frying; and (2) after battering and/or breading and, depending on product style, after frying; so that it served as its own control. These weights were used to calculate actual percent fish flesh (APFF) and considered to be generated by the on-line method. Collaborators weighed official test samples (1) before scraping; and (2) after scraping. These weights were used to calculate determined percent fish flesh (DPFF) and considered to be generated by the modified AOAC method. APFF and DPFF were the primary data for statistical analysis. Recoveries ranged from 71.75 to 106.40%. Repeatability (method precision indicator for a single collaborators) relative standard deviation (RSDr) values ranged from 1.04 to 8.37%. Corresponding reproducibility (method precision indicator among collaborators) relative standard deviation (RSDR) values ranged from 1.41 to 11.95%. The DPFF mean was lower than the APFF mean for 30 (83.3%) of 36 products. For 29 of these 30 products, the differences between method means (APFF minus DPFF) ranged from 0.38 to 6.51%. For the remaining product within this group (C/06, fillet blocks, fully cooked portions), the difference between method means was 21.73%. For the remaining 6 (16.67%) of 36 products, the DPFF mean was greater than the APFF mean. The differences between method means ranged from -0.03 to -2.76%. RSD values were considered acceptable (i.e., RDSr < 9% and RSDR < 12%) for all products studied. The modified method for determining FFC in frozen coated fish products has been adopted first action to replace AOAC Official Method 971.13.

Design and analysis of qualitative collaborative studies: minimum collaborative program.

Collaborative studies involving qualitative data are usually conducted under design constraints to fulfill the requirements for quantitative studies. The data from these qualitative studies are often analyzed in a manner that ignores the fact that collaborative studies involve matching (i.e., each laboratory analyzes a portion of each test sample). This report presents some design considerations and analysis procedures for qualitative collaborative studies that take into account that the design involves matching. Suggestions are offered as to the number of laboratories and test samples to use in the minimum collaborative program, and analysis procedures for outier screening are detailed. Method performance is assessed through such indicators as sensitivity, specificity, false positive, and false negative rates. Methods for estimating the error of the performance indicator rates are explained, and procedures are given for estimating false positive and false negative rates for lot defect rates that may occur in practice.

Statistical sampling approaches.

This article describes basic sampling principles and the application of statistical sampling techniques to specific problems encountered in the Food and Drug Administration (FDA). Concepts are emphasized, and theory is minimized. The basic principles of sampling from a normal and binomial population, including confidence interval calculation and sample size determination, are briefly reviewed. Stratified, random, systematic, and judgment sampling are explained. Operating characteristic curves for attribute (and perhaps variable) sampling for acceptance of lots are derived and applied to specific FDA problems. The advantages and disadvantages of single and multiple sampling plans and plans which address multiple classes of criteria such as major and minor defects are discussed. Sampling schedules such as MIL-STD-105D and Canada's Government Specifications Board CGSB-105-GP-1 are reviewed to familiarize readers with the principles involved in these plans and to give them an idea of how they could be applied to FDA problems.

Microbiological Quality of Crabmeat During Processing.

Duplicate samples of crab and crabmeat (body meat and claw meat) were collected four times a day for two consecutive days at seven in-line locations (plus finished product claw and body meat) along the processing lines of 47 crabmeat plants located along the Atlantic Ocean and Gulf of Mexico coasts of the United States. All the plants adhered to Good Manufacturing Practice, as determined by visual inspection. Two sanitation inspections and sample collections were conducted at 5-month intervals to reflect seasonal variation. In all, 8,477 in-line samples and 2,459 finished product units of blue crab and crabmeat and 522 in-line samples and 128 finished product units of red crab and Maine crab and crabmeat were analyzed microbiologically. Geometric mean aerobic plate count at 35°C (APC 35) values increased from 1,200 CFU/g before pick to 20,000 CFU/g in the finished product (body meat). For claw meat, APC 35 values increased from 15,000 CFU/g before pick to 24,000 CFU/g in the finished product. Aerobic plate count at 30°C (APC 30) values were consistently higher (2-fold or less) than APC 35 values. Coliform counts in both finished products were ≥19/g in approximately 60% of the units. Coliforms exceeded 500/g in 3.8 and 3.2% of the finished product units for body meat and claw meat, respectively. Geometric mean Escherichia coli counts were <3 for all sample sites and finished products, with only 3.3 and 2.7% of the units showing detectable E. coli for body meat and claw meat, respectively. Geometric mean values for Staphylococcus aureus were 16.8/g for finished body meat and 16.0/g for finished claw meat; approximately 20% of the units of both finished products had S. aureus values >100/g. S. aureus counts increased significantly after picking.

Performance of disturbed hyperactive and nonhyperactive children on an objective measure of hyperactivity.

Twenty hyperactive emotionally disturbed children (6-11 years) and a matched sample of nonhyperactive emotionally disturbed children were selected from the population of a therapeutic day treatment facility on the basis of teacher ratings. They were administered the Matching Familiar Figures Test-20 and were rated on several scales of impulsivity and/or hyperactivity. Each subject was required to perform on the Delay Task of the Gordon Diagnostic System, which required them to inhibit behavioral responding on a temporally based schedule (DRL-6) in order to win points. Children classified as hyperactive, whether by one or more criteria, were relatively unable to refrain from emitting a high number of nonreinforced responses. Moreover, these performance differences persisted regardless of age or IQ and were stable over the 8 minutes required to complete the test.

Comparison of Selective Plating Media for Enumeration of Bacillus cereus in Foods.

Enumeration of Bacillus cereus on raw sprouts of mung beans and wheat was compared in three agars: mannitol-egg yolk-polymyxin (MYP), polymyxin pyruvate-egg yolk-mannitol-bromthymol blue, and trypticase-soy-polymyxin blood. Ten different strains of B. cereus were used to seed the sprouts. Rates of recovery for the three media were not significantly different. However, with MYP agar, B. cereus could be differentiated more easily from other microorganisms and required fewer confirmatory tests.

Sources of variance in the bioassay of protein value.

A factorial design was used to simultaneously evaluate the relative effect of 8 experimental factors and their interactions on the weanling rat bioassay of protein value: (1) source of protein: ANRC casein, lactalbumin, high-protein wheat flour; (2) protein level of the diet: 5 and 10%; (3) dietary fat level: 10 and 20% corn oil; (4) animal: ARS-Sprague-Dawley, from Taconic Farms; (5) age of animal: 21 and 28 days; (6) acclimation time: 2 and 4 days; (7) replication: 2 complete replications in time; and (8) duration of the test: food consumption and body weights were measured at 3, 7, 10, 14, 17, 21, 24, and 28 days after starting the test diet and converted to the ratio of grams of weight gained per gram of protein consumed for each weigh day. The official AOAC method for determining the protein efficiency ratio was followed with minor modifications. All 8 factors and many of their possible interactions appeared to significantly influence the measured ratios. When the ratios were adjusted by reference to the corresponding value for casein as a control, fat level, rat source, rat age, and acclimation time were no longer significant sources of variation. Plots of measured ratios and their coefficients of variation against time suggest that the optimum assay time varies with the protein but that the assay time should not be less than 21 days. The generally used 28-day assay time seems to offer no increase in precision over 21 days.

Microbiological Quality of Breaded Shrimp During Processing.

Duplicate samples of shrimp or breading materials were collected four times a day for two consecutive days at 12 locations along the processing lines of 33 shrimp-breading firms in the United States during 63 inspections. All firms were using good manufacturing practices. For stock shrimp, the geometric mean aerobic plate count at 35°C incubation (APC 35) was reduced from 2.1 × 106 to 3.3 × 105 colony-forming units (CFU)/g for the frozen finished product. At 35°C, an APC 35 of ≤106 CFU/g was found for 78% of the finished samples. At 30°C incubation, the mean APC was reduced from 7.8 × 106 CFU/g for the stock shrimp to 7.6 × 105 CPU/g for the finished product. Coliform mean counts were virtually static (64 to 83/g) up to the batter-breading steps; however, these counts reached 148 to 160/g at the first batter-breading step and remained constant until the breaded shrimp were frozen. Mean Escherichia coli and Staphylococcus aureus counts were ≤3 and ≤10/g, respectively, for all 12 in-line sampling locations. Salmonella organisms were found in one of 118 finished product samples tested for this pathogen.

Recovery of eggs of two parasitic nematodes, Ascaris sp. and Trichuris sp.: Interlaboratory study.

An interlaboratory study was conducted to determine the effectiveness of the Nacconol ether centrifugation method for recovering parasitic nematode eggs from 3 contaminated products: a crop (cabbage), a sludge fertilizer (Milorganite), and a sewage effluent (Minneapolis). Six replicate samples for each of the 3 products were seeded with eggs at 3 different levels: 200 Ascaris suis and 8 Trichuris muris; 15 A.suis and 15 T.muris; 8 A.suis and 180 T.muris. Recovery was low for all samples except sewage effluent, in which recoveries greater than 100% in 2 samples resulted from the misidentification of arthropod eggs as Ascaris sp. The average mean percent recovery for the other samples was 22.53. Repeatability for replicate samples and reproducibility of results by individual laboratories were poor, and the method is not recommended for quantitative estimates of nematode egg contamination of foods and food-contact materials. However, the Nacconol ether centrifugation method can be used as an all-or-none test. (Only 13% of 1146 counts were falsely negative.) Of 69 samples, only 4 were falsely negative for A.suis eggs and only 1 was falsely negative for T.muris eggs in counts of 6 replicates.

Interlaboratory evaluation of the AOAC method and the A-1 procedure for recovery of fecal coliforms from foods.

An interlaboratory evaluation was made of the 96 h AOAC method and the 24 h A-1 procedure for the enumeration of fecal coliforms in samples of yellow corn meal, rye flour, mung beans, raw ground beef, and raw oyster homogenate. Results indicated that the efficiency of the A-1 procedure, measured in terms of recovery of fecal coliforms, and the reproducibility of that recovery were dependent on the particular food being analyzed. Accordingly, until its efficiency can be more fully demonstrated, the A-1 procedure is recommended only as a screening procedure for fecal coliforms in foods.

Comparison of modified A-1 method with standard EC test for recovery of fecal coliform bacteria for shellfish.

This study is one of a series in which variations of the A-1 method for the detection and enumeration of fecal coliforms and Escherichia coli in seawater and foods were evaluated. The tests were conducted jointly by the Food and Drug Administration and state and provincial laboratories that support shellfish control programs in the United States and Canada as part of the National Shellfish Sanitation Program's Microbiology Task Force activities. Results showed significantly higher recovery of fecal coliforms from naturally contaminated shellfish by the AOAC official A-1-M method than by the American Public Health Association standard method. There was no significant difference in recovery of E. coli by the 2 methods.

Recovery of parasitic nematodes from fish by digestion or elution.

Two methods, digestion and elution, were used to recover parasitic nematodes from 470 flatfish belonging to species in the family Pleuronectidae. Samples of similar fish were collected from market lots; half of each sample was subjected to digestion, and half was subjected to elution (sedimentation). The edible (flesh) and the inedible (viscera) portions of each fish were analyzed separately. The total number of nematodes recovered by digestion was 1,110, which was not significantly greater than the 922 nematodes recovered by elution. However, digestion recovered 1,062 nematodes of the anisakine genera Anisakis and Phocanema, which are potentially pathogenic for human consumers of raw of semiraw fish. This number is significantly greater than the 608 pathogenic nematodes recovered by elution. Digestion also recovered 242 more nematodes from the edible flesh than did elution. Conversely, more nonpathogenic nematodes were recovered by elution. Approximately half the fish (240) had been collected in Boston markets, and the other half (230) had been collected in San Francisco markets. Fish from San Francisco each contained an average of eight nematodes, and those from Boston contained an average of less than one nematode per fish.

pH Determination in acidified foods: collaborative study.

A proposed method for determining pH of acidified foods has been developed and subjected to collaborative study. The method appears to be both accurate and precise. Five samples consisting of pimientos, marinated pimientos, 2 pH buffer solutions, and chocolate syrup were sent to each of 12 collaborators along with a copy of the method. Two of the collaborators were FDA District laboratories while the remainder were representatives from industry, universities, and state health agencies. Many different types of pH meters and combinations of electrodes were used by the collaborators. The tabulated results from the collaborators are presented. The method has been adopted official first action.