Determination of Fat in Raw and Processed Milks by the Gerber Method: Collaborative Study

Abstract The Gerber method is used worldwide as a simple and rapid method for determining fat in raw and processed milks. However, the volume of the test portion used in the method has not been internationally agreed upon. A collaborative study was conducted to evaluate performance of the Gerber method using either a weighed test portion (11.13 g) or by a 10.77 mL test portion delivered by pipet. For each method, laboratories received 10 test samples: 5 raw and 5 pasteurized homogenized milks, 2 of which were blind duplicate pairs. Eleven and 10 laboratories participated in the evaluation of aliquot addition by weight and pipet, respectively. Mojonnier ether extraction (Method 989.05) was used as the reference method. Interlaboratory study statistics were similar between methods of test portion addition and between raw and processed materials; therefore, summary interlaboratory study statistics were pooled. The fat content of milk samples ranged from 0.96 to 5.48%. Absolute reproducibility and repeatability were not affected by fat level, and pooled statistical performance (invalid and outlier data removed) was (g fat/100 g milk) sr = 0.026, sR = 0.047, r = 0.074, and R = 0.132. Relative standard deviations increased with decreasing fat content, and were summarized by fat level: 1–2% fat milk, mean = 1.437, RSDr = 1.809%, RSDR = 3.271%; 2–6% fat milk, mean = 4.156, RSDr = 0.626%, RSDR = 1.131%. Compared with ether extraction, test results by the Gerber method were slightly lower (0.02% fat) using a weighed test portion and significantly lower (0.06% fat) using a 10.77 mL volume addition by pipet. A trend toward underestimating fat content at lower fat concentrations (1–2% fat) was observed with the weighed test portion but not when a pipet was used. The Associate Referee recommends that the Gerber method using a weighed test portion be adopted as First Action with applicability limited to whole milk.

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Modification of Babcock Method to Eliminate Fat Testing Bias Between Babcock and Ether Extraction Methods (Modification of AOAC Official Methods 989.04 and 995.18): Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/80.4.845 ◽

1997 ◽

Vol 80 (4) ◽

pp. 845-859 ◽

Cited By ~ 2

Author(s):

Joanna M Lynch ◽

David M Barbano ◽

J Richard Fleming

Keyword(s):

Standard Deviation ◽

Collaborative Study ◽

Reference Method ◽

Official Method ◽

Test Results ◽

Testing Bias ◽

Ether Extraction ◽

Relative Standard ◽

Aoac Official ◽

Aoac Official Method

Abstract The Babcock test for determining fat in milk (AOAC Official Method 989.04) and in cream (AOAC Official Method 995.18) gives consistently higher test results than does the modified Mojonnier ether extraction reference method (AOAC Official Methods 989.05 and 995.19). To decrease the density of material in Babcock columns and thus lower test results, the Babcock method was modified by lowering the temperatures used at various points in the method, from about 57.5° to 48°C. Using the AOAC collaborative study format, 9 laboratories tested 9 pairs of blind duplicate raw milk samples (fat range 2.5–5.7%) and 9 pairs of blind duplicate heat-treated cream samples (fat range 30–45%) using the temperature modified (TM) Babcock method. The ether extraction test was conducted as the reference method. The statistical performance (invalid and outlier data removed) of the TM Babcock method was, for milk: percent fat value of 4.110, repeatability standard deviation (sr) value of 0.037, reproducibility standard deviation (sR) value of 0.047, repeatability relative standard deviation (RSDr) value of 0.901% and reproducibility relative standard deviation (RSDR) value of 1.147%; and for cream: percent fat value of 37.555, sr value of 0.258, sR value of 0.353, RSDr value of 0.687% and RSDR value of 0.940%. The TM Babcock method performance was acceptable but not as good as that achieved in previous studies of the unmodified method. For the TM Babcock and ether methods, respectively, average percentages fat were 4.110 and 4.114 for milk, and 37.555 and 37.485 for cream. Temperature modification statistically eliminated the testing bias between methods. The tem-perature modifications of the Babcock methods for determination of fat in milk and cream (989.04 and 995.18) have been adopted revised first action by AOAC INTERNATIONAL.

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Combination of Total Solids Determined by Oven Drying and Fat Determined by Mojonnier Extraction for Measurement of Solids-Not-Fat Content of Raw Milk: Collaborative Study

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/72.5.719 ◽

1989 ◽

Vol 72 (5) ◽

pp. 719-724 ◽

Cited By ~ 2

Author(s):

Jenny L Clark ◽

David M Barbano ◽

Chapman E Dunham

Keyword(s):

Standard Deviation ◽

Collaborative Study ◽

Raw Milk ◽

Fat Content ◽

Oven Drying ◽

Total Solids ◽

Ether Extraction ◽

Relative Standard ◽

The Mean ◽

Forced Air

Abstract Each of 9 laboratories analyzed 9 pairs of blind duplicate raw milk samples for fat, using the Mojonnier ether extraction method (16.E13-16.E17), and for total solids, using a new direct forced air oven method. Solids-not-fat was determined by subtracting percent fat from percent total solids. The solids-not-fat content of the samples tested in the collaborative study ranged from 8.48 to 9.36%. The average repeatability standard deviation (sr) and the average reproducibility standard deviation (sR) for the solids-not-fat method were 0.019 and 0.041, respectively. Average repeatability (RSDr) and reproducibility (RSDR) relative standard deviations were 0.218 and 0.466%, respectively. The mean repeatability value (r) was 0.055; the mean reproducibility value (R) was 0.117. The difference between milk total solids determined by direct forced air oven drying and milk fat determined by Mojonnier ether extraction has been approved for interim official first action for determination of solids-not-fat content of milk.

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Comparison of Babcock and Ether Extraction Methods for Determination of Fat Content of Milk: Collaborative Study

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/71.5.898 ◽

1988 ◽

Vol 71 (5) ◽

pp. 898-914 ◽

Cited By ~ 5

Author(s):

David M Barbano ◽

Jenny L Clark ◽

Chapman E Dunham

Keyword(s):

Extraction Method ◽

Collaborative Study ◽

Raw Milk ◽

Extraction Methods ◽

Fat Content ◽

Absolute Level ◽

Ether Extraction ◽

Relative Standard ◽

Standard Deviations ◽

The Difference

Abstract Eleven collaborating laboratories analyzed 18 blind duplicate pairs of raw milk samples for fat by the Babcock method and by a modified Mojonnier ether extraction method in 7 round robins conducted over a 14 month period. Ten laboratories used the Babcock method and 10 used the modified Mojonnier method. Fat content of samples ranged from approximately 2.7 to 5.6%. Mean test value of samples analyzed was approximately 3.9% fat. Average standard deviations of within-laboratory repeatability (Sr) of the Babcock and ether extraction methods were 0.029 and 0.015% fat, respectively. Average standard deviations of between-laboratory reproducibility (SR) of the Babcock and ether extraction methods were 0.039 and 0.020% fat, respectively. Average repeatability relative standard deviations (RSDr) for the Babcock and ether extraction methods were 0.742 and 0.396%; average reproducibility relative standard deviations (RSDR) were 1.014 and 0.512%, respectively. Mean repeatability values (r) and reproducibility values (R) were 0.081 and 0.111% for Babcock and 0.044 and 0.056% for ether extraction, respectively. The ether extraction method demonstrated consistently better within- and between-laboratory agreement. The overall mean test value for the Babcock method was significantly higher (0.021% fat) than that for ether extraction. The difference between Babcock and ether extraction fat test results was different for different farms. In addition, the mean difference between percent fat determined by the Babcock and ether extraction methods was different in different months. There was no correlation in the difference between Babcock and ether extraction methods with the absolute level of fat in the samples in the range of 2.7 to 5.6% fat. The modifications of the AOAC Babcock method and the modified Mojonnier ether extraction method have been approved interim official first action.

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Comparison of Babcock and Ether Extraction Methods for Determination of Fat Content of Cream: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/79.4.907 ◽

1996 ◽

Vol 79 (4) ◽

pp. 907-916 ◽

Cited By ~ 2

Author(s):

Joanna M Lynch ◽

David M Barbano ◽

J Richard Fleming

Keyword(s):

Extraction Method ◽

Collaborative Study ◽

Extraction Methods ◽

Fat Content ◽

Official Method ◽

Ether Extraction ◽

Aoac Official ◽

The Difference ◽

Aoac Official Method

Abstract A modified Mojonnier ether extraction method for determination of the fat content of cream was developed based on the method for milk (AOAC Official Method 989.05). The cream Babcock method (AOAC Official Method 920.111 B-C) was modified to harmonize with the milk Babcock method (AOAC Official Method 989.04) and to clarify procedural details. Using the AOAC collaborative study format, 10 laboratories tested 9 pairs of blind duplicate heat-treated cream samples with a fat range of 30-45% using both methods. The statistical performance (invalid and outlier data removed) was as follows: mean % fat = 37.932, sr = 0.125, sR = 0.151, RSDr = 0.330, RSDR = 0.398, r = 0.354, and R = 0.427 for the ether extraction method. For the Babcock method, mean % fat = 38.209, sr = 0.209, SR = 0.272, RSDr = 0.548, RSDR = 0.712, r = 0.592, and R = 0.769. Average test results for fat from the Babcock method were 0.277% (absolute fat) greater than for the Mojonnier ether extraction method. The difference between methods, as a percentage of the average fat content of the samples, was 0.73%. This agrees with differences observed between the 2 methods for milk when 10 to 17 laboratories tested 7 milk samples in blind duplicate at bimonthly intervals over a 4-year period (average difference 0.029% fat, 0.78% as a percentage of average fat content). The Mojonnier ether extraction and Babcock methods for fat in cream have been adopted by AOAC INTERNATIONAL. The new Babcock method replaced the AOAC Official Method 920.111 B-C.

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Immunoaffinity Column Cleanup with Liquid Chromatography Using Post-Column Bromination for Determination of Aflatoxins in Hazelnut Paste: Interlaboratory Study

Journal of AOAC International ◽

10.1093/jaoac/88.2.526 ◽

2005 ◽

Vol 88 (2) ◽

pp. 526-535 ◽

Cited By ~ 18

Author(s):

Hamide Z Senyuva ◽

John Gilbert

Keyword(s):

Liquid Chromatography ◽

Standard Deviation ◽

Relative Standard Deviation ◽

Aflatoxin B1 ◽

The United States ◽

Interlaboratory Study ◽

Immunoaffinity Column ◽

Test Portion ◽

Relative Standard

Abstract An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for determination of aflatoxin B1 and total aflatoxins in hazelnut paste at European regulatory limits. The test portion was extracted with methanol–water (6 + 4). The extract was filtered, diluted with phosphate-buffered saline (PBS) solution to a specified solvent concentration, and applied to an immunoaffinity column containing antibodies specific to aflatoxins. The aflatoxins were removed from the immunoaffinity column with methanol, and then quantified by reversed-phase LC with post-column derivatization (PCD) involving bromination. The PCD was achieved with electrochemically generated bromine (Kobra Cell®) followed by fluorescence detection (except for one participant who used pyridinum hydrobromide perbromide for bromination). Hazelnut paste, both naturally contaminated with aflatoxins and blank (<0.1 ng/g) for spiking by participants with aflatoxins, was sent to 14 collaborators in Belgium, The Netherlands, Spain, Turkey, the United Kingdom, and the United States. Test portions were spiked at levels of 4.0 and 10.0 ng/g for total aflatoxins by participants using supplied total aflatoxins standards. Recoveries for total aflatoxins and aflatoxin B1 averaged from 86 to 89%. Based on results for naturally contaminated samples (blind duplicates at 3 levels ranging from 4.0 to 11.8 ng/g total aflatoxins), the relative standard deviation for repeatability (RSDr) ranged from 2.3 to 3.4% for total aflatoxins and from 2.2 to 3.2% for aflatoxin B1. The relative standard deviation for reproducibility (RSDR) ranged from 6.1 to 7.0% for total aflatoxins and from 7.3 to 7.8% for aflatoxin B1. The method showed exceptionally good within-laboratory and between-laboratory precision for hazelnut paste, as evidenced by HORRAT values, which in all cases were significantly below target levels, the low levels of determination for both aflatoxin B1 and total aflatoxins.

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Combined Phenyl Silane and Immunoaffinity Column Cleanup with Liquid Chromatography for Determination of Ochratoxin A in Roasted Coffee: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/84.2.444 ◽

2001 ◽

Vol 84 (2) ◽

pp. 444-450 ◽

Cited By ~ 33

Author(s):

A Catherine Entwisle ◽

Alison C Williams ◽

Peter J Mann ◽

Joanne Russell ◽

Philip T Slack ◽

...

Keyword(s):

Liquid Chromatography ◽

Standard Deviation ◽

Ochratoxin A ◽

Relative Standard Deviation ◽

Collaborative Study ◽

Immunoaffinity Column ◽

Roasted Coffee ◽

Test Portion ◽

Low Level ◽

Relative Standard

Abstract A collaborative study was conducted to evaluate a liquid chromatography (LC) method for ochratoxin A using sequential phenyl silane and immunoaffinity column cleanup. The method was tested at 3 different levels of ochratoxin A in roasted coffee, which spanned the range of possible future European regulatory limits. The test portion was extracted with methanol and sodium bicarbonate by shaking for 30 min. The extract was filtered, centrifuged, and then cleaned up on a phenyl silane column before being eluted from the washed column with methanol–water. The eluate was diluted with phosphate-buffered saline (PBS) and applied to an ochratoxin A immunoaffinity column, which was washed with water. The ochratoxin A was eluted with methanol, the solvent was evaporated, and the residue was redissolved in injection solvent. After injection of this solution onto a reversed-phase LC apparatus, ochratoxin A was measured by fluorescence detection. Eight laboratory samples of low-level naturally contaminated roasted coffee and 2 laboratory samples of blank coffee (< 0.2 ng/g ochratoxin A at the signal-to-noise ratio of 3:1), along with ampules of ochratoxin A calibrant and spiking solutions, were sent to 15 laboratories in 13 different European countries. Test portions of the laboratory samples were spiked at levels of 4 ng/g ochratoxin A, and recoveries ranged from 65 to 97%. Based on results for spiked blank material (blind duplicates) and naturally contaminated material (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) ranged from 2 to 22% and the relative standard deviation for reproducibility (RSDR) ranged from 14 to 26%. The method showed acceptable within- and between-laboratory precision, as evidenced by HORRAT values, at the low level of determination for ochratoxin A in roasted coffee.

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Multifunctional Column Coupled with Liquid Chromatography for Determination of Aflatoxins B1, B2, G1, and G2 in Corn, Almonds, Brazil Nuts, Peanuts, and Pistachio Nuts: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/77.6.1512 ◽

1994 ◽

Vol 77 (6) ◽

pp. 1512-1521 ◽

Cited By ~ 129

Author(s):

Mary W Trucksess ◽

Michael E Stack ◽

Stanley Nesheim ◽

Richard H Albert ◽

Thomas R Romer

Keyword(s):

Liquid Chromatography ◽

Standard Deviation ◽

Relative Standard Deviation ◽

Collaborative Study ◽

The United States ◽

Test Portion ◽

Brazil Nuts ◽

Pistachio Nuts ◽

Relative Standard

Abstract An AOAC/IUPAC collaborative study was conducted to evaluate the effectiveness of a multifunctional column for the determination of aflatoxins. The test portion is extracted with acetonitrile–water (9 + 1), the extract is filtered, and the filtrate is passed through the column. The aflatoxins in the eluate are determined by reversed-phase liquid chromatography after derivatization with trifluoroacetic acid. Naturally contaminated corn, almonds, Brazil nuts, peanuts, and pistachio nuts spiked with total aflatoxins at 5,10,20, and 30 ng/g were sent to 12 collaborators in the United States, Denmark, France, Japan, and Switzerland. Eleven collaborators completed the study. Average recoveries of total aflatoxins for each spike level for the various commodities (excluding Brazil nuts at 5 ng/g) were 93,97,95, and 95%, respectively; the repeatability relative standard deviation (RSDr) ranged from 6.0 to 23.2% and the reproducibility relative standard deviation (RSDR) ranged from 12.0 to 69.4%. The multifunctional column coupled with a liquid chromatographic method for determination of aflatoxins in corn, almonds, Brazil nuts, peanuts, and pistachio nuts has been adopted first action by AOAC INTERNATIONAL.

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Evaluation of the iQ-Check®Salmonella II Assay in Select Foods: Collaborative Study, First Action 2017.06

Journal of AOAC International ◽

10.5740/jaoacint.17-0426 ◽

2018 ◽

Vol 101 (4) ◽

pp. 1043-1057

Author(s):

Patrick Bird ◽

M Joseph Benzinger ◽

Benjamin Bastin ◽

Erin Crowley ◽

James Agin ◽

...

Keyword(s):

Statistical Analysis ◽

Collaborative Study ◽

Reference Method ◽

The United States ◽

Probability Of Detection ◽

Milk Chocolate ◽

Inoculum Level ◽

Test Portion ◽

Dog Food ◽

Interlaboratory Reproducibility

Abstract The iQ-Check Salmonella II Real-Time PCR test kit utilizes Salmonella-specific oligonucleotide probes and primers for the rapid and specific detection of Salmonella species in select food types. The alternative method was evaluated by using 375 g test portions in an unpaired study design for two matrices, milk chocolate and dry dog food. Each matrix was compared with the U.S. Food and Drug Administration Chapter 5 Salmonella reference method. Fourteen technicians from 12 laboratories, including academia and industry, located within the United States and Canada participated in the collaborative study. Three levels of contamination were evaluated for each matrix: an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2–2 CFU/test portion), and a high inoculum level (2–5 CFU/test portion). The statistical analysis was conducted according to the Probability of Detection (POD) statistical model. The results obtained for the low inoculum level test portions produced a difference in the candidate presumptive and confirmatory results (dLPOD) value with a 95% confidence interval of −0.05, (−0.15, 0.06) for the milk chocolate and 0.10, (−0.01, 0.21) for the dry dog food. The dLPOD results indicate an equivalence between the candidate method and reference method for the matrices evaluated, and the method demonstrated acceptable interlaboratory reproducibility as determined in the collaborative evaluation. False positive and false negative rates were determined for each matrix and produce values of <2%. Based on the data generated, the method demonstrated acceptable interlaboratory reproducibility data and statistical analysis.

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Determination of Moisture and Fat in Meats by Microwave and Nuclear Magnetic Resonance Analysis: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/91.4.802 ◽

2008 ◽

Vol 91 (4) ◽

pp. 802-810 ◽

Cited By ~ 23

Author(s):

Timothy P Leffler ◽

Cindy R Moser ◽

Bobbie J McManus ◽

John J Urh ◽

Jimmy T Keeton ◽

...

Keyword(s):

Nuclear Magnetic Resonance ◽

Collaborative Study ◽

Meat Products ◽

Fat Content ◽

Microwave Drying ◽

Processed Meat ◽

Nmr Spectrometry ◽

Relative Standard ◽

Aoac Method ◽

Total Fat

Abstract Ten laboratories participated in a collaborative study to determine the total moisture and fat in raw and processed meat products by microwave drying and nuclear magnetic resonance (NMR) spectroscopy. Meat products were prepared following the AOAC Method and analyzed using CEM Corp.'s SMART Trac Moisture and Fat Analysis system. SMART Trac provides moisture results by measuring the weight loss on drying by microwave energy. The dried sample is then analyzed by NMR spectrometry for fat content. Moisture and fat results are displayed and reported by the SMART Trac as a percentage (g/100 g). Microwave drying is an AOAC-approved reference method (Method 985.14), Moisture in Meat and Poultry Products. NMR spectrometry is a secondary technique used to determine the concentration of various constituents in biological, organic, or chemical samples. The study design was based on Youden's matched pair principle for collaborative tests. For the purposes of this study, 10 laboratories each tested 10 Youden matched pairs, for a total of 20 samples. The study samples represented a range of products processed daily in plant operations. Included were raw meat samples (beef, pork, chicken, and turkey) as well as processed meats (beef hot dog, pork sausage, and ham). The total moisture content of the undiluted samples, as received for the purposes of this study, was determined by AOAC Method 950.46 and ranged from 54.03 to 74.99. The total fat content of the undiluted samples was determined by AOAC Method 960.39 and ranged from 1.00 to 29.79. Statistical analysis of study results for total moisture yielded a relative standard deviation for repeatability (RSDr) range of 0.14 to 0.95 and a relative standard deviation for reproducibility (RSDR) range of 0.26 to 0.95. Statistical analysis for total fat yielded similar RSDr and RSDR range of 0.74 to 4.08. Results for turkey had higher RSDr and RSDR values, both at 12.6, due to low fat content and possibly to the separation of the samples observed by some of the collaborators. Results demonstrate that microwave drying with NMR is a rapid, practical method providing results equivalent to AOAC Methods 950.46 (Forced Air Oven Drying) and 960.39 (Soxhlet Ether Extraction) in raw and processed meat products.

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Evaluation of the Solus One Salmonella Assay in Select Foods: Collaborative Study: First Action 2020.03

Journal of AOAC International ◽

10.1093/jaoacint/qsaa071 ◽

2020 ◽

Vol 103 (6) ◽

pp. 1568-1581

Author(s):

Benjamin Bastin ◽

M Joseph Benzinger ◽

Erin S Crowley ◽

James Agin ◽

Raymond Wakefield

Keyword(s):

United States ◽

Statistical Analysis ◽

Collaborative Study ◽

Control Level ◽

Reference Method ◽

The United States ◽

Probability Of Detection ◽

Inoculum Level ◽

Test Portion ◽

The Matrix

Abstract Background The Solus One Salmonella immunoassay utilizes Salmonella specific selective media and automated liquid handling, for the rapid and specific detection of Salmonella species in select food types. Objective The candidate method was evaluated using 375 g test portions in an unpaired study design for a single matrix, instant non-fat dry milk (NFDM) powder. Method The matrix was compared to the United States Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 5 Salmonella reference method. Eleven participants from 10 laboratories within academia and industry, located within the United States, Mexico, South Africa, Germany, and the United Kingdom, contributed data for the collaborative study. Three levels of contamination were evaluated for each matrix: an uninoculated control level [0 colony forming units (CFU)/test portion], a low inoculum level (0.2–2 CFU/test portion) and a high inoculum level (2–5 CFU/test portion). Statistical analysis was conducted according to the Probability of Detection (POD) statistical model. Results Results obtained for the low inoculum level test portions produced a dLPOD value with a 95% confidence interval between the candidate method confirmed (both alternative and conventional confirmation procedures) and the reference method of 0.07 (−0.02, 0.15). Conclusions The dLPOD results indicate equivalence between the candidate method and the reference method for the matrix evaluated and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. False positive and false negative rates were determined for the matrix and produce values of <2%. Highlights Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis.

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