The US peanut industry can use up to three 21.8 kg samples per lot to determine if shelled peanut lots are acceptable or unacceptable due to aflatoxin content. If a lot is accepted by the first 21.8 kg sample (1AB≤8 ng/g) prepared with the USDA/AMS Subsampling mill (DM), then some peanut buyers request that the sheller prepare the second sample (2AB) and in some cases the 3AB sample (called special samples in the trade) with a vertical cutter mixer (VCM) type mill. These requests to specifically use the VCM instead of the DM to prepare official (1AB) and special (2AB or 3AB) samples is based in part on a perception that analytical results associated with a test portion taken from the 21.8 kg sample comminuted with a DM does not detect the full magnitude of aflatoxin in the 21.8 kg sample and that negative aflatoxin certificates (lot acceptance) are more likely to occur when samples are prepared with a DM than a VCM. Analysis of aflatoxin test results from two shellers along with Monte Carlo simulation indicate that differences between the 1AB and special sample test results are due to the use of a cut-off limit (≤ 8 ng/g associated with the 1AB) requested by the buyer as part of the acceptance criteria and not due to any bias associated with the DM. Operating characteristic curves were used to demonstrate that the performance of the USDA/AMS aflatoxin sampling plan is about the same regardless of the use of a DM or a VCM for sample preparation. The performances are similar because the DM, with an 1100 g test portion, account for only 8% of the total variability of the aflatoxin test procedure (sampling and analysis account for about 92%). A sampling plan that requires two 21.8 kg samples to test less than a limit, regardless of mill used to prepare the two samples, has a very low risk of accepting bad lots above the FDA limit of 20 ng/g, but has a very high risk of rejecting good lots, which makes for an extremely high economic burden on the sheller.
Aflatoxin is a toxic and carcinogenic compound produced by fungi such as
The Agricultural Marketing Service of the US Department of Agriculture (AMS/USDA) and the peanut industry estimate aflatoxin in all domestic raw shelled peanut lots before being shipped by the seller to a buyer. In the peanut industry the seller is typically a sheller, but the buyer can vary from exporters and importers to food manufacturers. The accepted aflatoxin test procedure used by USDA/AMS to estimate the aflatoxin concentration in a bulk lot of raw shelled peanuts consist of sampling, sample preparation, and analytical steps. It is important to understand the role and importance that each step has in the overall aflatoxin test procedure. These three steps are briefly described below.
When possible, automatic sampling equipment is used to select samples from moving streams of shelled peanuts. A diverter cup cuts through a moving a stream at specified intervals removing approximately 50 to 100 small incremental samples from the beginning to the end of the lot. For a 20 metric ton lot (about 44,053 lbs), the diverter cup takes an incremental sample about every 400 kg (50 cuts) to 200 kg (100 cuts). The incremental samples are accumulated to form an aggregate sample of approximately 70 to 75 kg. Using an USDA/AMS approved divider; three 21.8-kg (48-lb) laboratory samples are taken from the aggregate sample for aflatoxin analysis (the remainder of the aggregate sample is used as a grade sample).
Each 21.8-kg laboratory sample is comminuted in a USDA/AMS approved mill and an 1100 g test portion is removed for aflatoxin analysis. Typically, two types of mills are approved by AMS to comminute the laboratory sample. A USDA/AMS Subsampling mill (often called the Dickens Mill or DM for short), developed by the Agricultural Research Service (ARS) of USDA for the USDA/AMS, comminutes and automatically subsamples the 21.8-kg laboratory sample and provides an 1100 g comminuted test portion for aflatoxin analysis (
Because the 1100-g test portion is considered to be a large test portion size, a water slurry method was developed by USDA/ARS for AMS that provides additional particle size reduction and saves on organic solvents ( Total error of the mycotoxin-test procedure is the sum of sampling, sample preparation, and analytical errors. The term error denotes variability as measured by the variance.
1600 ml of water for three minutes. A 196 g portion of the water blend (containing 80 g of peanuts) is removed from the slurry blend. Then the appropriate amount of an organic solvent is added to the 196 g portion of water and peanuts and blended for an additional 30 seconds. Two aliquots (called A and B) are removed from the blend and each aliquot is quantified for aflatoxin and the results are averaged to obtain a sample test result. The aflatoxin is extracted from the peanut solvent blend and quantified using an approved USDA/AMS analytical methods such as thin layer chromatography (TLC), immunoassay, or high performance liquid chromatography (HPLC).
The USDA/AMS uses a sequential sampling plan where three 21.8-kg laboratory samples are removed form each lot and tested for aflatoxin (called 1AB, 2AB, and 3AB). If the 1AB laboratory sample test result is less than or equal to 8 ng/g total aflatoxins, the lot is accepted into the domestic market with no further tests. If the 1AB laboratory sample test result is greater than 45 ng/g, the lot is rejected with no further tests. If the 1AB sample test result is between 8 and 45 ng/g, the 2AB is analyzed for aflatoxin and the two sample test results are averaged (Avg 1AB/2AB = (1AB+2AB)/2). If the average of the two sample test results is less than or equal to 12 ng/g, the lot is accepted with no further tests. If the average of the two sample test result is greater than 22 ng/g, the lot is rejected with no further tests. If the average of the two sample test result is between 12 and 22 ng/g, the 3AB is analyzed for aflatoxin and the three sample test results are averaged (Avg 1AB/2AB/3AB = (1AB+2AB+3AB)/3). If the average of the three sample test results is less than or equal to 15 ng/g, the lot is accepted, else the lot is rejected. The sequential test was designed to save on the amount of sample required to accept or reject a lot. Lots with very low or very high levels of aflatoxin are accepted or rejected on the first 1AB sample. Lots with aflatoxin concentration near the USDA/AMS limit of 15 ng/g may require two or all three samples to be tested to make a decision to accept or reject the lot.
Some buyers of raw shelled peanuts will request that all official 21.8-kg laboratory samples used to accept or reject a lot be comminuted with a VCM. In that case, USDA/AMS still requires that an 1100 g test portion be removed from the 21.8 kg laboratory sample comminuted with a VCM for aflatoxin analysis. Some buyers also request that lots accepted by the first 21.8 kg laboratory sample (1AB≤8 ng/g) where the DM was used to prepare the official sample undergo additional testing before the buyer considers the lot acceptable for purchase. If the first 21.8-kg sample tests 8 ng/g or less when the sample is prepared by the DM, then the seller prepares the 2AB and/or 3AB samples (called Specials or special samples in the trade) with a VCM and the sample test result must be less than an accept/reject limit defined by the buyer. Accept/reject limits defined by the buyer may vary, but are generally in the 5 to 10 ng/g range. Once a lot has been accepted by the official USDA/AMS test (1AB≤8ng/g), any additional testing is strictly between the seller and the buyer. The buyer may also request that a 50 g test portion (not the USDA/AMS required 1100 g) be taken from the special sample prepared with a VCM for aflatoxin analysis.
Sellers and buyers have observed that on some occasions when the official 1AB prepared with the DM tested 8 ng/g or less, the corresponding Specials prepared with the VCM tested greater than 8 ng/g or some limit defined by the buyer. They also noticed that over many lots, the average aflatoxin among all 1AB samples prepared with the DM that tested below the USDA/AMS limit of 8 ng/g was lower than the average aflatoxin among all corresponding Special samples prepared with the VCM. These observations led to several concerns on part of both sellers and buyers about sample test results when the sample was prepared with the DM versus the VCM. There are concerns that (a) analytical results associated with 21.8-kg samples prepared with the DM may be lower than analytical results from subsequent tests of the 21.8 kg special samples prepared with a VCM and (b) that lots may be more likely to receive negative certificates (more likely to accept the lot) when a 21.8-kg sample is prepared by the DM than when prepared by the VCM. These observations have led some sellers and buyers to believe that analysis of 21.8-kg samples prepared by the DM produces results that are biased to the low side of the true 21.8-kg sample concentration. The DM can't be biased to the low side of the true sample concentration unless aflatoxin is destroyed during the comminution process and/or the extraction efficiency associated with the analytical method that is used to quantify aflatoxin in the test portion is less for the DM after the water slurry process than for the VCM.
The objective of this study was to use actual sample test results supplied by two commercial shellers that were prepared by the DM and VCM along with sampling theory to determine why buyers and sellers are observing that (a) a greater percentage of 1AB samples prepared by the DM test 8 ng/g or less than Special samples prepared by the VCM and (b) why the average aflatoxin among official sample test results (1AB) prepared with the DM are lower than the average aflatoxin among Special samples prepared with the VCM.
As described above, the USDA/AMS aflatoxin test procedure consists of a sampling, sample preparation, and analytical steps. Even when using accepted sampling, sample preparation, and analytical procedures that minimize bias, there is variability associated with each of the steps of the aflatoxin test procedure. Because of the variability associated with each step of the USDA/AMS aflatoxin test procedure for peanuts, the true aflatoxin concentration in a bulk lot can't be determined with 100% certainty by measuring aflatoxin in laboratory samples taken from the lot. As shown in
Among the various statistical measures of variability, only the variance is additive. Therefore, the total variance (s2
t) associated with a specific aflatoxin test procedure for peanut is the sum of the sampling, sample preparation, and analytical variances (
To understand how much each step of the aflatoxin test procedure contributes to the total variability and to be able to predict confidence limits associated with estimating the true lot aflatoxin concentration,
Studies (
Because of this extreme range in aflatoxin concentrations among a few contaminated kernels in a lot, variation among replicated laboratory sample test results tends to be large. The sampling variance, s2
s, associated with testing shelled peanuts is shown in
where ns is the laboratory sample size in number of shelled kernels and C is the aflatoxin concentration in ng/g total aflatoxins. If sample size is expressed in units of mass (kg), the mass can be converted to number of kernels knowing the count per unit mass.
Once the laboratory sample has been taken from the lot, the sample must be prepared for aflatoxin quantification. Since it is not practical to extract the aflatoxin from a large laboratory sample of kernels, the sample is comminuted in a suitable mill and the aflatoxin is extracted from a small test portion (sometimes called a subsample) taken from the comminuted laboratory sample. If the commodity is a granular product such as shelled peanuts, it is essential that the entire laboratory sample be comminuted in a suitable mill before a test portion is removed from the laboratory sample. Removing a test portion of whole seed from the laboratory sample before the comminuting process is a sample size reduction process and eliminates the benefits associated with the larger size laboratory sample of kernels.
Due to the aflatoxin distribution among contaminated particles in the comminuted laboratory sample, there is also variability among replicated test portions taken from the same comminuted laboratory sample. The sample preparation variance is not as large as the sampling variance due to the large number of comminuted particles in the test portion. An example of sample preparation variance for aflatoxin and shelled peanuts is shown below in
where nss is the comminuted test portion size in g. The sample preparation variance in
Once the test portion is removed from the comminuted laboratory sample, the aflatoxin is extracted from the test portion by blending the test portion with a suitable solvent. Analytical methods usually involve several steps such as solvent extraction, clean-up, dilution, and quantification. As a result, there can be variation among replicated analyses on the same solvent/test portion blend. The analytical variance (s2 a) associated with various analytical methods are shown below for TLC, immunoassay, and HPLC, respectively.
where na is the number of aliquots taken from the peanut solvent blend and quantified for aflatoxin by TLC (
where na is the number of aliquots taken from the peanut solvent blend and quantified for aflatoxin by immunoassay (
where na is the number of aliquots taken from the peanut solvent blend and quantified for aflatoxin by HPLC (
Two shellers in the southeastern United States (called sheller #1 and #2) provided one or more aflatoxin test results (1AB, and Specials) from each of 1677 and 2289 lots, respectively, of runner peanuts harvested in 2010. Along with the aflatoxin test result for each sample, the type mill used to comminute each of the 21.8-kg laboratory samples was also identified in the database. For each sheller, the database was sorted into two groups where the lots in group 1 had the 1AB and Special samples prepared by the DM and VCM, respectively, and group 2 had lots where both 1AB and Specials were prepared by the VCM. In both groups, the 1AB samples tested 8 ng/g or less. The percent of lots in each group where Specials tested 8 ng/g or less was determined (remember all 1AB samples tested 8 ng/g or less). The average aflatoxin among all 1AB and Special samples was determined for each group. Comparing DM to VCM results from group 1 illustrates what sellers and buyers are observing when the DM and the VCM are used to prepare 1AB and Special samples from the same lot. Comparing the results from group 1 to group 2 would give an indication if the DM behaved differently from the VCM when the VCM was used to prepare samples 1AB and Specials.
One possible problem with using actual sample test results in the above analysis is that lot concentrations vary over a wide range in each group and the aflatoxin distribution among lot concentrations may be different between group 1 and group 2 for each sheller. It would be desirable to compare the aflatoxin test results for 1AB and Specials for a large number of lots at the same aflatoxin concentration. Therefore, Monte Carlo simulation methods (
The forty lots were sorted in a similar manner to that described for actual data. The percentage of official and special samples testing 8 ng/g or less and the average aflatoxin among the official and special samples was computed as above for actual sample test results. One advantage of the Monte Carlo simulation is that 2AB and 3AB sample test results are shown when 1AB test greater than 8 ng/g unlike the database of actual sample test results (2AB and 3 AB would not be tested if the 1AB prepared by the DM tested greater than 8 ng/g).
A full-log plot of the sample preparation variance for the DM and VCM are shown in
Sample preparation variance associated with the USDA/AMS Subsampling mill (DM) and vertical cutter mill (VCM) and an 1100 g test portion.
Since sample preparation is just one of the three steps associated with the aflatoxin test procedure for peanuts, it is important to understand what the contribution of the sample preparation step is to the total variability of the aflatoxin test procedure. The total variance (s2
t) associated with the USDA/AMS aflatoxin sampling plan to detect aflatoxin in a peanut lot at 15 ng/g (C = 15 ng/g) when using the DM to prepare the sample is the sum of
When using the DM to prepare the 21.8 kg sample (
Contribution of the sampling, sample preparation, and analytical steps to the total variability associated with USDA/AMS aflatoxin test procedures when using 21.8 kg sample, USDA/AMS Subsampling mill (DM), 1100 g test portion, and analyzing aflatoxin in two aliquots using HPLC.
When the seller uses a VCM instead of the DM to comminute the 21.8 kg laboratory sample at 15 ng/g (C = 15 ng/g), the sample preparation variance for an 1100 g test portion (nss = 1100 g) is reduced from 7.9 (DM,
Assuming the distribution among sample test results is Normal, the range among replicated sample test results associated with the aflatoxin test procedure described above when a VCM is used to prepare the sample and lot concentration is 15 ng/g is ± 18.8 ng/g (18.8 = 1.98√90.1 for 95% confidence limits). Use of a VCM instead of the DM reduces the range of sample test results about the true lot concentration of 15 ng/g from ±19.5 to ±18.8 ng/g, respectively.
Using a 50 g test portion when an additional 21.8 kg special sample is comminuted outside the USDA/AMS testing program risks increasing the sample preparation variance associated with the VCM to a value greater than that associated with the DM and an 1100 g test portion. If a 50 g test portion is taken from a special 21.8 kg sample comminuted with a VCM, the sample preparation variance increases from 0.8 (for an 1100g test portion) to 16.9 (for C = 15 ng/g in
The 1100 g test portion defined by USDA/AMS was originally specified to lower the sample preparation variance associated with the DM. It would be interesting to determine the test portion size associated with a VCM such that the VCM sample preparation variance is the same as the sample preparation variance for the DM with 1100 g test portion. This can be calculated by setting
From the database provided by sheller#1, 531 lots were accepted by the USDA/AMS plan on the first sample (1AB≤8 ng/g). Of these 531 official 1AB samples that tested 8 ng/g or less, 115 and 416 were comminuted with a DM and VCM, respectively. Buyers requested that the 2AB sample from each of these 531 lots be tested for aflatoxin using the VCM. Depending on the buyer's specified limit (typically in the range of 5 to 10 ng/g total aflatoxin), the lot was accepted by the buyer if the special sample (2AB) tested less than or equal to the buyer limit. As a result, 115 lots had the 1AB and 2AB prepared by the DM and VCM, respectively, and the remaining 416 lots had both the 1AB and 2AB prepared by the VCM. These results allowed for sample test results to be compared for 115 lots where two different mills were used on the 1AB and 2AB (DM vs. VCM) and 416 lots where the same mill (VCM) was used on the 1AB and 2AB (VCM vs. VCM).
Average aflatoxin and percent lots testing 8 ng/g or less by the official 1AB and special samples (2AB) by type mill for sheller #1.
Comparing group 2 to group 1 results in
The average aflatoxin among the 2AB samples was lower for the 416 lots (VCM vs. VCM) than the 2AB for the 115 lots (DM vs. VCM). The special samples prepared by the VCM in
A second sheller (Sheller #2) provided sample test results in a similar data format as sheller #1 and the results are shown in
Average aflatoxin and percent lots accepted by the official 1AB and special samples for sheller #2.
The fact that the VCM vs. VCM lots behave in a similar manner to the DM vs. VCM lots (both
From the database provided by Sheller #1, 93 lots had two special samples (Special 1 = 2AB and Special 2 = 3AB) tested for aflatoxin in addition to having the official 1AB tested for aflatoxin. For each lot, Special 1 (2AB) and Special 2 (3AB) samples were prepared by with a VCM. Without considering the 1AB, these two special samples could also be used to demonstrate the effects of a sample cut-off limit on these 93 lots and show that the results are similar to
Summary of aflatoxin test results for 93 lots with two special samples before sorting into categories using a cut-off limit of 8 ng/g.
A sample cut-off limit of 8 ng/g or less was chosen to screen the 93 2AB samples (Special 1) since it is the first USDA/AMS accept limit. A total of 63 of the 93 lots had 2AB≤8 ng/g (not shown). Thirty lots had 2AB>8 ng/g. The 2AB and 3AB results among these 63 lots are summarized in
Summary of aflatoxin sample test results when a cut-off limit of 8 ng/g is used on special sample 1.
To expand on information in
Forty replicates of three 21.8 kg samples (1AB, 2AB, and 3AB) are taken from a lot at 10 ng/g using Monte Carlo simulation methods. By taking samples from a lot at a constant concentration (10 ng/g), we can remove the effect that sampling many lots with varying aflatoxin concentrations have on the comparison of the two mill (groups 1 and 2 in
Monte Carlo simulation used to sample a lot at 10 ng/g using the USDA/AMS aflatoxin test procedure (21.8 kg sample, VCM to prepare samples, 1100 g test portion, and HPLC to quantify aflatoxin in two aliquots.
Replicates where 1AB sample test results were 8 ng/g or less are shown in bold/italics along with the respective specials (2AB and 3AB). The average aflatoxin among the 40 1AB, 2AB, and 3AB along with the number of times that 1AB and 2AB and 3AB each tested 8 ng/g or less is shown at the bottom of the table. Since all samples are randomly chosen from the lot, the average among each of the 40 samples should be reasonably close to the true lot concentration of 10 ng/g and the number of samples testing 8 ng/g or less should also be about the same for 1AB, 2AB, and 3AB. The average among all 40 1AB, 2AB, and 3AB samples are 10.7, 10.3, and 10.3 ng/g, respectively. The more samples chosen from the lot, the closer the sample average should be to the lot average of 10 ng/g. The number of times that the 40 1AB, 2AB, and 3AB samples test 8 ng/g or less is 18 (45.0%), 22 (55.0%), and 23 (57.5%), respectively.
The domain of all sample test results (40 sets of 3 sample test results) in
Because of the total variability (s2
t ) associated with the aflatoxin test procedure, some lots will be misclassified by the sampling plan. Some good lots (true lot concentration below the USDA/AMS limit of 15 ng/g) will be rejected by the sampling plan (samples will test greater than the USDA/AMS limit). This is often called the seller's risk. Also, some bad lots (true lot concentration above the USDA/AMS limit of 15 ng/g) will be accepted by the sampling plan (samples will test less than the USDA/AMS limit). This is often called the buyer's risk. The magnitude of these two risks are a function of the aflatoxin sampling plan design (variability of the aflatoxin test procedure and the accept/reject limit) and can be estimated from operating characteristic (OC) curves that are calculated knowing the variability and distribution among sample test results (
Two OC curves are plotted in
Operating characteristic curves show the chances of accepting peanut lots by the USDA/AMS aflatoxin sampling plan when samples are prepared by the USDA/AMS Subsampling mill (DM) and a vertical cutter mixer (VCM) type mills.
Theoretically, the smaller the total variance of the aflatoxin test procedure, the steeper the OC curve about the final accept/reject limit of 15 ng/g (
When a lot is accepted on the first official USDA/AMS sample (1AB≤8 ng/g) and the buyer request that a second special sample (2AB) must also test less than a defined limit for acceptance of the lot, the performance of that sampling plan is very different from the USDA/AMS sequential sampling plan shown in
Operating characteristic curves show the chances of accepting peanut lots by sampling plans that require either one or two 21.8 kg samples to test 8 ng/g or less when samples are prepared by the USDA/AMS Subsampling mill and the vertical cutter mixer type mills.
From
With the USDA/AMS sequential sampling plan (
There are almost no benefits (lower variability) associated with using a VCM to prepare samples when both 21.8 kg samples must test 8 ng/g or less to accept or reject a lot (
When the Official USDA/AMS 1AB sample is prepared for analysis by either the DM or the VCM, the average aflatoxin among official samples testing ≤ 8 ng/g (or any other limit) will be less than the average aflatoxin among special samples. The difference in the average aflatoxin between official and special sample test results is not due to the type of mill used to prepare samples, but due to the use of a cut-off limit that restricts official sample test results to values below the cut-off limit while not restricting sample test results of the special samples. Even when the same mill is used to prepare both the official and special samples, the average aflatoxin among official samples is less than that of the special samples. The cut-off limit restricts high values for the official sample (1AB) from being included while there is no restriction on the special sample values.
The only way that a test portion prepared by the VCM will measure more aflatoxin than a test portion prepared by the DM is for (a) the VCM creates aflatoxin, (b) the DM destroy aflatoxin, or (c) the analytical method has a higher extraction efficiency associated with the test portions prepared with a VCM than with a DM after the water-slurry process. The last option is the most plausible, but not likely since the 1100 g test portion from the DM is further comminuted in the blender during the water slurry preparation and has decreased particle size even further.
An unintended consequence of a sampling plan that uses the VCM and test portions less than 104 g to prepare 21.8 kg samples is the total variance of the aflatoxin test procedure is larger than a similar sampling plan that uses a DM and an 1100 g test portion. If the test portion associated with the VCM is greater than 104 g, there will be slightly fewer lots misclassified by the sampling plan than one that uses a DM with 1100 g test portion. However, since the sample preparation variance of the DM and 1100 g test portion accounts for only about 8% of the total variability of the USDA/AMS aflatoxin test procedure, the decrease in the number of misclassification is minimal for one 21.8 kg sample and non existent when two 21.8 kg samples are used to accept or reject a lot.
Another consequence of requiring two 21.8 kg samples (the special and official samples) to both test less than a limit (ie., the USDA/AMS limit of 8 ng/g) is to have a sampling plan design that has a very low buyer's risk (bad lots accepted), but a very high seller's risk (good lots rejected). In years where aflatoxin levels are elevated, the large seller's risk can impose a large economic burden on the seller. The performance of a sampling plan that requires two 21.8 kg samples to both test 8 ng/g or less is approximately the same regardless of the type mill used to prepare the samples.
Each mill has it strengths and weaknesses, which provide a basis for choosing a specific mill to prepare samples. Since the contribution of the sample preparation step to the total variability of the aflatoxin test procedure is small, either mill can be chosen without significantly affecting the total variability of the overall aflatoxin test procedure. A more effective method of reducing the total variability of the aflatoxin test procedure is to select more samples from the lot since the sampling step contributes about 88% of the total variability.
The use of trade names in this publication does not imply endorsement by the N.C. Agricultural Research Service of the products named nor criticism of similar ones not mentioned.