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Aflatoxin sampling and determination in bulk maize for export
by Pin Pithaya-Acharlyakul
INTRODUCTION
Societe Generale de Surveillance (SGS), a worldwide independent inspection company with headquarters in Geneva, Switzerland, is represented in more than 140 countries by 155 affiliated companies, and has 152 laboratories and 1,000 offices.
SGS has been established in Thailand for more than 39 years. Here, there are over 800 staff with a wide range of sophisticated laboratory facilities to cope with the trade of the country.
SGS (Thailand) services cover:
For practical reasons, determination as to the level of aflatoxin contamination in a maize consignment must be carried out before shipment. This will allow opportunity for selection of acceptable cargo for loading. To reject cargo after loading is economically impractical.
Sampling and screening process will enable exporters to segregate their stocks, according to the level of aflatoxin, for their own trade planning. Sampling is carried out during:
In principle, sample increments are drawn at regular intervals during the transfer. From trucks of about 13 metric tonnes (t) per load, 3 increments are drawn during tipping - one from the rear, one from the middle, and one from the front. On the conveyor belt which transfers maize after drying from the dryer to the storage bin or godown, increments are drawn at every interval of about 10 t.
Sample increments are then combined to form a gross sample for every 100 t. Each gross sample is mixed well and divided into the required number of representative samples each weighing about 2 kg. Analysis can be performed either on every 100 or 500 t basis depending on the reqirements.
Once the results of the analysis is known for each individual lot, the silo operator can then release or transfer the cargo from temporary storage and group the maize as per maximum aflatoxin level, i.e., 20 ppb, 30 ppb, 50 ppb, etc., for shipment.
The results of the aflatoxin analysis for each stock has validity for a maximum of 2 weeks prior to shipment, on condition that both the temperature and moisture are well maintained.
The elements of time/speed and expenses are of prime importance to traders and exporters. Usually, they would require sampling and aflatoxin determination on their accumulated stocks prior to loading. The maize is stored in bulk either in silo bins or in flat warehouses (godowns) as stock piles.
SCOPE OF AFLATOXIN DETERMINATION AND SAMPLING PROCEDURES FOR MAIZE SHIPMENT
Representative samples from stocks either in silo bins or stockpiles are examined for aflatoxin levels prior to loading. Results of aflatoxin levels in the stocks found within the contractual specification are valid within 2 weeks from the date of sampling and with the conditions that temperature and moisture of the stocks are well maintained and that SGS seals are intact.
GENERAL PROCEDURE
Increments are systematically drawn from the stock and composited on the basis of 200 t per subgross sample. Each sub-gross sample is thoroughly mixed and reduced by an appropriate device (a riffle divider) to a minimum of 2 kg representative subsamples. The number of sub-samples depends on the size of the stocks, i.e.:
SAMPLING METHODS
Ex-silo bins
In silo bins where sampling is feasible from top and bottom of the bins, 2/5 of the total number of samples is prepared from increments drawn from the top of the bin by a pneumatic probe. The rest, 315 of the total number of samples, is prepared from increments drawn from the bottom of the bin by a scoop during recycling of the contents of the bin.
For example, the calculation of the number of samples from a silo bin with a volume of 1,800 t maize follows:
1,800 t/200 = 9 sub-samples
Number of sub-samples to be prepared from the top of the bin:
9 x 2/5, or 4 sub-samples
Number of sub-samples to be prepared from the bottom:
9 x 3/5 = 5 sub-samples
Top Sampling. Increments are drawn by a pneumatic probe extracting sample material from surface down to 5-7 meters depth or a minimum 1/4 depth of the bin contents. Each sub-sample is prepared from increments drawn from one location during probing.
Therefore, the 4 sub-samples are to be prepared from increments drawn separately from 4 different locations.
Bottom Sampling. To recycle about 10% of the bin contents, calculation is:
1,800 t x 10% = 180 t
Increments are systematically drawn at regular intervals from the bottom of the bin during recycling and number of sub-samples to be prepared from each subgross sample composited for every 180 t is:
180 t/5 = 36 t
Thus:
5th sub-sample to be prepared from increments drawn from 1st 36 t recycling
6th sub-sample to be prepared from increments drawn from 2nd 36 t recycling
7th sub-sample to be prepared from increments drawn from 3rd 36 t recycling
8th sub-sample to be prepared from increments drawn from 4th 36 t recycling
9th sub-sample to be prepared from increments drawn from 5th 36 t recycling.
In silo bins where sampling is feasible only from the bottom, 20% of the bin contents is recycled. For example, in a silo bin with 1,000 t of maize:
Number of sub-samples = 1,000 t/200 = 5 sub-samples
Recycling 20% of 1,000 t = 200 t
Sub-samples to be prepared from increments drawn and composited for every 200 t/5 = 40 t.
Sampling procedures to be applied are similar to the previously mentioned bottom sampling.
Ex-stockpiles in bulk
Stockpiles may vary in size, tonnage, height, width and length. They may range from 1,000 t to over 10,000 t.
To sample a stockpile which is over 1,500 t in size, it should be divided into sectors or "sub-lots" of approximately 1,000 t each. Each sub-lot is to be treated separately for sampling and analysis.
Thus, number of samples to be prepared from a sub-lot:
1,000 t/200 = 5 sub-samples
Increments are to be drawn by a pneumatic probe extracting sample material from surface either perpendicularly down to the bottom of the pile or at right angles from the surface toward the centre of the pile, depending on the structure or shape of the stockpile.
Five sub-samples are to be individually prepared from each probing at a designated location.
SAMPLE PREPARATION FOR LABORATORY ANALYSIS
The second stage of the sampling process is to prepare final samples for analysis of aflatoxin content. It is essential that at all stages, the equipment involved in the sample preparation must be clean and free from previous sample residues to avoid any contamination.
The following procedures are designed to systematically treat all the sub-samples to constitute a representative "final sample" for a silo bin or a stockpile sub-lot for aflatoxin analysis.
For each silo bin or stockpile sub-lot, mix all the sub-samples into a gross sample.
Divide the gross sample by a riffle divider to prepare 4 representative final samples of about 2 kg each:
1 sample for analysis
2 samples reserved for second and third analysis, if necessary
1 sample retained for reference
Crush the sample for analysis into an appropriate size so it can pass a 1-mm sieve. Mix and reduce the sample by a sample divider to a minimum weight of 500 grams (9). Mix and reduce the sample by "increment method" to a minimum weight of 50 9 for laboratory treatment and analysis of aflatoxin.
The Increment Method. After thoroughly mixing the sample, spread the sample into a rectangle with an appropriate even thickness. Divide the rectangle into 20 equal cells. Take sample material from each cell with an appropriate scoop to the bottom of the cell.
Following the principle of Gafta Rules, if aflatoxin analysis fails on the first sample (exceeding specification to a certain limit but not over 50%), a second analysis can be performed on the second sample. If the results of the second analysis are lower than specifications and when averaged with the first analysis produces a result within the specifications, then the lot is considered acceptable. On the other hand, if the second analysis is still higher than the specifications but lower than the first analysis, a third analysis can be performed on the third sample. If the average of the three analyses is within specifications, the lot is still considered acceptable. Otherwise, it is rejected.
For example, aflatoxin analysis for a specification of 50 ppb maximum shows the following:
1st analysis = over 75 ppb, rejected and does not qualify for second analysis.= up to 75 ppb, allowed for second analysis
2nd analysis = less than 25 ppb, the lot is accepted = over 25 but less than 75 ppb, allowed for third analysis
3rd analysis = average with previous 2 analyses is 50 ppb or below, lot can be accepted = average with previous 2 analysis is over 50 ppb, lot is rejected
METHODS OF ANALYSIS
Rapid screening method
This is applicable to yellow corn and can be used outside the laboratory (AACC Method 45-14, AOAC Method 26.020/1984).
Aflatoxin is extracted by aqueous methanol followed by clarification with zinc acetate. Aflatoxin is then trapped in the florisil layer in a minicolumn. Fluorescence in the column, indicating the presence of aflatoxin, is detected visually under ultraviolet (UV) lamp and compared with the standards for quantification.
This method only gives results indicating higher or lower amounts than the standard. The whole process takes only 10 minutes.
Velasco fluorotoxin meter
This is a rapid method, but is not recognised by standard organisations.
Aflatoxin is extracted by aqueous acetone followed by clarification with ferric gel. Aflatoxin is then trapped in the florisil layer in a minicolumn and is quantified by the Neotec Velasco Fluorotoxin Meter. Results from this method are reasonably accurate, taking about 1.5 hours to complete.
Thin layer chromatographic method (TLC)
This is applicable to corn (AACC Method 45-05, AOAC Method 26.049/1984).
Aflatoxin is extracted by aqueous methanol followed by column chromatography for clarification. Aflatoxin is the detected by TLC and quantified by a densitometer. Results from this method are accurate and most acceptable. This method will take 3-4 hours to complete.
High performance liquid chromatographic method (HPLC)
This is a highly sophisticated method used for umpire (?) analysis, research and development and trouble shooting purposes. The following sampling and sample preparation procedures are designed for shipment certification by aflatoxin analysis utilising the TLC method.