METHOD DEVELOPMENT FOR THE DETERMINATION OF …

METHOD DEVELOPMENT FOR THE DETERMINATION OF AFLATOXINS IN FOOD IN ASIA : USE OF COMPLEMENTARY TECHNIQUES TO TAKE CARE OF NUANCES INVOVED

SHRIRAM INSTITUTE FOR INDUSTRIAL RESEARCH19, UNIVERSITY ROAD, DELHI-110 007

Dr. MANJEET AGGARWALASST. DIRECTOR & CHIEF

Difuranocoumarins

Difurocoumarocyclopentanone (B1, B2, M1)

AflatoxinsAflatoxins: Types and Properties: Types and Properties

Difurocoumarolactone (G1, G2)

•Fluoresce strongly in UV ligh

•Degrade in presence of moisture & elevated temperature

•B1 & G1 get converted to B2 & G2 in presence of mineral acids

•Hydrogenation of B1 & B2 yields G1 & G2

•Highly soluble in methanol

Properties are important for developing analytical techniques

Fungi (AspergillusFlavus/ Aspergillus Parasiticus)

AflatoxinsAflatoxins: Types of Food Contaminated: Types of Food Contaminated

Herbs & Spices

Cereals & grains

Meat & poultry products

Milk & Milk products

Nuts & Oil seeds

Food Products

Animal FeedB1 B2 G1 G2

M1 M2

All food consumed in the form of seeds & the products thereof may be contaminated due to aflatoxinsM1 occurs only in food of animal origin due to conversion process of B1, B2, G1 & G2 in animal’s bodyLevel of carcinogenecity B1>G1>B2>G2

PRODUCT MAXIMUM LEVEL (µg/Kg)

Groundnuts & dried fruits & their processed products for

human consumption

Raw groundnuts before human consumption

2 ppb-B1

4 ppb-Total

Nuts & dried food

USFDA EU

Permissible Levels of Aflatoxins

Cereals for human consumption

Milk & Milk based products

Herbs & Spices

CODEX

8 ppb-B115 ppb-Total


2 ppb-B14 ppb-Total

50 ppt-M1


20 ppb-Total

20 ppb-Total

20 ppb-Total

500 ppt-M1

20 ppb-Total

20 ppb-Total

15 ppb-Total

Not specified

Not specified

Not specified

500 ppt-M1

Not specified

Limits for USFDA are 5 to 10 times higher than EU Norms

Aflatoxins:Worldwide limits for aflatoxin B1 in food

Majority of the countries have not accepted the limits of 2 ppb as per EU norms and follow US FDA Norms

AflatoxinsAflatoxins:Worldwide limits for total :Worldwide limits for total aflatoxinsaflatoxins in foodin food

Majority of the countries have not accepted the limits of 4 ppb as per EU norms

Majority of the countries follow the EU normsM1 is not yet mandatory in many of the countries

AflatoxinsAflatoxins:Worldwide limits for :Worldwide limits for aflatoxinaflatoxin M1 in milkM1 in milk

AflatoxinsAflatoxins:Worldwide limits for :Worldwide limits for aflatoxinaflatoxin B1 in feedB1 in feed

Only EU specifies the limit for animal feedNo correlation between the limits of B1 in animal feed & other food products as per USFDA norm

AflatoxinsAflatoxins:Worldwide limits for total :Worldwide limits for total aflatoxinsaflatoxins in feedin feed

Only EU specifies the limit for animal feedNo correlation between the limits of total aflatoxin in animal feed & other food products as per USFDA norms

HOMOGENIZATION

SAMPLE PREPARATION

EXTRACTION

CLEAN UP

DERIVATISATION

MEASUREMENT

Typical Steps Involved in Analysis ofTypical Steps Involved in Analysis of Aflatoxin Aflatoxin

Care needs to be taken at each step to reduce probability of nuances

Each step needs validation for different foods because of unique matrix

Solid phase extraction

Sampling, Sample handling storage

of sample

Extraction(Residue isolation from the matrix)

Clean up

Representative homogenoussample. Container free fromcontaminants .

Selectively isolating the components of interest from the matrix

Separation of residues of interest from interfering substances

Solvent compatible with analyte w.r.t. - Extraction efficiencyStability of analyte

Pure grade solvent

Selectively isolate the analyte&avoid potential interferencs from matrix

Adsorption chromatography

STEPS RELEVANCEMETHODOLOGY

Immunoaffinity columns

AflatoxinsAflatoxins: Procedure and Relevance of Steps: Procedure and Relevance of Steps

Each sub-sample to be ground separately andmixed thoroughly to achieve complete homogenization

Important to know the constituents of the matrix to develop the method at each step

Residual Analysis: Procedure and Relevance of Steps Residual Analysis: Procedure and Relevance of Steps

Quantitative Estimation of aflatoxin residuesEstimation

Confirmation of results

Most important complementary techniques for accuracy and misleading

results

−HPLC−HPTLC−LC-MS− Immunoassay

using standard reference materials

− LC-MS− Chemical derivatization

All the above steps are crucial to obtain reliable & reproducibleresults with confirmation and avoid all doubts from otherartifacts from the matrix

Biological

Immunological

Analytical

One of the earlier usedtechniques. Not in use atpresent

TECHNIQUE APPLICABILITYMEASUREMENT

AflatoxinsAflatoxins: Measurement Techniques Used: Measurement Techniques Used

Enzyme linked Immunoassay techniquesRadio-immunoassay Techniques

Thin layer Chromatography (TLC)High Performance Thin layer Chromatography (HPTLC)

Liquid Chromatography-Mass Spectroscopy (LC-MS)

High Performance liquid Chromatography (HPLC)

More widely used forqualitative purpose

Widely used. Both forqualitative and quantitativepurpose

---

Each technique has its own nuancesComplementary techniques are required to take care of nuancesConstant improvement in the measurement techniques depending upon regulatory requirements

AflatoxinsAflatoxins: Analysis by Thin Layer Chromatography: Analysis by Thin Layer Chromatography

One of the first measurement techniques

Shows a spot of 1ng/g

More of qualitative or screening technique

Lacks Precision, Selectivity & Specificity for complex matrices

Needs to be complemented using HPLC or LC-MS

AflatoxinsAflatoxins: Factors for Analysis by TLC: Factors for Analysis by TLCTLC plates :

Taking care of various factors would produce reliable results

Quality of silica gel w.r.t particle sizeAll silica gel may not necessarily separate the 4 toxinsRetention times may change with quality of silica gelLayer thickness and hardness

Spotting :

Size of spottingEffect of light & relative humidity (subdued light & 60% RH)

Development of chromatogram :

Nature & polarity of mobile phaseQuantitation :

Nature of solvent remaining in silicaSufficient drying of plate

Aflatoxins: Analysis using HPLC method

Precise, Selective & Sensitive quantification technique for

alfatoxin analysis

Sample needs to be free from interference

Sample to be carefully extracted, cleaned up & derivatized

Involves use of both UV-Visible & fluorescence detector

HPLC is both a qualitative & quantitative technique. Presence & elution of artifacts at similar retention times as that of analytes can again be a cause of concern.In such cases results need to be complemented using another suitable column or LC-MS/MS

AflatoxinsAflatoxins: Analysis of M1 using different clean: Analysis of M1 using different clean--up up techniques :A Case Studytechniques :A Case Study

Selection of suitable clean up techniques is important foraccuracy and precision of results

A. Clean up using SPE C18

B. Clean up using SPE immuno-affinity column

BA

AflatoxinsAflatoxins: B1 using HPLC: B1 using HPLC--UV detector: A Case StudyUV detector: A Case Study

A. B1 Reference std, C18 symmetry long column (250 X 4.6 mm)

C. Wheat extract, Spherisorb C18 long column (250 X 4.6 mm)B. Wheat extract , C18 symmetry long column (250 X 4.6 mm)

D. Wheat extract, Phenomenex C18 long column (250 X 4.6 mm)E. B1 Reference std., C18 symmetry short column (150 X 4.6 mm)F. Wheat extract, C18 symmetry short column (150 X 4.6 mm)

Selection of column is important for precision & reliability of resultsAlso use of two different columns can be used to complement eachother for the precision of the results

Aflatoxins: Analysis by LC-MS/MS

Qualitative & quantitative techniques

Provides conformation of identity even at residual levels

Can be used as an ultimate technique for simultaneous determination

of all aflatoxins

Serves as the best complementary technique to any other techniques for determination

AflatoxinsAflatoxins Determination in Sample of Herb: A Case Determination in Sample of Herb: A Case studystudy

AflatoxinsAflatoxins Determination in Sample of Herb: A Case studyDetermination in Sample of Herb: A Case study

Analysis by TLCLarge number of spots observed on the TLC plate

Some colored spots corresponding to reference standard

of B1, B2 ,G1& G2 obtained in the samples at same Rf

value a that in standard

Spots not very clear

Overlaying with other spots observed along with the spots

of interest

Indications of aflatoxins was doubtful using TLC Complementary technique using HPLC required for confirmation of identity

Reference standards for B1 , B2 , G1 & G2

AflatoxinsAflatoxins’’ Sample of Herb: A Case studySample of Herb: A Case study

Analysis by HPLC


Analysis by HPLC

Sample extracted, clean-up using both C18 & immunoaffinity SPE Column,derivatized and injected on C18 Column

Interference from the matrix did not allow resolution and quantitation of peaks in HPLC


Analysis by LC-MS

Reference standards for B1 , B2 , G1 & G2 using methanol-water

Solvent system needs to be changed to obtain better resolution of peaks

Aflatoxins: Analysis by LC-MS

Reference standards for B1 , B2 , G1 & G2 using buffer solution.Separation of peaks obtained in buffer solvent

Aflatoxins: Analysis by LC-MS/MS

Cleaned sample extract was injected onto LC-MS/MS using different solvent conditionsSeparation of peaks obtained in buffer solution Sample did not indicate the presence of any of the aflatoxinsConfirmation was done using ion-ratios

LC-MS/MS is not only a precise & accurate quantitative technique at residue levels but also a confirmatory technique to detect low levels of residues in a complex matrix

AflatoxinsAflatoxins: Analysis by ELISA technique: Analysis by ELISA technique

Convenient, Rapid, Cost effective

Qualitative and screening technique

Not suitable for quantitation at low levels due to matrix

interference

Qualitative results may vary from one kit to the other

To take care of the nuances-The positive results by ELISAneed to be complemented by HPLC or LC-MS-MS

Food Commodity Results found +ve by ELISA HPLC for conformation & quantitation

Study conducted in Korea Determination of Aflatoxin B1

RiceBarleySweet CornCorn flakesMaizeWheat FluorBeerSoybeanSoya sauceSoybean pasteHot Soy pasteSoybean oil

8/1346/134

5/78/923/501/71/70/71/7

13/560/463/7

Total 49/554

0/80/60/50/82/30/10/1-

1/12/13

-0/3

Total 5/49In actual fact only five samples showed the presence of aflatoxin B1 by HPLC whereas ELISA showed 49 samples positive.HPLC was performed after suitable extraction, partitioning and derivatization

Aflatoxins: B1 by ELISA & Complemented by HPLC:A Case study

Aflatoxins: Analysis by LC-MS

Peak not separated using methanol & water solvent

Conclusion: Future Needs

Better understanding of nuances of the analytical methods

Use of complementary or confirmatory methods

Validation of methods for wide range of matrices & low limits

Development of better multi-mycotoxin methods

THANK YOU

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