Describes various methods for testing of cereals and their products

1. Analytical tests
for cereals and their products
Devinder Dhingra
​PhD (Process Engineering) | M Tech (Post Harvest Engineering)
Principal Scientist, Indian Council of Agricultural Research
currently in Fiji, on deputation

2. Grains (cereals
and pulses)
57%
Milk
7%Eggs
1%
Meat
1%
Fish
1%
Fruit
2%
Vegetables
6%
Sugar
8%
Oil
17%
Availability of Energy from different Foods in India (2014-15)

3. Analytical testing …….
Key references:
Codex Alimentarius (Codex) for consumers, food processors, national
food control agencies and the international trade.
National Food Control Agency in India: FSSAI (Food Safety Standards
Authority of India)
Analytical testing is done to check that the product meets the
(i) product standards and
(ii) product safety standards; as prescribed by the regulatory agencies
3

4. Food Analysis – Analytical testing
4
Regulatory
Agencies
Food Analysis
Laboratory
Client
Warehousing / Food
processor / importer/
exporter/ researcher /
food control agency
Consumer

5. Analytical testing of food - inter-disciplinary
● Physical testing – Wholesomeness, appearance, colour, foreign matter
(organic and inorganic), damaged grains, broken grains, immature /
shrivelled grains, weevilled grains, grains of other variety, other food grains
and moisture content
● Food Chemistry – Nutritional parameters / proximate composition
● Food Microbiology – biological safety (pathogens)
● Food Toxicology – contaminants (heavy metals), toxins, residues, additives
● Food Rheology – Cooking / baking / processing / performance
characteristics
● Functional testing - body’s reaction to wide range of foods, additives, spices..
● Molecular biology (including GMO) - product authenticity
● Sensory testing / evaluation - organoleptic analysis
● Food adulteration -
5

6. Analytical testing – common steps and requirements
➔Food products to be tested
➔Purpose – trade / labelling / baking / safety / regulatory
➔Parameters to be tested – based on product & purpose
➔Sampling methods – sample size
➔Test methods / Analytical methods - IS / ISO / APHA / FDA / AOAC /
AACC / EPA / In-house method
➔Range of testing / limits of detection
➔Equipment required / calibration of equipment
➔Consumables – Building – Manpower
➔Accredited Laboratory / In-house Laboratory 6

7. Analytical Testing methods
● AOAC International: Association of Official Analytical Collaboration (AOAC)
International – Official Methods of Analysis
● AACC – Approved Methods of the American Association of Cereal Chemists
● Bureau of Indian Standards - Food and Agriculture Department has 28
committees and 2083 standards (product specifications, methods of tests
etc.) (e.g. IS 4333: Part 5:1970 Determination of uric acid in food grains)
● International Organization for Standardisation (ISO)
● APHA: American Public Health Association
● FDA
● EPA United States Environmental Protection Agency
● In-house developed methods
7

8. Analytical methods
Important characteristics of analytical methods
➔Applicability
➔Selectivity
➔Calibration
➔Accuracy and recovery
➔Precision
➔Linearity and range
➔LoQ
➔LoD
➔Sensitivity
➔Ruggedness / Robustness
8

9. 9
Applicability: of the method should be studied using various
samples, ranging from pure standards to mixtures with complex
matrices. In each case, the recovery of the analyte (s) of interest
should be determined and the influences of suspected
interferences duly stated.
Any restrictions in the applicability of the technique should be documented
in the method.
Specificity / Selectivity: The ability to measure accurately and
specifically the analyte of interest in the presence of other
components that may be expected to be present in the sample
matrix.

10. Analytical methods
10
Calibration: In analytical chemistry, calibration is defined as
the process of assessment and refinement of the accuracy and
precision of a method, and particularly the associated
measuring equipment (i.e., an instrument), employed for the
quantitative determination of a sought-after analyte
Accuracy: It is the closeness of test results to the true value
(min 9 observations, 3 conc. 3 replications)
Precision: is the degree of agreement among individual test
results when an analytical method is used repeatedly to
analyse multiple samplings of a homogeneous sample.

11. ● Linearity and range: The ability of the method to elicit test results
that are directly proportional to analyte concentration within a
given range. In chromatography, calibration curves at a minimum
of five concentrations are recommended.
Linearity is determined by the analysis of samples with analyte concentrations spanning the
claimed range of the method. The results are used to calculate a regression line against analyte
concentration using the least squares method. It is convenient if a method is linear over a
particular range but it is not an absolute requirement. For those methods where linearity is not
attainable, an algorithm for calculations specific for that analyte / matrix combination may be
employed.
11

12. Limit of detection (LoD)
Lowest concentration of an analyte in a sample that can be
detected, but not quantified.
Non-instrumental method: Determine LoD by analysing samples at known
concentrations and establishing the minimum level at which the analyte
can be reliably tested.
Instrumental method: LoD is determined as signal to noise ratio, usually
2:1 or 3:1 or LoD = 3.3 (SD/S) where S is the slope of the calibration curve
12
Analytical methods

13. Limit of quantification (LoQ):
Lowest concentration of the analyte in a sample that can be
determined (quantified) with acceptable precision and accuracy
under the stated operational conditions of the method.
Non instrumental method: Determine LoQ by analysing samples at
known concentrations and establishing the minimum level at
which the analyte can be reliably detected and quantitated.
Instrumental method: Signal to noise ratio, usually 10:1 or 10(SD/S)
13

14. ● Robustness: It is the capacity of the method to remain unaffected
by small, deliberate variations in method parameters; a measure of
the reliability of a method. (e.g. in LC solvent concentration, buffer
concentration, injection volume/ flow rate/temp etc.)
● Sensitivity: The ability to demonstrate that two samples have
different amounts of analyte is an essential part of many analyses.
A method's sensitivity is a measure of its ability to establish that
such a difference is significant. LLoQ
14

15. Uncertainty of measurement
15
Uncertainty of measurement: An estimate attached to a
measurement, which characterizes the range of values within
which the true value is asserted to lie (ISO/ DIS 3543-1)
Major sources of uncertainty
• Sampling and sub-sampling / lack of sample homogeneity
• Extraction / digestion/ sample preparation
• Inherent instability of reference standard and reference material
• Calibration of equipment and instrument
• Variation of environmental and supply condition
• Operator variation
• Non-repeatability of result
• Reference standards

16. Uncertainty of Measurement: Type A
16
● Number of measurements (n) = 10
● Calculate mean & SD (x ) = 79.4 mg/ kg
● Standard deviation or standard uncertainty (σ ) = 2.042
● Relative standard deviation (RSD) = 0.0257 (σ/ mean)
● Now, if measured value is 80 mg/ kg
● Calculated uncertainty of measurement = −
● 1×80×RSD = 1x80x0.0257 = 2.056
● 2×80×RSD = 2×80×0.0257 = 4.112 (95%)
● 3×80×RSD = 3×80×0.0257 = 6.168 (99%)
● Measured value is represented as
● 80±2.056 mg/kg (67 % level of confidence)
● or 80±4.112 mg/kg (95% level of confidence)
● or 80± 6.168 mg/kg (99 % level of confidence)

17. Type B Uncertainty
Data is given in terms of tolerance interval
Pipette: 10cc ± 0.05 cc
Standard uncertainty or standard deviation = 0.05/√3= 0.028 cc
Measurement of 10 cc will lie between 9.972 cc to 10.028 cc
Digital Instruments:
Digital balance: Least count 1 mg ± 0.5 mg tolerance
Eg. Measured result is 80.512 g
Standard uncertainty = 0.5/√3 = 0.288 mg or 0.29 mg
Result = 80.512 g ± 0.29 mg
Least count = 0.1 mg (±0.05 mg)
Standard uncertainty = 0.05/ √3 =0.029 mg
Result = 80.5123 g ± 0.029 mg

18. Combining Uncertainties
i. Uncertainty of measurement = ±0.028 cc (U1)
ii. For class B pipette, when measurements are made over a
temperature range of 15 to 25◦C, the
Uncertainty of measurement = ±0.003 cc (U2)
iii. Uncertainty due to random factors = 0.01 cc (U3)
Total uncertainty : √U1
2+ U2
2 + U3
2 = ±0.029 cc

19. Analysis of cereals and their products
19

20. Physical parameters of food grains (FSSAI)
20
Parameter Wheat Maize Rice Jowar & Bajra
Moisture 14 % 16 % 16 % 16 %
Foreign matter
(mineral matter/ animal
origin)
1 %
(0.25 & 0.1%)
1 % 1 % 1 %
Other edible grains 6 % 3 % - 3 %
Damaged grains 6 % 5 % 5 % 6 %
Weevilled grains 10 % by count 10 % 10 % 6 %
Total of fm / dg / oeg 12 % 9 % 6 % 10 %
Uric acid 100 mg/kg 100 mg/kg 100 mg/kg 100 mg/kg
Triticum aestivum, Triticum durum, T vulgare,
T Sphaerococcum, T dicoccum, T compactum only
Zea Mays Oryza sativa Sorghum Vulgare
Pennisetum-typhyoideum

21. Explanation
21
(a) Foreign Matter – any extraneous matter other than food grain comprising of
(i) inorganic matter (metallic pieces, sand, gravel, dirt, pebbles, stones,
lumps of earth, clay, mud, animal filth, mud etc)
(ii) Organic matter – husk, straws, weed seeds, other inedible grains, paddy
in case of rice;
(b) Poisonous, toxic and/or harmful seeds – means any seeds which may
have deleterious effect on (i)health, (ii)organoleptic properties or (iii)
technological performance such as Dhatura, corn cockle, Akra
(c) Damaged grains – sprouted, internally damaged bcz of heat, microbe,
moisture, ergot affected grains, karnal bunt grains.
(d) Weevilled grains – partially or wholly bored by insects
(e) Other edible grains – any other edible grain

22. 22
• Directorate of Marketing & Inspection, Ministry of Agriculture
and Farmers Welfare, Government of India
• Department of Food & Public Distribution, Govt. of India, Krishi
Bhawan, New Delhi
• Bureau of Indian Standards, Manak Bhavan, New Delhi, India
• US Standards for Grain, USDA, Agricultural Marketing Service

23. Ergot affected grains and karnal bunt
Damaged grains

24. 24
Parameters Methods Range
Moisture (Loss in Weight
determined as specified)
AOAC (19th Edition)
925.09 & 925.10
0.1 % to 20.0 %
Argemone Seeds IS 4333 (Part 1): 1996 0.1 % to 3 %
Khesari Dal IS 4333 (Part 1): 1996 0.1 % to 2 %
Insect damaged by weevilled
grain
IS 4333 (Part 1): 1996 0.1 % to 5 %
Other Edible grain IS 4333 (Part 1): 1996 0.1 % to 10 %
Insect IS 4333 (Part 1): 1996 0.1 % to 10 %
Rodent hair & Excreta IS 6261: 1971 0.1 % to 5 %
Recommended Methods - Scope of testing

25. Grain quality parameters
● Moisture content - direct methods / indirect methods
● Thousand kernel weight – 32 g – 42 g (wheat)
● Test weight (kg/hl) - Schopper Chondrometer – 70-80 kg/hl
● Foreign matter - hand pick and weigh / dockage tester
● Broken/shrunken grains (matter that passes through a 0.064
inch x 3/8-inch oblong hole sieve)
● Damaged grains - damaged by insect, fungus / black tipped,
heat, frost, immature grains - hand pick and weigh
25

26. Processing characteristics - wheat
● Gluten quantity and quality -
○ Wet Gluten Content
○ Dry Gluten Content
○ Water Binding of Gluten
○ Gluten strength by Gluten Index
 Methods: AACC 38-12.02, ICC/No. 137/1, 155 & 158, ISO 21415-
2, 21415-4, GBT 5506.2, 5506.4, IRAM 15864, CCAT Method 13
■ Falling Number- determination of alpha amylase activity in wheat
flour - Falling Number system - 240-640 (lower the Falling Number
higher is the alpha amylase activity) ( time in seconds)
 Methods: AACC/No. 56-81.03, ICC/No. 107/1, ISO/DIS 3093 26

27. Sample divider, mill, gluten and Falling number equipment
27

28. Farinograph Extensograph Amylograph
28

29. 29
Rheological Behavior of Flour by Farinograph:
Constant Flour Weight Procedure (AACC Method 54-21.02)
Constant dough weight procedure (AACC Method 54-22.01)
Principle: In a temperature-controlled measuring kneader, a rotating Sigma
kneading blade subjects the sample to defined mechanical stress. The
kneading resistance having an effect on the blades, which depends on the
sample’s viscosity, is measured as a torque value by the highly precise
measurement electronics. The software then registers and records this online
as a function of time, in a clear to understand coloured chart.
Dough characteristics - water absorption, dough development time,
dough stability, tolerance index and softening of dough

30. graph for a gluten rich bread flour, as its stability time is relatively
long and the MTI is still above the 500 BU line. A weaker flour, such
as a cake or pastry flour with a much lower gluten content would
have a much steeper decline after peak time. 30
1 arrival /
hydration time
2 peak time
3 MTI [5 min from
peak]
4 departure time
5 stability

31. 31
Water absorption – 58 % - 64 % (higher water absorption & retention,
indicates strong flour) amt of water reqd to reach 500 BU
DDT- Dough development time – 3 – 6 minutes – higher time indicates
strong flour
Dough stability – 7 – 12 minutes – Higher dough stability time indicates
that the dough can withstand mixing for a longer period.
Mixing Tolerance Index (MTI) - 90 BU -71.67 BU - Generally, higher the
tolerance index value, weaker is the flour.
Softening of dough (SD) - 43.33 BU, indicates strong flour since flours
that have lower SD are stronger and the ones having higher SD values are
weaker.
Rheological behaviour

32. Extensographs
32
Dough stretched after 45, 90 and 135 minutes of rest times

33. Amylograph
33

34. Oxidising, bleaching and maturing agents
● Benzoyl peroxide - AACC 48-08.01
● Bromates - AACC 48-42.01
● Ammonium persulphate - AACC 48-62.01
● Azodicarbonamide in premix - AACC 48-71.02
34

35. Some Quality parameters – rice
● Hull and bran color
● Grain characteristics
○ Size, shape weight, test weight, Color (long/medium/short grain)
○ Translucency vs Chalkiness
● Milling Quality
o Yield of head rice
o Total yield of milled rice
o Milling uniformity
● Cooking and Processing quality factors
 Amylose content; Alkali spreading value; Protein content;
Gelatinization temperature; Water-uptake capacity; Parboil-
canning stability; Brewing cook ability of rice with diastase
●Basmati /non-basmati
35

36. Rice authentication - DNA fingerprinting
(PCR, capillary Gel electrophoresis)
36

37. Methods - Chemical testing – Cereals - product standards
37
PARAMETERS METHODS RANGE
Moisture IS 1011:2002 /
IS 12711: 1989
0.1 % to 10 %
Acid insoluble ash IS 1011:2002 /
IS 12711: 1989
0.1 % to 1.0 %
Acidity of extracted fat
(as oleic acid),
percent by mass, max
IS 1011:2002 /
IS 12711: 1989
0.1 % to 5 %
Alcoholic acidity IS 12711: 1989 0.1 % to 1.5 %
Free / Total gossypol - 1 to 100 mg/kg
Scope of testing of laboratory

38. Chemical testing - Bakery products
38
Parameter Method Range
Acid insoluble ash in dilute HCl
(on dry basis)
IS 1011: 2002 0.01 % to 0.5 %
Acidity of extracted fat
(as Oleic Acid)
IS 1011: 2002 0.1 % to 5 %
Added colouring matter In–house methods 1 % to 100 %
Salt (as Sodium Chloride) IS 7874 (Part 2):
1975 (RA 2004)
0.1 % to 15 %
Artificial sweeteners (singly) In–house methods 0.1 % to 20 %
Aspartame In–house methods 100 mg/kg to
1000 mg/kg
Acesulphane –K In–house methods 50 mg/kg to
500 mg/kg

39. List of FSSAI prescribed methods for cereal products (issued 2018)
39
Parameter Products Method
Protein Durum wheat, Maida, Sorghum
flour, Durum wheat semolina,
Whole durum wheat semolina,
Whole maize (corn) flour, De-
germed maize corn flour, Textured
Soy Protein, Soymilk, Tofu, Tempe
ICC 105/1
(Codex Stan 234-1999)
Protein Decorticated millet grain,
Pearl millet flour
AOAC 920.87 – whole &
decorticated pearl millet
grains
(Codex Stan 234-1999)
Crude
protein
Wheat protein products
including wheat gluten
AOAC 979.09
(Codex Stan 234-1999)

40. 40
List of FSSAI prescribed methods for cereal products (issued 2018)
Parameter Product Method
Fat Pearl millet flour, Sorghum flour,
Soybean, Whole maize (corn) flour,
Tempe, Textured Soy protein,
Soymilk/tofu, Decorticated millet
grain
AOAC 945.38F; 920.39C
(Codex Stan 234-1999)
Acidity / Starch Edible sago flour IS 899
Titratable acidity (
as lactic acid)
Tofu Determination of acidity –
FSSAI manual of methods of
analysis of F&Vs

41. 41
Parameter Product Method
Hexane Textured Soy
protein
• Rapid quantitative determination of residual
oils by GC – J American Oil Chemists
Society Vol 52: 118-120
Yellow
pigment
Durum wheat • Spectrophotometric determination of yellow
pigment content and evaluation of
carotenoids by HPLC in durum wheat grain,
J Agric Food Chem. 2002 Nov6; 50(23):
6663-8
• AOAC-950.34- Pigment in flour
List of FSSAI prescribed methods for cereal products (issued 2018)

42. Analytical tests – food safety
42
Microbiological testing
Testing for metal contaminants
Testing for mycotoxins
Testing for residues (pesticide residues)
Regulatory limits, prescribed methods

43. Microbiological analysis - methods
43
Bacterial Count
(Total Plate Count)
IS 5402: 2012 / ISO 4833: 2003,
FDA-BAM Chapter 3,
APHA (4th Edition) Chapter 7-7.6
Coliforms IS 5401 (Part 1): 2002/ ISO 4832: 1991;
IS 5401 (Part 2): 2012/
ISO 4831: 2006; FDA-BAM Chapter 4, APHA
(4th Edition) Chapter 8-8.7
Yeasts & Molds IS 5403: 1999, APHA Chapter 20-20.51,20.52,
FDA-BAM Chapter18; DGHS manual

44. Microbiological analysis - pathogens
44
Escherichia coli IS 5887 (Part 1): 1976, FDA-BAM Chapter 4,
APHA (4th Edition) Chapter 8-8.8,8.91,8.92
Bacillus cereus IS 5887 (Part 6): 2012; ISO 7932: 2004;
FDA-BAM Chapter 14
Faecal streptococci
(Enterococci)
IS 5887(Part 2): 1976, APHA Chapter 9-9.5
Staphylococcus
aureus
IS 5887 (Part 2): 1976; IS 5887 (Part 8/Sec I
& II): 2002/ ISO 6888: 1999,
FDA-BAM Chapter 12,
APHA Chapter 39-39.5

45. Microbiological analysis - pathogens
45
Salmonella IS 5887 (Part 3): 1999 / ISO 6579: 2002;
FDA-BAM Chapter 5,
APHA (4th Edition) Chapter 37-37.123,124,125,126
Shigella IS 5887 (Part 7): 1999; FDA-BAM Chapter 6,
APHA (4th Edition) Chapter 38-38.23,24,25
Clostridium
perfringens
IS 5887 (Part 4): 1999;
APHA (4th Edition) Chapter 33 & 34,
FDA-BAM-Chaper16 & 17

46. Microbiological analysis - pathogens
46
Vibrio cholerae IS 5887 (Part 5): 1976
APHA (4th Edition) Chapter 40-40.5,
FDA-BAM Chapter 9
Detection/25
ml or 25 g
Vibrio
parahaemolyticus
IS 5887 (Part 5): 1976
FDA-BAM Chapter 9
Detection/25
ml or 25 g
Listeria Species IS 14988 (Part 1 & 2): 2001
FDA BAM Chapter 10,
APHA (4th Edition) Chapter 36-36.5
Qualitative 1g,
25g.
Test For Commercial
Sterility
APHA (4th Edition) Chapter 61-61.51 Qualitative
(Pass/ Fail)

47. Biological testing Lab equipment
47
S. No. Equipment Purpose
1. Autoclaves Sterilisation of media, pipette tips, glassware, waste
2 Drying cabinet For drying of glassware and other items and keep them
3 Stomacher Sample preparation
4 Bio safety cabinet cat. II Handling pathogens
5 BOD Incubators Incubation of samples
6 General Incubators Incubation of samples
7 Deep freezer -80°c for storage of reference cultures
8 Deep freezer -18°c for storage of test samples
9 Laboratory Refrigerators Storage of samples
10 Automatic pipetting and dispersing unit For making serial dilutions
11 Volumetric Automatic pipettes Accurate measurement of volume for dilutions and plating
12 Automatic Colony counter Colony counting
13 Trinocular microscope with CCD camera and
imaging software and accessories
Microscopic Evaluations, identification of cysts, parasites,
bacteria, fungi etc.
14 Hot plate with magnetic stirrer Media Preparation
15 Digital electronic balance 220g/0.1mg 220g/0.1mg
16 Hot water bath 10 litre

48. 48
Contaminants
Toxins
and
Residues

49. Contaminants, Toxins and Residues
Metal elements (in foods not specified) upper limit; FSSAI
Lead - 2.5 ppm
Copper - 30 ppm
Arsenic - 1.1 ppm
Tin - 250 ppm
Zinc - 50 ppm
Cadmium - 1.5 ppm
Mercury - 1.0 ppm
Methyl Mercury - 0.25 ppm (calculated as the element)
Chromium - Refined Sugar - 20 ppb
Nickel - 1.5 ppm
All hydrogenated, partially hydrogenated, inter-esterified vegetable oils and fats such as vanaspati,
bakery and industrial margarine, bakery shortening
49

50. Methods of analysis of metal elements
Single element methods - colorimetric methods - UV-VIS spectrophotomer
Hg - Mercury analyser
Methods based on - Atomic Absorption Spectrophotometer (AAS)
Pb, Cd - GFAAS (Graphite furnace AAS)
Cu, Fe, Zn - Flame AAS
Sb, As, Hg, Se - hydride generator with AAS
Inductively Coupled Plasma - Optical Emission spectrometer (ICP-OES)
Inductively Coupled Plasma - Mass Spectrometer (ICP-MS)
50

51. Methods of analysis of metal elements
References:
1. AOAC Method 2015.01 for Heavy Metals in Food by Inductively
Coupled Plasma - Mass Spectroscopy (ICP-MS) is applicable to the
analysis of a wide variety of matrix types.
2. AOAC SMPR 2012.007: Standard Method Performance
Requirements for Determination of Heavy Metals in a Variety of
Foods and Beverages (As, Pb, Hg, Cd) based on ICP-MS
3. US FDA Elemental Analysis Manual
4. Manual of Methods of Analysis of Foods - Metals, by FSSAI
5. AOAC International: Association of Official Analytical Collaboration
(AOAC) International – Official Methods of Analysis
51

52. Mycotoxins
52

53. 53
Materials commonly contaminated with mycotoxins

54. 54

55. Regulatory limits of mycotoxins
55
Toxin Article of food Limit 𝞵g/kg (FSSAI)
Aflatoxin Cereals, pulses and
products
15.0
Ready to eat products 10.0
Oilseeds, nuts 15.0
Spices 30.0
Aflatoxin M1 Milk 0.5; 0.05 (EU)
Patulin Apple juice 50.0
Ochratoxin A Wheat, barley and rye 20.0
Deoxynivalenol Wheat 1000.0
US FDA and EU have more stricter regulatory requirements for mycotoxins.

56. Classification of mycotoxins
56
Group - Characteristics
Aflatoxins B1,B2,G1,G2,M1 Named after their fluorescent
properties; Highly polar
Zearalenone 𝞪 - zearalenol, 𝞫 - zearalenol
𝞪 - zearalenone 𝞫 - zearalenone
Greenish fluorescent
compound, Non-polar
Ochratoxins A, B Medium polarity, Fluorescent
compounds
Fumonisins B1,B2,B3,B4 Non-fluorescent compound,
High polarity
Type A
trichothecenes
Deoxynivalenol, Nivalenol,
Fusarenon-X
Polar, complex structures,
Non-macrocyclic
Type B
trichothecenes
T2 toxin, HT-2 toxin Polar, complex structures,
Macrocyclic

57. Mycotoxin analysis methods
57
Method /Advantages Disadvantages Mycotoxin/
Matrix
LoD LoQ
TLC; Simple,
inexpensive, rapid, good
selectivity, accurate
identification
Poor sensitivity
and precision
DON/
wheat flour
30
ng.mL-1
100
ng.mL-1
HPLC- FLD: Official
methods available,
selective, sensitive
Expensive,
derivatization
may be needed
AFB1/
Spices
0.04
ng.mL-1
0.15
ng.mL-1
LC-MS: simultaneous
analysis of multiple toxins
expensive AFB1/
Wheat grain
2
𝞵g.kg-1
3.5
𝞵g.kg-1

58. Mycotoxin analysis methods
58
Method Advantages Disadvantages Mycotoxin/
Matrix
LoD LoQ
GC simultaneous
analysis of
multiple toxins
expensive,
derivatization reqd,
non-linear
calibration, matrix
interference
DON/
Pasta
0.5
𝞵g.kg-1
1
𝞵g.kg-1
ELISA Low use of
organic solvents
possible false
positive/negative
OTA/
Corn
4
ng.mL-1
ns
Spectral
Analysis
can be used in-
situ
spectra overlapping Fumonisin/
Corn
100
𝞵g.kg-1
ns

59. 59
Mycotoxin(s) Document Scope Technique
AFB1, AFB2, AFG1,
AFG2
AOAC 991.31 Corn, raw peanuts HPLC-FLD (IAC)
AFB1, AFB2, AFG1,
AFG2
AOAC 2005.08 Corn, raw peanuts HPLC-FLD (IAC)
AFB1, Total AFLAs ISO 16050:2003 Cereals HPLC-FLD
DON AOAC 986.18 Wheat GC
DON EN 15891:2010 Cereals, and foods HPLC-UV
DON AOAC 986.17 Wheat TLC
AFB1, AFB2, AFG1,
AFG2
AOAC 994.08 Corn, almonds, nuts HPLC-FLD
FB1, FB2, FB3 AOAC 995.15 Corn HPLC-FLD
OTA AOAC 991.44 Barley, wheat, rye, corn HPLC-FLD
Documented Methods for analysis of mycotoxins

60. 60
HPLC – FLD with Kobra Cell

61. Kobra Cell
61

62. Immuno Affinity Column (IAC)
62

63. 63
Mycotoxin(s) Document Scope Technique
AFB1 AOAC 990.32 Corn, roasted peanuts ELISA
AFB1, AFB2, AFG1,
AFG2
AOAC 990.34 Corn, cotton seed,
peanuts
ELISA
Total FBs AOAC 2001.06 Corn ELISA
ZEN AOAC 994.01 Corn, wheat, feed ELISA
T-2, HT-2 EN 16923:2017 Cereals, cereal based
foods
LC-MS/MS
CIT EN 17203:2018 Cereals, red yeast rice LC-MS/MS
Documented Methods for analysis of mycotoxins

64. Mycotoxins
References
1. FSSAI Manual of Methods of Analysis of foods - Mycotoxins
(available online)
2. AOAC Official Methods, Chapter 49, 20th Edition, 2016 AOAC
International
3. Food Microbiology William C. Frazier, Dennis C. Westhoff
64
AOAC International: Association of Official Analytical Collaboration
(AOAC) International – Official Methods of Analysis

65. Residues (Insecticides, Herbicides, Fungicides)
65

66. Residues (Insecticides, Herbicides, Fungicides)
● 567 formulations of 272 insecticides - registered in India.
● More than 150 combinations are also registered
● Worldwide, more than 1200 active substances are registered for
pesticides production. Pesticides can be classified into more than
100 classes/groups, for example, Carbamates, Triazines,
Pyrethroids, Organophosphates, Organochlorines, Phenoxy
alkane Pesticides, Pesticides based on glyphosate….
● Organochlorines, organophosphates, carbamates and pyrethroids
● Tolerance limits of the residues (insecticides, herbicides,
fungicides) on crop, food commodities are prescribed under
regulations. Also called MRLs 66

67. FSSAI Tolerance Limits - Residues
67
Name of the
insecticide
Crop/commodity Tolerance limit
(mg/kg or ppm)
Aldrin, Dieldrin Food grains 0.01
Milled foodgrains Nil
Fenitrothion Food grains 0.02
Milled foodgrains 0.005

68. 68
Residue from Instrument Standard LOQ (𝜇g/l)
Organochlorine pesticides GC-MS/ECD 0.005 - 0.01
Organophosphorous pest. GC-MS/ECD 0.01 - 0.05
Nitrile Pesticides GC-MS/ECD 0.01
Pyrethroids GC-MS/ECD 0.1
Phosphine Headspace GC-MS 1.5
Carbamate pesticides LC-MS/MS 0.01-0.02
Dinitrophenol pesticides LC-MS/MS 0.02-0.04
Imidazolinone pesticides LC-MS/MS 0.01

69. 69
The lastest high end mass spectrometers, along with gas chromatographs /ultra-high
performance liquid chromatographs (UHPLC), equipped with modules of online
concentration techniques for clean-up and pre-concentration are now routinely used in
the analysis of pesticide residues.
In the case of substances requiring a very low limit of detection or with compounds
with low sensitivity, it is necessary to pre-concentrate the sample prior to analysis.
Pre-concentration steps can be performed manually, such as offline SPE, liquid-liquid
extraction followed by evaporation of organic solvents, or by the QuEChERS
technique.
When needed, sample preparation includes a step for derivatisation. Certain types of
compounds need to be treated with a derivatisation agent prior to analysis to make
them more “suitable” for the selected instrumentation. Examples are compounds like
glyphosate, amitrole and gluphosinate etc.

70. Mass spectrometer
70

71. Mass spectrometer scans
71

72. FullscanMS
72
m/z 269.9

73. 73

74. SIM
74

75. Product ion scans or daughter ion scans
75

76. Product ion or daughter ion scans
76

77. SelectedReactionMonitoring
77

78. Fullscanproductions
daughterions
78
m/z 181.2

79. SelectivityofSRM
79

80. Selectedreactionmonitoring
daughter/productions
80

81. Measurement of residues on cereals and products
References
● Guidance document on analytical quality control and method validation
procedures for pesticide residues and analysis in food and feed,
SANTE/11813/2017 (available online)
● FSSAI Manual of Methods of Analysis of foods - Pesticide residues
(available online)
● AOAC International: Association of Official Analytical Collaboration
(AOAC) International – Official Methods of Analysis
● Joint FAO/WHO Food Standard Programme. Codex Alimentarius
Commission. Report of the thirty fifth session of the Codex Committee on
Pesticide Residues, Rotterdam, The Netherlands. 31st March - 5th April
2003. pp. 46-55.
QuEChERS (quick, easy, cheap, effective, rugged, and safe)
81

82. 82
References….
• EN 15662: 2018 Foods of plant origin. Multimethod for the determination
of pesticide residues using GC- and LC-based analysis following
acetonitrile extraction / partitioning and clean-up by dispersive SPE.
Modular QuEChERS-method. The method has been collaboratively studied
on a large number of commodity/ pesticide combinations.
• Tamer M A M Thabit , Dalia I H Elgeddaw Determination of Phosphine
Residues in Wheat and Yellow Corn with a New Developed Method
Using Headspace and SIM Mode GC-MS. J AOAC Int. 2018 Jan
1;101(1):288-292. doi: 10.5740/jaoacint.17-0186. Epub 2017 Sep 22
Measurement of residues on cereals and products

83. LABORATORY EQUIPMENT
83

84. 84
Equipment Purpose
HPLC – PDA/DAD
(High Performance Liquid
Chromatography with Photo Diode
Array detector)
Used for analysis of vitamins & assay of fine chemicals & raw materials. This
can be used for analysis of plant growth regulators & mycotoxins in food
samples at very low level.
HPLC – FLD / RID
(High Performance Liquid
chromatography with Fluorescence
detector/ Refractive Index
detector)
Used for analysis of compounds having florescent activity. used for analysis
of Aflatoxins, Anthracene, antibiotics, vitamins and pesticides etc.
Kobra Cell Derivatization of Aflatoxins to give the fluorescent compound which is
analyzed on HPLC-FLD.
ICP – OES
(Inductively coupled Plasma –
Optical Emission Spectrometer)
Used for the detection of trace metals. Also minerals in premix samples, food
samples, animal feed can be analysed with accuracy with ICP-OES.

85. 85
Microwave Digester Microwave digestion is a common technique used by elemental analysis to
dissolve heavy metals in the presence of organic molecules prior to analysis by
inductively coupled plasma, atomic absorption, or atomic emission measurements
by microwave irradiation. Due to microwave irradiation, thermal decomposition of
samples & solubility of heavy metals can be increased.
Gas Chromatography with
Flame ionization Detector &
Head Space
Used for analysis of volatile constituents in food samples which includes VOC's &
residual solvents. Well suited for analysis of hydrocarbons such as methane,
ethane, acetylene & also for fatty acid profile, cholesterol analysis.
GC with ECD and auto
injector
Analysis of halogenated contaminants in water and waste water, and
agrochemical residue analysis (pyrethroids / organochlorine pesticides)
LC-MS/MS Pesticide residues, veterinary drugs, antibiotics
GC-MS/MS Pesticide residues
UV-VIS spectrophotometer Used in analytical methods
FTIR (Fourier Transform
Infrared spectrophotometer)
with ATR & KBr pellet
accessory
Rapid Determination of functional groups, adulteration etc. Used to obtain an
infrared spectrum of absorption, emission of a solid & liquid. This is particularly
useful for structural identification of samples.

86. 86
Refrigerated centrifuge Laboratory procedures
Kjeltec Assembly (Semi Automatic) Estimation of N2 content for protein
Solvent Distillation Unit (semi Automatic) Estimation of fat/oil in samples
Muffle furnace Estimation of ash
Hot Air Oven Estimation of moisture content
Vacuum Oven with pump Estimation of moisture content
Hot water bath General purpose heating
Rotary vacuum evaporation with vacuum pump Concentration of samples evaporation of solvents
Bench top pH Meter Measurements of pH
Abbes Refractometer Refractive index & Sugar concentration
Analytical balance Weighing
Analytical balance Weighing
Automatic pipettes Volumetric measurements
Lovibond Tintometer Measurement of color
Sample mill Grinding of solid samples
Ultra Sonicator Sample dispersion, preparation
Millipore “Milli Q-Ultrapure water system Water for analysis
Gas cylinders and regulators For GC
Digital Polarimeter Conc and purity measurement oil, sugar etc. (angle of rotation)
Fume hood Working with volatile chemicals
KF titrator

87. ● Farinograph
● Mixograph
● Extensograph
● Risograph
● Alveograph
● Falling number
● Viscoamylograph
● Rapid visco analyzer
● Mixolab
87

88. Thank you
88