MANUAL OF METHODS

OF

ANALYSIS OF FOODS

BEVERAGES: TEA,
COFFEE, CHICORY
TABLE OF CONTENTS

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 TABLE OF CONTENTS

S. No. METHOD NO. METHOD (TITLE) PAGE No.

1. FSSAI 04A.001:2021 Determination of moisture 01
FSSAI 04A.002:2021 Determination of Moisture for roasted coffee and chicory
2. 02
mixture - Vacuum Oven method (Reference method)
FSSAI 04A.003:2021 Determination of Moisture for soluble (instant) coffee
3. 03
powder - Vacuum Oven method (Reference method)
4. FSSAI 04A.004:2021 Determination of Total ash 04
FSSAI 04A.005:2021 Determination of Total ash (Alternate method) for Roasted
5. 05
and Ground Coffee
6. FSSAI 04A.006:2021 Determination of Total ash in Instant Tea In Solid Form 06
7. FSSAI 04A.007:2021 Determination of water soluble ash 07
8. FSSAI 04A.008:2021 Determination of Ash insoluble in dilute hydrochloric acid 08
9. FSSAI 04A.009:2021 Determination of alkalinity of soluble ash : Coffee 09
10. FSSAI 04A.010:2021 Determination of alkalinity of soluble ash : Tea 10
11. FSSAI 04A.011:2021 Determination of aqueous extract 11
12. FSSAI 04A.012:2021 Determination of Caffeine Content (Bailey Andrew Method) 12-14
FSSAI 04A.013:2021 Determination of Caffeine
13. 15-17
(Alternate Chromatographic – Spectrophotometric Method)
FSSAI 04A.014:2021 Determination of Caffeine
14. 18-19
Alternate HPLC method
15. FSSAI 04A.015:2021 Determination of adulterants (Microscopic Examination) 20-22
16. FSSAI 04A.016:2021 Determination of presence of Chicory in Coffee 23
17. FSSAI 04A.017:2021 Determination of Solubility in boiling water 24
18. FSSAI 04A.018:2021 Determination of Solubility in Cold water 25
19. FSSAI 04A.019:2021 Determination of Crude Fibre in Tea 26-27
20. FSSAI 04A.020:2021 Determination of Total Catechins in Tea : HPLC Method 28-34
21. FSSAI 04A.021:2021 Determination of Added color 35-36
22. FSSAI 04A.022:2021 Determination of Iron Filings in Tea 37-38
23. FSSAI 04A.023:2021 Determination of Extraneous matter 40

Note: The test methods given in the manual are standardized / validated and were taken from national
or international methods or recognized specifications, however it would be the responsibility of the
respective testing laboratory to verify the performance of these methods onsite and ensure that it gives
proper results before putting these methods in to use.

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 Determination of Moisture

Method No. FSSAI 04A.001:2021 Revision No. & Date 0.0

Scope This method is applicable for Tea, Kangra Tea, Green Tea, Instant Tea, Coffee,
Soluble Coffee Powder, Decaffeinated roasted and ground coffee, Decaffeinated
soluble coffee powder, Chicory and coffee – chicory mixture Form and
Decaffeinated coffee – chicory mixture

Caution Once sample is opened, seal it in airtight manner after taking test portion

Principle Moisture is the weight lost due to evaporation of water present in a sample. The
sample is dried under controlled conditions to remove moisture during the
analysis. To determine moisture content, the difference in sample weight before
and after drying is calculated.
Apparatus/Instrument 1. Aluminium dish (About 7.5 cm in dia and 2.5 cm deep)
2. Air Oven
3. Desiccator
4. Stop Clock
5. Weighing Balance
Materials and Reagents Desiccants (for Desiccators)
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get
a homogenous sample. Store sample in a tightly stoppered bottle, withdraw
portions for analytical determinations.
Method of analysis 1. Weigh accurately about 5 g of sample in a pre-weighed aluminium dish.
2. Dry the sample in an air oven at 100 ±2 °C for 5 to 6 h.
3. Cool in a desiccator and weigh.
4. Dry again for 30 min, cool in a desiccator and weigh.
5. Repeat the process of heating and cooling in a Desiccator until the difference
in two successive weighings is less than 1 mg.
6. Record the lowest weight. Carry out the analysis in duplicate.
Calculation with units of W 1 – W2
expression Moisture (%) = x 100
(by weight) W1 – W
Moisture % (M)
Where,
W = Weight in g, of empty Aluminium dish
W1 = Weight in g, of empty Aluminium dish + sample before drying
W2 = Weight in g, of empty Aluminium dish + dried sample
Reference  IS: 3077 – 2009 (A Specification for Roasted and Ground Coffee)

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Approved by Scientific Panel on Methods of Sampling and Analysis

Determination of Moisture for roasted coffee and chicory mixture -

Vacuum Oven method (Reference method)

Method No. FSSAI 04A.002:2021 Revision No. & Date 0.0

Scope Roasted coffee, chicory and coffee – chicory mixture
Caution Once sample is opened seal it in air tight manner after taking test portion.
Principle Moisture is the weight lost due to evaporation of water present in a sample. The
sample is dried in a vacuum oven under controlled conditions of pressure and
temperature to remove moisture by passing dry air. To determine moisture content,
the difference in sample weight before and after drying is calculated
Apparatus/Instrument 1. Aluminium dish (7 cm diameter and about 3 cm height) with close fitting cover
2. Vacuum oven – connect with pump capable of maintaining partial vacuum in
oven with pressure equivalent to 25 mm Hg and provided with thermometer
passing into the oven in such a way that the bulb is near the test sample.
Concentrated H2SO4 gas drying bottle with oven to admit dry air when releasing
vacuum
3. Desiccator
4. Stop Clock
5. Weighing Balance
Materials and Reagents 1. Conc. Sulphuric Acid
2. Desiccants (for Desiccator)
Preparation of Reagents NA
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get a
homogenous sample. Store sample in a tightly stoppered bottle, withdraw portions
for analytical determinations.
Method of analysis 1. Accurately weigh about 5 g of sample, in a dish previously dried at 98 –100
°C, cooled in desiccator and weighed with cover soon after attaining room
temperature.
2. Place in oven, lean cover against dish and heat to constant weight (about 5.5
hr at 98 – 100°C at pressure equal to 25 mm Hg.
3. During heating allow slow current of air (about two bubbles / second through
H2SO4) into oven.
4. Carefully admit dry air into oven to bring to atmospheric pressure.
5. Cover dish, transfer to desiccator and weigh soon after room temperature is
attained.
6. Repeat the operation until the difference between two successive weighing is
less than 1 mg. Record the lowest mass.
7. Report percent loss in weight as moisture.
Calculation with units of W1 – W2
expression Moisture (%) = x 100
(by weight) W1 – W
Where,
W = Weight in g, of empty Aluminium dish.
W1 = Weight in g, of empty Aluminium dish + sample before drying.
W2 = Weight in g, of empty Aluminium dish + dried sample.

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Reference A.O.A.C 21st edn, Official Method of Analysis(2019) Method no. 968.11 Moisture
(Loss on Drying in Roasted Coffee, Vacuum Oven method 1
Approved by Scientific Panel on Methods of Sampling and Analysis
Determination Of Moisture For Soluble (Instant) Coffee Powder -
Vacuum Oven Method (Reference Method)

Method No. FSSAI 04A.003:2021 Revision No. & Date 0.0

Scope Soluble (Instant) Coffee powder
Caution Once sample is opened seal it in air tight manner after taking test portion.
Principle Moisture is the weight lost due to evaporation of water present in a sample. The
sample is dried in a vacuum oven under controlled conditions of pressure and
temperature to remove moisture by passing dry air. To determine moisture content,
the difference in sample weight before and after drying is calculated
Apparatus/Instrument General Apparatus and Glassware
1. Aluminium dish 7 cm diameter and about 3 cm height with close fitting cover.
2. Vacuum oven – connected with pump capable of maintaining partial vacuum in
oven with pressure equivalent to 25 mm Hg and provided with thermometer
passing into the oven in such a way that the bulb is near the test sample. Connect
H2SO4 gas drying bottle with oven to admit dry air when releasing vacuum
3. Desiccator.
4. Stop Clock.
5. Weighing Balance
Materials and Reagents 1. Conc. Sulphuric acid
2. Desiccants (for desiccator)
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get a
homogenous sample. Store sample in a tightly stoppered bottle, withdraw portions
for analytical determinations.
Method of analysis 1. Accurately weigh about 5 g of sample in a dish, previously dried at 98 –100 °C,
cooled in desiccator and weighed with cover soon after attaining room
temperature.
2. Place in an oven, lean cover against dish and heat to constant weight (about 16 h)
at 70 ± 1°C at pressure equal to 37.5 mm Hg.
3. During heating, admit slow current of air (about one bubble / second through
H2SO4) into oven.
4. Carefully admit dry air into oven to bring to atmospheric pressure.
5. Cover dish, transfer to desiccator and weigh soon after room temperature is
attained.
6. Repeat the operation until the difference between two successive weighing is
less than 1 mg. Record the lowest mass.
7. Report % loss in weight as moisture.
Calculation with units of (M1 – M2)
expression Moisture (%) = x 100
(M1 – M0)
Where
M0 = Weight of empty dish
M1 = weight of dish + sample before drying
M2 = Weight of dish + sample after drying
Reference A.O.A.C 21st edn, Official Method of Analysis (2019) Method no. 979.12 Moisture
(Loss on Drying) in Roasted Coffee – applicable to instant coffees.

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Approved by Scientific Panel on Methods of Sampling and Analysis
Determination Of Total Ash

Method No. FSSAI 04A.004:2021 Revision No. & Date 0.0

Scope Tea, Kangra Tea, Green Tea, Instant Tea, Coffee, Soluble Coffee Powder,
Decaffeinated roasted and ground coffee, Decaffeinated soluble coffee powder,
Chicory and coffee – chicory mixture and Decaffeinated coffee – chicory mixture
Caution Once sample is opened, seal it in airtight manner after taking test portion
Wear heat resistant gloves and face protection while doing analysis
Principle Ash is the inorganic residue remaining after destruction of organic matter at a
temperature of 550 ± 10 °C. Sample is weighed before and after heat treatment to
estimate total ash.
Apparatus/Instrument 1. Silica / Platinum dish
2. Burner
3. Muffle furnace
4. Desiccator
5. Weighing balance
Materials and Reagents 1. Desiccants (for Desiccator)

Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get a
homogenous sample. Store sample in a tightly stoppered bottle, withdraw portions
for analytical determinations.
Method of analysis 1. Weigh accurately about 5 g of sample in a tarred silica / platinum dish.
2. Char the material carefully on a burner. (Instead of Bunsen burner, hot plate can
also be used for charring of samples).
3. Transfer the dish to a muffle furnace.
4. Ash at a temperature of 550 ± 10 °C until the ash is free of Carbon.
5. Heat the dish again at 550 ± 10 °C for 30 min.
6. Cool the dish in a desiccator and weigh.
7. Repeat this process of heating for 30 min, cooling in a desiccator and weighing
until the difference between two successive weighing is less than 1 mg.
8. Record the lowest weight.
Note: – Preserve the dish containing this ash for the determination of acid insoluble
ash.
Calculation with units of (W2 – W) x 100 x 100
expression Total ash (% on dry weight) =
(W1 – W) x (100 – M)
Where,
W1 = Weight in g of empty Silica dish. + sample
W2 = Weight in g of empty Silica dish + ash
W = Weight in g of empty Silica dish
M = Moisture % of the sample

Reference  I S: 3077 – 2009(A Specification for Roasted and Ground Coffee Appendix F
 I S 13854: 1994 (ISO 1575: 1987) Tea – Determination of Total Ash

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Total Ash
(Alternate Method for Roasted and Ground Coffee)

Method No. FSSAI 04A.005:2021 Revision No. & Date 0.0

Scope Roasted and ground coffee
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. Wear heat resistant gloves and face protection while doing analysis
Principle Ash is the inorganic residue remaining after destruction of organic matter at a
temperature of 550 ± 10 °C and sample is weighed before and after ash to estimate
total ash.
Apparatus/Instrument 1. Silica / Platinum dish
2. Muffle furnace (programmable)
3. Desiccator
4. Weighing balance
Materials and Reagents 1. Desiccants (for desiccator)

Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get a
homogenous sample. Store sample in a tightly stoppered bottle, withdraw portions
for analytical determinations.
Method of analysis 1. Use a programmable muffle furnace that allows a gradual increase in
temperature to 550 °C. [Charring with a Bunsen burner and inserting the
sample into the furnace at 550 °C. The charring technique is often prone to
losses and can have superheating of samples as they enter the furnace causing
them to ‘explode’ (not in a dramatic way) and lose sample or contaminate
surrounding samples. It’s very quick but not as accurate as using a
programmable muffle. It’s also safer to use a programmable furnace for the
analyst- handling crucibles at 550 °C, is prone to risk].
2. Ash at a temperature of 550 ± 10 °C until the ash is free of Carbon.
3. Heat the dish again at 550 ± 10 °C for 30 min.
4. Cool the dish in a desiccator and weigh.
5. Repeat this process of heating for 30 min, cooling in a desiccator and weighing
until the difference between two successive weighing is less than 1 mg.
6. Record the lowest weight.
Calculation with units of (W2 – W) x 100 x 100
expression Total ash (% on dry weight) =
(W1 – W) x (100 – M)
Where,
W1 = Weight in g of empty Silica dish + sample
W2 = Weight in g of Silica dish + ash
W = Weight in g of empty Silica dish
M = Moisture % of the sample
Reference  IS: 3077 – 2009 (A Specification for Roasted and Ground Coffee Appendix F

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination Of Total Ash ( Instant Tea In Solid Form)

Method No. FSSAI 04A.006:2021 Revision No. & Date 0.0

Scope Instant tea in solid form.
Caution Concentrated hydrochloric acid is corrosive, has an irritant vapour and causes
burns. Wear mask and gloves during handling
Principle Ash is the inorganic residue remaining after destruction of organic matter at a
temperature of 550 ± 10 °C and sample is weighed before and after ash to estimate
total ash.
Apparatus/Instrument 1. Dish: approximately 50 ml capacity made of platinum, porcelain or any other
material unaffected by the conditions of the test.
2. Furnace: capable of being controlled at 550°C ± 25°C.
3. Hot-plate thermostatically controlled.
4. Desiccator, containing an efficient desiccant.
Materials and Reagents Hydrochloric acid, concentrated (Analytical grade).
Sample Preparation Thoroughly mix the instant tea sample as received, by shaking or inverting the
sealed sample container.
Method of analysis 1. Preparation of the dish: Ensure that the dish is completely clean, and then heat
it in the furnace at 550 °C ± 25 °C for at least 30 min. Cool in the desiccator.
Alter cooling to room temperature, weigh to the nearest 0,001 g.
2. Weigh about 2 g of the prepared test sample into the prepared dish. Spread the
sample evenly over the base of the dish.
3. Add, drop by drop, to the test portion contained in the dish, sufficient
(approximately 1 ml) of the concentrated hydrochloric acid solution to wet it
completely.
4. Place the dish on the cool hot-plate, set the control to medium and heat for 30
min. Raise the hot-plate temperature to the highest setting in three successive
steps, allowing the test portion to heat at each stage for 30 min. keep the test
portion at the highest setting until no fuming has occurred for at least 5 min.
5. Place the dish containing the test portion in the furnace at 550°C ± 25°C for 16
h. Remove, leave to cool and add a few drops of water to moisten and disperse
the ash.
6. Evaporate-to dryness on the hot-plate as before, and then return to the furnace
for a further 30 min.
7. Remove, cool to room temperature in the desiccator and weigh to the nearest
0,001g. Determine the mass of the total ash.
NOTE - instant tea ashed under these conditions should give a grey/white ash.
8. Carry out two determinations on the same test sample.
Calculation with units of The total ash, expressed as a percentage by mass of the sample on a dry basis, is given
expression by the formula
𝑚1 100
× 100 ×
𝑚𝑜 𝑅𝑆
Where,
mo is the mass, in grams, of the test portion;
m1 is the mass, in grams, of the total ash;
RS is the dry matter content, expressed as a percentage by mass, of the test sample. It
is equal to 100 minus the moisture content.
Reference IS 13860:1993 (ISO 7514:1990): Instant tea in solid form - Determination of total ash.

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination Of Water Soluble Ash

Method No. FSSAI 04A.007:2021 Revision No. & Date 0.0

Scope Roasted coffee, Tea, Kangra Tea, Green Tea, Coffee Roasted /unroasted
ground/green, Decaffeinated roasted and ground coffee
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. Wear gloves and face protection while doing analysis.
Principle Water Soluble Ash is the part of the total ash dissolved by water. Difference
between Total ash and water in-soluble ash is calculated as water soluble ash.
Apparatus/Instrument General Apparatus and Glassware
1. Beakers, 2. Silica dish, 3. Watch glass, 4. Filter Paper (Whatman No. 42 or
its equivalent)and 5. Red litmus
Materials and Reagents 1. Total ash after ashing of sample
2. Distilled water
Preparation of Reagents NA
Sample Preparation Continue after ashing of sample

Method of analysis 1. Transfer the total ash with the aid of about 25 mL distilled water into a
beaker.
2. Cover with a watch glass and boil for 5 min.
3. Filter through an ash less filter paper (Whatman No. 42 or its equivalent).
4. Collect the filtrate in a 150 mL beaker.
5. Wash the filter paper 4 -5 times with hot water until the filtrate no longer
turns red litmus blue and collect the washings in the same beaker. (Note:
Reserve the entire filtrate for the determination of alkalinity of soluble ash)
6. Dry the ash less paper with residue in an oven in a silica dish and transfer to
muffle furnace and ignite at 550 °C for 2 h.
7. Cool in a desiccator and weigh (W3).
8. Repeat the process till the difference in two consecutive weighing is less
than 1 mg. Record the lowest weight.
Calculation with units of (W3 – W) x 100 x 100
expression Water in-soluble ash on dry wt. basis (%) =
(W1 – W) x (100 – M)
Where,
W3 = Weight in g of Silica dish + water insoluble ash.
W = weight in g of empty dish.
W1 = weight in g of Silica dish with material.
M = Percentage of Moisture
Water soluble ash percent by wt = A – B
Where, A = Total ash percent by wt
B = Water insoluble ash percent by wt
Water soluble ash
Water soluble ash of total ash = x 100
(Percent by wt) Total ash

Reference  IS: 3077 – 2009 (A Specification for Roasted and Ground Coffee)
 IS 13855: 1993 ( ISO 1576:1988) Tea – Determination of Water soluble ash and
Water insoluble Ash
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination Of Ash Insoluble in Dilute Hydrochloric Acid

Method No. FSSAI 04A.008:2021 Revision No. & Date 0.0

Scope Tea, Kangra Tea, Green Tea, Instant Tea, Coffee Roasted /unroasted ground/green,
Chicory, coffee – chicory mixture, Instant Coffee - Chicory Mixture, Decaffeinated
Roasted and Ground coffee-chicory, Decaffeinated Instant coffee-chicory mixture

Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. Concentrated hydrochloric acid is corrosive, has an irritant vapour and causes
burns. Wear mask and gloves during analysis
Principle The proportion of ash that is not hydrolyzed by acid is known as the acid insoluble
ash(silica and oxalates). Acid insoluble ash is evaluated by dissolving total ash in
dilute hydrochloric acid (5N)and ignited in muffle furnace @ 550 °C .
Apparatus/Instrument General Apparatus and Glassware: 1. Beakers, 2. Silica dish, 3. Watch glass, 4. Filter
Paper (Whatman No. 42 or its equivalent) and 5. Red litmus

Materials and Reagents 1. Total ash after ashing of sample

2. Conc. Hydrochloric acid
3. Distilled water
Preparation of Reagents 1. Hydrochloric acid (5N) - Hydrochloric acid (10 mL) is dissolved in 25 mL
distilled water.
Sample Preparation Continue after ashing of the sample.
Method of analysis 1. Boil the total ash with 25 mL of 5N Hydrochloric acid for 5 min, covering the
Silica dish with a watch glass to prevent spattering.
2. Filter through ash less filter paper (Whatman No. 42 or equivalent).
3. Wash the entire residue with hot water (> 85 °C) until the filtrate does not turn
blue litmus paper to red.
4. Dry the ash less paper with the residue in silica dish and transfer to muffle
furnace and ignite at 550 °C for 2 h.
5. Repeat the process of igniting in the muffle furnace, cooling and weighing at 30
min intervals until the difference in two successive weighing is less than 1 mg.
6. Cool in a desiccator and weigh (W4).
Calculation with units of (W4 – W) x 100 x 100
expression Ash insoluble in dilute HCl (%) =
(on dry wt.) (W1 – W)x (100 – M)
Where,
W4 = weight of empty dish + acid insoluble ash
W1 = weight of dish + sample
W = weight of dish
M = Percent moisture
Reference  IS: 3077 – 2009 A Specification for Roasted and Ground Coffee
 IS 13857: 1993 ( ISO 1577: 1987) Tea – Determination of Acid insoluble Ash
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination Of Alkalinity Of Soluble Ash: Coffee

Method No. FSSAI 04A.009:2021 Revision No. & Date 0.0

Scope Coffee Roasted /unroasted ground/green, Decaffeinated roasted and ground
coffee
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. Concentrated hydrochloric acid is corrosive, has an irritant vapour and
causes burns. Wear mask and gloves during analysis
Principle Alkalinity of soluble ash, indicate the amount of acid required to neutralize the
aqueous extract of the total ash. The ash obtained mixed with water and heated to
boiling and filtered through ash less filter paper. The filtrate of water soluble ash
is titrated against 0.l N HCl using methyl orange as an indicator to calculate
alkalinity of soluble ash.
Apparatus/Instrument General Apparatus and Glassware
1. Calibrated Burette
2. Dropper
Materials and Reagents 1. Methyl orange indicator
2. Conc. Hydrochloric Acid
Preparation of Reagents 1. Methyl orange indicator (0.1% w/v) - 0.1 g of methyl orange dissolved in
100 mL of distilled water.
2. Hydrochloric acid (0.1 N) – Concentrated (1 mL) diluted to 116.5 mL
with distilled water.
Sample Preparation 1. Filtrate reserved during the determination of water soluble ash

Method of analysis 1. To the filtrate reserved during the determination of water soluble ash, add 3-4
drops of methyl orange indicator (0.1% w/v in water).
2. Titrate with 0.1 N hydrochloric acid to an orange end point. Note down the titre
value.
Calculation with units of Titre value x Normality of HCl
expression Alkalinity of soluble ash % per =
g of sample (on dry wt.) Wt. of sample (W1– W) x (100 – M)
Where,
W = weight of empty dish
W1 = weight of dish + sample
M = % Moisture of the sample
Reference  IS: 3077 – 2009 (A Specification for Roasted and Ground Coffee)

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination Of Alkalinity Of Soluble Ash : Tea

Method No. FSSAI 04A.010:2021 Revision No. & Date 0.0

Scope Tea/ Instant Tea, Kangra Tea, Green Tea
Caution Concentrated hydrochloric acid is corrosive, has an irritant vapour and causes
burns. Wear mask and gloves during analysis
Principle Alkalinity of soluble ash, indicate the amount of acid required to neutralize the
aqueous extract of the total ash. The ash obtained mixed with water and heated to
boiling and filtered through ash less filter paper. The filtrate of water soluble ash
is titrated against 0.l N HCl using methyl orange as an indicator to calculate
alkalinity of soluble ash.
Apparatus/Instrument General Apparatus and Glassware
1. Calibrated Burette.
2. Dropper.
Materials and Reagents 1. Methyl orange indicator
2. Concentrated Hydrochloric acid (36%)
Preparation of Reagents 1. Methyl orange indicator - 0.1 g of methyl orange dissolved in 100 mL of
distilled water.
2. Hydrochloric acid (0.1 N) – Concentrated hydrochloric acid (1 mL)
diluted to 116.5 mL with distilled water.
Sample Preparation Filtrate reserved during the determination of water soluble ash

Method of analysis 1. To the filtrate reserved during the determination of water soluble ash, add 3-4
drops of methyl orange indicator (0.1% in water).
2. Titrate with 0.1 N hydrochloric acid to an orange end point. Note down the titre
value.
Calculation with units of Express the result as KOH (m/m) on dry basis:
expression 0.0056 x titer value x Normality HCl x 100 x 100
Alkalinity of =
soluble ash % Weight of sample x 0.1 x (100 – moisture %)
Reference I.S 13856: 1993 ( ISO 1578: 1975) - Tea Determination of Alkalinity of Water
soluble ash
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Aqueous Extract

Method No. FSSAI 04A.011:2021 Revision No. & Date 0.0

Scope Tea/ instant tea, Kangra tea, green tea, coffee roasted /unroasted ground/green,
decaffeinated roasted and ground coffee, chicory, coffee – chicory mixture,
decaffeinated roasted and ground coffee -chicory mixture
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. Wear gloves and face protection during analysis
Principle Sample is refluxed in water for one h and filtered the water soluble portion/ extract
and calculated as % Aqueous Extract.
Apparatus/Instrument General Apparatus and Glassware: 1. Flask -500 mL, 2. Water jacketed condenser
– 50 cm length, 3. Burner / hot plate, 4. Whatman No 1filter paper, 5. Pipette – 50
mL, 6. Aluminum dish, 7. Steam bath and 8. Hot air oven
Materials and Reagents 1. Distilled water
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get
a homogenous sample. Store sample in a tightly stoppered bottle, withdraw
portions for analytical determinations.
Method of analysis 1. Accurately weigh around 2 g of sample and transfer to a 500 mL flask.
2. Add 200 mL distilled water and connect the flask with a 50 cm long water
jacketed condenser. Reflux for one h over low flame with occasional mixing.
3. Cool, and filter through Whatman No. 1 filter paper or equivalent, wash three
times with 10 – 15 mL distilled water and finally make upto 250 mL in a
volumetric flask.
4. Shake well and pipette 50 mL of aliquot to a tarred aluminium dish.
5. Evaporate on a steam bath.
6. Transfer to 100 °C air oven and dry for two h.
7. Dry again for 30 min, cool in a desiccator and weigh.
8. Repeat this process of heating for 30 min, cooling in desiccator and weighing
until the loss in weight between two successive weighing is less than 1 mg.
9. Record the lowest weight.
(W2 – W1) x 250 x 100 x100
Calculation with units of Aqueous extract (%) = -----------------------------------------
expression (on dry wt.) W x 50 x (100 – M)
Where,
W= Weight of sample.
W1 = Weight of empty aluminium dish.
W2 = Weight of empty aluminium dish + dried extract.
M = Moisture %
Reference  IS: 3077 – 2009 (A Specification for Roasted and Ground Coffee)

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Caffeine Content (Bailey Andrew Method)

Method No. FSSAI 04A.012:2021 Revision No. & Date 0.0

Scope Coffee roasted /unroasted ground/green, decaffeinated roasted and ground
coffee, Soluble coffee Powder, decaffeinated Soluble coffee Powder, coffee
– chicory mixture, decaffeinated coffee – chicory mixture, Instant coffee –
chicory mixture and decaffeinated Instant coffee – chicory mixture
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. Wear gloves and face protection during Analysis
Principle Caffeine is a naturally occurring stimulant found in coffee. Caffeine from coffee
sample is extracted followed by digestion using Micro Kjeldhal flask. The
conversion factor is used to convert the estimated nitrogen to caffeine content.
Apparatus/Instrument 1. Erlenmeyer flask – 250 mL
2. Reflux condenser
3. Filter papers.
4. Volumetric flask – 50 mL.
5. Filtration set.
6. Separating funnels – 125 mL.
7. Kjeldahl flask (100 mL) and distillation assembly.
8. Beaker - 125 mL.
9. Burette. Space-1.0
Materials and Reagents 1. Magnesium oxide.
2. Distilled water.
3. Concentrated Sulphuric acid (98%).
4. Chloroform.
5. Potassium hydroxide.
6. Potassium sulphate.
7. Mercuric oxide.
8. Vaseline.
9. Sodium hydroxide.
10.Methyl red indicator.
Preparation of Reagents 1. Diluted sulphuric acid– Concentrated sulphuric acid (1 mL) diluted by
mixing with 9 mL of distilled water.
2. Potassium hydroxide solution (1%) - Potassium hydroxide (1 g) dissolved
in distilled water (100 mL).
3. Sulphuric acid (0.05 N) – conc. Sulphuric acid (1 mL) is added to 735 mL
distilled water.
4. Sodium hydroxide (concentrate) (1:2) - Sodium hydroxide (5 g) dissolved
in 10 mL distilled water.

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 5. Sodium hydroxide (0.1 M / 0.1 N) - Sodium hydroxide (0.4 g) dissolved
in distilled water (100 mL).
6. Methyl Red Indicator Solution: Dissolve 50 mg of methyl red in a mixture
of 1.86 mL of 0.1 M sodium hydroxide and 50 mL of ethanol (95 %, v/v).
After the solution is effected, add sufficient water to produce 100 mL
7. Methyl Red Indicator Solution: Dissolve 50 mg of methyl red in 100 mL
of 95% ethanol.
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get
a homogenous sample. Store sample in a tightly stoppered bottle, withdraw
portions for analytical determinations.

Method of analysis 1. Weigh accurately about 5 g of sample, transfer to a 250 mL Erlenmeyer

flask and add 3 g of magnesium oxide and 100 mL of distilled water.
2. Weigh the flask with contents and boil under a reflux condenser for 45
min, shaking occasionally.
3. Cool and weigh the flask again and add water till the original weight is
obtained.
4. Mix well and filter through a dry filter paper directly into a 50 mL
graduated flask until exactly 50 mL of the solution (equivalent to half the
quantity of the sample taken for test) is obtained.
5. Transfer the solution to a 125 mL separator. Wash the graduated flask
with 2 mL of water and add the washings to the separator.
6. Add 4 mL of dilute Sulphuric acid (1: 9).
7. Extract with five 10 ml portions of chloroform shaking vigorously for 1
minute for each extraction. Let the emulsion break, then drain the
chloroform into a 125 mL separator.
8. Add 5 mL of Potassium hydroxide solution (1%).
9. Shake vigorously for 1 min, let the emulsion break and drain the
chloroform through a cotton plug into a 100 mL Kjeldahl flask.
10. Extract the Pot hydroxide solution with 5 mL of chloroform and add to
the Kjeldahl flask.
11. To the digestion flask add 1.3 ± 0.5 g of potassium sulphate and 40 ± 5
mg mercuric oxide. Rinse down the neck of the flask with 3 mL
chloroform.
12. Place the flask on the digestion rack and concentrate chloroform to about
20 mL
13. Distil off chloroform. Add 2 ± 0.1 mL conc. sulphuric acid of Sp. gravity
1.84, digest for one h after the acid begins to boil.
14. Cool and add minimum quantity of water to dissolve the solids.
15. Cool and place a thin film of Vaseline at the rim of the flask.
16. Transfer the digest with a few boiling chips to the distillation apparatus
and rinse the flask five-six times with 1 – 2 mL distilled water.
17. Place a 125 mL beaker containing a known quantity of standard sulphuric
acid (0.05 N).

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 18. Add 6 mL of conc. sodium hydroxide solution (1:2) carefully through the
side of the still so that it does not mix, and assemble the distillation
apparatus taking care that the dip tube extends well within the standard
sulphuric acid solution contained in the beaker.
19. Mix the contents of the distillation flask and distill until all ammonia has
passed over into the standard sulphuric acid.
20. Shut off the heater and immediately detach the flask from the condenser.
21. Rinse the condenser thoroughly with water into the beaker. Wash the dip
tube carefully so that all traces of the condensate are transferred to the
beaker.
22. When all the washings have drained into the beaker, add 2-3 drops of
methyl red indicator and titrate with standard sodium hydroxide solution
(0.1 N).
23. Carry out a blank determination using reagents in the same proportion
without the sample.

Calculation with units of 486.96 (B- A) N

expression Caffeine on dry basis =
(%) by weight W (100 – M)

Where,
B= Volume of standard sodium hydroxide used to neutralize acid in the blank
determination
A= Volume of standard sodium hydroxide used to neutralize the excess acid
in the test with the sample
N= Normality of standard sodium hydroxide solution
W= Weight in g of the sample in the aliquot
M= Percentage of moisture in the sample
Note: - For soluble coffee (instant coffee) the quantity of sample for test should
be 1 g only.
Reference  IS: 3077 – 2009 (A Specification for Roasted and Ground Coffee)
 A.O.A.C 21st edn, Official Method of Analysis (2019) Method no.960.25
Caffeine in Roasted Coffee.

Approved by Scientific Panel on Methods of Sampling and Analysis

16 | P a g e
 Determination of Caffeine
(Alternate Chromatographic – Spectrophotometric Method)

Method No. FSSAI 04A.013:2021 Revision No. & Date 0.0

Scope Coffee roasted /unroasted ground/green, decaffeinated roasted and ground coffee,
Soluble coffee Powder, decaffeinated Soluble coffee Powder, coffee – chicory
mixture, decaffeinated coffee – chicory mixture, Instant coffee – chicory mixture
and decaffeinated Instant coffee – chicory mixture
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. Wear gloves and face protection during Analysis
Principle Caffeine is a natural stimulant most commonly found in tea, coffee, and cacao
plants Caffeine is separated using column chromatography using chloroform
solvent and optical density (OD) is measured using spectrophotometer at 276nm
using caffeine standard.
Apparatus/Instrument General Apparatus and Glassware
1. Glass columns – 25 x 250 mm size
2. UV – VIS Spectrophotometer – To record 250 – 350 nm range with
matched 1 cm cells.
Materials and Reagents 1. Ammonia solution
2. Concentrated Sulphuric acid (98%)
3. Diethyl ether
4. Chloroform
5. Celite 545
6. Caffeine
7. Sodium hydroxide
Preparation of Reagents 1. Ammonium hydroxide solution (1:2)– Ammonia (100 mL) is added to
distilled water (200 mL)
2. Sulphuric acid (4 N) – Concentrated sulphuric acid (10 mL) is diluted to 92
mL with distilled water.
3. Diethyl ether (Water Saturated) – Diethyl ether (100 mL) is mixed with
distilled water and shaken well. Top layer is diethyl ether saturated with water
and taken is extracted.
4. Chloroform – Chloroform (100 mL) is mixed with distilled water and shaken
well. Bottom layer is chloroform saturated with water and taken.
5. Caffeine standard solution (10, 20, 30 µg /mL in Chloroform) - Accurately
weigh 100 mg of caffeine (USP, anhydrous) into 100 mL volumetric flask,
dissolve in chloroform and make upto volume. Dilute 10 mL aliquot to 100
mL with chloroform. Further dilute 10, 20, and 15 mL aliquots to 100, 100
and 50 mL respectively with chloroform to obtain standard solutions of 10,
20, and 30 µg /mL
6. Sodium hydroxide (2 N) – Sodium hydroxide (8 g) dissolved in distilled water
(100 mL).

17 | P a g e
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get
a homogenous sample. Store sample in a tightly stoppered bottle, withdraw
portions for analytical determinations.
Method of analysis For Green/roasted Coffee
1. Accurately weigh about 1 g ground sample and transfer to 100 mL beaker.
2. Add 5 mL NH4OH (1:2) and warm on boiling water-bath for 2 min.
3. Cool, transfer to 100 mL volumetric flask and make up to volume with
water. To 5 mL aliquot of the turbid solution add 6 g celite 545 and mix
carefully.
For decaffeinated green/roasted coffee
1. Accurately weigh 1 g of ground sample.
2. Transfer to 100 mL beaker, add 5 mL NH4OH (1:2) and warm on boiling
water bath for 2 min. Add 6 g celite 545 and mix carefully.
For soluble Coffee
1. Proceed as in green/roasted coffee except 0.5 g sample and an aliquot of
3 mL
For decaffeinated soluble coffee
1. Proceed as in decaffeinated green/roasted coffee except 0. 5 g sample.
Column Chromatography
Acid column:
1. Place fine glass wool and plug into the base of 25 x 250 mm column.
2. Add 3 mL 4 N H2SO4 to 3 g celite 545 and mix well by kneading with
spatula. Transfer into the tube and tamp using gentle pressure and place
small glass wool above the surface.

Basic Column:
Layer I:
1. Mix 3 g celite 545 and 2 mL 2 N NaOH and place in 25 x 250 mm tube.
Transfer over glass wool plug as in Acid column.
Layer II:
1. Transfer sample plus celite 545 mixtures in about 2 g portions to tube directly
over layer I, taping before adding mixture portion of sample until homogenous
and compact layer is obtained.
2. Dry wash beaker with about 1 g celite 545, transfer to tube and tap to uniform
mass.
3. Dry wash beaker with wad of glass wool and transfer to top of basic column.
4. Mount basic column above acid column.
5. Pass 150 mL water saturated ethers sequentially through basic column to acid
column and discard ether. Then pass 50 mL water saturated ether through acid
column and discard ether.
6. Place 50 mL volumetric flask under acid column.
7. Pass 48 mL water saturated CHCl3 through acid column washing tip of basic
column with first portions.

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Calculation with units of 1. Dilute contents of volumetric flask (100 mL) to volume with water
expression saturated chloroform, mix, and read O.D at 276 nm against water saturated
chloroform CHCl3 blank, by scanning from 350 to 250 nm.
2. Determine O.D of standards and use this value to calculate the caffeine
percentage.
Reference A.O.A.C 21st edn, Official Method of Analysis(2019) Method no. 979.11
Caffeine in Roasted Coffee, Chromatographic – Spectrophotometer method.

Approved by Scientific Panel on Methods of Sampling and Analysis

19 | P a g e
 Determination of Caffeine
(Alternate method By HPLC)

Method No. FSSAI 04A.014:2021 Revision No. & Date 0.0

Scope Coffee roasted /unroasted ground/green, decaffeinated roasted and ground coffee,
Soluble coffee Powder, decaffeinated Soluble coffee Powder, coffee – chicory
mixture, decaffeinated coffee – chicory mixture, Instant coffee – chicory mixture
and decaffeinated Instant coffee – chicory mixture
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. The cartridge should not be dry during elution.
Principle Caffeine is a natural stimulant most commonly found in tea, coffee, and cacao
plants is usually extracted by C-18 cartridges and quantified by HPLC (absorbance
measured at 280 nm)
Apparatus/Instrument 1. General Apparatus and Glassware (Page 3 and Analytical Balance
(0.0001g)
2. Millipore filters (0.45 µm).
3. Bond C 18 cartridges technical details???
4. Volumetric flasks -10 mL.
5. HPLC system with UV-VIS
6. Column: Spherisorb ODS, C 18, 5 um packed column 25 cm long x 4 mm
internal Dia.
Materials and Reagents 1. Distilled water.
2. Sodium acetate.
3. Tetrahydrofuran.
4. Standard Caffeine
Preparation of Reagents 1. Sodium acetate (0.005 M)
2. Standard Caffeine solutions: Caffeine (0.2, 0.4, 0.6, 0.8 and 1.0 mg) in 10
mL mobile phase (0.005 M Sodium acetate: tetrahydrofuran – 95: 5 at pH
5).
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get
a homogenous sample. Store sample in a tightly stoppered bottle, withdraw
portions for analytical determinations.
Method of analysis 1. Dissolve 1 g of sample in 100 mL hot water
2. Filter 20 mL through a Millipore filter (0.45 µm) under vacuum.
3. To a Bond Elute C 18 cartridge or equivalent under vacuum.
4. Elute the caffeine with 5 mL of mobile phase (0.005 M Sodium acetate:
tetrahydrofuran – 95: 5 at pH 5).
5. Collect in a 10 mL flask and make upto volume.

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 6. Inject 20 µL into a Spherisorb ODS, C 18, 5 um packed column 25 cm
long x 4 mm internal dia.
7. Elute with the mobile phase at 1 mL/min, read the absorbance at 280 nm.
8. Calibrate with standard Caffeine solution, 0 - 1 mg Caffeine in 10 mL
mobile phase.
Note: For routine purposes the HPLC step can be eliminated and the absorbance
of eluent from the cartridge measured at 280 nm in a spectrophotometer.
Calculation with units of 1. Calibration curve of Caffeine is prepared using absorbance standard
expression solutions of caffeine (280 nm) solutions versus concentration.
2. Caffeine in sample solution is determined using the calibration curve.
Reference Pearson’s Composition and Analysis of Foods 9th edn, 1991, page 373
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of adulterants
(Microscopic Examination)

Method No. FSSAI 04A.015:2021 Revision No. & Date 0.0

Scope Coffee roasted /unroasted ground/green, soluble coffee powder and coffee –
chicory mixture, instant coffee - chicory mixture

Caution 1. Roasted cereals such as barley, oats and wheat and soya may be mixed
with coffee and coffee and chicory as coffee substitutes.
2. Once sample is opened, seal it in airtight manner after taking test portion
Principle Sample is first heat treated to extract color present in the sample and
microscopically examined to check the presence of any adultrant.
Apparatus/Instrument General Apparatus and Glassware
1. Filtration set.
2. Microscope.
3. Microscopic slide.
Materials and Reagents 1. Sodium hydroxide.
2. Distilled water.
3. Glycerine.
4. Chloral hydrate.
5. Phloroglucinol.
6. Hydrochloric acid.
Preparation of Reagents 1. Sodium hydroxide (2%) - Sodium hydroxide (2 g) is dissolved in distilled
water (100 mL)
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get
a homogenous sample. Store sample in a tightly stoppered bottle, withdraw
portions for analytical determinations.
Method of analysis 1. Boil about 1 g of sample with 50 mL of 2% sodium hydroxide for about
2 - 3 min.
2. Dilute and filter then wash the residue with water till the filtrate is free of
alkali.
3. Repeat till the residue gives no colour with water (treatment with calcium
chloride solution and then washing with water may be done in case, decant
still shows some colouring matter).
4. Place a drop of residue material in glycerine on a clear microscopic slide.
5. Place a cover slip on the drop of the suspension and see under microscope.
Alternatively
1. Boil sample with water so that most of the colour is extracted.
2. Drain and replace with chloral hydrate. Heat until sufficiently cleared.
3. Wash out chloral hydrate and stain with phloroglucinol/ hydrochloric
acid. The microscopic structure as shown in the photomicrograph given
below can be seen:

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23 | P a g e
Calculation with units of Coffee is characterized by longitudinal and transverse schlerenchymatous fibres
expression (from pericarp)
Chicory has large vessels upto 115 microns across which have short pits.
Reference  IS: 3077 – 2009(A Specification for Roasted and Ground Coffee)
 FAO Manuals of Food Quality Control 14 /8 pages 318 and 319

Approved by Scientific Panel on Methods of Sampling and Analysis

24 | P a g e
 Determination of Presence of Chicory in Coffee

Method No. FSSAI 04A.016:2021 Revision No. & Date 0.0

Scope Coffee roasted /unroasted ground/green, soluble coffee powder
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. Concentrated hydrochloric acid is corrosive, has an irritant vapour and
causes burns. Wear mask and gloves during analysis
Principle Chicory contains inulin, which hydrolyses to laevulose. Coffee contains no inulin.
The presence of chicory is shown by a positive reaction with Seliwanoff's reagent.
Apparatus/Instrument General Apparatus and Glassware
1. Filtration set.
Materials and Reagents 1. Neutral lead acetate
2. Conc. HCl.
3. Resorcinol
4. Hydrochloric acid.
5. Distilled water.
Preparation of Reagents 1. Neutral lead acetate (10%) – Neutral lead acetate (10 g) dissolved in water
(100 mL).
2. Seliwanoff reagent – Dissolve 0.05 g of resorcinol in 100 mL of mixture
of hydrochloric acid: distilled water (1:2).
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get
a homogenous sample. Store sample in a tightly stoppered bottle, withdraw
portions for analytical determinations.
Method of analysis 1. Clarify 25 mL of 2% aqueous extract of the sample with neutral lead
acetate and filter.
2. To 5 mL of filtrate add 5 mL of Seliwanoff reagent and 1 mL of conc.
HCl.
3. Boil for 2 min.
4. Appearance of distinct red color on standing shows the presence of
Chicory in coffee.

Calculation with units of Absent/Present of chicory in coffee

expression
Reference FAO Manuals of Food Quality Control 14 / 8 pages317 and 318
Approved by Scientific Panel on Methods of Sampling and Analysis

25 | P a g e
 Determination of Solubility in boiling water

Method No. FSSAI 04A.017:2021 Revision No. & Date 0.0

Scope Soluble (Instant) Coffee powder, Decaffeinated soluble coffee powder, Instant
Coffee - Chicory Mixture, Decaffeinated Instant coffee- chicory mixture
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. Wear gloves and face protection during analysis
Principle Instant coffee / coffee-chicory powder are dissolved in hot water and solubility
time is recorded.
Apparatus/Instrument General Apparatus and Glassware
1. Beaker -500 mL
2. Heating equipment.
3. Weighing balance.
4. Stop clock.
5. Stirring equipment.
Materials and Reagents Instant coffee powder.
1. Instant coffee- chicory powder.
2. Freshly boiled water.
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get
a homogenous sample. Store sample in a tightly stoppered bottle, withdraw
portions for analytical determinations.
Method of analysis 1. Weigh 2.5 g of instant coffee powder/coffee- chicory powder in a 500 mL
beaker.
2. Then pour 150 mL of freshly boiled water, stir. Check the solubility time
of sample. The product should dissolve in 30 sec.
Calculation with units of Record the time taken by the sample to get dissolved in boiled water.
expression
Reference IS 3309:2016 Soluble Coffee -Chicory Powder― Specification
Approved by Scientific Panel on Methods of Sampling and Analysis

26 | P a g e
 Determination of Solubility in Cold water

Method No. FSSAI 04A.018:2021 Revision No. & Date 0.0

Scope Soluble (Instant) Coffee powder, Decaffeinated soluble coffee powder, Instant
Coffee - Chicory Mixture, Decaffeinated Instant coffee- chicory mixture
Caution Once sample is opened, seal it in airtight manner after taking test portion

Principle Instant coffee / coffee-chicory powder are dissolved in cold water and solubility
time is recorded.
Apparatus/Instrument General Apparatus and Glassware

1. Beaker-500 mL
2. Weighing balance
3. Stop Clock
4. Stirring equipment

Materials and Reagents Instant coffee powder/coffee- chicory powder

Distilled water.
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get
a homogenous sample. Store sample in a tightly stoppered bottle, withdraw
portions for analytical determinations.
Method of analysis 1. Weigh 2.5 g of instant coffee powder/coffee- chicory powder in a 500 mL
beaker.
2. Pour 50 mL of water (16 ± 2 °C) and stir. The product should dissolve in
3 min with moderate stirring, leaving no appreciable sediments.
Numbering
Calculation with units of Record the time taken by the sample to get dissolved in cold water
expression
Reference IS 2791:2016 Soluble Coffee Powder― SPECIFICATION
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Crude Fibre in Tea

Method No. FSSAI 04A.019:2021 Revision No. & Date 0.0

Scope Tea, Kangra Tea, Green Tea
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. Wear gloves and face protection during analysis
Principle Crude fiber is determined gravimetrically after chemical digestion and
solubilization of other materials present. The fiber residue weight is then corrected
for ash content after ignition. The loss in mass resulting from ashing is called the
crude fibre content
Apparatus/Instrument General Apparatus and Glassware
1. Condenser – Use condenser that will maintain constant volume of
refluxing solutions.
2. Digestion Flask-700-750 mL, Erlenmeyer flask is recommended.
3. Filtering cloth–Use filtering cloth such character that no solid matter
passes through when filtering is rapid. Fine linen or dress linen with about
18 threads/cm or 45 threads per inch (i.e. the aperture size 0.14 mm and
thread thickness 0.42 mm) or its equivalent may be used (Whatman filter
Paper No. 54 or equivalent may also be used).
4. Muffle Furnace maintained at 525 ± 20 °C.
Materials and Reagents 1. Sulphuric acid.
2. Caustic soda (free from sodium carbonate).
Preparation of Reagents 3. Sulphuric acid (1.25%, v/v) - Sulphuric acid (1.25 g) dissolved in
distilled water (100 mL) (w / v).
4. Caustic soda (1.25%, w/v) - Caustic soda (1.25 g) dissolved in distilled
water (100 mL) (w / v).
Sample Preparation Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get
a homogenous sample. Store sample in a tightly stoppered bottle, withdraw
portions for analytical determinations.
Method of analysis 1. Weigh accurately 2 g fat free of prepared sample.
2. Dry in an air oven maintained at 100 ± 2 °C for 4 h.
3. Transfer to the digestion flask. Add 200 mL of boiling 1.25% sulphuric
acid. Immediately connect to the condenser and heat (it is essential that
the solution boils within one minute and boiling continues briskly for
exactly 30 min). Rotate flask frequently until sample at sides is thoroughly
wetted, taking care to keep material from remaining on the sides of the
flask.
4. Immediately filter through linen in fluted funnel, and wash with boiling
water until washings are acid free.

28 | P a g e
 5. Wash the residue back into the flask with 200 mL of boiling 1.25%
Caustic soda solution using wash bottle marked to deliver 200 mL.
6. Connect flask to reflux condenser and boil briskly, exactly for 30 min.
7. After 30 min remove flask immediately, filter via prepared asbestos mat
and carefully transfer, all the residue into the Gooch crucible with hot
water. Wash the residue thoroughly with hot water until the filtrate is
alkali free. Then, wash with about 10 mL alcohol.
8. Dry the Gooch crucible at 110 °C to constant weight. Cool and weigh
(W1).
9. Transfer the Gooch crucible to a muffle furnace controlled at 525 – 550
°C and ash the material.
10. Cool, weigh (W2). Loss in weight represents crude fibre.

Calculation with units of ( W1– W2) x 100 x 100

expression Crude fibre % =
(on dry weight) Wt. of sample x (100-Moisture content)

Reference  IS 16041:2012- Tea — Determination of Crude Fibre Content, IS 10226

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Catechins in Tea — HPLC Method

Method No. FSSAI 04A.020:2021 Revision No. & Date 0.0

Scope Green Tea, Instant Tea and Black Tea
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. The cartridge should not be dry during elution.
Principle Catechin is a plant secondary metabolite of Flavonoids family is extracted from
the tea by Methanol- Acetonitrile mixture and the extract is quantified on HPLC
at 278nm.
Apparatus/Instrument 1. Analytical Balance (± 0.0001 g).
2. Water Bath (70±1°C.)
3. Dispenser — set at 5 ml for methanol/water extraction mixture Centrifuge
— capable of 3500 rev/min.
4. Vortex Mixer —
5. Extraction Tubes —Centrofuged tubes 15ml capacity,
6. Graduated Tubes — glass, 10ml capacity with 0.1 ml graduations.
7. Automatic Pipettes — (10-100ul, 1ml, 10ml)
8. Filters — membrane filter 0.45 pm pore size.
9. HPLC with ultraviolet detector (wavelength of 278 nm)
NOTES
1. Phenyl bonded phases give additional selectivity over reversed phase packings, and
result in improved resolution of the catechins.
2. In this standard the chromatographic conditions and composition of the mobile phase
specified are suitable for a Phenomenex Lures 5 µm Phenyl-Hexyl column of dimensions
250 mm x 4.6 mm fitted with a Phenomenex Security Guard 4 mm x 3.0 mm Phenyl-
Hexyl cartridge. If other types of column are used, an alternative mobile phase and
alternative chromatographic conditions may be necessary.

30 | P a g e
Materials and Reagents 1. Water —HPLC grade
2. Acetonitrile — HPLC Grade.
3. Methanol - HPLC Grade
4. Glacial Acetic Acid — HPLC Grade.
5. EDTA (Ethylenediaminetetraacetic Acid Disodium Salt, Dihydrate)
6. L-ascorbic Acid — Free acid.
7. Methanol/Water Extraction Mixture, 70 percent v/v Methanol — Add 700
ml of the methanol to a 1 litre mark volumetric flask. Dilute to the mark
with water and mix.
8. HPLC Mobile Phase.
8.1 Mobile Phase A — Add 180 ml of acetonitrile and 40 ml
acetic acid to a 2 litre mark volumetric flask. Dilute to the
mark with water, mix, and filter through a filter of 0.45 µm
pore size.
8.2 Mobile Phase B — Add 800 ml acetonitrile to a 1 Iitre mark
volumetric flask. Dilute to the mark with water, mix and filter
through a filter of 0.45 µm pore size.

Preparation of Reagents 1. Stabilizing Solution- Weigh, to the nearest 0.01 g, 0.25 g of EDTA and
0.25g of ascorbic acid into a 1 litre mark volumetric flask and dissolve in
approximately 500 ml water. Add 100 ml acetonitrile dilute to the mark
with water and mix. Prepare fresh stabilizing solution on the day of use.
2. Stock Standard Solutions
2.1 Weigh standards (>20mg) on an analytical balance in a
volumetric flask and dissolved in stabilizing solution, gently
warming (if required, 40°C maximum).The cool solution is
diluted to the mark with stabilizing solution. Same procedure
shall be followed for the preparation of the following stock
standard solution.
2.2 Gallic Acid Stock Standard Solution — corresponding to
2.00 mg/ml.
2.3 Caffeine Stock Standard Solution — corresponding to 2.00
mg/ml.
2.4 (+) –Catechin, (C), Stock Standard Solution —
corresponding to 1.00 mg/ml.
2.5 (–)-Epicatechin, (EC), Stock Standard Solution —
corresponding to 1.00 mg/ml,
2.6 (–) –Epigallocatechin, (EGC), Stock Standard Solution —
corresponding to 2.00 mg/ml.
2.7 (–) –Epigallocatechingallate, EGCG, Stock Standard
Solution — corresponding to 2.00 mg/ml.
2.8 (–) –EpicatechingaIlate, ECG, Stock Standard Solution —
corresponding to 2.00 mg/ml.

31 | P a g e
 3. Dilute Gallic Acid Standard Solution — corresponding to 200 µg/ml.
Using a pipette transfer 10 ml of the gallic acid stock standard solution to
a 100 ml one-mark volumetric flask. Dilute to the mark with stabilizing
solution and mix.
4. Mixed Working Standard Solutions
4.1 Prepare three mixed working standard solutions, with
concentrations selected to cover the range of compositions
typically found in tea.
4.2 Following Table 1, carefully pipette the given aliquots of
dilute gallic acid standard solution and stock standard
solutions into three separate 20 ml one-mark volumetric
flasks, dilute to volume with stabilizing solution and mix.
These mixed working standard solutions correspond to the
nominal concentrations shown in Table 1. Use the actual
standard weights taken to obtain the actual concentrations at
each standard level.
4.3 Pipette 1.0ml aliquots of each mixed standard solution into
labeled small amber glass vials, gently flush with nitrogen
prior to sealing and store frozen at –20°C. NOTES
i. Mixed working standard solutions are stable for at least 2
months when stored frozen at –20”C.
ii. Only thaw sufficient mixed working standard solution
vials for each chromatographic run. Discard any remaining
solution, do not re-freeze

Table 1: Composition of Mixed Working Standard Solutions Standard 1 to

Standard 3
Sr. Component Solution Aliquot, ml
No.
Standard 1 Standard 2 Standard 3

i. Gallic acid 200 µg/ml dilute 0.5 1.0 2.5

stock standard
solution
ii. Caffeine 2.00 mg/ml stock 0.5 1.0 1.5
standard solution
iii. +C 1.00 mg/ml stock 1.0 2.0 3.0
standard solution
iv. EC 1.00 mg/ml stock 1.0 2.0 3.0
standard solution
v. EGC 2.00 mg/ml stock 1.0 2.0 3.0
standard solution
vi. EGCG 2.00 mg/ml stock 1.0 2.0 4.0
standard solution

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 vii. ECG 2.00 mg/ml stock 0.5 1.0 2.0
standard solution

Table 2: Nominal Concentrations in Mixed Working Standard Solutions

Standard 1 to Standard 3

Sr. No. Component Nominal concentration

Standard 1 Standard 2 Standard 3

i. Gallic acid 5 10 25
ii. Caffeine 50 100 150

iii. +C 50 100 150

iv. EC 50 100 150

v. EGC 100 200 300

vi. EGCG 100 200 400

vii. ECG 50 100 200

Sample Preparation Sample is prepared by grinding a small quantity of the sample and reject it, then
quickly grind an amount slightly greater than that required for the specified tests
and for the determination of dry matter content. Store all samples in well sealed
containers, protected from light, and cool.
NOTE — Grinding of instant tea is only required for samples with a coarse granular structure.
Method of analysis 1. Determination of Dry Matter Content: Calculate the dry matter content
from the moisture content (loss in mass at 103°C/1hr)determined on a
portion of the test sample
2. Test Portion
2.1 Instant tea: Weigh, to the nearest 0.0001 g, 0.5 g of the test sample
into a 50 ml one-mark volumetric flask.
2.2 Green and Black Tea: Weigh, to the nearest 0.0001 g, 0.2 g of the
test sample into an extraction tube.
3. Extraction
3.1 Instant tea:
3.1.1 a. Add, to the instant tea in the flask from 2.1, approximately
25 ml hot water (maximum temperature of 60°C), mix to
dissolve the sample and allow to cool to room temperature.
3.1.2 Add, 5 ml acetonitrile, dilute to the mark with water and mix.
3.2 Green and Black Tea:
3.2.1 Place the methanol/water extraction mixture contained in the
dispenser into the waterbath set at 70°C, and allows 30 min
for the extraction mixture to reach temperature.
3.2.2 Place the extraction tube containing the tea sample into the

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 water bath set at 70°C. Add 5 ml hot methanol/water
extraction mixture from the dispenser, stopper the tube and
carefully mix on the vortex mixer. NOTE — it is important to mix
samples thoroughly to ensure complete extraction.
3.2.3 Continue heating the extraction tube in the water bath for 10
min, mixing on the vortex mixer at 5 min and 10 min.
3.2.4 Remove the extraction tube from the water bath, and allow
cooling to room temperature. Remove stopper and place in
the centrifuge at 3500 rev/min
3.2.5 Carefully decant the supernatant into a graduated tube.
3.2.6 Repeat extraction steps 3.2.2 to 3.2.5. Combine extracts,
make up to 10ml with cold methanol/ water extraction
mixture and mix contents.
NOTE — The extract from 3.2.6 is stable for at least 24 h if stored at 4°C.
Allow extract to reach room temperature before proceeding with the assay.
Resuspension of the small amount of fine particulate material settled during
storage is not necessary.
4. Dilution: Using a pipette, transfer 1.0 ml of the sample extract into a
graduated tube and dilute to 5 ml with stabilizing solution. Mix solution
then filter through 0.45 µm filter.
5. Determination
5.1 Adjustment of the Apparatus: Set up the chromatography in
accordance with the manufacturer’s instructions and adjust it as
follows:
5.1.1 Flow rate of the mobile phase: 1.0 ml/min
5.1.2 Binary gradient conditions: 100 percent mobile phase A for
10 min, then over 15min a linear gradient to 68 percent
mobile phase A, 32 percent mobile phase B and hold at this
composition for 10 min. Then reset to 100 percent mobile
phase A and allow to equilibrate for 10min before next
injection.
5.1.3 Temperature of the column: 35 ± 0.5°C.
Notes: 1 Column temperature control is recommended
(chromatography column oven or recirculating water jacket) if
major drifts in retention times are to be avoided. UV detector
setting: wavelength 278 nm.
2 Ensure that the detector sensitivity range selected is able to keep
all peaks from the highest mixed working standard (Standard 3)
within the scale of the data collection system used.
5.2 HPLC Analysis
5.2.1 Once the flow rate of the mobile phase and temperature are
stable, condition the column with a blank gradient run. Then
inject onto the column 10 µl of each of the mixed working
standard solutions Standard 1 Standard 2 and Standard 3
followed by an equal volume of the diluted test solution.

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 Repeat injection of the mixed working standard solutions at
regular intervals (typically after six test solutions). Collect
and record the data for the peaks of all standards and test
samples.
5.2.2 After each day’s use and prior to storage, wash the column
with approximately 50 percent acetonitrile, replacing the
column sealing plugs after disconnection.

Calculation with units of 1. Identify and measure the peak areas or heights (area is preferable) for all
expression standards and test samples. Construct linear calibration graphs for all
components in the standards of concentration (~g/ml) against peak areas
or heights and obtain the individual standard response factors (RF)
automatically using a data collection/integration system or manually from
a selected point on the calibration graph.

𝐶𝑠𝑡𝑑
𝑅𝐹 =
𝐴𝑠𝑡𝑑 𝑜𝑟 ℎ𝑠𝑡𝑑

Where, RF = standard response factor Cstd = concentration of the standard

(µg/ml); Astd = peak area of the standard; and hstd = peak height of the standard.
2. Calculate response factors for all the individual components, that is Gallic
acid, caffeine and the individual catechins EGC, +C, EC, EGCG and
ECG. Calibration information obtained from a data collection/integration
system will include an intercept value when the calibration is not forced
through the origin and this should be included in the calculation.
3. The concentration of the individual components expressed as a percentage
by mass on a sample as received basis is given by the formula:

𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 (𝑚/𝑚)(𝑎𝑠 𝑟𝑒𝑐𝑒𝑖𝑣𝑒𝑑 𝑏𝑎𝑠𝑖𝑠)

Vd
= ( Asamp or hsamp ) × RF
10,000m

Where,
Asamp = peak area for the test sample;
hsamp = peak height for the test sample;
RF = response factor for the individual component;
V = sample extraction volume (50 for instant tea or 10 for leaf tea);
d = dilution factor (see 4 in method of analysis), typically 5; and
m = mass, in g, of the test sample.
Percent total catechins (m/m) (as received basis) = (percent EGG) +
(percent +G) + (percent EG) + (percent EGGG) + (percent EGG).

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 Percent total catechins (m/m) (dry matter basis)
Percent total catechins m/m(as received basis) × 100
=
w
Where,
w = dry matter content of the test sample, determined in accordance with
step 1 in method of analysis.
Reference IS 15344:2003 (Green Tea - Specification)

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Added Color

Method No. FSSAI 04A.021:2021 Revision No. & Date 0.0

Scope Tea, Coffee and Chicory products
Caution 1. Once sample is opened, seal it in airtight manner after taking test portion
2. Wear gloves and face protection while doing analysis.
Principle Presence of added colors in foods, involve preliminary treatment of the food
(Acidic/Alkali) and extraction of the color from the prepared solution of the food.

Apparatus/Instrument General Apparatus and Glassware

1. Pipette
2. Beaker
3. Flask.
4. Soxlet extractor.
5. Whatman No.1 filter paper.
6. Woolen thread.
Materials and Reagents 1. White knitting wool.
2. Petroleum ether.
3. Distilled water.
4. Ammonia (0.88 sp. gr).
5. Acetic acid.

Preparation of Reagents 1. White knitting wool: - Extract pure white wool in a soxhlet extractor with
petroleum ether for 2-3 h to remove fat. Boil in very dilute solution of sodium
hydroxide and then in water to free it from alkali.
2. Paper: Whatman No. 1 chromatographic paper or equivalent.
3. 1 mL (0.88 sp. gr) ammonia + 99 mL water.
4. Acetic acid solution in water (1:3).

Sample Preparation 1. Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix well to get
a homogenous sample. Store sample in a tightly stoppered bottle, withdraw
portions for analytical determinations.
2. Preliminary treatment of food: Assuming that an acidic colour is present, the
preliminary treatment involves removing interfering substances and obtaining the
dye in acid solution prior to boiling with wool. To test the presence of basic color,
treat the sample with ammonia to make alkaline solution prior to boiling with
wool.
Method of analysis Acidic Dyes

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 1. Introduce about 20 cm length of woolen thread into a beaker containing about
35 mL of the prepared acidified solution of the sample and boil for a few min
till the woolen thread is dyed.
2. Take out the woolen thread and wash it with tap water.
3. Transfer the washed woolen thread to a small beaker containing dilute
ammonia and heat again. If the color is stripped by the alkali, the presence of
an acid coal-tar dye is indicated.
4. Remove the woolen thread. Make the liquid slightly acidic and boil with a
fresh piece of woolen thread. Continue boiling until the color is taken by the
woolen thread.
5. Extract the dye from the woolen thread again with a small volume of dilute
ammonia, filter through a small plug of cotton and concentrate the filtrate
over a hot water bath.
6. This double stripping technique usually gives a pure color extract. Natural
colors may also dye the wool during the first treatment, but the color is not
usually removed by ammonia.
Basic dyes
1. Basic dyes can be extracted by making the food alkaline with ammonia,
boiling with wool and then stripping with dilute acetic-acid.
2. At present, all the permitted water soluble coal-tar dyes are acidic; hence an
indication of the presence of a basic dye suggests that an unpermitted color
is present.

Calculation with units of Present/Absent

expression
Reference Manual Methods of Analysis for Adulterants and Contaminants in Food, I.C.M.R
1990 Page 56
Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Iron Filings in Tea

Method No. FSSAI 04A.022:2021 Revision No. & Date 0.0

Scope
Tea, Kangra Tea.
Caution NA
Principle
Iron filings or Iron particles may appear during the manufacturing/processing
of tea and affects its quality. This method follows the gravimetric estimation
of iron particles after separation using a magnet.
Apparatus/ Magnet (Strength: ~ 1000 gauss)
Instruments
Materials and Reagents Analytical balance, Magnet, white sheet,
Preparation of Reagents Not Applicable
Sample Preparation  Step-1: Take whole unit pack (250 g) sample and homogenize/ mix
properly and spread the mixed sample in thin layer (~ 5 mm) on white sheet.

Method of analysis
 Step-2: Collect the five sub-lot fractions of sample from five different
regions (4 corners and centre) in total weighing 50 g. Remaining 200 g sample
shall return to pack.

 Step-3: Combine and mix all 5 sub-lot fractions into one.

 Step-4: From the above, weigh and use 20 g of sample for next step.
Spread it to very thin layer (close to uni-layer; around 2 - 3 mm) on white sheet.

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  Step-5: Slowly move the magnet (~ 1000 gauss strength) over thinly
spread (around 2 - 3 mm height) tea sample, as above in the flow manner
indicated in below diagram. Repeat this manual magnet movement multiple
times over 10 min duration. Collect the iron particles sticking to magnet each
time of movement and pool in petri dish (Note: magnet should pass just above
the surface of Tea powder).

 Step-6: Spread the collected iron pieces (which may contain few tea
particles also along, due electrostatic attraction) on white paper and use magnet
movement (2nd time), above the distance of 0.5 - 1.0 cm from the spread layer
on paper. This second action of magnet collects only iron particles, leaving tea
sample on paper.
 Step-7: Take the weight of the collected iron particles, sticking on
magnet, using analytical balance.
 Step-8: Repeat the entire process in triplicate for averaging.

Calculation with units of Calculation (mg/Kg) : Weight of the iron filings (mg) X 1000
expression Weight of the sample(g)

Reference IS 3633:2003 - Black Tea – Specification, 2nd Revision

Approved by Scientific Panel on Methods of Sampling and Analysis

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 Determination of Extraneous matter

Method No. FSSAI 04A.023:2021 Revision No. & Date 0.0

Scope Tea, Coffee and Chicory

Caution NA

Principle Sample is examined visually/using magnifying lens for extraneous matter like
strings, stones, dirt, wood, glass and metallic pieces, twigs, bark and stems.
Apparatus/Instrument NA

Materials and Reagents Magnifying lens

Sample Preparation Mix whole sample Properly

Method of analysis Mix the whole sample and test visually for extraneous matter. The sample should
be free from extraneous matter like strings, stones, dirt, wood, glass and metallic
pieces
Calculation with units of Presence/Absence
expression
Reference IS: 3077 – 2009 A Specification for Roasted and Ground Coffee
Approved by Scientific Panel on Methods of Sampling and Analysis

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