File No.

11014/07/2021-QA
Food Safety and Standards Authority of India
(A statutory Authority established under the Food Safety and Standards Act, 2006)
(Quality Assurance Division)
FDA Bhawan, Kotla Road, New Delhi – 110002
_________________________________________________________________________________________________________
Dated, the 5th August, 2024

Order

Subject: FSSAI Manual of Methods of Analysis of Foods- Honey and other beehive
products - reg.

The FSSAI Manual of Methods of Analysis of Foods- Honey and other beehive
products which has been approved by the Food Authority in its 44th meeting held on
19.06.2024 is enclosed herewith.

2. The approved methods shall be implemented with immediate effect. The notified lab
shall include the new methods in their respective scope of accreditation within six months
from the date of issue of this order.

3. Since the process of updation of test methods is dynamic, any changes happening
from time to time will be notified separately. Queries/concerns, if any, may be forwarded to
email: [email protected].

Encl: as above

Digitally signed by Dr.

Dr. SATYEN SATYEN KUMAR PANDA
KUMAR PANDA Date: 2024.08.05
11:43:08 +05'30'

(Dr. Satyen Kumar Panda)

Advisor (QA)

To:

1. All FSSAI Notified Laboratories

2. All State Food Testing Laboratories
3. CEO, National Accreditation Board for Testing and Calibration Laboratories (NABL)
 फा. सं. 11014/07/2021 – क्यूए
भारतीय खाद्य सुरक्षा और मानक प्राधिकरण
(खाद्य सुरक्षा और मानक अधिधनयम, 2006 के अंतर्गत स्थाधित एक वैिाधनक प्राधिकरण)
(गुणवत्ता आश्वासन धवभाग)
एफडीए भवन, कोटला रोड, नई धिल् ली-110002
___________________________________________________________________________________ ___
धिनांक: 05 अगस्त, 2024

आिे श

धवषय: खाद्य पिार्थों के धवश्लेषण के तरीक ं की एफएसएसएआई मैनुअल – शहि और अन्य

मिुमक्खी उत्पाि - के संबंि में ।

खाद्य ििाथों के धवश्ले षण के तरीकों की एफएसएसएआई मैनुअल - शहि और अन्य मिुमक्खी

उत्पाि, धिसे खाद्य प्राधिकरण ने 19.06.2024 को आयोधित अिनी 44वी ं बैठक में अनुमोधित धकया है ,
इसके साथ संलग्न है ।

2. अनुमोधित धवधियां तत्काल प्रभाव से लार्ू धकये िायेंर्े। अधिसूधित प्रयोर्शाला इस आिे श के
िारी होने की तारीख से छह महीने के भीतर मान्यता के अिने संबंधित िायरे में नई धवधियों को शाधमल
करे र्ी।

3. िूंधक िरीक्षण धवधियों के अद्यतन की प्रधरियया र््ा्क है , समय-समय िर होने वाले धकसी भी
िररवतगन को अलर् से अधिसूधित धकया िाएर्ा। प्रश्न/धिंताएं , यधि कोई हों, ईमेल: sp-
[email protected], िर अग्रेधषत की िा सकती हैं ।

संलग्नक: उिरोक्त अनुसार

Dr. SATYEN Digitally signed

by Dr. SATYEN
KUMAR KUMAR PANDA
Date: 2024.08.05
PANDA 11:43:42 +05'30'

(डॉ. स्ेन कुमार िंडा)

सलाहकार (र्ुणवत्ता आश्वासन)

प्रधत:

1. सभी एफएसएसएआई अधिसूधित प्रयोर्शालाएं

2. सभी राज्य खाद्य िरीक्षण प्रयोर्शालाएं
3. सीईओ, राष्ट्रीय िरीक्षण और अं शशोिन प्रयोर्शाला प्र्ायन बोडग
 MANUAL OF METHODS OF ANALYSIS OF FOODS
HONEY& OTHER BEE HIVE PRODUCTS
1 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS

AUGUST 2024
8 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
9 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
10 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 LIST OF CONTRIBUTORS

Dr. Ajit Dua

Chief Executive Officer, Punjab Biotechnology Incubator, Mohali

Dr. Vandana Awasthi

Scientist, Punjab Biotechnology Incubator, Mohali

Dr. Sanjivan Alkesh

Assistant Scientific Officer, Punjab Biotechnology Incubator, Mohali

Mr. Amit Aggrawal

Assistant Scientific Officer, Punjab Biotechnology Incubator, Mohali

Ms. Anju Thakur

Junior Scientific Officer, Punjab Biotechnology Incubator, Mohali

Dr. Kaushik Banerjee

Director, National Research Centre for Grapes, Pune

Dr. Rajesh Nair

Dy. Managing Director, National Dairy Development Board CALF Ltd. Anand

Dr. Satyen K Panda

Advisor (Quality Assurance), Food Safety and Standards Authority of India

Ms. Sweety Behera

Director (Quality Assurance), Food Safety and Standards Authority of India

Mr. Balasubramanian K
Joint Director (Quality Assurance), Food Safety and Standards Authority of India

Dr. Dinesh Kumar

Assistant Director (Tech.), Food Safety and Standards Authority of India

Ms. Priyanka Meena

Technical Officer, Food Safety and Standards Authority of India

Ms. Gurpreet Kaur

Technical Officer, Food Safety and Standards Authority of India

11 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 TABLE OF CONTENTS
S. No. METHOD NO. METHOD PAGE No.
A. Honey
1. FSSAI 04B.001:2024 Determination of Specific gravity 2-3
Determination of Moisture (Vacuum Oven Drying
2. FSSAI 04B.002:2024 4-5
method)
3. FSSAI 04B.003:2024 Determination of Moisture (By Refractometer) 6-7
Determination of Total Reducing Sugars, Sucrose And
4. FSSAI 04B.004:2024 8-11
Fructose-Glucose Ratio (Titrimetric method)
5. FSSAI 04B.005:2024 Determination of Sucrose and F/G Ratio (HPLC method) 12-13
6. FSSAI 04B.006:2024 Determination of Total Ash 14-15
7. FSSAI 04B.007:2024 Determination of Acidity as Formic acid 16-17
8. FSSAI 04B.008:2024 Determination of Free Acidity 18-19
9. FSSAI 04B.009:2024 Determination of Hydroxy Methyl Furfural (HMF) 20-21
10. FSSAI 04B.010:2024 Determination of Diastase Activity 22-24
11. FSSAI 04B.011:2024 Determination of Water insoluble matters 25
12. FSSAI 04B.012:2024 Determination of Pollen and Plant Elements 26-27
13. FSSAI 04B.013:2024 Determination of Proline 28-29
14. FSSAI 04B.014:2024 Determination of Electrical Conductivity 30-32
Determination of 2-Acetylfuran-3-Glucopyranoside (2-
15. FSSAI 04B.015:2024 33-36
AFGP) as Marker for Rice Syrup
Determination of C4 sugar, ∆δ 13C Protein-Honey by
16. FSSAI 04B.016:2024 EA/LC-IRMS and ∆δ 13C Fru-Glu, ∆δ 13C Max , Foreign 37-44
Oligosaccharides by LC-IRMS
B. Bees Wax
17. FSSAI 04B.017:2024 Determination of Solubility 46
18. FSSAI 04B.018:2024 Determination of Melting point range, o C 47- 48
19. FSSAI 04B.019:2024 Determination of Acid value 49-50
20. FSSAI 04B.020:2024 Determination of Peroxide value, Max 51-52
21. FSSAI 04B.021:2024 Determination of Saponification value 53-54
22. FSSAI 04B.022:2024 Determination of Carnauba wax 55
Determination of Ceresin, paraffins and
23. FSSAI 04B.023:2024 56
other waxes
24. FSSAI 04B.024:2024 Determination of Fats, Japan wax, Rosin and Soap 57
25. FSSAI 04B.025:2024 Determination of Glycerol and other polyols 58-59
26. FSSAI 04B.026:2024 Determination of Ash 60-61
27. FSSAI 04B.027:2024 Determination of Total Volatile matter 62

12 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 C. Royal Jelly
Determination of Moisture (Vacuum Oven Drying
28. FSSAI 04B.028:2024 64-65
method : Reference method)
29. FSSAI 04B.029:2024 Determination Moisture (Karl Fisher method) 66
30. FSSAI 04B.030:2024 Determination of Moisture (Lyophilization method) 67
Determination of 10-DHA ( HPLC-UV External Standard:
31. FSSAI 04B.031:2024 68-69
Reference method)

Determination of 10-DHA (HPLC-UV Internal standard

32. FSSAI 04B.032:2024 70-71
method)
Determination of Protein : Kjeldahl method (Automatic)
33. FSSAI 04B.033:2024 72-74
(Reference method)
Determination Protein : Kjeldahl method (Alternative
34. FSSAI 04B.034:2024 75-77
method)
35. FSSAI 04B.035:2024 Determination of Sugar (Titrimetric method) 78-80
Determination of Sugars : Fructose, Glucose, Sucrose,
36. FSSAI 04B.036:2024 Erlose, Maltose and Maltotriose (HPLC-Reference 81-82
method)
Determination of Sugars: Fructose, Glucose, Sucrose,
37. FSSAI 04B.037:2024 Erlose, Maltose and Maltotriose (Gas Chromatography 83-84
method)
38. FSSAI 04B.038:2024 Determination of Total acidity 85
39. FSSAI 04B.039:2024 Determination of Total lipid 86-87
40. FSSAI 04B.040:2024 Determination of δ13C/ δ12C Isotopic Ratio 88
41. FSSAI 04B.041:2024 Determination of Furosine 88-90

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.

13 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 A. Honey

14 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Specific gravity

Method No. FSSAI 04B.001:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew

1. Honey sample must be kept at moisture free place in air tight jar
Caution
2. Mix the sample thoroughly before taking test portion for analysis

Specific gravity is the ratio of the density of a substance to that of a

standard substance. Specific gravity of honey calculated by the ratio of
Principle
weight of a given volume of the honey at 27±1°C to the weight of an equal
volume of water at 27±1°C with the help of Specific gravity bottle.

1. Specific gravity bottle

Apparatus/Instruments 2. Thermostatically controlled water bath-maintained at 27±1°C
3. Weighing balance
4. Sieve (No. 40)
Materials and Reagents NA

Preparation of Reagents NA

A) Liquid or Strained honey: If honey is free from granulation, mix

thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. cool the honey sample rapidly as soon
it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
Sample Preparation
determination. If foreign matter such as wax, sticks, bees, particles of
comb etc. is present, heat honey to 40°C and filter through cheesecloth
in hot water funnel before weighing, test portions for analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A and
filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove wax.

1. Clean and thoroughly dry the specific gravity bottle and weigh.
2. Fill it up to the mark with freshly boiled and cooled distilled water
Method of analysis
which has been maintained at 27 ± 1°C and weigh.
3. Remove the water, dry bottle again and fill it with the honey
sample maintained at the same temperature.
4. Weigh the bottle again.
C–A
Specific gravity at 27 °C =
B–A
Calculation with units of
Where
expression
C = mass, in g, of the specific gravity bottle with the honey sample;
A = mass, in g, of the empty specific gravity bottle; and

2 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 B = mass, in g, of the specific gravity bottle with water

Inference
NA
(Qualitative Analysis)

Reference IS 4941:1994
AOAC (920.180) 21st edition-2019

Approved by Scientific Panel on Methods of Sampling and Analysis

3 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Moisture (Vacuum Oven Drying Method)

Method No. FSSAI 04B.002:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew.
Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for analysis.
Principle Honey sample is heated in a vacuum oven under controlled conditions of
pressure and temperature to remove moisture by passing dry air. Sample
is weighed before and after drying to estimate moisture.
Apparatus/Instruments 1. Flat-Bottom Dish- of nickel or other suitable material not affected
by boiling water; 7 cm to 8 cm in diameter and not more than 2.5
cm deep.
2. Sand- Passing through 500-microns IS Sieve but retained on 180-
micron IS Sieve. It shall be prepared by digestion with
concentrated hydrochloric acid, followed by thorough washing
with water till free form chlorides. It shall be dried and ignited to
dull red heat.
3. Vaccum Oven
Materials and Reagents NA
Preparation of Reagents NA
Sample Preparation A) Liquid or Strained honey: If honey is free from granulation, mix
thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. Cool the honey sample rapidly as soon
it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
determination. If foreign matter such as wax, sticks, bees, particles of
comb etc. is present, heat honey to 40°C and filter through cheesecloth
in hot water funnel before weighing, test portions for analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A and
filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove wax.
Method of analysis 1. Heat the dish containing 20 g of the prepared sand and a stirring rod in
the oven for one hour.
2. Allow to cool in a desiccator for 30-40 mins.
3. Weigh accurately 2 g of the material into the tared dish.
4. Add 5 mL of distilled water in dish and thoroughly mix sand with the
sample by stirring with the glass rod having a widened flat end,
smoothing out lumps and spreading the mixture over the bottom of the
dish.
5. Place the dish on boiling water-bath for 30 mins.
6. Wipe the bottom of the dish and transfer it with the glass rod, to the
vaccum oven maintained at a temperature between 60 °C and 70 °C

4 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 and at a pressure not more than 50 mm of mercury.
7. After 2 h, remove the dish and transfer to a desiccator, allow it to cool
and then weigh.
8. Replace the dish in the oven for a further period of one hour, remove
and transfer to desiccator, cool and weigh again.
Repeat the process of heating, cooling and weighing after every hour till
consecutive weighing do not differ by more than 0.5 mg.
Calculation with units of 100 (M1 - M2)
expression
Moisture, % by mass =
M1 – M
Where
M1 = mass, in g, of the contents of the dish before drying
M2 = mass, in g, of the contents of the dish after drying
M = mass, in g, of the empty dish with the sand and the glass rod
Inference NA
(Qualitative Analysis)
Reference IS 4941:1994
AOAC (920.180) 21st edition-2019
Approved by Scientific Panel on Methods of Sampling and Analysis

5 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Moisture (By Refractometer)

Method No. FSSAI 04B.003:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew.

Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Ensure that the prism of the refractometer is clean and dry.

Principle The method is based on the principle that refractive index increases with
solids content. The moisture content value is determined from the
refractive index of the honey by reference to a standard table.

Apparatus/Instruments Refractometer

Materials and Reagents NA

Preparation of Reagents NA

Sample Preparation A) Liquid or Strained honey: If honey is free from granulation, mix
thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. Cool the honey sample rapidly as
soon it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
determination. If foreign matter such as wax, sticks, bees, particles
of comb etc. is present, heat honey to 40°C and filter through
cheesecloth in hot water funnel before weighing, test portions for
analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A and
filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove
wax.

Method of analysis 1. Clean and dry the refractometer before use.

2. Determine the refractometer reading of honey at 20 °C and
calculate the percentage of moisture from the values given in
Table 1.
3. If the determination is made at a temperature other than 200C,
correct the reading according to the Note in Table 1.

Calculation with units of Table 1. Relationship Between Refractive Index

expression and Moisture Content of Honey
Refractive Moisture Refractive Moisture
Index @20 ° C Index @20 ° C
1.504 4 13.0 1.488 5 19.2

6 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 1.503 8 13.2 1.488 0 19.4
1.503 3 13.4 1.487 5 19.6
1.502 8 13.6 1.487 0 19.8
1.502 3 13.8 1.486 5 20.0
1.501 8 14.0 1.486 0 20.2
1.501 2 14.2 1.485 5 20.4
1.500 7 14.4 1.485 0 20.6
1.500 2 14.6 1.484 5 20.8
1.499 7 14.8 1.484 0 21.0
1.499 2 15.0 1.483 5 21.2
1.498 7 15.2 1.483 0 21.4
1.498 2 15.4 1.482 5 21.6
1.497 6 15.6 1.482 0 21.8
1.497 1 15.8 1.4815 22.0
1.496 6 16.0 1.4810 22.2
1.496 1 16.2 1.480 5 22.4
1.495 6 16.4 1.480 0 22.6
1.495 1 16.6 1.479 5 22.8
1.494 6 16.8 1.479 0 23.0
1.494 0 17.0 1.478 5 23.2
1.493 5 17.2 1.478 0 23.4
1.493 0 17.4 1.477 5 23.6
1.492 5 17.6 1.477 0 23.8
1.492 0 17.8 1.476 5 24.0
1.491 5 18.0 1.476 0 24.2
1.491 0 18.2 1.475 5 24.4
1.490 5 18.4 1.475 0 24.6
1.490 0 18.6 1.474 5 24.8
1.489 5 18.8 1.474 0 25.0
1.489 0 19.0
NOTE - Temperature correction for refractive
index = 0.000 23 per 0C. If the reading is
made at a temperature above 20°C, add the
correction; if made below, subtract the
correction

Inference NA
(Qualitative Analysis)

Reference IS 4941:1994
AOAC (920.180)21st edition-2019

Approved by Scientific Panel on Methods of Sampling and Analysis

7 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Total Reducing Sugars, Sucrose And Fructose-
Glucose Ratio (Titrimetric Method)

Method No. FSSAI 04B.004:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew
Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
Principle This method is the modification of the Lane and Eynon procedure,
involving the reduction of Soxhlet’s modification of Fehling’s solution
by titration at boiling point against a solution of reducing sugar in honey
by using methylene blue as internal indicator. The difference in the
concentrations of invert sugar multiplied by 0.95 to give the apparent
sucrose content.
Apparatus/Instruments 1. Weighing balance
2. Volumetric Flask-250 mL
3. Volumetric Flask-1000 mL
4. Burrete-50 mL
Materials and Reagents 1. Copper Sulphate Solution (Solution A)
2. Potassium Sodium Tartrate (Rochelle Salt) (Solution B)
3. Hydrochloric Acid (12 N)
4. Standard Invert Sugar Solution
5. Methylene Blue Indicator
Preparation of Reagents 1. Soxhlet Modification of Fehling’s Solution- Prepare by mixing
equal volumes of Solution A and solution B immediately before
using.
2. Copper Sulphate Solution (Solution A) - Dissolve 34.639 g
copper sulphate crystals (CuSO4.5H2O) in 500 mL distilled
water, and filter through glass wool or filter paper.
3. Standardization of Copper Sulphate Solution- Using separate
pipette, pipette out accurately 5 mL of Solution A and 5 mL of
Solution B into a conical flask of 250 mL capacity. Heat this
mixture to boiling on an asbestos gauze and add standard invert
sugar solution from a burette, about one millilitre less than the
expected volume which will reduce the Fehling’s solution
completely (about 48 mL). Add one millilitre of methylene blue
indicator while keeping the solution boiling. Complete the
titration within three minutes, the end point being indicated by
change of color from blue to red. From the volume of invert
sugar solution used, calculate the strength of the copper sulphate
solution by multiplying the titrate value by 0.001 (mg/ml of the
standard invert sugar solution). This would give the quantity of
invert sugar required to reduce the copper in 5 ml of copper
sulphate solution.
4. Potassium Sodium Tartrate (Rochelle Salt) Solution
(Solution B)- Dissolve 173 g of potassium sodium tartrate and

8 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 50 g of sodium hydroxide in water and makeup volume to 500
mL. Let the solution stand for a day and filter.
5. Hydrochloric Acid- Sp gr 1.18 at 20 °C (approximately 12 N)
6. Standard Invert Sugar Solution- Weigh accurately 0.95 g
sucrose and dissolve it in 500 mL of water. Add 2 mL of
concentrated hydrochloric acid, boil gently for 30 mins and keep
aside for 24 h. then neutralize with sodium carbonate and make
the final volume to 1000 mL. 50 mL of this solution contains
0.05 g invert sugar.
7. Methylene Blue indicator- 0.2 percent in water.
Reagents for Fructose-Glucose Ratio
1. Iodine Solution- 0.05 N
2. Sodium Hydroxide Solution- 0.1 N
3. Sulphuric acid- concentrated
4. Standard Sodium Thiosulphate Solution- 0.05 N.
Sample Preparation A) Liquid or Strained honey: If honey is free from granulation, mix
thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. cool the honey sample rapidly as
soon it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
determination. If foreign matter such as wax, sticks, bees, particles
of comb etc. is present, heat honey to 40°C and filter through
cheesecloth in hot water funnel before weighing, test portions for
analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A
and filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove
wax.
Method of analysis Procedure for Total Reducing Sugar
1. Place one gram (W) of the prepared sample of honey into 250-
mL volumetric flask and dilute with 150 mL of water.
2. Mix thoroughly the contents of the flask and make the volume to
250 mL with water.
3. Using separate pipettes, take accurately 5 mL each of solution A
and solution B, in a porcelain dish or in conical flask.
4. Add 12 mL of honey solution from burette and heat to boiling
over asbestos gauze.
5. Add one millilitre of methylene blue indicator and while
keeping the solution boiling complete the titration, within three
minutes.
6. The end point being indicated by change of color from blue to
red.
7. Note the volume (H) in mL of honey solution required for the
titration.

9 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Procedure for Sucrose
1. To 100 mL of the stock honey solution add one millilitre 1.0
millilitre of concentrated hydrochloric acid and heat the solution
to near boiling.
2. Keep aside overnight. Neutralize this inverted honey solution
with sodium carbonate and determine the total reducing sugars
as described.
Procedure for Fructose-Glucose Ratio
1. Pipette 50 mL of honey solution in a 250 mL stopped flask.
2. Add 40 mL of iodine solution and 25 mL of sodium hydroxide
solution.
3. Acidify with 5 mL of sulphuric acid and titrate quickly the
excess of iodine against standard sodium thiosulphate solution.
4. Conduct a blank using 50 mL of water instead of honey
solution.

Calculation with units of 250 x 100 x S

expression
Total reducing sugar, percent by mass =
HxM
Where
S = strength of copper sulphate solution;
H = volume, in ml, of honey solution required for titration; and
M = mass, in g, of honey

Calculation for Sucrose

Sucrose, percent by mass = [(reducing sugars after inversion, percent by
mass) – (reducing sugars before inversion, percent by mass)] x 0.95
Calculation for Fructose-Glucose Ratio
(B – S) x 0.004502 x 100
Approximate glucose, percent by mass (w) =
a
where
B = volume of sodium thiosulphate solution required for the blank,
S = volume of sodium thiosulphate solution required for the sample, and
a = mass of honey taken for test.

Approximate total reducing

Sugar, percent - w
Approximate fructose, percent by mass (x) =
0.925
True glucose, percent by mass (y) = w – 0.012 x

10 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Approximate reducing sugars,
percent - y
True fructose, percent by mass (z) =
0.925
True reducing sugars, percent by mass = y + z

True fructose, percent by mass (z)

Fructose-glucose ratio =
True glucose, percent by mass (y)

Inference NA
(Qualitative Analysis)
Reference IS 4941:1994
AOAC (920.180)21st edition-2019, IHC(2009)
Approved by Scientific Panel on Methods of Sampling and Analysis

11 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Sucrose and F/G Ratio (HPLC Method)

Method No. FSSAI 04B.005:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew
Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for analysis.
3. Always wear gloves and mask while doing sample analysis.
Principle Weighed sample dissolved in water and diluted with Acetonitrile,
injected on HPLC-RID for the separation and quantification of sugars.
Apparatus/Instruments 1. Liquid chromatography- equipped with Refractive Index Detector
(RID)
2. Column- 300 x 4 (id) mm or µ-Bondapak or Carbohydrate or 250 x
4.6 (id) mm carbohydrate column or column containing amine modified
silica gel or equivalent
3. Syringe filters- 0.45 µm filters stable in organic solvents
Materials and Reagents 1. Acetonitrile
2. Ultra pure water
Preparation of Reagents 1. Mobile phase- LC grade Acetonitrile diluted with ultra-pure water
(83+17) or (75+25) or (80+20): Degas mobile phase daily by
magnetic stirring 15 min under vacuum.
2. Sugar standard solutions- Weigh 3.804 g fructose, 3.10 g glucose,
and 0.602 g sucrose standards in 100 mL volumetric flask and
dissolve in 50 mL water and make up the volume with Acetonitrile
or in 100ml water or Methanol: Water (25:75).
Sample Preparation A) Liquid or Strained honey: If honey is free from granulation, mix
thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. Cool the honey sample rapidly as
soon it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
determination. If foreign matter such as wax, sticks, bees, particles
of comb etc. is present, heat honey to 40°C and filter through
cheesecloth in hot water funnel before weighing, test portions for
analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A
and filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove
wax.
Method of analysis 1. Weigh 5.0 g test portion in small beaker and transfer to 50 mL
volumetric flask with 25 mL water.
2. Mix well and dilute to volume (to make final volume 50mL) with
Acetonitrile or 100ml water or Methanol: Water (25:75).
3. Filter through 0.45 µm filter.
4. Inject 10 µl standard solutions into instrument and establish retention

12 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 times, measure peak heights, and check reproducibility. Repeat same
for test solution.
5. Run Time: 20 min
6. Flow rate: 1.0 ml/min (3.45 Mpa; ca 500 psi);
7. Column temperature: ambient (ca 23 °C)
Calculation with units of Calculate glucose, fructose, and sucrose from integrator values or from
expression peak heights as follows:
Weight percent sugar = 100 x (PH/PH’) x (V/V’) x (W’/W)
Where PH and PH’ = peak heights (or integrator values) of test solution
and standard, respectively; V and V’ = ml test and standard (50 and
100) solutions, respectively; and W and W’ = g test portion (5.000) and
standard, respectively.
Inference NA
(Qualitative Analysis)
Reference AOAC 977.20
AOAC (920.180) 21st edition-2019, IHC(2009)
Approved by Scientific Panel on Methods of Sampling and Analysis

13 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Total Ash

Method No. FSSAI 04B.006:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew
Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
Principle The honey is ashed at a temperature 600 °C ± 20 and the residue
weighed.
Apparatus/Instruments 1. Muffle -Furnace
2. Silica Crucible
Materials and Reagents NA
Preparation of Reagents NA
Sample Preparation A) Liquid or Strained honey: If honey is free from granulation, mix
thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. cool the honey sample rapidly as
soon it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
determination. If foreign matter such as wax, sticks, bees, particles
of comb etc. is present, heat honey to 40°C and filter through
cheesecloth in hot water funnel before weighing, test portions for
analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A
and filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove
wax.
Method of analysis 1. Weigh accurately 5 g to 10 g of the honey sample in a silica or
platinum dish,
2. Add a few drops of pure olive oil to prevent spattering, heat
carefully over a low flame until swelling ceases.
3. Ignite in a muffle furnace at 600 ± 20 °C till white ash is
obtained.
4. Cool the dish in a desiccator and weigh.
5. Incinerate to constant weight.
Calculation with units of 100 (M2 – M)
expression
Ash, percent by mass =
M1 – M
Where
M2 = mass, in g, of the dish with the ash;

14 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 M = mass, in g, of the empty dish; and
M1 = mass, in g, of the dish with the material taken for the test.
Inference NA
(Qualitative Analysis)
Reference IS 4941:1994
AOAC (920.180)21st edition-2019
Approved by Scientific Panel on Methods of Sampling and Analysis

15 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Acidity as Formic acid

Method No. FSSAI 04B.007:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew
Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
Principle The acidity is obtained by adding an excess of sodium hydroxide to the
honey solution and developed pink color of Phenolphthalein indicator
observed as end point.
Apparatus/Instruments 1. Burette
2. Conical Flask-50 mL
Materials and Reagents 1) Standard Sodium Hydroxide Solution- 0.05 N.
2) Phenolphthalein Indicator Solution
Preparation of Reagents 1) Standard Sodium Hydroxide Solution- 0.05 N.
2) Phenolphthalein Indicator Solution- Dissolve 0.5 g of
Phenolphthalein in 100 mL of 50 percent ethyl alcohol (v/v)
Sample Preparation A) Liquid or Strained honey: If honey is free from granulation, mix
thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. cool the honey sample rapidly as
soon it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
determination. If foreign matter such as wax, sticks, bees, particles
of comb etc. is present, heat honey to 40°C and filter through
cheesecloth in hot water funnel before weighing, test portions for
analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A
and filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove
wax.
Method of analysis 1) Take 10 g of the sample in a suitable titration flask and dissolve it in
75 mL of carbon dioxide-free water.
2) Mix thoroughly.
3) Titrate against standard sodium hydroxide solution using 4 to 6
drops of carefully neutralized phenolphthalein solution (pink color
of indicator should persist for at least 10 seconds).
4) Determine blank on water with indicator and correct the volume of
standard sodium hydroxide solution used.
Calculation with units of 0.23 x V
expression
Acidity (as Formic acid), percent by mass = M

16 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Where,
V = corrected volume of 0.05 N sodium hydroxide solution required for
titration; and
M = mass, in g, of the sample taken for the test.
Inference NA
(Qualitative Analysis)
Reference IS 4941:1994
AOAC (920.180) 21st edition-201
Approved by Scientific Panel on Methods of Sampling and Analysis

17 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Free Acidity

Method No. FSSAI 04B.008:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew

Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.

Principle The free acidity is the acidity titratable with sodium hydroxide up to the
equivalence point.

Apparatus/Instruments 1. Burette

Materials and Reagents 1. Conical Flask-50 mL

Preparation of Reagents 1. NaoH-0.05M

Sample Preparation A) Liquid or Strained honey: If honey is free from granulation, mix
thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. Cool the honey sample rapidly as
soon it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
determination. If foreign matter such as wax, sticks, bees, particles
of comb etc. is present, heat honey to 40°C and filter through
cheesecloth in hot water funnel before weighing, test portions for
analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A and
filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove
wax.

Method of analysis 1. Take 10 g of the sample in a suitable titration flask and dissolve it in
75 mL of carbon dioxide-free water.
2. Stir with magnetic stirrer; immerse electrodes of pH meter in
solution, and record pH.
3. Titrate with 0.05M NaOH at rate of 5.0 mL/min.
4. Stop addition of NaOH at pH 8.5.

Calculation with units of Calculate as milliequivalent/kg

expression
Free acidity = (ml 0.05 M NaOH from burette – ml blank) x 50/g test
portion

18 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Inference NA
(Qualitative Analysis)

Reference AOAC 962.19

AOAC (920.180)21st edition-2019

Approved by Scientific Panel on Methods of Sampling and Analysis

19 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Hydroxy Methyl Furfural (HMF)

Method No. FSSAI 04B.009:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew

Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for analysis.
3. Always wear gloves and mask while doing sample analysis.

Principle The determination of the Hydroxy Methyl Furfural (HMF) content is

based on the determination of UV absorbance of HMF at 284 nm. In
order to avoid the interference of other components at this wavelength the
difference between the absorbance of a clear aqueous honey solution and
the same solution after addition of bisulphite is determined. The HMF
content is calculated after subtraction of the background absorbance at
336 nm.

Apparatus/Instruments UV Spectrophotometer ( 284 and 336 nm wavelength)

Materials and Reagents 1. Carrez solution I

2. Carrez solution II
3. Sodium bisulfite solution

Preparation of Reagents 1. Carrez solution I- Dissolve 15 g Potassium ferrocyanide K4Fe

(CN)6. 3H2O and dilute to 100 mL with water.
2. Carrez solution II- Dissolve 30 g Zinc acetate dehydrate Zn
(CH3COO)2.2H2O and dilute to 100 mL with water.
3. Sodium bisulfite solution- 0.20% Dissolve 0.20 g Sodium
bisulfite (NaHSO3) and dilute to 100 mL with water. Dilute 1 + 1
for dilution of reference solution if necessary. Prepare fresh
solution daily.

Sample Preparation A) Liquid or Strained honey: If honey is free from granulation, mix
thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. cool the honey sample rapidly as
soon it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
determination. If foreign matter such as wax, sticks, bees, particles
of comb etc. is present, heat honey to 40°C and filter through
cheesecloth in hot water funnel before weighing, test portions for
analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A and
filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove

20 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 wax.

Method of analysis 1. Accurately weigh ca 5 g honey in small beaker and transfer with
total of ca 25 mL H2O to 50 ml volumetric flask.
2. Add 0.50 ml Careez solution I, mix and add 0.50 mL Careez
solution II, mix and dilute to volume with water. Drop of alcohol
may be added to suppress foam.
3. Filter through paper, discarding first 10 mL filtrate.
4. Pipet 5 mL filtrate into each of two 18 x 150 mm test tubes.
5. Add 5.0 mL H2O to 1 tube (test solution) and 5.0 mL NaHSO3
solution to other (reference). Mix well (Vortex mixer is useful)
and determine A of test solution against reference at 284 and 336
nm in 1 cm cells.
6. If A is > 0.6, dilute test solution with H2O and reference solution
with 0.1% NaHSO3 solution to same extent and correct A for
dilution.

Calculation with units of Hydroxymethyl furfural(HMF) = (A284 – A336) x 14.97 x 5

expression
mg 100 g honey g test sample
Factor = 14.97 = (126/16830) (1000/10) (100/5)
Where 126 = molecular weight HMF; 16830 = molar a of HMF at 284
nm; 1000 = mg/g; 10 =centiliters / L; 100 = g honey reported; 5 =
nominal test portion weight.

Inference NA
(Qualitative Analysis)

Reference AOAC official Methods 980.23

AOAC (920.180)21st edition-201

Approved by Scientific Panel on Methods of Sampling and Analysis

21 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Diastase Activity

Method No. FSSAI 04B.010:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew

Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
4. Don’t heat the sample before use.

Principle Diastase is an enzyme that is found naturally in honey and degrades over
time, especially when exposed to heat. For determination of Diastase
activity, Buffered soluble starch-honey solution is incubated and time
required to reach specified end point is determined photometrically.
Results are expressed as ml 1% starch hydrolyzed by enzyme in 1 g
honey in 1 h.

Apparatus/Instruments 1. Reaction vessel- Attach sealed side arm, 18 x 60 mm, to 18 x

175 mm test tube. Lower side of side arm is attached 100 mm
from bottom of tube, making 45° angle with lower portion of
tube.
2. Visible Photo Spectrometer- With 660 nm red filter or 600 nm
interference filter and 1 cm cells.

Materials and Reagents 1. Iodine stock solution

2. Iodine solution- 0.0007 M
3. Acetate buffer solution
4. Sodium chloride solution-0.5 M
5. Starch solution-2%

Preparation of Reagents 1. Iodine stock solution - Dissolve 8.80 g resublimed I2 in 30-40

mL H2O containing 22.0 g KI, and dilute to 1 L with H2O.
2. Iodine solution (0.0007 M) - Dissolve 20 g KI and 5.00 mL I2
solution, (a) in H2O and dilute to 500 mL. Prepare fresh every
second day.
3. Acetate buffer solution (1.59 M) (pH 5.3) - Dissolve 87 g
NaCH3COO.3H2O in 400 mL H2O, add ca 10.5 mL CH3COOH
in H2O, and dilute to 500 mL. Adjust pH to 5.30 with
NaCH3COO or CH3COOH, if necessary.
4. Sodium chloride solution (0.5 M) - Dissolve 14.4 g NaCl in
H2O and dilute to 500 mL.
5. Starch solution- Weigh 2.000 g soluble starch (special for
diastatic power determination) and mix with 90 mL H2O in 250
ml Erlenmeyer. Rapidly bring to boiling point, swirling solution
as much as possible. Reduce heat and boil gently 3 min, cover,
and let cool to room temperature. Transfer to 100 ml volumetric

22 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 flask and dilute to volume. Observe details closely to limit
variation in absorbance (A) values of starch-I2 blank
Standardization of Starch: - Pipet 5 mL starch solution into 10
mL H2O and mix well. Pipet 1 mL of this solution into several 50
mL graduates containing 10 mL dilute I2 solution. Mix well, and
determine water dilution necessary to produce A value of 0.760 ±
0.02. This is standard dilution for starch preparation used. Repeat
when changing starch source.

Sample Preparation A) Liquid or Strained honey: If honey is free from granulation, mix
thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. Cool the honey sample rapidly as
soon it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
determination. If foreign matter such as wax, sticks, bees, particles
of comb etc. is present, heat honey to 40°C and filter through
cheesecloth in hot water funnel before weighing, test portions for
analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A and
filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove
wax.

Method of analysis 1. Weigh 5 g test portion into 20 mL beaker, dissolve in 10-15 mL

H2O and 2.5 mL buffer solution, and transfer to 25 mL
volumetric flask containing 1.5 mL NaCl solution.
2. Dilute to volume. (Solution must be buffered before addition to
NaCl).
3. Pipet 5 mL starch solution into side arm of reaction tube and 10
mL test solution into bottom of tube, with care not to mix.
4. Place tube in water bath for 15 min at 40 ± 0.2 °C.
5. Then mix contents by tilting tube back and forth several times.
6. Start stopwatch. At 5 min, remove 1 mL aliquot with 1 mL
serological pipette and add rapidly to 10.00 mL dilute I2 solution
in 50 mL graduate tube
7. Mix and dilute to previously determined volume and determine
A in photometer.
8. Note time from mixing of starch and honey to addition of aliquot
to I2 as reaction time. (Place 1 mL pipette in reaction tube for
reuse when later aliquots are taken.)
9. Continue taking 1 mL aliquots at intervals until A value of
<0.235 is obtained.
10. Table given below shows absorbance values with corresponding
end point times.
Absorbance values with corresponding end point
Absorbance End Point

23 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 0.7 >25
0.65 20-25
0.6 15-18
0.55 11-13
0.5 9-10
0.45 7-8

Calculation with units of Plot A against time (min) on rectilinear paper; draw straight line through
expression starting A and as many points as possible. From graph, determine time
diluted 24eflon24n-I2 mixture reaches A of 0.235.
Divide 300 by this time to obtain diastase number (DN).
[Notes: A 5 min reading is sufficient for approximating end point of test
solutions of high DN (>35) if another value is taken soon enough to
obtain A of ca 0.20. For accurate results, repeat determination, taking test
solutions each min from start. With test solutions of low DN, another
reading at 10 min will permit prediction of end point by plotting the data.
No additional readings need be taken until within few minutes of end
point. Only two such readings are needed. The 5 min value will not
accurately predict low DN.]

Inference NA
(Qualitative Analysis)

Reference AOAC Official Method 958.09

AOAC (920.180)21st edition-201

Approved by Scientific Panel on Methods of Sampling and Analysis

24 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Water insoluble matters

Method No. FSSAI 04B.011:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew

Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for analysis.
3. Always wear gloves and mask while doing sample analysis.
Principle The insoluble matter is collected on a crucible of specified pore size and the dried
residue is weighed after being washed free of soluble material.
Apparatus/Instruments 1. Analytical balance, to 0.1mg.
2. Sintered glass crucible, pore size 15 to 40 microns.
3. Drying oven at 135 ± 10 0C.
Materials and Reagents NA

Preparation of Reagents NA

Sample Preparation Homogenize the sample before weighing.

Method of analysis 1. Accurately weigh approximately 20 grams of honey and dissolve in about
200 ml of water at about 80 0C. Mix well.
2. Dry a crucible in the oven and leave to obtain ambient temperature in a
desiccator containing an efficient desiccant such as silica gel. Weigh the
crucible.
3. Filter the sample solution through the crucible.
4. Wash carefully and extensively with warm water until free from sugars.
5. Check by adding to some filtrate in a test tube some 1% phloroglucinol in
ethanol.
6. Mix and run a few drops of concentrated sulphuric acid down the sides of
the tube. Sugars produce a colour at the interface.
7. Dry the crucible at 135°C for an hour, cool in the desiccator and weigh.
8. Dry again for 30 minute intervals until constant weight is obtained.
Calculation with units of
expression % Insoluble Matter in g/100 m1= M1 x 100
M

where M1 = Mass of dried insoluble matter and

M = Mass of honey taken
Inference NA
(Qualitative Analysis)
Reference Harmonized Methods of the International Honey Commission (2009)

Approved by Scientific Panel on Methods of Sampling and Analysis

25 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Pollen and Plant Elements

Method No. FSSAI 04B.012:2024 Revision No. & Date 0.0

Scope Honey

Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis, as heterogeneous distribution of pollen in honey

Principle Pollens present in honey are separated as sediment by centrifugation

followed by staining with Basic Fuchsin. Stained pollens are then
observed and counted under microscope using haemocytometer.

Apparatus/Instruments 1. Microscope 10x10, 10x40 magnification capacity

2. Haemocytometer (1 mm square x 0.1 mm depth).
3. Centrifuge with rotor for 10,50 ml tubes
4. Weighing balance
Materials and Reagents 1. Basic Fuchsin (0.5 percent alcoholic solution)

Preparation of Reagents 1. 0.5 percent alcoholic solution: weigh 0.5gm of basic fuchsin in
95% ethanol

Sample Preparation 1. Weigh accurately 10g of honey in a small clean beaker. Dissolve
the honey in 50ml of distilled water. For honey rich in sediments,
the quantity of honey may be reduced to 5g or 1g and dilution
and calculation may suitably be altered.
2. Transfer this carefully to a 100mL measuring cylinder and fill the
cylinder with distilled water upto 100mL mark.
3. Centrifuge 10mL of this stock solution in 15mL centrifuge tube
at 3000 rev/min for 5 minutes.
4. Decant cautiously the supernatant liquid without disturbing the
sediment, taking care to leave one millilitre of the liquid with the
sediment in the tube.
5. Then, shake well the sediment and completely transfer to a
collecting tube. Repeat centrifuging for all the stock solution of
honey and sediments in the same collection tube.
6. To these sediments in the collection tube, add a drop of 0.5
percent alcoholic basic fuchsin solution and stir the sediment
well.
7. Then centrifuge it and draw of the supernatant liquid and
disperse the sediment in one millilitre of the solution.

Method of analysis 1. Shake well the sediments and place a drop of this solution on the
one millimeter squares on the haemocytometer and place a cover
slip.
2. Count pollens present in one millimeter square at the

26 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 magnification of 100 X.
3. Repeat this counting ten times and take 10 different counts with
the dispersed sediment.

Calculation with units of The average number of pollens counted over the haemocytometer is for
expression the volume 0.1 mm (1 mm square X 0.1 mm depth).
For this, calculate the pollens present in one millilitre, which is
equivalent to their absolute number present in X g of honey taken for
analysis. Express the results as the number of pollens in 1g of honey

Inference Not Applicable

(Qualitative Analysis)

Reference IS 4941:1994

Approved by Scientific Panel on Methods of Sampling and Analysis

27 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Proline

Method No. FSSAI 04B.013:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew

Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.

Principle Proline, predominant free amino acid of honey, reacts with acid
ninhydrin solution to form colored compound. Interference from other
amino acids is negligible, ≤5%.

Apparatus/Instruments 1. Spectrophotometer
2. Reaction tubes- 18 x 130 mm borosilicate scew-cap tubes with
28eflon liners

Materials and Reagents 1. Ninhydrin solution

2. L-(-)-Proline

Preparation of Reagents 1. Ninhydrin solution (3%) - Dissolve 3.0 g Ninhydrin in 100 mL

peroxide-free ethylene glycol monomethyl ether. Store solvent,
not reagent, over Zn metal in amber bottle.
2. L-(-)-Proline- Dry in vaccum oven and store in desiccator.
Prepare standard solutions as follows:
a. Stock solution- 0.5 mg/mL H2O. Dilute 25 mg Proline to
50 mL with H2O and refrigerate it.
b. Working solution- 50 µg/mL. Dilute to 10 mL stock
solution to 100 mL with H2O. Prepare working solution
fresh daily

Sample Preparation A) Liquid or Strained honey: If honey is free from granulation, mix
thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. cool the honey sample rapidly as
soon it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
determination. If foreign matter such as wax, sticks, bees, particles
of comb etc. is present, heat honey to 40°C and filter through
cheesecloth in hot water funnel before weighing, test portions for
analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A and
filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove

28 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 wax.

Method of analysis 1. Weigh 2.5 g honey into to 50 mL volumetric flask and makeup
50 mL volume with H2O.
2. pipette 0.5 mL into beach of three reaction tubes, add 0.25 mL
HCOOH and 1.00 mL Ninhydrin solution.
3. Cap tightly, shake well and place in boiling water for 15 min.
4. Cool 5 min in 22 °C water bath, remove cap, and pipette 5 mL
aqueous Isopropanol (1 + 1) into each.
5. Mix well and determine A at 520 nm against blank of H2O
carried through method.
6. Read all tubes within 35 min of cooling.
7. Correct for color of honey by determining A of solution
containing 0.5 mL prepared honey solution, 1.25 mL H2O and
5.00 mL Isopropanol (1 + 1).
8. Subtract value from that of reacted test solution before
calculating.

Calculation with units of Prepare calibration curve as in determination, using Proline standard
expression solution instead of honey.
Absorbance (A) of 0.5 mL of solution of 50 µg proline/mL is ca 0.35 in
10 mm cell.
Calculate Proline mg/100 g honey.

Inference NA
(Qualitative Analysis)

Reference AOAC Official Method 979.20

AOAC (920.180)21st edition-2019

Approved by Scientific Panel on Methods of Sampling and Analysis

29 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Electrical Conductivity

Method No. FSSAI 04B.014:2024 Revision No. & Date 0.0

Scope All types of Honey including Carvia Callosa and Honey dew

Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.

Principle The determination of the electrical conductivity is based on the

measurement of the electrical resistance, of which the electrical
conductivity is the reciprocal.
The electrical conductivity of a solution of 20 g dry matter of honey in
100 ml distilled water is measured using an electrical conductivity cell.

Apparatus/Instruments 1. Conductivity meter,

2. Conductivity cell, platinized double electrode (immersion electrode).
3. Thermometer with divisions 0.10 0C.
4. Water bath, thermostatically controlled at a temperature of 20°C ±
0.5oC.
5. Volumetric flasks, 100 mL and 1000 mL.
6. Beakers, tall form.
Materials and Reagents 1. Potassium chloride solution

Preparation of Reagents 1. Potassium chloride solution (0.1M) - Dissolve 7.4557 g of potassium

chloride (KCl), dried at 130 °C, in freshly distilled water in a 1000
mL flask and fill to volume with distilled water. Prepare fresh on the
day of use.

Sample Preparation A) Liquid or Strained honey: If honey is free from granulation, mix
thoroughly by stirring or shaking before weighing portion. If
granulated, place closed sample container in water bath without
submerging and heat at sample at 60°C for 30 min until liquefied.
Occasional shaking is essential. cool the honey sample rapidly as
soon it liquefies and mix thoroughly before taking test portion for
determination. Do not heat honey sample intended for diastase
determination. If foreign matter such as wax, sticks, bees, particles
of comb etc. is present, heat honey to 40°C and filter through
cheesecloth in hot water funnel before weighing, test portions for
analysis.
B) Comb Honey: Cut across top of comb, if sealed and separate
completely from comb by straining through No. 40 sieve. When
portions of comb or wax pass through sieve, heat product as in A and
filter through cheese cloth. If honey is granulated in comb, heat
sample until wax is liquefied and after this stir, cool, and remove
wax.
Dissolve an amount of honey, equivalent to 20.0 g anhydrous honey, in
distilled water. Transfer the solution quantitatively to a 100 mL

30 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 volumetric flask and make up to volume with distilled water.

Method of analysis Determination of the cell constant

If the cell constant of the conductivity cell is not known, proceed as
follows:
1. Transfer 40 ml of the potassium chloride solution to a beaker.
2. Connect the conductivity cell to the conductivity meter, rinse the
cell thoroughly with the potassium chloride solution .
3. Immerse the cell in the solution, together with a thermometer.
4. Read the electrical conductance of this solution in mS after the
temperature has equilibrated to 200C.
5. Calculate the cell constant K, using the following formula:
K = 11.691 x 1/G
Where:
K = the cell constant in cm-1.
G = the electrical conductance in mS, measured with the
conductivity cell.
11.691= the sum of the mean value of the electrical conductivity of
freshly distilled water in mS.cm- and the electrical conductivity of a
0.1M potassium chloride solution, at 20 °C.
6. Rinse the electrode thoroughly with distilled water after the
determination of the cell constant. When not in use keep the
electrode in distilled water in order to avoid ageing of the platinum
electrode.
Note:

If necessary, a 1 in 5 w/v dilution of a smaller amount of honey can

be used.
7. Pour 40 ml of the sample solution into a beaker and place the beaker
in the thermostated water bath at 20 °C.
8. Rinse the conductivity cell thoroughly with the remaining part of the
sample solution.
9. Immerse the conductivity cell in the sample solution. Read the
conductance in mS after temperature equilibrium has been reached.
Note:
a. Most conductivity meters are direct current. In order to avoid
false results due to polarization effects, measurement time
should as short 1as possible.
b. If the determination is carried out at a different temperature,
because of lack of thermostated cell, then a correction factor
can be used for calculation of the value at 20 °C:
i. For temperatures above 20 °C : subtract 3.2 % of the
value per °C
ii. For temperatures above 20 °C : subtract 3.2 % of the
value per °C
iii. For temperatures below 20 °C : add 3.2 % of the value
per °C

31 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 iv. For temperatures below 20 °C : add 3.2 % of the value
per °C
c. Data from measurements corrected with the above factors
values have not been validated in ring trials.
d. However there were no significant differences between
conductivity of 50 honeys, measured at 20 °C and at
temperatures varying from 20 to 26 °C after applying the above
correction factor (5)

Calculation with units of Calculate the cell constant K, using the following formula:
expression
Calculate the electrical conductivity of the honey solution, using the
following formula:
SH = K . G
Where:
SH = electrical conductivity of the honey solution in mS.cm-1
K = cell constant in cm-1
G = conductance in mS
Express the result to the nearest 0.01 mS.cm-1. G = the electrical
conductance in mS, measured with the conductivity cell.

Inference NA
(Qualitative Analysis)

Reference Harmonised Methods of the International Honey Commission (2009),

AOAC (920.180)21st edition-201

Approved by Scientific Panel on Methods of Sampling and Analysis

32 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of 2-Acetylfuran-3-Glucopyranoside (2-AFGP) as Marker
for Rice Syrup

Method No. FSSAI 04B.015:2024 Revision No. & Date 0.0

Scope All types of Honey

Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for analysis.
3. Always wear gloves and mask while doing sample analysis.

Principle The method involves the dilution of honey with water; a clean-up through
HLB cartridge and subsequent analysis by Liquid Chromatography- Mass
Spectrometry (LC-MS/Ms).

Apparatus/Instruments 1. High performance LC or Ultra-high-performance LC (UHPLC) system

2. Mass spectrometer: Triple-quadrupole mass spectrometer or equivalent
MS/MS instrument.
3. Column: Agilent Eclipse plus C18 (100 mm x 4.6 mm, 3.5 µm)/Waters
Acquity UPLC HSS PFP (100 x 2.1 mm, 1.8 µm) or equivalent.

Materials and Reagents 1. Centrifuge Tubes (15 mL)

2. Analytical balance (Readability 0.0001 g)
3. Vortex mixer
4. Micro pipettes 20-200 µL and 100-1000 µL
Glassware & others:
1. Injection vials
2. Volumetric flask Class A, 10 mL and 1 mL
3. Glass tubes 15 mL capacity
4. Hydrophilic syringe filters (0.22 µm)
5. Hydrophilic-Lipophilic-Balanced (HLB) water-wettable, reversed-phase
sorbent cartridge or equivalent should be used for sample preparation.
Chemicals:
1. Acetonitrile (MS Grade)
2. Methanol (MS Grade)
3. ASTM Type I Water/HPLC grade: Resistivity, min, 18.2 MΩ cm (at 25
°C)
4. Standard: 2-acetylfuran-3-glucopyranoside (AFGP)

Preparation of Reagents 1. Stock Solution: Accurately weigh standard AFGP and add methanol as
solvent make a stock solution of approximate 1.0 g/L (1000 mg/L which is
same as 1 mg/mL) in a volumetric flask.
2. Intermediate Standard Solution: Prepare the intermediate standards of
concentration of 10.0 mg/L (10000 µg/L) and 1.0 mg/L (1000 µg/L) by
subsequent dilution with water.

33 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Concentration Vol. of stock Vol. of water Final conc. (g/L)
of stock solution (µL) (µL)
standard (g/L)
1.0 100 900 0.1
0.1 100 900 0.01
0.01 100 900 0.001

3. Working Standard (WS) Solution for calibration curve: Prepare the

working standards from the intermediate standard (0.001 g/L) by dilution
with water as shown below.
Working Volume of Volume of Total volume
standard intermediate water (µL) (µL)
concentration standard (µL)
(µg/L (ppb)
100 100 900 1000
200 200 800 1000
300 300 700 1000
400 400 600 1000
800 800 100 1000
1000 100 0 1000
Note: If sample preparation is carried out using HLB cartridge the dilution
must be carried out with methanol

Sample Preparation A. By dilution

1. Weigh 1 g ± 0.01 g of honey sample in a 15 ml centrifuge tube.
Note (If the honey samples have particles centrifuge it at 5000 g for 5
minutes or pass through a nylon mesh (100-150 micron).
2. Add 1 ml water and shake vigoursly.
3. Dilute 1:5 if necessary.
4. Vortex the tubes for 5 minutes and rotospin for 5 minutes.
5. Centrifuge the tubes at 7000 x g for 5 min.
6.Collect upper clean extract and filter it through syringe filter (0.22 µm)
7.Use for LC-MS/MS
B. Using HLB cartridge
1. Take 1 g of honey sample in 15 mL centrifuge tube.
(If the honey sample has particles centrifuge it at 5000 g for 5 min or pass
through a nylon mesh (100-150 micron).
2. Add 5 mL ASTM Type I water and mix in a vortex for 3 min.
3. Make the volume up to 10 mL with water.
4. Take a 500 mg/6 cc HLB cartridge, condition it with methanol first then
followed with water.
5. Pass the honey solution through the cartridge with constant speed and
without applying any external pressure.
6. Elute the cartridge using 5.0 mL methanol.

34 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 7. Collect the elute in a clean tube.
8. Filter using 0.2 µm syringe filter prior to LC analysis.

Method of analysis A. HPLC/UPLC configuration:

1. Set up the HPLC/UPLC system with the configuration shown below
a. Column: C18 (100 mm x 4.6 mm, 3.5 µm)/(100 x 2.1 mm, 1.8 µm)
or equivalent
b. Injection volume: 10 µL
c. Flow rate:0.5mL/min
d. Elution: Gradient
e. Solvent A: Water containing 0.1 % Formic acid
f. Solvent B: Acetonitrile containing 0.1% Formic acid
II. Form Gradients by high-pressure mixing of two mobile phases, A and B,
using the gradient programme shown below:
Gradient programme for HPLC/UPLC*
Time (min) Solvent A (%) Solvent B (%)
Start 95 5
7 10 90
7.01 5 95
10 5 95
11 95 5
13 Stop
*Gradient can be suitably modified and optimized to obtain best
peak shape and resolution
III. After verifying equilibration of the HPLC/UPLC system, inject the
working standards followed by a reagent blank, control sample, and sample
extracts. Injected working standards after the analysis of the last sample
extract.
B. Mass spectrometer instrument settings:
Set up the mass spectrometer with instrument settings listed below
Gas temp. (°C) 300
Gas Flow (1/min) 10
Nebulizer (psi) 50
Sheath Gas Heater (°C) 300
Sheath Gas Flow (L/min) 10
Capillary (V) 3500
V Charging 500
Note: These settings are suitable for the 6460 triple-quadrupole (Agilent
Technologies) mass spectrometer. Optimal tuning on alternative instrument
will differ. Tune the instrument to obtain the precursor and product ions.
Follow the manufacturer’s instruction or alter conditions to obtain the best
resolution of AFGP peaks.
Mass analysis parameters for AFGP
AFGP ion Precursor Product Dwell Fragmentor #CE Cell Polarity
ion (m/z) ion (m/z) time (ms) (V) Acceleration

Analyte 311.07 185 100 162 9 7 Positive

qualifier
Analyte 311.07 148.9 100 162 13 7 Positive
quantifier
#CE: Collision Energy

35 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Peak Identification
a. Peak shape and response ratio of extracted ion chromatograms of sample
should be similar to those obtained from calibration standard
b. The retention time of the AFPG in the extract should correspond to that of
the calibration standard with a tolerance of ± 0.1 min.
c. Identification in MRM mode largely relies on the correct selection of ions.
d. Chromatographic peaks of different selected ions for the analyte must fully
overlap.
e. Ion ratio from sample should be within ± 30% (relative) of average of
calibration standards from same sequence

Calculation with units of Acquire the chromatograms and prepare the calibration curve. Calculate the
expression regression by plotting peak height response r for each working standard vs
AFGP concentration. Carry out a regression analysis R2 = 0.999
Calculate the concentration of AFGP in the sample using the equation
y = mx + c
Where, y = Area under the curve for AFGP in sample
x = Concentration of Analyte
m = Slope of the calibration curve
c = value of y intercept
The curve can also be directly taken from instrumental software. If the analyte
concentration in sample is greater than the calibrated standards, the sample
elute should be appropriately diluted and analyzed.

Inference If concentration of AFGP is < 1.0 mg/kg, results are reported as Absent/kg
(MRPL 1mg/kg). If marker concentration is > 1.0 mg/kg, results to be
(Qualitative Analysis)
reported as Present/kg.

Reference 1. 2-Acetylfuran-3-Glucopyranoside as a Novel Marker For the Detection of

Honey adulterated with Rice syrup. Xue Xiaofeng, Wang Qiang, Li Yi,
Wu Liming, Chen Lanzhen, Zhao Jhing and Liu Fengmao. J. Agric. Food
Chem., 2013, 61, 7488-7493p.
2. Rapid screening of multiclass syrup adulterants in honey by Ultra –
Performance Liquid Chromatography/Quadrupole Time of Flight Mass
Spectrometry, Du Bing, Wu Liming, Xue Xiaofeng, Chen Lanzhen, Zhao
Jing and Cao Wei. J. Agric. Food Chem, 2015, 63(29), 6614-6623.
3. Guidance document on analytical quality control and method validation
procedures for pesticide residues and analysis in food and feed;
SANTE/11813/2017

Approved by Scientific Panel on Methods of Sampling and Analysis

36 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of C4 sugar,∆δ13C protein-Honey by EA-IRMS and
∆δ CFructose – Glucose , ∆δ13C max Foreign Oligosaccharide by LC-IRMS
13

Method No. FSSAI 04B.016:2024 Revision No. & Date 0.0

Scope Honey
Caution 1. Honey sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis and
reference material handling.
4. Keep the eluent and reagent bottles under constant Helium purge
to prevent CO2 contamination from ambient air.
5. Phosphoric acid and Sulphuric acid are highly corrosive.
6. Prepare the oxidation reagents fresh daily, store in dark brown
bottle.
7. Many of the routine gases for IRMS are hazardous. The
laboratory should have an atmospheric monitoring system to
warn of dangerous levels of gases.
Principle The method involves the determination of the relative isotopic ratios (δ13
C) of
1) protein isolated from honey by EA-IRMS and
2) δ13 C values of every individual sugar present in honey within a single
HPLC run by LC-IRMS.
Isotopic ratios are measured relative to a working gas calibrated using
internationally accepted standards and are reported using the delta
notation (δ) and expressed as ‘per mill (%0)’.
The delta notation is defined as
δ13 C(0/00) sample = [R(sample)/R(standard)-1] x 100
Where R represents the ratio 13CO2/12CO2. The 13C/12C carbon isotope
ratios reported as δ13 C values are related to Vienna Pee Dee Belemnite
(VPDB) according to the AOAC Official Method 998.12.
δ is the 13C/12C ratio of the sample related to the 13C/12C ratio of a
reference material to ensure international compatibility of data sets. The
unit of expression is, per mill (%0).
The CO2 produced from combustion of the protein fraction is isolated
from the sample is analyzed to give δ13 Cprotein%0 by IRMS.
The δ13 C %0 values of fructose, glucose, disaccharides, and trisaccharide
and any other oligosaccharides present in honey are determined by LC-
IRMS. The sugars are separated by LC using a cation exchange column.
All individual sugars eluting from LC column pass into the LC/IRMS
interface. Here the carbon from organic samples in the mobile phase is
converted into CO2 by a wet chemical oxidation process using sodium
peroxodisulfate either in the presence or absence of phosphoric acid.
CO2 and O2 both diffuse through, which are subsequently dried in an
online gas drying unit. The individual CO2 peaks are subsequently
admitted to the IRMS, which directly gives the δ13 C values for each
individual sugar; δ13 Cfru%0, δ13 Cglu%0, δ13 Cdisaccharide%0, δ13 Ctrisaccharide%0

37 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 and δ13 C %0 of any other oligosaccharides (see chromatograms below).
The schematic of a typical LC-IRMS is shown below.
The difference in the carbon isotope ratio between other δ13 Cfru%0 and
δ13 Cglu%0 gives ∆δ13 Cfru-glu%0,
δ13 Cmax is the maximum difference observed between all possible
isotopic ratios measured (∆δ13 Cfru-disaccharides / ∆δ13 Cfru-trisaccharides / ∆δ13 Cfru-
protein/ ∆δ Cfru-disaccharides / ∆δ13 Cglu-trisaccharides / ∆δ13 Cglu-protein / ∆δ13
13

Cdisaccharides-trisaccharides / ∆δ13 Cdisaccharides-protein / ∆δ13 Ctrisaccharides-protein )

The peak area (%) for foreign oligosaccharides is calculated from the
areas appended in the LC chromatogram.
Apparatus/Instruments 1.An integrated EA-IRMS instrument equipped with an automated
combustion system and mass spectrometer designed or modified for
isotope ratio measurement at natural abundance
2.An integrated LC-IRMS comprising of HPLC/UPLC and in line
oxidation reactor for aqueous oxidation of LC elute and a mass
spectrometer designed or modified for isotope ratio measurement at
natural abundance
3.LC comprises of a binary pump, autosampler, column oven (set at 80
°C), and cation exchange column (Ca2+, 300 x 7.7 mm, 8 µm or
equivalent)
4.Analytical microbalance: 0.0001 g
5.Micropipette: 10-100 µL, 20-200 µL and 100-1000 µL
6.Volumetric flasks: 10 mL Class A
7.Vortex mixer
8.Sonicator
9.Centrifuge (capable of 10,000 x g)
10.Water Bath (80 °C)
11.Convection oven
12.Centrifuge tubes (50 mL, 15 mL)
13.Spatula
14.Forceps (Blunt end and pointed curved end)
15.Tin capsules
16.Capsule holding tray
17.Nylon stocking material (100-150 mesh)
18.Syringe filters (0.45 µm and 0.22 µm)
19.Vaccum concentrator
Materials and Reagents 1. Stable Isotope reference standard

Certified Reference Standards Δ13C (‰)

Sucrose -10.449
Casein -26.98
NBS 22 Oil -30.031
Beet Sugar -26.027

38 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Galactose -21.415
Fructose -10.985
Glucose -10.97
Cane Sugar -11.64
In-house standards for normalization and verified against above listed
standards:
a. D-(-)-fructose ≥99% pure
b. D-(+)-glucose monohydrate ≥99.5% pure
c. D-(+)-sucrose ≥99% pure
d. D-(+)-maltose monohydrate ≥99% pure
e. D-(+)-raffinose pentahydrate ≥99% pure

2. Ultra-pure water (Electrical Resistivity, Min.,18.18 MΩcm, at 25 °C)

3. Phosphoric acid (H3PO4) (purity ≥ 99%)
4. Sodium peroxodisulfate (Na2S2O8, Sodium persulfate) (purum p. a. ≥
99%)
5. Sodium tungstate dihydrate (Na2WO4.2H2O) (puriss. p. a. ≥ 99%)
6. Sulfuric acid (p. a. 98%)
7. Tin capsules
8.CO2 (working standard reference gas): 99.999% Pure
9.O2 (flash combustion gas): > 99.999% Pure
10.Helium: 99.999% Pure
Preparation of Reagents Reagents for protein isolation
1. 10% aqueous solution of Sodium tungstate: Dissolve 10 g of
Na2WO4.2H2O in 100 mL of pure water. Prepare fresh daily
2. 0.335 M H2SO4: Dilute 1.88 mL concentrated H2SO4 to 100 mL with
ultra-pure water
Chemical oxidation reagents
1. 20% Sodium peroxodisulfate: Dissolve 200 g sodium peroxodisulfate
in 1000 mL ultra-pure water in a brown glass bottle using an
ultrasonic bath. Use a water-jet pump for vaccum degassing to
remove all dissolved CO2.
2. 1.5 M H3PO4 in water: Weigh 147.0 g of crystalline H3PO4. Dissolve
in ~250 mL of ultra-pure water and make up to 1 L with water.
LC reagents
Ultra-pure water: (Electrical Resistivity, Min.,18.18 MΩcm, at 25 °C)

Sample Preparation 1. EA-IRMS analysis

A. Preparation of Standards for EA-IRMS
a. Weigh protein standard (Casein), approximately between 0.1-
0.2mg, with the help of spatula in the tin capsule
b. Fold the tin capsule with the help of the forceps in such a way so as
39 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 to remove air.
c. Gently fold it from all the sides and place the folded tin capsule in
the carousel and start the sequence of operation following the
manufacturer’s instruction
B. Sample preparation for EA-IRMS:
a. Prepare in triplicate
b. Strain honey through 100-150 mesh nylon stocking material to
remove insoluble material.
c. Add 4 mL H2O to 10-12 g honey (in triplicate) in a 50 mL
centrifuge tube and mix well to get a homogeneous solution
d. Prepare fresh by mixing 2.0 mL 10% Na2WO4 solution and 2.0
mL 0.335 M H2SO4 in a small test tube.
e. Add this mixture immediately to the diluted honey solution and
mix well.
f. Swirl the tube in ca 80 °C water bath until a visible flocculants
(precipitate) forms with a clear supernatant.
Note: If no visible flocculants forms, or if supernatant remains cloudy,
add 2 mL aliquots of 0.335 MH2SO4 with repeating heating between
additions.
g. Fill tube with water, mix, centrifuge for 5 min at 1500 x g
h. Decant supernatant.
i. Repeat washing, mixing, and centrifuging steps nine times with ca
40 mL portions of water, thoroughly dispersing the pellet each time.
j. Dry protein at least for 3 h in ca 75 °C oven
k. Weigh approximately 0.1-0.2 mg isolated protein in tin capsules.
l. Gently fold the tin capsule with the help of forceps and place it
m. For δ 13Choney%0, weigh filtered honey approximately 0.1-0.2 mg in
tin capsules and follow step at (l).
Precautions:
a. Decant the supernatant immediately after centrifugation to avoid
the mixing of pellet with the supernatant
b. Protein washing must be done very carefully to avoid any loss of
pellet with the water
c. Fold the tin capsules gently to avid the leakage or loss of sample
d. Be careful during tin capsule folding to avoid air trapping.
e. Repetitive addition of Sulfuric acid Could lead to protein
burning hence will cause more positive delta C values of
Protein.
C. Sample analysis on EA-IRMS
a. Placed the weighed casein standard, weighed protein and honey
sample on the carousel of EA-IRMS for determining δ 13Cprotein, δ
13
Choney
b. Operate the instrument as per manufacturer’s instructions after
calibration with CO2 reference gas.

40 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 2. LC-IRMS analysis
A. Preparation of Standards for LC-IRMS
1. Prepare a solution of Fructose, Glucose, Sucrose and Raffinose
containing 250mg/L of each in ultra-pure water.
2. Filter the solution through 0.22 µm syringe filter

B. Sample preparation for LC-IRMS analysis:

1. Strain honey through a 100-150 mesh size nylon stocking material
2. In triplicate accurately weigh about 200 mg sample in a 15 mL
centrifuge tube. Mix well with 5 mL of Ultra-pure water.
3. Sonicate the mixture and make the volume up to 10 mL with water in
a 10 mL volumetric flask.
4. Filter through 0.22 µm syringe filter into HPLC injection vials.
Note: Prepare sample solutions fresh everyday

C. Sample analysis on LC-IRMS

a. Introduce CO2 reference gas pulse three times (20s each) at the
beginning of each run.
b. The constant flow rate during this period gives the peaks a flattop
appearance.
c. A level of CO2 corresponding to 2-5 V (depending on the
instrument) at m/z 44 is used to calibrate the system
d. Inject standard mixture (10µL) of fructose, glucose, disaccharide
and trisaccharide. Repeat 10 times to obtain the mean and standard
deviation for the δ13C ‰ of individual sugars.
e. Inject Honey sample (10µL) in triplicate
f. The IRMS chromatogram provides details of the δ13C‰ of each of
the sugars in the sample and the area under the curve of each of the
resolve sugars.
g. The ∆δ13 C fru- glu, ∆δ13 Cmax and foreign oligosaccharide content are
calculated from the chromatogram data.
Method of analysis 1. EA-IRMS conditions:
a. EA conditions (vario ISOTOPE cube, Elementar, UK)
Temperature: Oxidation tube:950°C
Reduction tube: 650°C
Pressure:1300-1400mbar
He flow: 230ml/min
CO2 flow: 230mL/min
O2 flow: 18mL/min
b. IRMS conditions (Isoprime)
Ion Source: CEI High Vacuum: 5e-6

41 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Turbo speed: 100% TCD temperature:59°C
Focus point:>0.5 Accelerating voltage:4000v
Extraction voltage:76.00v Half plate differential (v): -121.00
Z-plate voltage (v): -53 Trap current (µA): 200
Electron volts (9ev): 75 Ion Repellor voltage(v): -9
Magnet current:4000
The gas cylinders (associated valves etc) which supply working gases to
the IRMS must be stored in a temperature-controlled environment.

2. LC-IRMS conditions:
a. LC conditions
1.Column: Ca2+ (300 x 7.7 mm, 8 µm)
2.Solvent: Ultra-pure water
3.Flow rate: 0.3 mL/min
4.Column Oven temperature: 80 °C
5.Injection volume: 10 µL
b. Interface for wet oxidation (Isoprime Liquiface,Elementar,UK)
1.Reactor temperature: 95 °C
2.Oxidation reagent: 20% Sodium peroxodisulfate (Purge the
solution with helium gas before use)
3.Flow rate: 60 µL/min
Note: Some instruments use 20% Sodium peroxodisulfate and 1.5 M
H3PO4 for wet oxidation. Follow the manufacturer’s instructions.
c. IRMS Parameters (Isoprime IRMS):
Ion Source: CEI High Vaccum: 5e-6
Turbo Speed: 100% TCD temperature: 59 °C
Focus point: >0.5 Accelerating Voltage: 4000v
Extraction Voltage:76.00v Half Plate Differential(v): - 121.00
Z-Plate Voltage(v): -53.00 Trap current(µA): 200.00
Electron Volts (9ev): 75.00 Ion Repellor Voltage(v): -9.00
Magnet Current: 4000
Calculation with units of 1. ∆δ C protein-honey: Subtract the δ13Cprotein (%0) value given in the
13

expression chromatogram from the δ13Choney (%0) value. Report as ∆δ13Cprotein-

honey%0.

2. C4 Sugar (%):
δ 13CProtein – δ 13CHoney x 100
δ 13CProtein – (-9.7)

Where, -9.7 is the average δ13C value for corn syrup, ‰. Report negative
values from this calculation as 0%. Product is considered to contain
significant C4 sugars (primarily corn or cane) only at or above of 7%.
42 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 3. ∆δ13Cfru-flu%0
Subtract the δ13CGlu (%0) value given in the chromatogram from the
δ1313CGlu (%0) value. Report as ∆δ13Cfru-flu%0.
4.∆δ13Cmax%0
Extract the δ13C (%0) values of fructose, glucose, disaccharides and
trisaccharides from the LC-IRMS profile.
Extract the δ13C %0 of protein from EA-IRMS profile and tabulate as
shown
A B A-B
δ C %0
13
δ C %0
13
∆δ13C %0
Fructose Disaccharide
Fructose Trisaccharide
Fructose Protein
Glucose Disaccharide
Glucose Trisaccharide
Glucose Protein
Disaccharide Trisaccharide
Disaacharide Protein
Trisaccharide Protein

The highest value observed in column three gives ∆δ13C max%0

5.Foreign oligosaccharides (% peak area)
Extract the area of individual peaks and calculate using the formula
Foreign oligosaccharide (area%)= Sum of the peak area of all peak(s) other than Fructose, Glucose,
Disaccharides and Trisaccharides
____________________________ x 100
Total peak area

Inference NA
(Qualitative Analysis)
Reference 1. AOAC Official Method 998.12 C-4 Plants Sugar in Honey. Internal
Standard Stable Carbon Isotope ration Method First Action 1998
2. Improved detection of honey adulteration by measuring differences
between 13C/12C stable carbon isotope ratios of protein and sugar
compounds with a combination of elemental analyzer – isotope ratio
mass spectrometry and liquid chromatography – isotope ratio mass
spectrometry (δ13C-EA/LCIRMS). Lutz Elfein, Kurt-Peter
Raezke;Apidologie 2008, 39 (5), 574-587.
3.Liquid chromatography coupled to isotope ratio mass spectrometry: A
new perspective on honey adulteration detection. Ana I. Cabanero, Jose
L. Recio, Mercedes RupeaRez; J. Agric. Food Chem. 2006, 54, 9719-
9727.
4.LC-IRMS: Authenticity control of honey using Thermo Scientific LZ
IsoLink LC-IRMS. Andreas W.Hilkert, Michael krummen, Dieter
Juchelka; Thermo application note 30024.

43 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 5.”Scientific support to the implementation of a Coordinated Control
plan with a view to establishing the prevalence offraudulent practices in
the marketing of honey” N° SANTE/2015/E3/JRC/S12.706828.E Aries,
J. Burton,L. Carrasco, O. De Rudder, and A. Maquet. JRC Technical
Report 2016, JRC104749, 38 p.
Approved by Scientific Panel on Methods of Sampling and Analysis

44 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 B.BEE WAX

45 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Solubility

Method No. FSSAI 04B.017:2024 Revision No. & Date 0.0

Scope Bees wax
Caution 1.Sample must be kept at moisture free place in air tight jar.
2.Mix the sample thoroughly before taking test portion for analysis.
3.Always wear gloves and mask while doing sample analysis.
4.Keep the sample at dry and cool place.
Principle Solubility of bees wax is determined by adding known amount of
sample into known volume of various solvent i.e. Alcohol, Ether and
Water.
Apparatus/Instruments Conical Flask
Materials and Reagents 1. Ethanol
2. Ether
3. Water
Preparation of Reagents NA
Sample Preparation Melt the sample, if necessary, and filter it through a dry filter paper to
remove any traces of moisture.

Method of analysis A. Procedure: Transfer a known amount of the sample into a flask
containing known amount of the specified solvent, shake for not less
than 30 sec and not more than 5 min.

Descriptive term Parts of solvent required for 1

part of solute
Very soluble Less than 1

Freely soluble From 1 to Less than 10

Soluble From 10 to Less than 30
Sparingly soluble From 30 to Less than 100
Slightly soluble From 100 to Less than 1,000
Very slightly soluble From 1,000 to Less than 10,000
Practically insoluble or More than 10,000
in soluble

B. Solubility in Ethanol:
1. Transfer a 1 mL sample into a calibrated 10-mL glass-stoppered
cylinder graduated in 0.1-mL subdivisions
2. Add slowly, in small portions, ethanol, the concentration and
quantity of which are specified in the monograph.
3. Maintain the temperature at 20°C.
A clear solution, free from foreign matter should be obtained.
Calculation with units of NA
expression
Inference NA
(Qualitative Analysis)
Reference JECFA INS 901 and JECFA combined compendium of food additives
specification volume 4

Approved by Scientific Panel on Methods of Sampling and Analysis

46 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Melting Point

Method No. FSSAI 04B.018:2024 Revision No. & Date 0.0

Scope Bees wax
Caution 5. Sample must be kept at moisture free place in air tight jar.
6. Mix the sample thoroughly before taking test portion for
analysis.
7. Always wear gloves and mask while doing sample analysis.
8. Keep the sample at dry and cool place.
Principle Bees wax softens or become sufficiently fluid to slip or clear at
given temperature which is determined by capillary-slip method.

Apparatus/Instruments 1. Thermometer of a suitable type, with an accuracy of 0.1 °C and

graduated at every 0.1 °C.
2. Test Tube- with a centrally bored cork to take the thermometer.
The cork shall have a slit so as to permit circulation of air.
3. Water Bath, with the thermometer.
Materials and Reagents NA
Preparation of Reagents NA
Sample Preparation Before determining the melting range of a substance, the sample
should be dried under the conditions specified for Loss on Drying in
the individual monograph. If a temperature is not specified in the
monograph, the sample should be dried for 24 h in a desiccator.
Method of analysis 1. Transfer a quantity of the dried powder to a dry capillary-
tube about 10 cm long and sealed at one end (thickness of
the wall, 0.10-0.15 mm; i.d. 0.9-1.1 mm) and pack the
powder by tapping the tube on a hard surface so as to form
a tightly-packed column 2-4 mm in height.
2. Attach the capillary-tube and its contents to a standard
thermometer so that the closed end is at the level of the
middle of the bulb, and heat in a suitable apparatus
containing an appropriate liquid (liquid paraffin or silicone
oil) and fitted with a stirring device and an auxiliary
thermometer.
3. Regulate the rise in temperature during the first period to 3°
per min.
4. When the temperature has risen to 5° below the lowest
figure of the range for the substance being tested, heat more
slowly: if no other directions are given, the rate of rise in
temperature should be 1-2° per min, Unless otherwise
directed.
5. Read the temperature at which the substance is observed to
form droplets against the side of the tube and the
temperature at which it is completely melted, as indicated

47 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 by the formation of a definitive meniscus.
Calculation with units of Before starting the determination of the melting range, adjust the
expression auxiliary thermometer so that the bulb touches the standard
thermometer at a point midway between the graduation for the
expected melting range and the surface of the heating material.
When the substance has melted, read the temperature on the
auxiliary thermometer. Calculate the correction to be added to the
temperature reading of the standard thermometer from the following
formula:
0.00015 N(T - t)
in which
T is the temperature reading of the standard thermometer,
t is the temperature reading of the auxiliary thermometer and
N is the number of degrees of the scale of the standard thermometer
between the surface of the heating material and the level of the
mercury.
The statement "melting range, ao - bo" means that the corrected
temperature at which the material is observed to form droplets must
be at least ao, and that the material must be completely melted at the
corrected temperature bo.
Inference NA
(Qualitative Analysis)
Reference JECFA INS 901 and JECFA combined compendium of food
additives specification volume 4

Approved by Scientific Panel on Methods of Sampling and Analysis

48 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Acid Value

Method No. FSSAI 04B.019:2024 Revision No. & Date 0.0

Scope Bees wax
Caution 1. Sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
4. Keep the sample at dry and cool place.
Principle Acid Value is the number of milligrams of potassium hydroxide (KOH)
necessary to neutralize the fatty acids in 1 gram of sample. Acid value is
determined by directly titrating the alcoholic solution of test sample with
aqueous potassium hydroxide solution.
Apparatus/Instruments 1.Burette
2. Erlenmeyer flask
Materials and Reagents 1) Neutral ethanol
2) Neutralized diethyl ether/ethanol or petroleum spirit/ethanol
3) Phenolphthalein
4) 0.5 N KOH
Preparation of Reagents 1) Neutral ethanol-95%
2) Neutralized diethyl ether/ethanol or petroleum spirit/ethanol
3) Phenolphthalein-Dissolve One gram of phenolphthalein indicator in
100 mL of ethyl alcohol
Sample Preparation Melt the sample, if necessary, and filter it through a dry filter paper to
remove any traces of moisture.

Method of analysis 1. Weigh accurately about 5 g of sample into a 500- mL Erlenmeyer

flask.
2. Add 75-100 mL of hot neutral ethanol.
3. Heat and agitate the sample solution.
4. For some samples, it may be necessary to use as the solvent a 1:1
mixture of neutralized diethyl ether/ethanol or petroleum
spirit/ethanol.
5. Add 0.5 mL of phenolphthalein and titrate immediately, while
shaking, with 0.5 N KOH until the pink colour persists for at least
30 sec.
6. For acidity less than 2% by weight, 0.1 N KOH should be used for
the titration.
7. For acidity less than 0.2% by weight, it is necessary, in addition, to
first neutralize the carbon dioxide in the reaction vessel.
Calculation with units of Acid value = (56.1 x T x N) / W
expression
Where
T is the titre (ml);

49 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 N is the normality of potassium hydroxide solution; and
W is the weight of sample (g).
Inference NA
(Qualitative Analysis)
Reference Food Chemical Codex 2016
Approved by Scientific Panel on Methods of Sampling and Analysis

50 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Peroxide Value

Method No. FSSAI 04B.020:2024 Revision No. & Date 0.0

Scope Bees wax
Caution 1. Sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
4. Keep the sample at dry and cool place.
Principle The peroxide value is a measure of the peroxides contained in a sample
of wax, expressed as milli-equivalents of peroxide per 1000 grams.

Apparatus/Instruments 1. Burette (50 mL)

2. Conical Flask(250 mL)
Materials and Reagents 1. Chloroform
2. Acetic Acid
3. Potassium Iodide Solution
4. 0.01N sodium thiosulfate
5. Starch-1%
Preparation of Reagents 1. Potassium Iodide Solution-Saturated: Prepare saturated solution of
potassium iodide in boiled distilled water and store in dark.
2. Acetic Acid- Chloroform solution: Mix three parts by volume of
glacial acetic acid with 2 parts by volume of chloroform.
Sample Preparation Melt the sample, if necessary, and filter it through a dry filter paper to
remove any traces of moisture.

Method of analysis 1. Weigh accurately 5 g of the sample into a 200 mL conical flask.
2. Add 30 mL of a 2:3 solution of chloroform and acetic acid and
close the flask with a stopper.
3. Heat with warm water and swirl to dissolve the sample.
4. Cool to room temperature and add 0.5 mL of saturated potassium
iodide solution.
5. Close the flask with the stopper and shake vigorously for 60 ± 5
sec. Add 30 mL of water and titrate immediately with 0.01 N
sodium thiosulfate using starch as indicator.
6. Carry out a blank determination.
Calculation with units of Peroxide value = (a-b) x N x 1000/W
expression
where
a = Volume (ml) of sodium thiosulfate used for the sample
b = Volume (ml) of sodium thiosulfate used for the blank
N = Normality of the sodium thiosulfate
W = Weight of sample (g)

51 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Inference NA
(Qualitative Analysis)
Reference JECFA INS 901 and JECFA combined compendium of food additives
specification volume 4,
IS:548(Part 1)-1964
Approved by Scientific Panel on Methods of Sampling and Analysis

52 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Saponification Value

Method No. FSSAI 04B.021:2024 Revision No. & Date 0.0

Scope This method is applicable for Bees wax
Caution 1. Sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
4. Keep the sample at dry and cool place.
Principle The test sample is saponified by refluxing with a excess of alcohol
potassium hydroxide solution. The alkali consumed for saponification is
determined by titrating the excess alkali with standard hydrochloric acid.
Apparatus/Instruments 1. Conical Flask-250 to 300 mL capacity made of alkali-resistant glass.
2. Reflux Air Condenser-at least 65 cm long.
Materials and Reagents 1. Methyl Ethyl Ketone
2. Rectified Spirit
3. Alcoholic potassium Hydroxide solution
4. Phenolphthalein Indicator Solution
5. Standard Hydrochloric Acid
Preparation of Reagents 1. Methyl Ethyl Ketone- This shall be stored in dark
2. Rectified Spirit-Neutral to phenolphthalein indicator
3. Alcoholic potassium Hydroxide solution-
Dissolve 30 g of potassium hydroxide in rectified spirit and make up to 1
litre. Allow to settle overnight in a dark place, decant the clear liquid and
keep in a bottle closed tight with cork or rubber stopper.
4. Phenolphthalein Indicator Solution-
Dissolve 0.1 g of phenolphthalein in 60 mL of rectified spirit and dilute
with water to 100 mL
5. Standard Hydrochloric Acid-0.5 N
Sample Preparation Melt the sample, if necessary, and filter it through a dry filter paper to
remove any traces of moisture.

Method of analysis 1. weigh accurately into a 250 mL flask a sample of such size (usually
about 4-5 g) that the titration of the sample solution after
saponification will require between 45 and 55% of the volume of 0.5
N hydrochloric acid required for the blank.
2. Add 50.0 ml of ethanolic potassium hydroxide from a pipette and
allow the pipette to drain for a definite period of time.
3. Prepare and conduct blank determinations simultaneously with the
sample and similar in all respects.
4. Connect an air condenser to each flask and boil gently but steadily,
with occasional mixing, until the sample is completely saponified.
(This usually

53 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Requires about 1 h for normal samples).
5. After the flasks and condensers have cooled somewhat but not
sufficiently for the contents to gel, wash down the inside of the
condensers with a few mL of distilled water.
6. Disconnect the condensers, add about 1 mLof phenolphthalein to
reach flask, and titrate with 0.5 N hydrochloric acid until the pink
colour has just disappeared.
Calculation with units of Saponification value = [56.1 x N (A - B)] / W
expression
Where
A is mL of HCl required for the titration of the blank;
B is mL of HCl required for the titration of the sample;
W is the weight of sample in g; and
N is normality of the HCl.
Inference NA
(Qualitative Analysis)
Reference Food Chemical Codex 2016
Approved by Scientific Panel on Methods of Sampling and Analysis

54 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Carnauba Wax

Method No. FSSAI 04B.022:2024 Revision No. & Date 0.0

Scope Bees wax
Caution 1. Sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
4. Keep the sample at dry and cool place.
Principle When the sample is dissolved in n-butanol and boiling followed by
cooling is done, loose mass of fine needle-like crystals separates from
clear mother liquor. Further examination is done under microscope.
Apparatus/Instruments 1. Microscope
Materials and Reagents 2. n-butanol
Preparation of Reagents NA.

Sample Preparation NA.

Method of analysis 1. Transfer 100 mg of the sample into a test tube, and add 20 mL of
n-butanol.
2. Immerse the test tube in boiling water, and shake the mixture
gently until the sample dissolves completely.
3. Transfer the test tube to a beaker of water at 60 °C, and allow the
water to cool to room temperature.
4. A loose mass of fine, needle-like crystals separates from clear
mother liquor.
5. Under the microscope, the crystals appear as loose needles or
stellate clusters, and no amorphous masses are observed,
indicating the absence of carnauba wax.
Calculation with units of NA
expression
Inference NA
(Qualitative Analysis)
Reference JECFA INS 901
Approved by Scientific Panel on Methods of Sampling and Analysis

55 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Ceresins, Paraffins and other waxes

Method No. FSSAI 04B.023:2024 Revision No. & Date 0.0

Scope Bees wax
Caution 1. Sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
4. Keep the sample at dry and cool place.
Principle The sample is refluxed with a known excess of alcohol potassium
hydroxide solution, which lead to solution become clear at given
temperature. Any kind of precipitation indicate the presence of Ceresins,
paraffins and other waxes
Apparatus/Instruments 1. Round-bottomed flask
2. Reflux condenser
3. Thermometer
Materials and Reagents 1. Alcoholic Potassium hydroxide
2. Aldehyde-free ethanol
Preparation of Reagents 1. Alcoholic Potassium hydroxide- Approximately 0.5 N, prepared by
dissolving potassium hydroxide in rectified spirit.
2. Aldehyde-free ethanol- To 125 mL alcohol contained in 1000 mL
flask, add 375 mL of dinitrophenylhydrazine solution, heat on a water
bath under a reflux condenser for twenty-four hours, remove the alcohol
by distillation, dilute to 100 ml with a 2 percent v/v solution of sulphuric
acid, and set aside for 24 hours
Sample Preparation NA
Method of analysis 1. Transfer 3.0 g of the sample to a 100 mL round-bottomed flask.
2. Add 30 mL of a 4% w/v solution of potassium hydroxide in
aldehyde-free ethanol and boil gently under a reflux condenser for
2 h.
3. Remove the condenser and immediately insert a thermometer.
Place the flask in water at 80 °C and allow to cool, swirling the
solution continuously.
4. Observe any kind of precipitation before the temperature reaches
65 °C, although the solution may be opalescent.
Calculation with units of NA
expression
Inference Any kind of precipitation indicate the presence of Ceresins, paraffins
and certain other waxes
(Qualitative Analysis)
Reference JECFA INS 901,
IS 4028:1992
Approved by Scientific Panel on Methods of Sampling and Analysis

56 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Fats, Japan wax, Rosin and Soap

Method No. FSSAI 04B.024:2024 Revision No. & Date 0.0

Scope Bees wax
Caution 1. Sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
4. Keep the sample at dry and cool place.
Principle When Sample is boiled in solution of sodium hydroxide, followed by
cooling, filtration and acidification with hydrochloric acid. Any kind of
precipitation indicates the presence of Fats, Japan wax, rosin and soap.
Apparatus/Instruments NA
Materials and Reagents 1. Sodium hydroxide Solution
2. Dilute Hydrochloric Acid
Preparation of Reagents 1. Sodium hydroxide Solution- 10 percent (m/v).
2. Dilute Hydrochloric Acid – approximately 4 N.
Sample Preparation NA
Method of analysis 1. Boil 1 g of the sample for 30 min with 35 ml of a 1 in 7 solution of
sodium hydroxide, maintaining the volume by the occasional
addition of water, and cool the mixture.
2. The wax separates and the liquid remains clear.
3. Filter the cold mixture and acidify the filtrate with hydrochloric
acid.
4. There should be no precipitation.
Calculation with units of NA
expression

Inference Any kind of precipitation indicate the presence of Fats, Japan wax, rosin
and soap
(Qualitative Analysis)
Reference JECFA INS 901,
IS 4028-1992
Approved by Scientific Panel on Methods of Sampling and Analysis

57 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Glycerol and other polyols

Method No. FSSAI 04B.025:2024 Revision No. & Date 0.0

Scope Bees wax

Caution 1. Sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
4. Keep the sample at dry and cool place.

Principle After 30 minutes of refluxing with ethanolic potassium hydroxide, the

acidic filtrate is combined with decolorized fuchsin chemical to produce
a blue colour.

Apparatus/Instruments 1. Round-bottom flask

2. Beaker

Materials and Reagents 1. Ethanolic potassium hydroxide

2. Sulfuric acid
3.Sodium periodate
4. Decolourized fuchsin solution

Preparation of Reagents 1) Decolourized fuchsin solution-

Dissolve 0.1 g of basic fuchsin in 60 mL of water. Add a solution of 1 g
of anhydrous sodium sulfite (Reagent grade) in 10 mL of water. Slowly
and with continuous shaking of the solution add 2 mL of hydrochloric
acid. Dilute to 100 mL with water. Allow to stand protected from light
for at least 12 h, decolourize with activated charcoal and filter. If the
solution becomes cloudy, filter before use. If on standing the solution
becomes violet, decolourize again by adding activated charcoal. Store
protected from light.

Sample Preparation NA

Method of analysis 1. To 0.20 g of the sample in a round-bottom flask, add 10 mL of

ethanolic potassium hydroxide TS, attach a reflux condenser to
the flask and heat in a water bath for 30 min.
2. Add 50 mL of dilute sulfuric acid cool and filter.
3. Rinse the flask and filter with dilute sulfuric acid TS.
4. Combine the filtrate and washings and dilute to 100.0 mL with
dilute sulfuric acid TS.
5. Place 1.0 mL of the solution in a tube, add 0.5 mL of a 1.07 %
(w/v) solution of sodium periodate.
6. Mix and allow standing for 5 min.

58 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 7. Add 1.0 mL of decolorized fuchsin solution and mix. Any
precipitate disappears.
8. Place the tube in a beaker containing water at 40 °C.
9. Allow to cool while observing for 10 to 15 min. Any bluish-
violet colour in the solution is not more intense than a standard
prepared at the same time in the same manner using 1.0 mL of a
0.001 % (w/v) solution of glycerol in dilute sulfuric acid.
10. Bluish-Violet color should not be more intensive than a standard.

Calculation with units of NA.

expression

Inference More intensive Bluish-Violet color than standard indicate the presence of
Glycerol and other polyols.
(Qualitative Analysis)

Reference JECFA INS 901

Approved by Scientific Panel on Methods of Sampling and Analysis

59 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Ash

Method No. FSSAI 04B.026:2024 Revision No. & Date 0.0

Scope Bees wax

Caution 1. Sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
4. Keep the sample at dry and cool place.

Principle The sample ashed at a temperature 650 °c for 1 hr and the residue
weighed and calculated as ash content.

Apparatus/Instruments 1. Platinum Dish- having a capacity of 100 mL

2. Muffle Furnace
3.Dessicator

Materials and Reagents NA

Preparation of Reagents NA

Sample Preparation NA

Method of analysis 1. Heat the platinum dish to redness, cool to room temperature in a
desiccator and weigh.
2. Take about 50 g of the material in a watch glass and weigh
accurately.
3. Transfer about three quarters of this quantity to the platinum
dish and heat on a Bunsen burner so that the material burns
gently at the surface.
4. When about half of the material is burnt away, stop heating, cool
and add the remainder of the material.
5. Weigh the watch glass again and find, by difference, the exact
mass of sample transferred to the platinum dish.
6. Heat again till the material is completely charred.
7. Incinerate in a muffle furnace at 550 °C to 650 °C for 1 h.
8. Cool to room temperature in a desiccator and weigh.
9. Repeat incineration, cooling and weighing until the difference
between two successive weighing is less than one milligram.

Calculation with units of

expression
Ash, percent by mass = M2 x 100
M1

60 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Where
M2 = mass in g of the ash; and
M1 = mass in g of the material taken for the test

Inference NA
(Qualitative Analysis)

Reference IS 4028:1992

Approved by Scientific Panel on Methods of Sampling and Analysis

61 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Total Volatile matter

Method No. FSSAI 04B.027:2024 Revision No. & Date 0.0

Scope Bees wax
Caution 1. Sample must be kept at moisture free place in air tight jar.
2. Mix the sample thoroughly before taking test portion for
analysis.
3. Always wear gloves and mask while doing sample analysis.
4. Keep the sample at dry and cool place.
Principle Total volatile matter is determined by weighing the sample before and
after drying and determining the difference.
Apparatus/Instruments 1. Oven
2. Desiccator
Materials and Reagents NA
Preparation of Reagent NA
Sample Preparation NA
Method of analysis 1. Weigh accurately about 10 g of the material in a suitable dish,
previously dried and weighed, and place it in an oven maintained at
105 ± 2 °C for 6 h.
2. Cool the dish in a desiccator and weigh with the lid on.
3. Heat the dish again in the oven for 30 min.
4. Repeat the process until the loss in mass between two successive
weighing is less than one milligram. Record the constant mass
obtained.
Calculation with units of 100 (M1 - M2)
expression Total volatile matter at 105 °C, percent by mass =
M 1 – M3
Where
M1 = mass in g of the dish with the material before heating;
M2 = mass in g of the dish after heating; and
M3 = mass in g of the empty dish.
Inference NA
(Qualitative Analysis)
Reference IS 4028-1992
Approved by Scientific Panel on Methods of Sampling and Analysis

62 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 C. Royal Jelly

63 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Moisture (Vacuum Oven Drying Method : Reference
Method)

Method No. FSSAI 04B.028:2024 Revision No. & Date 0.0

Scope Royal Jelly

Caution Properly mix the sample before analysis and it should be free from bubbles.

Principle Royal jelly sample is heated in a vacuum oven under controlled conditions of
pressure and temperature to remove moisture by passing dry air. Sample is
weighed before and after drying to estimate moisture.

Apparatus/Instruments 1. Vacuum drying oven

2. Weighing dish, (height 25 mm to ~30 mm, of diameter 35 mm to 50 mm).
3. Analytical Balance, (weighing to the nearest 0, 0001 g).
4. Desiccator

Materials and Reagents Desiccants

Preparation of Reagents NA

Sample Preparation Homogenize the sample before weighing

Method of analysis 1. Weigh approximately 0.5 g of royal jelly sample in the weighing dish
which is dried to constant weight, spread the sample evenly weigh
accurately.
2. Put the dish with sample in the vacuum drying oven .
3. Dry for 4 h at 75 °C under the pressure between 0,000 Mpa and
0,005 Mpa.
4. Take out the weighing dish and put it in the desiccators.
5. Weigh after it has been cooled for 30 min.
6. Re dry for 2 h and repeat the process until the weight difference
between two consecutive times is no more than 2 mg, until a constant
weight is achieved.

Calculation with units of

expression
W1 – W2
Moisture (%) = x 100

(by weight) W1 – W

Where,
W = Weight in g, of Aluminium dish.
W1 = Weight in g, of Aluminium dish + sample before drying.
W2 = Weight in g, of Aluminium dish + dried sample until constant weight

64 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Inference NA
(Qualitative Analysis)

Reference IS/ISO 12824:2016

Approved by Scientific Panel on Methods of Sampling and Analysis

65 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination Moisture (Karl Fisher Method)

Method No. FSSAI 04B.029:2024 Revision No. & Date 0.0

Scope Royal Jelly
Caution Properly mix the sample before analysis and it should be free from
bubbles.
Principle The Karl Fisher reaction is based upon the oxidation of sulfur dioxide by
iodine with the consumption of water in a buffered solution. Water reacts
with iodine and sulphur dioxide to form sulphur trioxide and hydrogen
iodide. An endpoint is reached when all the water is consumed. The
water content is then calculated from the amount of reagent added.
Apparatus/Instruments Karl Fisher
1. Karl Fischer titration system, Mettler DL 18 titrator or equivalent.
2. Analytical balance, capable of weighing, to the nearest 0,00001 g.
3. Hydranal Composite 5 R.D.H. as titrating solution or equivalent.
Materials and Reagents 1. Methanol
Preparation of Reagents NA
Sample Preparation Homogenize the sample before weighing
Method of analysis 1 Prior to titration of a sample, each working day, the titre of the
employed one-component reagent (e.g. Hydranal (R)-Composite
5) is determined.
2 A suitable water standard (e.g. Hydranal R - Water Standard
10,0, ultrapure water or terpine hydrate with a moisture content
well defined at 10,46%) is determined in triplicate in the
employed titration medium.
3 Weigh a 1 mL syringe. Weigh approximately 30 mg of the royal
jelly sample in the syringe.
4 Introduce the sample into the titration of the titrator containing
about 40 mL in methanol.
5 Weigh again the syringe.
6 The weighing of royal jelly exactly introduced in the titration
cell in calculated by the difference of the two weighings of the
syringe.
7 After 600s of stirring, the moisture content is determined and
automatically calculated by the titrator in % and mg/kg.
8 The determined titre shall be taken into account for the
calculation of the water content in the sample.
Calculation with units of After 600s of stirring, the moisture content is determined and
expression automatically calculated by the titrator in % and mg/kg.

Inference NA
(Qualitative Analysis)
Reference IS/ISO 12824:2016
Approved by Scientific Panel on Methods of Sampling and Analysis

66 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Moisture (Lyophilization Method)

Method No. FSSAI 04B.030:2024 Revision No. & Date 0.0

Scope Royal Jelly
Caution Properly mix the sample before take it and no bubble shall be there.
Principle Lyophilization or freeze drying is a process in which moisture content is
removed from a product after it is frozen and placed under a vacuum,
allowing the ice to change directly from solid to vapor without passing
through a liquid phase. After completion of lyophilization % loss of
moisture is calculated.
Apparatus/Instruments 1 Analytical balance, capable of weighing, to the nearest 0,00001 g.
2 Centrifuge tubes
3 Lyophilizer
4 Freeze
Materials and Reagents NA
Preparation of Reagents NA
Sample Preparation Homogenize the sample before weighing
Method of analysis 1. Weigh accurately centrifuge tube with its cap.
2. Weigh exactly around 1 g of royal jelly in it.
3. Lyophilize at least 36 h without the cap.
4. After completion of lyophilization process, put the cap and weigh
the sample immediately.
Calculation with units of
expression The percentage of dry matter is calculated using
% dry matter = 100 x (m1 – m0)/m
Where
m1 = is the mass of the tube after the lyophilization process with the cap,
in grams;
m0 = is the mass of the empty tube with its cap, in grams;
m = is the mass of the sample, in grams
The moisture content in royal jelly is calculated using
% moisture content = 100 - % dry matter
Inference NA
(Qualitative Analysis)
Reference IS/ISO 12824:2016
Approved by Scientific Panel on Methods of Sampling and Analysis

67 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of 10-HAD (HPLC-UV External Standard: Reference
method)

Method No. FSSAI 04B.031:2024 Revision No. & Date 0.0

Scope Royal Jelly
Caution Properly mix the sample before take it and no bubble shall be there.
Principle 10-HDA is a bio-active compound found in royal jelly. Lyophilized royal
jelly sample is extracted with phosphate buffer and 10HDA is detected by
HPLC-UV at 216 nm.
Apparatus/Instruments 1.HPLC with UV detector
2.Column: Zorbax SB-CN 150 x 3.0 mm; 3.5 µm or equivalent
3.Ultrasonic bath
4.Homogenizer
5. Analytical balance (0.00001 g)
Materials and Reagents 1. Reference standard: 10-HDA (purity above 99%)
2. Ultrapure water
3. Methanol
4. Sodium di-hydrogen phosphate monohydrate
5. Orthophosphoric acid (H3PO4)
Preparation of Reagents 1. 10-HDA, Standard stock solution = 0.13 mg/ml
2. External calibration: Prepare a standard calibration curve of 10-HAD
standard with different levels 1 g/100mL, 1.5 g/100mL, 2.0 g/100mL,
2.5 g/100mL corresponding to the sample with different levels as
prepared for the standard.
3. Phosphate buffer (25 mM, pH 2.5): Weigh 6.90 g sodium di-hydrogen
phosphate monohydrate (M= 137.99 g/mol) into 2L measuring flask,
dissolve in approximately 1800 ml H2O, adjust pH to 2.5 with 85%
H3PO4 and fill up to volume with water.
4. Extraction solution (25 mM phosphate buffer): Mix 550 ml 25 mM
phosphate buffer, pH 2.5 with 450 ml methanol, equilibrate to room
temperature.
5. Sample solvent (2 mM phosphate buffer): Mix 700 ml 25 mM
phosphate buffer pH 2.5 with 300 mL methanol, equilibrate to room
temperature
Sample Preparation 1. Weigh approximately 80 mg lyophiliated royal jelly or 200 mg fresh
royal jelly into a 50 ml centrifuge tube.
2. Add 40 ml extraction solution. Homogenize for approximately 10 to 20
seconds using an ultrasonic bath at 15000 rpm until royal jelly material
is emulsified. Treat for 10 min in ultrasonic bath.
3. Pipette 1 ml of the homogeneous extract into a 10 mL measuring flask
and fill up to volume with sample solvent.
4. Filter an aliquot of the diluted extract through membrane filter (0.45
µm)
5. Inject 20 µl into the instrument.
6. Measure the concentration against an external standard calibration
curve.

68 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Method of analysis Chromatography Conditions:
Detection wavelength: 216 nm
Mobile Phase A: 25 mM phosphate buffer pH 2.5
Mobile Phase B: Methanol
Gradient:
34% B, 0.2 – 2 min
34 - 43 % B, 2.0-9.0 min
43 - 80% B, 9.0- 10 min
34% B, 10.1- 16.0 min
Calculation with units of 1) Standard calibration curve
expression Determine the equation of the straight line for a plot of peak area
versus purity corrected concentration (µg/mL) of the 10-HDA
standard solutions of the form:
y= ax+b
where
y is the area of the 10- HDA peak
a is the slope of the standard curve
x is the purity corrected concentration of the standard
b is the y- intercept of the standard calibration curve
2) Using the 10-HDA peak area from the sample, calculate the
amount of the 10- HDA in the measuring solution from the
calibration curve as follows:
xʹ = (yʹ - b)/a
where
xʹ is the concentration (µg/mL) of the 10- HDA in the
measuring solution of the sample;
yʹ is the area of the 10- HDA peak in the sample.
The 10- HDA content (C10-DHA) in royal jelly ( sample in g/100g)
is calculated by
(C10-HDA) = xʹ × 40/m
Where
xʹ is the calculated concentration (µg/mL) of the 10- HDA in the
measuring solution of the sample;
40 is the dilution factor considering the extraction volume of 40
ml, the pipette volume used for dilution (1 mL) and the volume of
the measuring flask used for dilution (10 mL)
m is the actual mass of the royal jelly sample, in mg.
Inference NA
(Qualitative Analysis)
Reference IS/ISO 12824 : 2016
Approved by Scientific Panel on Methods of Sampling and Analysis

69 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of 10-HDA (HPLC-UV Internal standard)

Method No. FSSAI 04B.032:2024 Revision No. & Date 0.0

Scope Royal Jelly

Caution Properly mix the sample before take it and no bubble shall be there.

Principle Sample extraction is carried out with Hydrochloric acid (HCl) and the
supernatant obtained after centrifugation is analysed on HPLC-UV at 210
nm.

Apparatus/Instruments 1. HPLC with UV detector

2. Column: 250x 4.6 mm, fill amorphous silica gel with C18 bonded
stationary phase of 5 or 10 µm particle size
3. Ultrasonic bath
4. Mixer
5. Vortex mixer or equivalent
6. Analytical balance (0.00001 g)

Materials and Reagents 1. Double distilled water

2. Methanol
3. Anhydrous alcohol
4. Trans-2-hexenoic acid (as internal standard, purity >99%)
5. 10- HDA standard (purity >99%)
6. 10- HDA standard solution, HCl (c= 0.03 M)
7. Mobile phase (Methanol + 0.03M HCl + H2O) : 55+10+35 or
(Methanol +25 mM phosphate buffer pH 2.5) : 55+45

Preparation of Reagents 1. 10- HDA standard: Decompress and dry for 24 h in the vacuum
drying oven or desiccator with concentrated sulfuric acid before it
is used.
2. 10- HDA standard solution: Weigh accurately 12.5 mg of dried
10-HDA standard sample and dissolve it with anhydrous alcohol
and transfer it to a 25 ml volumetric flask, dilute to the mark with
anhydrous alcohol and mix evenly.
3. Internal Standard solution: Weigh accurately 650 mg of trans-2-
hexenoic acid dissolve with anhydrous alcohol and transfer it to a
1000 mL volumetric flask, dilute to the mark with anhydrous
alcohol and mix evenly. The concentration of the internal
standard solution obtained in solution is 0.65 mg/mL.
4. HCl (0.03 M): Take 100 mL of 0.1 M HCl, add 200 ml double
distilled water.

Sample Preparation Defreeze the sample at room temperature and stir evenly with glass
rod.

70 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Method of analysis 1. Weigh accurately and approximately 0.5 g and put in a 50 ml
volumetric flask that has been weighed already.
2. Add 1 mL of 0.03 M HCl and 2 mL water, put it on the vortex
mixer and mix to dissolve the sample.
3. Add anhydrous alcohol 30 ml while shaking lightly
4. Add 10 mL internal standard solution accurately. Dilute to the
mark with anhydrous alcohol and mix evenly.
5. Immediately put in the ultrasonic bath for 15 minutes or shake on
vortex mixer for 15 minutes.
6. Centrifuge at 3000 rpm for 10 minutes and filter with 0.45 µm
membrane filter if necessary. Then carry out the analysis test or
store in refrigerator if analysis could not be conducted
immediately.
7. Inject 10µL of sample into the instrument and measure by internal
standard method.
Wavelength: 210 nm
Column temperature: 35 ⁰C
Flow: 1 mL/min

Calculation with units of Determination of correction factor:

expression
Weigh 10-HDA standard solution 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 ml separately
and transfer them to respective 10ml volumetric flasks. Add accurately
2ml internal standard solution, dilute to the mark with anhydrous alcohol,
and mix evenly. Weigh respectively 10µl of these solutions, inject it into
the instrument. Plot the mass ratio of 10- HDA per internal standard
against the peak area ration of that, and draw a linear calibration curve.
The 10- HDA content in royal jelly, is calculated by:
X2 = F ×(Ai/As )× (ms/mi ) × 100

Where
X2 is the 10- HDA content in royal jelly, %;
F is the correction factor;
Ai is the peak area of tested group in sample;
As is the area of the internal standard in sample;
ms is the mass of the internal standard in grams;
mi is the mass of sample , in grams.

Inference NA
(Qualitative Analysis)

Reference IS/ISO 12824 : 2016

Approved by Scientific Panel on Methods of Sampling and Analysis

71 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Protein: Kjeldahl method (Automatic) (Reference
method)

Method No. FSSAI 04B.033:2024 Revision No. & Date 0.0

Scope Royal Jelly

Caution Properly mix the sample before take it and no bubble shall be there.

Principle Nitrogen is estimated in Royal Jelly using Kjeldahl method and

converted to protein In digestion step the organically bonded nitrogen is
converted into ammonium ions and these ammonium ions during
distillation converted into ammonia which is transferred into the receiver
by means of steam distillation and the ammonia is quantitavely captured
in Boric acid and the concentrarion of ammonia determined by acid base
titration.

Apparatus/Instruments 1. Analytical balance (0.0001 g)

2. Digestion block: Aluminium alloy block with adjustable
temperature device for measuring and controlling block
temperature (for eg. Tecator Digestion System 20, 1015 Digestor
or KjelDigestor K-449, SpeedDigestor K-439 or equivalent)
3. Digestion tubes (250 mL to 300 mL)
4. Distillation units: Foss Tecator 2200, Buchi KjelMaster K-375 or
equivalent to accept 250 mL to 300 mL)
5. Titration Flask (500 mL graduated Erlenmeyer flask)
6. Fume exhaust manifold (with PTFE rings seals, connected to a
water aspirator in a hooded sink)
7. Nitrogen free weighing boats
8. Pipetting dispenser

Materials and Reagents 1. Concentrated sulfuric acid, 95% to 98%, reagent grade
2. Catalyst.
3. Mixed indicator
4. Boric acid (H3BO3)
5. Sodium hydroxide
6. Hydrochloric acid standard solution(0.1 mol/L)

72 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Preparation of Reagents 1. Concentrated sulfuric acid (95% to 98%)
2. Catalyst: Weigh 7.0 g potassium sulfate and 0.4 g copper
sulfate.
3. Mixed indicator: Dissolve 100 mg methyl red in 100 ml
methanol and 100 mg bromocresol green in 100 ml methanol.
When potentiometric titration is used, no indicator is required.
4. Boric acid solution: 4% (w/v). Dissolve 400 g boric acid in 5 to
6 L hot deionized water. Mix and add more hot de-ionized water
to a volume of about 9 L.
Cool to room temperature, add 100 ml bromocresol green
solution and
70 ml methyl red solution, and dilute to a final volume of 10 L.
Adjust
the pH of the boric acid solution to 4.6 to 4.8 using 0.1
mol/NaOH or 0.1
mol/L HCL or 25 ml Sheer mixed indicator and dilute to a final
volume.
5. Sodium hydroxide solution. 32% (w/v). Weigh 32 g sodium
hydroxide, dilute to100 mL with distilled water.
6. Hydrochloric acid standard solution,0.1 mol/ L

Sample Preparation Homogenize the sample before weighing

Method of analysis Digestion: -

1. Weigh approximately 1 g of royal jelly sample into a tarred, N
free weighing boat and transfer carefully whole material into a
kjeldahl tube.
2. Add the catalyst, (7.0 g potassium sulfate and 0.4 g copper
sulfate) and add 12 mL of concentrated sulfuric acid, using
pipetting dispenser. Hold the mixture overnight.
3. Place fume manifold tightly on tubes, and turn water aspirator on
completely.
4. Place rack of tubes in preheated block (at 420 °C).
5. After 10 min, turn on water aspirator or scrubber. A
condensation zone should be maintained within the tubes. After
bulk of sulfur oxides fumes are produced during initial stages of
digestion, reduce vaccum source to prevent loss of sulphuric
acid.
6. Digest additional 50 min. Total digestion time is approximately
60 min.
7. Let tubes cool. Add deionized water to each tube to a total
volume of approximately 80 ml
Distillation: -
1. Place 32% NaOH in alkali tank of distillation unit.
2. Adjust volume dispensed to 50 mL.
3. Attach digestion tube containing diluted digest to distillation
unit, or use automatic dilution feature.

73 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 4. 60 ml H3BO3 solution are added to the receiving vessel with
indicator on receiving platform, and immerse tube from
condenser below surface of H3BO3 solution.
5. Steam distil until ≥150 mL distillate is collected. Remove
receiving flask.
Titration: -
1. Titrate H3BO3 receiving solution with standard 0.1 mol/L HCl to
violet or grey end point.
2. Record mL of HCl consumed to end point.

Calculation with units of The protein content in royal jelly is calculated by

expression
(Vs – Vb) x M x 14.01
N= x 6.25
m x 10
where
N = is the protein content in royal jelly, given by mass fraction, %;
Vs = is the volume of standardized acid consumed when the sample is
titrated, in mL;
Vb = is the volume of standardized acid consumed when blank titration
is made, in mL;
M = is the concentration of hydrochloric acid standard solution, in mol/l;
14.01 = is the atomic weight of N;
m = is the mass of sample, in grams;
10 = is the factor to convert mg/g to percent;
6.25 = is the factor to convert N to proteins.

Inference NA
(Qualitative Analysis)

Reference IS/ISO 12824:2016

Approved by Scientific Panel on Methods of Sampling and Analysis

74 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination Protein : Kjeldahl method (Alternative Method)

Method No. FSSAI 04B.034:2024 Revision No. & Date 0.0

Scope This method is applicable for Royal Jelly
Caution Properly mix the sample before take it and no bubble shall be there.
Principle Nitrogen is estimated in Royal Jelly using Kjeldahl method and
converted to proteins. In digestion step the organically bonded nitrogen
is converted into ammonium ions and these ammonium ions during
distillation converted into ammonia which is transferred into the receiver
by means of steam distillation and the ammonia is quantitavely captured
in Boric acid and the concentrarion of ammonia determined by acid base
titration.
Apparatus/Instruments 1. Kjeldahl nitrogen determination method digestion
equipment, 50 mL Kjeldahl flask (if far infrared digesting
furnace is used, a 50 mL digesting tube and retort funnel shall be
collocated).
2. Acid burette, 10 mL capacity
3. Analytical balance, Readability 0.00001 g.
4. Semimicro method distillation unit
Materials and Reagents 1. Concentrated sulfuric acid, 95% to 98%, reagent grade
2. Mixed Catalyst.
3. Mixed indicator
4. Boric acid solution
5. Sodium hydroxide
6. Sulfuric acid.
7. Hydrochloric acid standard solution (0.1 mol/L)
Preparation of Reagents 1. Concentrated sulfuric acid,w = 95%~98%.
2. Mixed catalyst of copper sulfate and potassium sulfate.
Weigh 1 g copper sulfate and 10 g potassium sulfate, put it in the
mortar, mix evenly, and grind finely to use.
3. Mixed indicator: Weigh two volumes of methyl red ethanol
solution (⍴ = 1 g/L) and three volumes of bromocresol green
ethanol solution (⍴ = 2 g/L), and mix evenly, or use mixed
indicator.
4. Boric acid absorption solution (⍴ = 20 g/L). Weigh 2.0 g boric
acid, put it in the 100 ml measuring cylinder, add 20 ml ethanol,
dilute to the mark with distilled water, shake until the boric acid
is dissolved, and put it aside for later use.
5. Sodium hydroxide solution (⍴ = 400 g/L) : Weigh 32 g sodium
hydroxide, and dilute to 100 ml with distilled water.
6. Dilute sulfuric acid : Using a pipette, take 5.7 mL concentrated
sulfuric acid, and dilute to 100 mL with distilled water.
7. Hydrochloric acid standard solution (0.1 mol/L) : Dilute to 10
times before using.
Sample Preparation Homogenize the sample before weighing

75 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Method of analysis Cleaning of distillation unit

1. Figure: Semimicro method distillation unit

Link distillation unit, add proper amount of distilled water and a few
drops of methyl red indicator in bottle A,
1. Add dilute sulfuric acid to make it acidic, add a few granules of
glass beads and zeolites.
2. Add 50 mL distilled water from funnel D, close nip G, open
condensate water, and boil the distilled water in bottle A.
3. When the vapor comes from the top of the condenser tube,
remove the fire, close nip H, and make the distilled water in
bottle C flow reversely to Bottle B.
4. Open nip G, discharge the distilled water in bottle B and close
nip B and G.
5. Immerge the top of the condenser tube in approximate 50 ml
distilled water, make the distilled water flow reversely to bottle
C from the top of the condenser tube and then flow to bottle B,
and discharge the distilled water with the above method.
6. Clean the apparatus twice or three times like this.
2) Digestion
1. Weigh approximately 1 g of royal jelly sample, put it on a
filter paper or a paraffin paper that is weighed, pack it well
after being weighed accurately, and put it in Kjeldahl flask or
a digesting tube.
2. Add 2 g of mixed catalyst of copper sulfate and potassium
sulfate, add 10 mL concentrated sulfuric acid slowly along
the bottle wall, mix sufficiently.
3. Put a small funnel at the bottle mouth, make the flask lean at
a 45° angle, heat slowly at comparative low temperature at
first, keep the temperature of the solution below the boiling
point, and increase the electric power gradually until the
boiling is stopped.
4. When the digestion solution is boiling, maintain this state and
watch out that the solution shall not overflow; heat another 30
min after the solution becomes clear green.

76 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 5. Transfer to a 100 ml volumetric flask after it is cooled, dilute
to the mark with distilled water and shake evenly for later
use.
3) Distillation
1. Weigh 10 mL boric acid of 20 g/L
2. Put it in a 100 mL conical flask, add five drops of mixed
indicator, immerge the top of the condenser tube in the solution,
3. Take 5 mL of the above digestion solution accurately, move to
reaction tube through funnel D, then add 10 mL sodium
hydroxide of 400 g/L
4. Clean the funnel D repeated with a little distilled water, close nip
G and add a few milliliters of distilled water in funnel D for the
purpose of closing tube.
5. Heat bottle A (dilute sulfuric acid shall be added a drop by drop
into the distilled water in the bottle so as to keep its acidity) and
distil the vapor.
6. When the boric solution starts to become cyan from wine red,
keep distilling for 10 min, lift the top of the condenser tube from
the solution, make the vapor continue to wash for 1 min, drip-
washing the top with a little distilled water and stop distillation.
4) Titration: -
1. The absorption solution shall be titrated with 0.01
mol/hydrochloric acid standard solution.
2. When the color changes from cyan to grey purple, the end point
has been reached.
Calculation with units of
expression The protein content in royal jelly is calculated by
(V1 – V0) x c1 x 0.014
X3= x 6.25 x 100
m4 x 5/100
where
X3 = is the protein content in royal jelly, given by mass fraction, %;
V1 = is the volume of 0.01 mol/L hydrochloric acid standard solution
consumed when the sample is titrated, in millilitres;
V0 = is the volume of 0.01 mol/L hydrochloric acid standard solution
consumed when blank titration is made, in millilitre;
c1 = is the concentration of hydrochloric acid solution, in mol/L;
0.014 = is the millimol mass of nitrogen, in grams;
m4 = is the mass of sample, in grams;
6.25 = is the coefficient of protein conversed from nitrogen.
Inference NA
(Qualitative Analysis)
Reference IS/ISO 12824:2016
Approved by Scientific Panel on Methods of Sampling and Analysis

77 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Sugar (Titration Method)

Method No. FSSAI 04B.035:2024 Revision No. & Date 0.0

Scope This method is applicable for Royal Jelly
Caution Properly mix the sample before take it and no bubble shall be there.
Principle This method is involving the reduction of solution A and Solution B
by titration at boiling point against a solution of reducing sugar in
honey by using methylene red as internal indicator last faded blue color
of the sample noted as final reading to calculate the Sugars.
Apparatus/Instruments Electric-headed thermostatic water bath: (±1°C).
Analytical balance: (0.0001g)
Materials and Reagents 1. Glucose standard Alkaline cupric tartrate solution A,
2. Alkaline cupric tartrate solution B,
3. Zinc acetate solution,
4. Potassium ferrocyanide,
5. Hydrochloric acid
6. Hydrochloric acid
7. Sodium hydroxide Methyl red indicator.
Preparation of Reagents 1. Glucose standard solution: Weigh accurately 1000 g pure
glucose (specific rotation is +52.5 ～+53°) with constant
weight after it is dried at the temperature from 98°C to 100°C,
dissolve with distilled water and add 5 mL hydrochloric acid
(c=6 mol/L) and dilute to 1000 mL with distilled water.
2. Alkaline cupric tartrate TS solution A: Dissolve 15 g copper
sulfate (CuSO4.5H2O) and 0.05g methylene blue, in 1000 mL
water, and store in a tightly stoppered bottle.
3. Alkaline cupric tartrate TS solution B: Weigh 50 g
potassium sodium tartrate and 75 g sodium hydroxide, dissolve
with distilled water, add 4 g potassium ferrocyanide, dilute to
1000 mL with distilled water when it is dissolved completely
and store in a tightly stoppered polyethylene plastic bottle.
Calibration of alkaline cupric tartrate TS solution: weigh
accurately 5 mL respectively from alkaline cupric tartrate TS
solution A and B, put them in 150 mL conical bottles, add 10
ml distilled water, add approximately 9 ml glucose standard
solution from burette, heat to the boiling point within 2 min and
keep adding glucose standard solution at the speed of one drop
per 2 s when it is boiling. The end point is reached when the
blue colour of the solution has just faded. Record the total
volume of the glucose standard solution consumed, operate
three times in parallel at the same time, take the mean value
and calculate the mass (mg) of the glucose equivalent to 10 ml
(5 ml per respectively from solution A and B) of alkaline cupric
tartrate TS solution.
4. Zinc acetate solution, ⍴ = 219 g/L. Weigh 21.9 g zinc acetate,
add 3 mL acetic acid, dissolve with distilled water and dilute to

78 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 100 mL.
5. Potassium ferrocyanide, ⍴ = 106 g/L.
6. Concentrated hydrochloric acid, w = 36 % ~ 38 %.
7. Hydrochloric acid, c = 6 mol/L. Weigh 50 ml hydrochloric
acid, add distilled water and dilute to 100 mL.
8. Sodium hydroxide solution, ⍴ = 200 g/ L Methyl red indicator,
⍴ = 1 g/L, ethanol solution.
Sample Preparation 1. Weight approximately 4 g of royal jelly sample; put it in a 100
mL volumetric flask.
2. Add 50 mL distilled water; shake till dissolution of the sample.
3. Then add 5 mL zinc acetate solution and potassium sodium
tartrate respectively and slowly, dilute to the mark with
distilled water, and mix evenly.
4. Allow standing for 30 min and filtrating with dried filter paper,
discard a few milliliters of initial filtrate. The filtrate is for later
use.
Method of analysis 1. Take accurately 50 mL of the above filtrate, put it in a 100 mL
volumetric flask, add 10 mL hydrochloric acid (c = 6 mol/L),
mix evenly, put it in an electric-heated thermostatic water bath,
hydrolyze for 10 min at the temperature from 68 °C to 70 °C,
leave it to room temperature by cooling with flowing water,
add two drops of methyl red indicator and mix evenly,
neutralize with sodium hydroxide (p = 200 g/L) until the
solution becomes yellow and dilute to the mark with distilled
water and mix evenly, which serves as sample solution and is
prepared for later use.
2. Take accurately 5 mL of alkaline cupric tartrate TS solution A
and B respectively.
3. Put them in 150 mL conical bottles, heat to the boiling point
within 2 min, at a speed that is fast at first and slow later.
4. Add sample solution drop by drop from the burette and keep
the solution in boiling state.
5. When the solution colour starts to lose, titrate at the speed of
one drop per 2 seconds.
6. The end point is reached when the colour blue has just faded.
7. Record the volume of the sample solution consumed.
Calculation with units of The total sugar content in royal jelly is calculated by:
expression

Where,

X4 is the total sugar content (counted by glucose), given by mass

fraction, %;
T is the titre value of alkaline cupric tartrate TS, the mass of which
10 ml alkaline cupric tartrate TS (5 ml
respectively from solution A and B) equals to glucose, in

79 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 milligrams;
m5 is the mass of the sample, in grams;
V2 is the volume of sample solution consumed in titration, in
milliliters.
Inference NA
(Qualitative Analysis)
Reference IS/ISO 12824 : 2016
Approved by Scientific Panel on Methods of Sampling and Analysis

80 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Sugars : Fructose, Glucose, Sucrose, Erlose,
Maltose and Maltotriose (by HPLC : Reference Method)

Method No. FSSAI 04B.036:2024 Revision No. & Date 0.0

Scope This method is applicable for the determination of Fructose, Glucose,

Sucrose, Erlose, Maltose, Maltotriose in Royal Jelly

Caution Properly mix the sample before take it and no bubble shall be there.

Principle Sugar is extracted from the sample by mixing it with methanol & water
and the supernatant collected after centrifugation is analysed on RID
detector for various Sugars estimation.

Apparatus/Instruments 1. HPLC with refractive index detector (RID)

2. Column: Amino modified phase
3. Ultrasonic bath
4. Centrifuge
5. Analytical balance (0.00001 g)

Materials and Reagents 1. Acetonitrile (HPLC grade)

2. Methanol (HPLC grade)
3. Ultra pure water
4. Sugar standards (≥98.0 % purity)

Preparation of Reagents Standard (M): Weigh exactly the sugar standard in order to obtain in
anhydrous sugar concentration of 1g/100mL. Transfer in a 100mL flask.
Add around 25mL of water and stir. Make up the volume with methanol.
F1: Dilute 10mL of solution M in a 20mL volumetric flask with a
mixture MeOH/H2O:75/25
F2: Dilute 5mL of solution M in a 20mL volumetric flask with a mixture
MeOH/H2O:75/25

Sample Preparation 1. Weigh accurately and approximately 2 g of royal jelly in a

beaker.
2. Add some milliliters of a solution MeOH/H2O: 75/25 under
magnetic stirring
3. Transfer in a 20 mL volumetric flask and complete with the
same solution MeOH/H2O
4. Centrifuge for 10 min at a speed of 4000 rpm.
5. Filter the supernatant before chromatographic injection.

Method of analysis 1. Mobile Phase : Acetonitrile : water (75:25)

2. Flow: 1 mL/min
3. Column Temp. : 30⁰C

Calculation with units of The concentration of the sugar i in sample is calculated using formula:
expression
Ci = ki + Ai

81 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Where
Ci is the concentration of the sugar I in sample; in mg/mL;
ki is the response factor of sugar i, which is calculated from the slope
of the calibration curve constructed by the area against concentration of
the standard solutions (M, F1, F2);
Ai is the area of sugar i in sample.
Total sugar in royal jelly is calculated using formula:
%Sugar i = Ci × 20/m × 100
Where
%Sugar i is the percentage of the sugar i in royal jelly;
Ci is the concentration of the sugar i in sample; in mg/mL;
M is the mass of the sample, in mg.

% Total sugar = % Sugar (Fructose + glucose+ Sucrose)

Inference NA
(Qualitative Analysis)

Reference IS/ISO 12824 : 2016

Approved by Scientific Panel on Methods of Sampling and Analysis

82 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Sugar : Fructose, Glucose, Sucrose, Erlose,
Maltose and Maltotriose (By Gas Chromatography)

Method No. FSSAI 04B.037:2024 Revision No. & Date 0.0

Scope Royal Jelly

Caution Properly mix the sample before take it and no bubble shall be there.

Principle Sugar is extracted from the sample by mixing it with Pyridine,

hexamethyldisilazane and Trimethylchlorosilane. and analyzed on FID
detector for various Sugars estimation.

Apparatus/Instruments 1. GC with flame ionization detector

2. Chromatographic column HP5-MS column (30 m x 0.25 mm x
0.25µm)
3. Analytical balance (0.00001 g)

Materials and Reagents 1. Hexamethyldisilazane (≥ 99% purity)

2. Trimethylchlorosilane (≥ 99% purity)
3. Pyridine (≥ 99.8 % purity) (anhydrous pyridine is obtained by
distillation over calcium hydride)
4. Sorbitol (internal standard) (≥ 99% purity)

Preparation of Reagents Anhydrous pyridine is obtained by distillation over calcium hydride.

Sample Preparation 1. Weigh accurately about 40 mg of lyophilized royal jelly and 1

mg of sorbitol.
2. Introduce them in a glass reactor and close tightly.
3. Then add 1 mL of anhydrous pyridine. Stir the mixture for 5
minutes with the reactor sealed.\
4. Then add 200 µl of hexamethyldisilazane and stir the mixture for
5 minutes.
5. Add 100 µL trimethylchlorosilane and stir for 30 minutes.
6. Leave the mixture for 20 h at room temperature with the reactor
sealed.

Method of analysis 1. Helium as carrier gas (5.0 grade) constant pressure of 22 psi
2. Injection volume: 2 µL
3. Injection and detector temperatures set at 280 ⁰C
4. Program of oven temperature: Maintain initial temperature (150
⁰C) for 5 minutes, then increase to 325 ⁰C at a rate of 3 ⁰C/min
5. Maintain the final temperature for 10 min.
6. Use reference standards or retention indics to identify the
different sugars. Determine the retention indics of each sugar by
injecting the standard with the same analytical and
chromatographic conditions.

Calculation with units of 1. Sugar Quantification-Determination of correction factor:

83 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 expression Sugar is quantified by internal standard (Sorbitol). A response
factor or mass correction factor is calculated for each sugar by
following fomula:
ki = ASI/Ai × Mi/MSI
Where, ki is the response factor of the sugar i
ASI is the area of the internal standard
Ai is the area of the standard of sugar i
MSI is the mass of the internal standard
Mi is the mass of the standard of sugar i
2. Calculations: The mass of the sugar I in the royal jelly sample is
calculated using formula.
Mi = ki ×Ai / ASI × mSI
where mi is the mass of the sugar I in the royal jelly sample in mg;
ki is the response factor of sugar I;
ASI is the area of the internal standard;
Ai is the area of sugar I in the royal jelly sample’
MSI is the mass of the internal standard, in mg.
The percentage of the sugar I in the royal jelly is calculated using the
formula:
% sugar I = % MS × mi / m sample
Where
mi is the mass of the sugar I in the royal jelly sample in mg;
m sample is the mass of the royal jelly sample, in mg;
%MS is the dry matter percentage.

Inference NA
(Qualitative Analysis)

Reference IS/ISO 12824 : 2016

Approved by Scientific Panel on Methods of Sampling and Analysis

84 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Total Acidity

Method No. FSSAI 04B.038:2024 Revision No. & Date 0.0

Scope Royal Jelly
Caution Properly mix the sample before take it and no bubble shall be there.
Principle The total acidity is the sum of the free acidity and the lactone acidity.
The total acidity is obtained by adding an excess of sodium hydroxide
to the honey solution and the end point is achieved when the pH-meter
indicates at pH 8.3.
Apparatus/Instruments 1. pH-meter pH value,to the nearest 0.1
2. Burette,10 mL
3. Analytical balance,capable of weighing to the nearest 0.0001 g.

Materials and Reagents Sodium hydroxide

Preparation of Reagents Sodium hydroxide,c = 0.1 mol/L
Sample Preparation Homogenize the sample before weigh
Method of analysis 1. Weigh 1.0 g royal jelly sample.
2. Put it in a 100 mL beaker, and add 75 mL boiled and cooled
distilled water.
3. Titrate with sodium hydroxide standard solution (c = 0.1
mol/L).
4. The end point is achieved when the pH-meter indicates at pH
8.3.
Calculation with units of The millilitre quantity of sodium hydroxide standard solution consumed
expression in titration is multiplied by the concentration value (mol/L) and divided
by the mass of sample, and then multiplied by 100. The acidity of
sample is determined.
Acidity [(1 mol/NaOH) ml/100 g] = (V x c x 100)/m
Where
V = is the volume of 0.1 mol/L NaOH standard solution consumed in
titration, in millilitres;
C = is the concentration of NaOH standard solution, in mol/L;
M = is the mass of sample, in grams.
Inference NA
(Qualitative Analysis)
Reference IS/ISO 12824 : 2016
Approved by Scientific Panel on Methods of Sampling and Analysis

85 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Total Lipid

Method No. FSSAI 04B.039:2024 Revision No. & Date 0.0

Scope Royal Jelly

Caution Properly mix the sample before take it and no bubble shall be there.

Principle Fat extractor uses the Diethyl ether reflux and siphon principle to
continuously extract the solid matter, and calculate the fat by difference
between the initial and final weight of beaker.

Apparatus/Instruments 1. Soxhlet extraction apparatus, with soxhlet extraction tube

(internal diameter ca. 40 mm), extraction bottle and condenser
tube.
2. Thimble Filter, of internal diameter 25 mm to 30 mm, length
100 mm to 120 mm.
3. Thermostatic bath
4. Drying Oven
5. Vaccum drying oven.

Materials and Reagents 2) Diethyl ether

3) Celite

Preparation of Reagents 1) Diethyl ether, of purity above 99.5%. Or use tert-buthylmethyl

ether (TBME) as alternative extraction solvent.

Sample Preparation Homogenize the sample before weighing.

Method of analysis 1. Weigh accurately approximately 2.5 g of royal jelly sample in a

beaker and add 3 g to 5 g of Celite.
2. Mix the sample and Celite well with a glass rod until the
mixture is equalized.
3. Transfer the mixture from the beaker to thimble filter and wipe
carefully the beaker and the glass rod with defatted cotton
impregnated with diethyl ether
4. Put the defatted cotton into upper half of thimble filter.
5. Dry in air the thimble filter until the smell of diethyl ether has
gone.
6. Dry the thimble filter for 2 h at 70 °C under the pressure in
vaccum drying oven.
7. Add 100 mL to 150 mL diethyl ether into an extraction bottle
which is dried until a constant weight, put the thimble filter into
extraction tube, and connect the extraction tube to a condenser
tube and the extraction bottle.
8. Extract lipid on a thermostatic bath at approximately 50 °C for
8 h.

86 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 9. After extraction, take the thimble filter out of the extraction
tube, evaporate almost all the diethyl ether in the extraction
bottle and completely evaporate it by evaporator or nitrogen
gas.
10. Wipe the outside of the extraction bottle.
11. Dry it in a drying oven at 105 °C for 1 h and weigh it after
cooling in a desiccator for 1 h.

Calculation with units of The total lipid in royal jelly is calculated by

expression
m7 – m6
X5 = x 100
m8
where
X5 = is the total lipid content, given by mass fraction, %;
m6 = is the mass of the extraction bottle which is dried until the constant
weigh, in grams;
m7 = is the mass of the extraction bottle after extraction and drying, in
grams;
m8 = is the mass of the sample, in grams.

Inference NA
(Qualitative Analysis)

Reference IS/ISO 12824 : 2016

Approved by Scientific Panel on Methods of Sampling and Analysis

87 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of δ13C/ δ12C Isotopic Ratio

Method No. FSSAI 04B.040:2024 Revision No. & Date 0.0

Scope Royal Jelly

Caution Properly mix the sample before take it and no bubble shall be there.
Samples must be folded properly in the tin capsule to avoid sample
leakage and air trap.

Principle Sample injected into the Elemental analyzer (EA) is combusted and
oxidized and the CO2 produced from combustion of the bulk royal jelly
is quantified in the form of carbon isotopic value of 13C/12C ratio by Ion
ratio mass spectrometer (IRMS)

Apparatus/Instruments 1. Elemental Analyzer

2. Ion ratio mass spectrometer (IRMS)
3. Analytical balance (0.00001g)
4. Tin capsules
5. Blunt ended forceps

Materials and Reagents 1. Chromium Oxide

2. Cobaltous/Cobaltic Oxide

Preparation of Reagents NA

Sample Preparation 100 to 1000 µg of royal jelly are loaded into a tin (or silver) capsule.

Method of analysis 1. Samples are dropped from a carousel-type auto sampler into a
reactor filled with chromium oxide and cobaltous /cobaltic
oxide.
2. Automated oxygen dosing ensures complete combustion of the
sample. Subsequent to combustion, NOx compounds are
reduced to N2 in a reactor filled with reduced copper.
3. All gas species are carried in a continuous helium stream and
separated on an isothermal GC column.H2O and SOx species
are removed by adsorption.

Calculation with units of The CO2 produced from combustion of the bulk royal jelly is analysed
expression for the 13C/12C ratio in a dedicated isotopic ratio mass spectrometer.

Inference NA
(Qualitative Analysis)

Reference IS/ISO 12824 : 2016

88 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 Determination of Furosine

Method No. FSSAI 04B.041:2024 Revision No. & Date 0.0

Scope Royal Jelly

Caution Properly mix the sample before take it and no bubble shall be there.

Principle Acid hydrolyzed sample is loaded on conditioned SPE cartridge and

finally eluted with hydrochloric acid and injected into the instrument for
final quantification.

Apparatus/Instruments 1. HPLC with UV detector (or DAD)

2. Analytical balance (0.00001 g)

Materials and Reagents 1. Sodium Acetate

2. Acetic Acid
3. Hydrochloric acid (HCl)
4. Syringe-tip filter: 0.45 µm PTFE seal or equivalent.
3. SPE cartridge: C18, 500 mg (SPE-PAK cartridge) or
equivalent.
4. Column: Reverse phase C-8, 25 cm x 4.6 mm, 5 µm or
equivalent
5. Vial: Amber glass vial

Preparation of Reagents 1. 0.06 M/L Sodium Acetate, pH 4.3 with acetic acid: 4.92g
Sodium acetate in 1000 mL of water.
2. 3 M/L HCl: Take 250 mL of 12 M HCl and make up with 1000
ml distilled water
3. 8 M/L HCl: Take 666 mL of 12 M HCl and make up with 1000
ml distilled water

Sample Preparation 1. An aliquot of sample (0.35 g) corresponding to about 30mg to

70mg of protein, is hydrolyzed with 8ml of 8 M/L HCl at 110
°C for 23 h.
2. After hydrolysis, collect 0.5 mL of hydrolysate.
3. SPE C18 cartridge conditioning: Conditioning the SPE
cartridge with 5 ml methanol followed with 10 mL ultrapure
water.
4. Load 0.5 mL hydrolysate sample on the SPE C18 cartridge.
5. Discard the eluate and Dry the cartridge in air.
6. Elute 1 mL x 4 of HCl 3M/L
7. Collect all the eluate in a 5 mL volumetric amber glass and
make up with 5 mL 3 M/L HCl solution.
8. Filter with syringe-tip filter (0.45µm) in amber glass vial.

89 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
 9. Inject on 50µl in a HPLC for analysis.
Protein Determination
Follow the method FSSAI 04B.033:2024/FSSAI 04B.034:2024

Method of analysis 1. Mobile phase:0.06 M/L sodium acetate, pH 4.3 acetic acid
2. Flow: 2 mL/min
3. Column Temperature: 30 °C
4. Detector: UV-280 nm
5. Injection volume:20 µL to 50 µL

Calculation with units of Quantification the Furosine by external calibration standard and express
expression the value as:
Furosine = mg Furosine/100g protein

Inference NA
(Qualitative Analysis)

Reference IS/ISO 12824 : 2016

Approved by Scientific Panel on Methods of Sampling and Analysis

90 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS
RAPID ANALYTICAL FOOD TESTING (RAFT) KIT/ EQUIPMENT

Alternate Rapid kits/equipments may be used to get quick results for screening and surveillance
purposes, provided the kit/equipment is approved by FSSAI. Details of the rapid food testing
kit/equipment approved by FSSAI are available at https://www.fssai.gov.in/cms/raft.php.

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92 | MANUAL OF METHODS OF ANALYSIS OF FOODS- HONEY & OTHER BEE HIVE PRODUCTS