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Report on Training Program on Method of Analysis of Fortificants in Cereals 19th June – 21st June 2019

Mysore, Karnataka Three-day training program was organised by Food Safety & Standards Authority of India (FSSAI) and Food Fortification Resource Centre (FFRC) for Food Analysts and laboratory personnel of FSSAI notified National Accreditation Board for Testing and Calibration Laboratories (NABL) on method of analysis of fortificants in cereals (iron, folic acid, vitamin B12) from 19th June – 21st June 2019 at CSIR-CFTRI, Gurgaon. On 10th May, FSSAI issued a letter seeking nomination from laboratories across the country. A total of fifteen FSSAI notified NABL labs from across India including the private and state food labs were shortlisted for the training. Participants from fifteen labs and one representative from FFRC attended the training. A list of participants in attached at Annexure 1.

Training Program In the past, it was reported that there was a considerable variation between the results in testing of fortificants when tested out at different labs. Therefore, need for harmonisation of procedures was generated. In this regard, the training was conducted to equip the lab personnel with requisite testing protocol of iron, folic acid and vitamin B12 in cereals so that they can adopt a common method and procedure in their labs. Training Schedule Day 1: 19th June: Training started with the inauguration and greeting of all the participants. A brief introduction about the history, strengths, certifications and activities of CFTRI was presented by Dr. Alok Srivastava (Head-Food Safety & Analytical Quality Control Laboratory). A brief introduction on Food Fortification under FSS Act, explaining the Fortification Regulation, need of fortification,

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quality assurance and surveillance was given by Dr. Alok and FFRC representative. Each day was given to one fortificant, starting with a lecture session followed by the practical. First day estimation of iron was performed as per the step by step procedure as attached at Annexure 2. It was assured that each participants have a hands on experience of sample extraction and use of microdigestor. After sample extraction, standard and extract was run through AAS and results were noted. Based on the readings and calculation, results achieved were 95% correct.

Day 2: 20th June The day started with the tour of the wheat flour milling plant (ISMT, CFTRI) and then lecture on the wheat flour fortification process. Thereon, the participants were given lecture on folic acid and vitamin B12 analysis. They were divided into two batches, one for folic acid and the other for Vitamin B12 analysis. For folic acid group, participants were given the tour of the lab and were explained on the use of immunoaffinity columns. Participants were given three samples: wheat flour, fortified wheat flour and premix. Sample preparation for extraction of folic acid in these samples was initiated. The step by step procedure as attached at Annexure 2 was followed. It was assured that each participants have a hands on experience of sample extraction. After sample extraction, standard and extract was run through HPLC. The day closed with all having hands on experience of HPLC. In the meantime, participants were given a tour of the pilot plants instument labs. Day 3: 21st June The day started with the recap of the day 2 and readings of vitamin B9 were taken. It was followed by laboratory testing and hands on experience on the method of analysis of Vitamin B12 in cereals. Sample extracts of rice flour, fortified rice flour and premix were prepared as per the AOAC method (attached at Annexure 2).

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The analysis concluded with the completion of sample preparation and running the extracts on HLPC instrument. A valedictory function was conducted and chaired by Mr. Raghavarao KSMS, Director, CFTRI and Dr. Bhaskar, Advisor, QA, FSSAI. The training closed with distribution of participation certificates and vote of thanks. Open House Discussion

The training included question and answer round after each lecture and session which are summarised

as follow:

Difference in results of testing of Vitamins was discussed where conditions such as

transportation, storage, sample handling and temperature conditions play an important role in

micronutrient retention. It was concluded by the laboratory participants that as per CODEX,

80-120% of recovery is acceptable

Participants raised the queries on chemicals being used (grade, suppliers, purity, chemical

composition), calculation and preparation of standard solution. Lot of queries was also raised

on using of AAS, HPLC, UHPLC including the solvent, columns, software, graph formation

and calculation.

Precautionary steps to be taken care while doing the analysis of fortificants were discussed

which are as follows:

a. Single trained analyst should handle a single sample at a time

b. Step by step addition and mixing of chemicals and water should take place

c. Accuracy in extraction and transfer of the chemicals/extract should be maintained

d. High precision in volume making up and extraction of samples is required

e. Sensitivity of testing should be noted and required precautions need to be taken

f. Correct formula and calculation methods needs to be applied

The questions/queries raised by participants on calculations, graph interpretation and recovery

factor while calculating the values of vitamins and minerals were answered by the faculty

members

All the participants of labs to perform the tests based on the training at their labs and share

any concerns regarding it by end of July with FSSAI and CFTRI.

Outcomes

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All the participants from fifteen labs got the hands on training on the iron, folic acid and

vitamin B12 analysis including the sample preparation and using HPLC & AAS. They agreed

to adopt the procedure as demonstrated.

Way Forward

FFRC/FSSAI to upload the report of fortificants in cereals on the website of FSSAI and

FFRC after necessary approvals

CFTRI to share the revised manual capturing all the necessary details of the procedure,

principle and results

FFRC to prepare manuals on fortificant testing using the harmonised procedure as shared by

CFTRI

FFRC/FSSAI to conduct more trainings on method of analysis of fortificants in the five

commodities

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Annexure 1 List of participants

1. Ms. Sakshi Jain, FFRC, FSSAI 2. Mr. Abhilash Babu G., Government Analyst Laboratory, Thiruvananthapuram 3. Ms. Kavitha Chethan, TUV SUD South Asia Pvt. Ltd., Bangalore 4. Mr. Kiran R. Dodtale, Envirocare Lab Pvt. Ltd. Thane 5. Dr. B. Vijaykumar, Export Inspection Agency-Kochi Laboratory 6. Mr. Jithin C.R., Geochem Laboratories Pvt. Ltd., Alappuzha, Kerala 7. Dr. Shalini Sharma, Eurofins Analytical Services, Bangalore 8. Ms. Jaya P.A., Biochemistry and Nutrition Division, Central Institute of Fisheries

Technology, Kochi 9. Mr. Ripu Daman Kumar, Composite Testing Laboratory, Solan, Himachal Pradesh 10. Mr. S Sundarambal, Department of Food & Drug Testing, Puducherry 11. Mr. Sheshagiri B.G., Divisional Food Laboratory, Mysore 12. Dr. Chetan T.P., Public Health Institute, Bangalore 13. Mr. Jaydevsing Nirmalsing Rana, Food and Drugs laboratory, Vadodara 14. Ms. Vijaya Lakshmi Kandregula, JNTUK, School of Food Technology, Kakinada 15. Mr. Amitava Chatterjee, Dept. of Health & Welfare, Kolkata 16. Ms. Vrushali S Shirke, Export Inspection Agency- Mumbai (Pilot Test House)

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Annexure 2:

FSSAI TRAINING PROGRAMME ON

METHOD OF ANALYSIS FOR FORTIFICANTS IN

CEREALS

Organized by

CSIR-CFTRI, Mysuru & FSSAI, New Delhi on

June 19 - 21, 2019

at Food Safety & Analytical Quality Control Laboratory,

CSIR-Central Food Technological Research Institute, Mysuru

Karnataka – 570020

FSSAI TRAINING PROGRAMME ON

Method of Analysis for Fortificants in Cereals

19.06.2019 – 21.06.2019

TABLE OF CONTENTS

1. Program overview 3

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2. Determination of Iron in fortified cereal using AAS 4

3. Determination of Vitamin B9 (Folic acid) in fortified cereal using HPLC 9

4. Determination of Vitamin B12 in fortified cereal using HPLC 12

FSSAI TRAINING PROGRAMME ON

Method of Analysis for Fortificants in Cereals

19.06.2019 – 21.06.2019

Venue: FSAQCL Lecture Hall L = Lecture P = Hands-on Practical

Date/Day Time Topic L/P Faculty

19.06.2019

WEDNESDAY

09:15 Registration

09.30 Food Fortification under Food Safety & Standards Act

L Alok Kumar Srivastava

11.00 Mineral Fortificants: AAS & ICP- Analytical Principles

L Devendra J Haware

11.30 Iron Analysis in cereals P Siva Shankara Reddy S

Sushmita

14.00 Iron Analysis in cereals – Continued

P -do-

20.06.2019

THURSDAY

09:30 Flour Fortification : Model Pilot Scale Demonstration

P Suresh D Sakhare

10.00 Vitamin (Folic acid & Vit. B 12): Analytical Principles

L Prasanna Vasu

11.00 Folic acid analysis in Cereals

P Mathen Mathew

Pooja D

14.00 Folic acid analysis in Cereals – Continued

P -do-

21.06.2019

WEDNESDAY

09:30 Vitamin B 12 analysis in Cereals P Kannan R

Asha Mary Joseph

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Harshitha

14.00 Vitamin B 12 analysis in Cereals – Continued

P -do-

16.30 Certificate Distribution and Valedictory Function

1.0 Title: Determination of Iron in fortified cereal using Atomic absorption

spectrophotometric method (AAS)

2.0 Principle: Organic matrix is destroyed by dry ashing in muffle furnace. Remaining ash is

dissolved in diluted acid and analyte is determined by atomic absorption spectrophotometry

(dry ashing). Products are digested with HNO3 and H2O2 (if required) under pressure in a

closed vessel heated by microwaves. Solution is diluted with H2O. Fe are determined by FAAS

(wet ashing).

3.0 Requirements:

3.1 Apparatus, glassware and consumables

3.1.1 Glassware- thoroughly clean all glassware by soaking overnight in 20% HNO3

(V/V). Rinse all glassware 3x with distilled- deionized or 18MΩ resistance H2O.

3.1.2 Evaporation dish- 100mL unetched Vycor (or Pt) flat-bottom, with pour spout;

capable of withstanding temperatures to 600 0C.

3.1.3 Atomic absorption spectrophotometer- With air–acetylene burner. Equipment

should be well maintained with good response per unit concentration, e.g., 0.200

abs or above 4 mg/L Cu.

3.1.4 Furnace- with pyrometer to control temperature range of 200-600 ± 10 0C.

3.1.5 Hollow cathode or electrodeless discharge lamps.—For Fe.

3.1.6 Microwave digestor

3.1.7 Drying oven.

3.1.8 Teflon digestion vessels -100 mL, withstanding a pressure of at least 1.4 MPa.

3.1.9 Volumetric flasks - 25, 50, 100, 250 and 1000 mL.

3.1.10 Funnels - Glass.

3.1.11 Plastic bottles - e.g., Polystyrene bottles with tightly fitting lids, 500 mL.

(All glassware and plasticware should be carefully cleaned and rinsed, e.g., with HNO3

or HCl, in order to avoid metal contamination).

3.2 Solvents and Reagents

(Reagents should be of at least analytical reagent grade (p.a.), preferably ultrapure

(suprapure) or equivalent).

3.2.1 Water - distilled or deionized, ≥18 MΩ⋅cm, for preparation of standard or test

solutions.

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3.2.2 Nitric acid – Unless specified otherwise, use redistilled or ultrapure.

3.2.3 Standard stock solution- commercially prepared, certified AA standard

(see2.6.01) for Fe, 965.09B €

3.2.4 Filter pulp- analysed ash-free.

3.2.5 Nitric acid- 1% dilute 10 mL concentrated HNO3, with water to 1 L.

3.2.6 Hydrogen peroxide - 30% (w/w), if required

3.2.7 Iron standard solution.—1 mg/mL. Dissolve 1.000 g Fe in 14 mL water + 7 mL

nitric acid, (b), in 1 L volumetric flask. Dilute to volume with water.

4.0 Procedure:

4.1 Cleaning procedure:

4.1.1 For glass and plasticware - Acid solution: 500 mL concentrated HNO3, + 4500

mL deionized water. Wash first with water and detergent. Rinse with tap water,

followed by deionized water, then with acid solution. Finally rinse 4–5 times with

deionized water.

4.1.2 For Teflon digestion vessels - Rinse with acetone, wash with deionized water,

keep vessels covered with 0.1M HNO3, for at least 30 min, rinse with deionized

water, and let vessels dry. Use separate vessels for different applications,

depending on the concentration of metals. If, however, the same digestion vessels

are used for heavily contaminated products, e.g., sludge, it may be necessary to

use a more severe cleaning procedure, e.g., heating vessels together with

concentrated HNO3. The instrument manual usually provides detailed instructions

for such cleaning procedures.

4.2 Pre-treatment - If product is to be analyzed fresh, proceed to Homogenization.

Otherwise, continue at Drying.

4.3 Drying - Dry to constant weight in drying oven at 105°C, or freeze-dry. Freeze-drying

is usually preferable because it renders the product less compact and easier to

homogenize. If final result is based on fresh weight, weigh test portion before and after

drying to obtain water content:

H2O = (Wf – Wd/ Wf ) ×100

where H2O, % = water content of the test portion (%); Wf = weight of the test portion

(g); Wd = weight after drying (g).

4.4 Homogenization - Homogenize products using non contaminating equipment. Check

for leached metals if the apparatus consists of metal parts.

5.0 Sample Preparation:

5.1 Ashing:

5.1.1 Wet ashing (Microwave Digestion) - Weigh 0.2–0.5 g dry material into

digestion vessel. Add 4 - 10 mL HNO3. Close vessels, place vessels in holder,

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place vessel holder in microwave oven, and close door. Set oven program

according to the parameters and start program. If water-containing materials are

used, maximum weight is restricted to 2 g, but dry matter content must never

exceed 0.5 g. For example, if product has a water content of 50%, take a

maximum of 1 g (= 0.5 g dry matter). If a product has a water content of 95%,

take 2 g (Remove digestion vessels from microwave oven and let cool

thoroughly before opening them. Open vessel and rinse down lid and walls into

container. Transfer solution to 25 mL volumetric flask and dilute to mark with

deionized water. Then, transfer solution to plastic container. Treat blanks in the

same way as tests. One blank should be included in every set.

5.1.2 Dry Ashing:

(Note: For liquid formulas, shake container before weighing).

Place test portion in previously cleaned Vycor evaporating dish (which may

contain 5g filter pulp for ease of handling.) Exact amount of composite required

will depend on concentration for mineral present. (For powders take ≥1.5 g). In

general 25mL will be adequate. If some minerals, in particular Fe, Cu or Mn, are

at very low levels, a larger aliquot (≤50mL) may be necessary.

5.2 Dry aliquots in 100 0C oven overnight or in microwave oven (programmed over ca 30

min). when dry , heat on hot plate until smoking ceases, and then place dish in 525 0C

furnace (carefully avoiding ignition ) for minimum time necessary to obtain ash that is

white and free from C, normally 3-5h, but ≤8h. Remove dish from furnace and let cool.

Ash should be white and free from C. if ash contains C particles (i.e., it is gray), wet

with H2O and add 0.5-3 mL HNO3. Dry on hot plate or steam bath and return dish to

525 0C furnace 1-2 h.

5.3 Dissolve ash in 5 mL 1M HNO3 warming on steam bath or hot plate 2-3 min to aid in

solution. Add solution to 50 mL volumetric flask and repeat with 2 additional portion

of 1M HNO3. Dilute to 50 mL with 1M HNO3. (Note: additional dilution may be

necessary to bring concentrations within the linear range of instrument.)

5.4 Dilution- If test solution needs to be further diluted (due to high metal concentrations),

dilute with 1% HNO3, in order to maintain same acid concentration prior to metal

determination (g).

5.5 High acid concentration is environmentally undesirable and may depress the analytical

signal. Reduce acid strength by diluting the test solution 1/2 with 0.1M nitric acid and

standard solutions 1/2 with 3M nitric acid. The tests and standards are thereby brought

to the same acid concentration. Matching of acid concentrations is important when a

calibration curve is used.

6.0 Determination:

6.1 Prepare calibration curve (concentration vs absorbance) for each mineral to be

determined, using wavelength and flame specified. Optimize flame parameters in

accordance with instrument manufacturer’s instructions. Prepare solutions for

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calibration of instrument to cover linear range of calibration curve. See instrument

instruction manual.

6.2 Assay portions in similar manner. Determine concentration of each mineral from its

calibration curve, and calculate concentration in test sample, taking into account test

portion size and dilutions.

6.3 Measurements must be within the linear range when the method of standard addition is

used. A standard addition curve consists of at least 3 points, of which at least 2 are

standards. The concentration of the highest standard should be 3–5 times the

concentration in the test solution. The lower standard should have a concentration

approximately half of the highest standard. A simplified version of the method of

standard addition is to use a matrix-matched standard curve, which is applicable to

products with the same matrix: The test and standard solutions are mixed and used to

make a standard addition curve. This curve is then parallel transferred to origin and is

used as the standard curve for the tests that followed and that have been diluted in the

same proportions. The matrix-matched standard curve and the test solutions will thus

have the same matrix concentration. On most modern instruments, this function is

included in the software.

6.4 Flame technique - The concentration of Zn, Cu, and Fe are usually at levels suitable for

determination by FAAS. When calibration curve is to be used, standards and test

solutions must have the same acid concentration. Since Fe may be strongly affected by

interferences from the matrix, use either the method of standard addition or matrix-

matched standards. When experiencing severe interferences, an oxidizing nitrous oxide

acetylene flame may be an alternative.

6.5 Program the autosampler to deliver a volume that gives as large an absorbance as

possible within the linear range and producing a background absorbance not larger than

approximately 0.5 absorbance units. Multiple injection may enhance the absorbance at

very low concentrations. Evaluate each new matrix by means of ash- and atomization-

curves in order to optimize the graphite furnace parameters.

7.0 Calculations and Evaluation of Results

7.1 Calculate the concentration (C) of metal in the test sample according to the formula:

C = [(a-b)df x 25]/m

where C = concentration in the test sample (mg/kg); a = concentration in the test

solutions (mg/L); df = dilution factor; b = mean concentration in the blank solutions

(mg/L); m = weight of the test portion (g).

7.2 If (a – b) is lower than the detection limit, DL, then (a – b) is replaced by DL for

calculation of the limit of detection in the test sample.

7.3 If the test solution has been diluted, the dilution factor (df) has to be taken into account.

If the test portion was dried and the result should be based on fresh weight, correct

according to the following:

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CFW = C × (100 - H2O %)/100

where CFW = concentration in the test portion corrected to fresh weight (mg/kg); H2O%

= the water content of the test portion (%).

7.4 When running replicates, the average of the results should be given with 3 significant

figures.

7.5 Detection limit.—The DL for each metal is calculated as DL = 3 × standard deviation

of the mean of the blank determinations (n = ≥20). A large number of blanks must be

analyzed before DL can be established. A DL is not static and will need to be re-

evaluated from time to time in accordance with changes in the blank levels.

References:

1) AOAC Official Method 985.35

2) FSSAI Manual of Methods of Analysis – Fortificants in food

1.0 Title: Determination of Vitamin B9 (Folic acid) in fortified cereal using HPLC

2.0 Principle: Folic acid is stable over a wide range of pH and is a relatively heat stable vitamin.

However, ascorbates may be added to the sample to prevent oxidative loss of the vitamin

throughout the sample preparation. Autoclaving of the sample and proteolytic/ amylolytic

enzymes denature proteins/ carbohydrates, inactivates endogenous enzymes and releases any

complexed vitamin. The vitamins are extracted into phosphate buffer and analysed using

HPLC.

3.0 Requirements:

3.1 Consumables/ Glassware

3.1.1 50 ml centrifuge tubes,

3.1.2 100 ml conical flasks,

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3.1.3 volumetric Flasks - 10mL, 25ml,

3.1.4 0.45µm membrane filters,

3.1.5 1 ml HPLC vials

3.1.6 Micropipettes 10-100µl, 100-1000µl capacity

3.2 Chemicals and Solvents:

3.2.1 Potassium hydrogen phosphate (PHP),

3.2.2 ascorbic acid,

3.2.3 α-amylase (30U/mg),

3.2.4 potassium hydroxide,

3.2.5 Hydrochloric Acid,

3.2.6 sodium pentane sulphonate,

3.2.7 acetic acid,

3.2.8 triethyl amine (TEA),

3.2.9 Methanol HPLC Grade,

3.2.10 Deionised water,

3.2.11 Certified Reference Material

3.2.12 Folic acid

3.3 Equipments:

3.3.1 HPLC with diode array detector,

3.3.2 Waterbath, orbital shaker,

3.3.3 Centrifuge,

3.3.4 pH-meter,

3.3.5 Incubator,

3.3.6 Vortex

4.0 Preparation of reagents and Vitamin B9 stock solutions:

4.1 Preparation of Mobile Phase: Take 1.8g sodium pentane sulphonate, add 735ml

distilled water. Add 5 mL of acetic acid, 0.9 mL TEA and make up to 1L with HPLC

methanol. Filter and degas for 15 minutes.

4.2 Preparation of standard stock solution

4.2.1 Stock solution for Vitamin B9: Transfer 25 mg of vitamin B9 in 25

mL volumetric flask, add minimum volume of 0.1N NaOH to

dissolve, adjust the pH 4.5 – 5.0 with 2.5 M sodium acetate/HCl and

make up with water, stored at 4oC in the dark.

4.2.2 Intermediate stock solution # 1 (100 µg/ml) : Transfer 1.0 ml from

stock to a 10 ml volumetric flask and make up the volume with water.

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4.2.3 Intermediate stock solution # 2 (10 µg/ml) : Transfer 1.0 mL from # 1

to a 10 mL volumetric flask and make up the volume with water.

4.2.4 Intermediate stock solution # 3 (1 µg/ml) : Transfer 1.0 mL from # 2

to a 10 mL volumetric flask and make up the volume with water.

4.2.5 Preparation of working standard solutions for vitamin B9

[Working Standards for Vitamin B9 (prepared in 1 ml HPLC vials)]

Standard

con.(ug/ml)

Vol. from intermediate

stock # 3 (ml)

Vol. of water

added (ml)

0.5 0.5 0.5

1.0 1.0 0.0

Standard

con.(ug/ml)

Vol. from intermediate

stock # 2 (ml)

Vol. of water

added (ml)

2.5 0.25 0.75

5.0 0.50 0.50

10.0 1.0 0.0

The choice of working standards may also be decided based upon the approximate

concentration of the vitamin in the sample.

5.0 Sample preparation for extraction of vitamin B9

5.1 Weigh 1 - 5g of sample into a 100 ml conical flask.

5.2 Add 100 mg ascorbic acid, 15 - 20 mL of 0.1M PHP (pH 8-9, may be adjusted with 1M

KOH), kept in orbital shaker for 60 min.

5.3 Adjust pH to 7.0 with 2 N HCl, Add 25 mg/g α-amylase

5.4 Mixture incubated at 65oC for 1 h, in a water bath, and the reaction is arrested by raising

to boiling temperature.

5.5 Cool the solution mixture to room temperature, centrifuge, supernatant is made up to 25

mL with 0.1 M PHP.

5.6 Centrifuge and filter through 0.45 µm membrane filters before HPLC injection.

6.0 Instrument Parameters

6.1 HPLC Conditions for Vitamin B9 Analysis

Column Phenomenex or Luna C18 or equivalent,

4.6 x 250mm, 5

Flow rate, Run Time 0.5 mL/min, 30 minutes

Injection Volume 10 µL

Column Oven Temperature 35 0C

Detector Photodiode Array Detector

Vitamin B2 λmax : 280 nm

Mobile phase As discussed under section 4.1

7.0 Confirmation Criteria:

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7.1 Retention time of the analyte must agree within 5% of that standard

8.0 Calculation:

Vitamin B9 (g /g) = Peak area of sample x Concentration of standard x vol. extract (ml) x dilution

Peak area of standard x weight of sample

References:

1) AOAC Official Method 2011.06

2) Ann Chromatogr Sep Tech. 2016; 2(1): 1017.

3) FSSAI Manual of Methods of Analysis – Fortificants in food

1.0 Title: Determination of Vitamin B12 in fortified cereal using HPLC

2.0 Principle: Vitamin B12 is extracted from the sample using sodium acetate buffer in the

presence of -amylase to hydrolyze starch, and sodium cyanide to convert into

cyanocobalamin (optional), at 100°C for 30 min. Extracts are passed through and

concentrated with an immunoaffinity column (IAC). Vitamin B12 (cyanocobalamin) is then

eluted from the IAC using methanol, and determined by high-performance liquid

chromatography with UV detection at A361 nm.

3.0 Requirements:

3.1 Apparatus and Materials:

3.1.1 Balances - With readability of 0.1 mg

3.1.2 Sonicator

3.1.3 Laboratory oven or water bath.

3.1.4 In-line water bath (with magnetic stirrers) or autoclave.

3.1.5 pH meter

3.1.6 Rotary shaker

3.1.7 Heating block - With nitrogen evaporation

3.1.8 Vortex or magnetic stirrer

3.1.9 Homogenizer

3.1.10 filter paper

3.1.11 Micropipet - Variable volume, 10 - 100 μL.

3.1.12 Immunoaffinity columns - EASY-EXTRACT VITAMIN B12

3.1.13 Immunoaffinity column rack

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3.1.14 Chromatographic system - Waters Alliance HPLC including quaternary solvent

system manager, Sample Manager, and PDA detector or high-performance

chromatography system of equivalent characteristics.

3.1.15 Chromatographic column - Waters Acquity UPLC® BEH C18, 1.7 μm, 2.1 ×

100 mm (Waters), or equivalent.

3.2 Glassware and consumables

3.2.1 Volumetric flasks - glass, 5, 10, 50, 100, 500, 1000 mL, clear glass

3.2.2 Pipets - Graduated glass, 10 mL, or volumetric glass, 9 mL.

3.2.3 Graduated cylinders - 50, 100, and 1000 mL.

3.2.4 Beakers - glass, 250 mL.

3.2.5 Flat-bottom round flasks or Erlenmeyer - glass, 250 mL.

3.2.6 Amber vials - Screw top, 7 or 4 mL

3.2.7 Micro LC vials - Amber or polypropylene vials

3.3 Chemicals and Solvents

3.3.1 Methanol - HPLC grade (Merck, Darmstadt, Germany).

3.3.2 Acetonitrile - HPLC grade (Merck).

3.3.3 Acetic acid, glacial - Merck.

3.3.4 Milli-Q water - Millipore.

3.3.5 Sodium cyanide puriss, optional

3.3.6 TFA or Formic acid for spectroscopy

3.3.7 Vitamin B12 (cyanocobalamin), approximately 99% - Sigma-Aldrich

3.3.8 Sodium acetate trihydrate p.a

3.3.9 Sodium hypochlorite for degrade excess sodium cyanide

3.3.10 α-Amylase from Bacillus subtilis - Approximately 50 units/mg (Sigma-Aldrich);

optional.

4.0 Preparation of Reagents and Standard Solutions

4.1 Sodium acetate solution 0.4 M, pH 4.0 - Into a 2000 mL volumetric flask, weigh 108.8 g

sodium acetate trihydrate. Add about 1800 mL water. Dissolve. Add 50 mL acetic acid,

and adjust pH to 4.0 with acetic acid. Dilute to volume with water.

4.2 Sodium cyanide solution, 1% (w/v) - Weigh 0.5 g sodium cyanide into a 50 mL amber

glass volumetric flask. Dilute to volume with water. Any excess of 1% sodium cyanide

solution must be destroyed by adding 1.5 mL of a 15% solution of sodium hypochlorite

per 1 mL sodium cyanide solution. Let react for 2 days in a fume hood. (Caution:

Sodium cyanide is highly toxic. Avoid contact with skin, and work in a fume hood.

Disposal of any unused solutions should comply with local regulations.)

4.3 Mobile phase A - To 1000 mL water, add 1 mL formic acid. Mix well.

4.4 Mobile phase B - To 1000 mL acetonitrile, add 1 mL formic acid. Mix well.

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4.5 Sample dilution solvent - Mix 98 mL mobile phase A with 2 mL mobile phase B,

starting gradient.

4.6 Vitamin B12 stock standard solution (1000 μg/mL) - Accurately weigh 5.0 mg vitamin

B12 into a 5 mL amber glass volumetric flask. Add about 2 mL water. Dissolve by

sonication and stirring for a few minutes. Make the volume with water to 5 mL.

Solution is stable for ≥6 months at –20°C. (Note: Vitamin B12 is sensitive to light.

Conduct operations under subdued light, or use amber glassware, or cover with

aluminium foil. Keep all solutions away from direct light.)

4.6.1 Vitamin B12 intermediate standard solution (50 μg/mL).—Pipet 250 μL vitamin

B12 stock standard solution into a 5 mL glass volumetric flask. Make up to

volume with water.

4.6.2 Vitamin B12 working standard solutions for calibration (0.78, 1.56, 3.1, 6.25,

12.5, 25, 50 μg/mL) - Pipet into six separate 1 mL Eppendorf tubes, 0.015, 0.03,

0.06, 0.125, 0.25, 0.5 1 mL vitamin B12 intermediate standard solution. Dilute to

1 mL volume with water. Or do a serial dilution from the highest concentration

to 1:1, up to 7 times.

5.0 Sample Preparation and Extraction

5.1 Sample reconstitution for powder samples - Weigh 10.0 g sample into a 250 mL

beaker. Add 40 mL water at RT. Cover with aluminium foil. Mix with a glass rod

until the suspension is homogeneous. Or stir on top of a magnetic stirrer.

5.2 Extraction. Add about 100 μL of α-amylase enzyme, mix thoroughly, beaker, and

incubate for 10 min at RT. Mix well. Transfer quantitatively the content of flask to a

50 mL plastic tube. Centrifuge at 5000 rpm for 10 min, and filter 10 mL supernatant

through a folded whatman No. 1 filter paper. In the case of high-fat products, and if

recovery is low, dilute the filtrate 1:3 in water before cleanup to improve recovery or

repeat the extraction by using a smaller sample portion.

5.3 Immunoaffinity cleanup - Let the immunoaffinity columns warm to room temperature

by removing them from refrigeration at least 30 min before use. Place each

immunoaffinity column on the rack. Open the caps and let the storage buffer drain by

gravity. Close the lower cap. Load the column with 3 mL clear filtrate and close the

upper cap. Place the column in a rotary shaker, and mix slowly for 10–15 min. Return

the column to the support and let stand for few minutes. Open the caps to let the

liquid drain by gravity. Repeat this loading and incubation for 3 times to load 9-10

mL of supernatant (to achieve low ppb level, pass the whole filtarate through IAC).

Wash the column with 10 mL water. With a syringe, insert air to dry the column.

Elute with 3 mL methanol, and collect eluate in a 4 or 7 mL reaction vial or in 1 mL

Eppendorf tubes (3 Nos.) for ease of drying in the vacuum concentrator. Evaporate

the eluate at 50°C using vacuum concentrator. Reconstitute the sample in 0.1 mL

sample dilution solvent or water. Mix on a Vortex mixer. Transfer to HPLC vial, and

inject into HPLC for analysis.

6.0 Analysis

6.1 Chromatographic conditions - Flow rate, 0.4 mL/min; injection volume, 10 μL;

detection, UV at 361 nm;

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6.2 Gradient elution

Time, min Mobile Phase A, % Mobile Phase B, %

0.0 98 2

2.0 98 2

15.0 40 60

15.1 0 100

17.0 0 100

17.1 98 2

22.0 98 2

6.3 System suitability test - Equilibrate the chromatographic system for at least 15 min.

Inject a working standard solution three to six times, and check peak retention times

and responses. Inject working standard solutions on a regular basis within a series of

analyses. The coefficient of variation should not be higher than 2%.

6.4 Analysis - Make single injections of standard and test solutions. Measure

chromatographic peak response (height or area).

6.5 Identification -Identify vitamin B12 peak in the chromatograms of the test solution by

comparison with the retention time and UV spectrum of the corresponding peak

obtained for the standard solution.

6.6 Calibration - Plot peak responses (area) against concentrations (in µg/mL). Perform

regression analysis. Calculate slope and intercept. Check the linearity of the

calibration (R2 > 0.99; standard error of calibration < 10%).

6.7 Quantitation - Calculate the concentration of vitamin B12, in μg/100 g of product as

reconstituted, as follows:

where A = response (height or area) of the peak obtained for the sample solution, I =

intercept of the calibration curve, S = slope of the calibration curve, V0 = volume of the

test solution (volume used to dissolve the test portion) in mL (40 mL), V2 = volume in

which the aliquot of sample solution is reconstituted after immunoaffinity cleanup (0.1

mL), m = weight of the test portion, as reconstituted, in g (10 g), and V1 = volume of the

aliquot of sample solution loaded onto the affinity column (9 mL). Deduct the amount

of vitamin B12 in the blank to the amount in the sample.

6.8 Reporting - Report results with two decimal points as cyanocobalamin, in μg/100 g of

reconstituted product.

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Reference: 1) AOAC Official Method 2011.09,

2) AOAC Official Method 2014.02,

3) FSSAI Manual of Methods of Analysis – Fortificants in food