KLE PHARMACY COLLEGE

FOOD ANALYSIS (MPA 104T)

VITAMINS
Presented by : Presented to :

Athira Chandran L Dr. P V Murali

1 1st Semester [Link] Associate Professor

Department of Pharmaceutical Analysis Department of Pharmaceutical Analysis
KLE College of Pharmacy KLE College of Pharmacy

21/09/2023
2 CONTENT
 Introduction
 Classification of vitamins
 Deficiency disorders
 Determination of vitamins
Determination of liposoluble vitamins
 Extraction of vitamin A
 Determination of vitamin A
 Extraction of vitamin D
 Determination of vitamin D
 Ascorbic acid Extraction
Determination of water soluble vitamins
 Summary 21/09/2023

 References
3 INTRODUCTION
Vitamins are defined as relatively low molecular weight compounds which
humans and for that matter, any living organism that depends on organic
matter as a source of nutrients, require small quantities for normal
metabolism.
Vitamins are substances that our body needs to grow and develop normally.
If it cannot be synthesized in sufficient quantities by an organism and must
be obtained from the food and supplements.
For example vitamin C as ascorbic acid we get from citrus fruits like
Lemon, Oranges etc.
Insufficient levels of vitamins results in deficiency diseases like scurvy and
pellagra, due to the lack of ascorbic acid (vitamin C) and niacin respectively.

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CLASSIFICATION OF VITAMINS
WATER SOLUBLE VITAMINS FAT SOLUBLE VITAMINS
Thiamine Vit- B1 Retinol/ Retinal/ Retinoids Vit-A
Riboflavin Vit-B2 Ergocalciferol/ Cholecalciferol Vit-D
Niacin/ Niacinamide Vit- B3 Tocopherols/Tocotrienols Vit-E
Pantothenic acid Vit-B5 Phylloquinone/ Menaquinones Vit-K
Pyridoxine/Pyridoxamine /Pyridoxal Vit- ----------
B6
Biotin Vit-B7/Vit-H ----------
Folic acid/ Folinic acid Vit-B9 ----------
Cyanocobalamin/ Hydroxy cobalamin/ ----------
Methyl cobalamin Vit-B12
Ascorbic acid Vit-C ----------- 21/09/2023
5 DEFICIENCY DISORDERS

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6 DETERMINATION OF VITAMINS
 The general principles are the same, but the extraction methods differ
depending on the matter being analyzed.
 Numerous determination techniques can be proposed:
determination by fluorimetry.
determination by colorimetry.
determination by liquid phase chromatography, with ultraviolet or
fluorometric detection.
determination by gas phase chromatography (vitamin E).
 microbiological determination.
The unit for vitamin is expressed in international units (IU), United
States Pharmacopoeia (USP) units and Daily Values (DV).
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7 DETERMINATION OF LIPOSOLUBLE
VITAMINS
 Weigh proper amount of the food substance and transfer to a 150 ml round
flask.
 Add 10 ml of 33% KOH and 40 ml ethanol , Cool very rapidly, then add 17
ml of 25% HCl, and cool again.
 Boil at reflux for 30 mins .
 Add 50 ml of petroleum ether and shake vigorously for 3 mins.
 Wait to obtain complete separation of the two phases .
 Evaporate to dry in a vacuum at a temperature of 35 0 C.
 Remove and filter the organic phase through anhydrous sodium phases .
 Redissolve the residue with a known volume of hexane for HPLC analysis. 21/09/2023
8 EXTRACTION OF VITAMIN-A
 Weigh 5-10 g of the previously crushed food substance into a 1 L round flask.
 Add 20 ml of a 50% NaOH solution and warm the mixture in a water bath.
 Then, add 100 ml of diethyl alcohol and 2 ml of a hydroquinone solution that was
obtained by dissolving 20 g in 100 ml of pure alcohol.
 Maintain the water bath at 90℃ for 30 minutes.
 Pour the contents of the round flask into a decanting vial and add 100 ml of water.
 Add 50 ml of ethylic ether and shake.
 Add 50 ml of petroleum ether. Shake and allow it to decant.
 Extract once or twice with 50 ml of petrol ether.
 Wash the ether phase three times with 100 ml of water.
 Filter, evaporate, and concentrate until 1 ml is obtained.
 It must be noted that all of these steps are conducted away from light. Moreover,
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9 DETERMINATION OF VITAMIN-A

 After extraction, the determination is carried out on the solvent of the liquid
extraction.
 Colorimetric Determination: Through this method, carotenoids and vitamin
A are determined.
 Determination of carotenoids: Carotenoids are determined at 450 nm. After
evaporating the ether phase of the extracted solution, dissolved the
extraction with 1 ml of hexane. Determine the O.D. of this phase at 450 nm.
 Determination of vitamin A: The hexane phase obtained earlier is taken
again and concentrated in a vacuum. Redissolve the extract in a chloroform.
Then, to the volume of chloroform, add four volumes of the trifluoroacetic
acid reagent prepared by mixing 1 v of trifluoroacetic acid with three
volumes of chloroform. Then, observe the OD at 620 nm. 21/09/2023
10 VITAMIN D

EXTRACTION OF VITAMIN D2/D3

 Weigh between 5 and 10 g of the sample food substance.
 Add 1 g of propanol, 90 ml of a mixture of 60 ml absolute ethanol, and 30
ml of a 50% potash solution.
 Extract three times, each time with 50 ml of petroleum ether. Wash the
ether extract and material three times with water.
 Filter, evaporate, and concentrate until 1 ml is obtained.
 Saponification with the alcoholic potash mixture is not necessary if the
products to be analyzed do not contain fats.

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DETERMINATION OF VITAMIN D

 Determination of Vitamins D2, D3, and their Metabolites

 If the sample contains all the vitamin D metabolites, then you can carry
out a liquid chromatography under the following conditions: -
 Column: fatty acid analysis column - Solvent: MeCN (acetonitrile),
55%
 Mixture of water/acetic acid (4 ml of acetic per liter) 45%
 Flow rate: 1 ml/min
 Wavelength: 265 nm
 Solvent temperature: 25℃
 Temperature of oven: 40℃
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 EXTRACTION OF VITAMIN E
12

 Weigh between 5 and 10 g of the food substance that you crush.

 Add 100 ml of ascorbic acid methanol solution obtained by mixing 0.5 g of
ascorbic acid, 4 ml of water, and 20 ml of ethanol brought to 100 ml with
methanol. Keep in boiling water for minutes. Then add 15 ml of a 70% KOH
solution .Place again in the water bath for 40 minutes
 Decant the contents of the flask into a separation flask vial, washing the flask with
50 ml of water.
 Add 120 ml of ethylic ether and stir the mixture. Decant and filter on Na2SO4.
Extract again with 120 ml of ethyl ether.
 Filter, evaporate, and concentrate to 1 ml. - Saponification with potash is not
necessary for non-fat products.
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 DETERMINATION OF VITAMIN E
13
 A number of methods can be used to determine this vitamin.
 1. Colorimetric Determination After extraction and evaporation, re-dissolve the residue
using n-heptane. Add 1 ml of dipyridyl solution, then determine the absorbance at 460
nm. Methods derived from this one have been recommended for use with ferric
chloride with a reading at 510 nm.
 [Link] by Liquid Chromatography Using the extract prepared as described
earlier, proceed with an HPLC determination under the following conditions:
 Column: Lichrosorb R P 8,25 cm ×, 4.6 mm, 5 µm
 Solvent: methanol/water (92:8)
 Flow rate: 1.5 ml/min
 Wavelength: 288 nm
 Solvent temperature: 25℃ 21/09/2023

 Temperature of oven: 40℃

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ASCORBIC ACID EXTRACTION

 In a breaker weigh to about 0.1 mg of a certain product quantity as a

function of the assumed vitamin C content of the sample food substance.
 Decant into a 50 ml volumetric flask using 0.4% metaphosphoric acid.
 Bring the volume to 50 ml with this solution. (For the analysis of a liquid,
pipette the sample directly into the flask and adjust the volume to 50 ml
with the 0.4% metaphosphoric acid solution.)
 Filter the solution a cellulose acetate membrane (0.2 µg), then pass the
filtered substance through a SEP-PAK C18 cartridge (supplied by Waters, a
division of Millipore). Eliminate the first 2 ml, then collect 5 ml for
analysis by RP-HPLC.

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 DETERMINATION OF WATER-SOLUBLE
15 VITAMINS
 Determination of Vitamin B1 Thiamine
Fluorimetric Determination
 After the action of potassium ferricyanide in the presence of potash,
determine fluorescence using a nm primary filter and a nm secondary
filter.
Microbiological Determination
 A number of lactobacilli can be used, such as Lactobacillus fermentum
and Lactobacillus viridiceus ATCC 1270 C, depending on the chemical
methods of determination. The statistical calculation of activity is
based on the six point method.
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Determination by Liquid Chromatography

 Chromatographic conditions Column: C 18, 25 cm × 4.6 mm, 5µm
 Solvent: methanol, 0.005 M sodium acetate adjusted to 4.5 pH (30-70)
 Flow rate: 1 ml/min
 Detection: excitation: 366 nm
 Emission: 435 nm

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Extraction & Determination of vitamin B2
 Extraction of vitamin B2
 Choose an appropriate solvent, often methanol or ethanol, to extract
riboflavin from the sample
 Mix the sample with the solvent and sonicate or shake it to facilitate
extraction.
 Centrifuge the mixture to separate the liquid from solid particles.
 Determination of vitamin B2
 Use a spectrophotometer or fluorescence detector to measure the
absorbance or fluorescence detector to measure the absorbance or
fluorescence of riboflavin at specific wavelength.
 Compare these measurements to a standard riboflavin solution to
determine the concentration of vitamin B2 in the sample. 21/09/2023
18 SUMMARY
 Vitamins are essential organic compounds required in small amounts for various
biochemical processes in the human body. They play crucial roles in maintaining
health and preventing deficiencies and diseases. Determination and extraction of
vitamins are important processes in both nutritional science and analytical chemistry.
 Extraction of Vitamins:
• Extraction methods depend on the type of vitamin and the source material (e.g., food,
plants, or pharmaceuticals).
• Common extraction techniques include solvent extraction, solid-phase extraction, and
supercritical fluid extraction.
• Solvent extraction involves using organic solvents like methanol or chloroform to
dissolve vitamins from the matrix.
• Solid-phase extraction employs specialized cartridges or sorbents to selectively
adsorb and elute vitamins. 21/09/2023
19 SUMMARY
 Determination of Vitamins:
• Vitamins can be determined using different techniques, including spectroscopy,
chromatography, and bioassays.
• High-performance liquid chromatography (HPLC) is a widely used method for
vitamin quantification due to its precision and sensitivity.
• Immunoassays, such as enzyme-linked immunosorbent assays (ELISA), are also
employed for vitamin detection.
• Biochemical assays and microbiological tests can assess the vitamin content in
foods and supplements.
 vitamins are essential nutrients that require accurate determination for assessing
their presence and concentration in various samples. Analytical techniques and
extraction methods are essential for obtaining reliable data on vitamin content.
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REFERENCES

 Vitamins: MedlinePlus
 Determination of riboflavin [Internet]. Bing. [cited 2023 Oct 3]. Available
from:
[Link]
=788519c742804968aa628c4c9a289f21&gs_lcrp=EgZjaHJvbWUyBggAEEU
YOTIGCAEQRRhA0gEJMTMyMjhqMGoxqAIAsAIA&FORM=ANNTA1&
PC=ASTS
.
 Onyekere, Peculiar & Egbuna, Chukwuebuka & Munir, Naveed & Daniyal,
Muhammad & Ahmed, Olatunde & Găman, Mihnea-Alexandru & Akram,
Muhammad. (2020). Vitamins and Minerals: Types, Sources and their
Functions. 10.1007/978-3-030-42319-3_9.
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REFERENCES

 Kumar Patle T, Shrivas K, Patle A, Patel S, Harmukh N, Kumar A.

Simultaneous determination of B1, B3, B6 and C vitamins in green
leafy vegetables using reverse phase-high performance liquid
chromatography. Microchemical Journal. 2022 Feb;107249.
 Santa Monica college. 10: Vitamin C Analysis (Experiment) [Internet].
Chemistry LibreTexts. 2017. Available from:
[Link]
ts/Wet_Lab_Experiments/General_Chemistry_Labs/Online_Chemistry
_Lab_Manual/Chem_11_Experiments/10%3A_Vitamin_C_Analysis_(
experiment)

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THANK YOU

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