DETERMINATION OF VITAMIN

B3 IN ANIMAL & PLANT BASED

FOOD AND B3 SUPPLEMENT BY
HPLC.
PRESENTATION CONTENT:
 Introduction
 Problem Statement
 Significance Of Study
 Research Gap
 Research Objective
 Literature Review
 Methodologies
INTRODUCTIO
N
 VITAMIN B3
 Vitamin B3 (niacin) is essential vitamins for all
living things and play a central role in energy
metabolism, skin health, digestive and nervous
system (Zhang et al., 2018)

 Water-soluble vitamin is present in two forms of

nicotinic acid and nicotinamide (niacinamide)

 Cannot be stored in the body. So, people need

to take this kind of vitamins daily through the
food sources or supplement and can make
small amounts from the amino acid tryptophan

 Excess of vitamin will excrete by urination.

 Can be founded in a various kind of animal and

plant based food such as meat, fish, egg,
cereal, peanut etc.
VITAMIN B3

Nicotinic Acid Nicotinamide

 • Nicotinic acid is  Nicotinamide is a water
corresponding acid soluble amide isotypes of
isotype vitamin B3
 • This nicotinic  The nicotinamide used in
acid is used clinically the treatment of psychiatric
as a pharmacological disorder.
agent in large doses to
lower serum lipids
and cholesterol
 B3 Supplement
 Niacin can be found in multivitamin or can available in
B-complex and supplement containing only niacin
(National Institutes of Health: Office of Dietary
Supplements (ODS), 2019).

 Available as a dietary supplement in the form of

nicotinic acid or nicotinamide.

 Consumers take the supplement because they have

allergies with animal or plant food based which contains
high of vitamin B3 or vegetarian or consumer who don’t
like to eat plant based food

 Sometimes the amounts in supplements are far beyond

the Recommended Dietary Allowance (RDA) up to
2,000 mg daily.
 PROBLEM
STATEMENT
01. Comparison Between The Amount of Vitamin B3 in
Natural Food with Vitamin B3 Supplement

 Many researcher had done analyzing the amount of Vitamin B3 in many types of animal and plant based
food which involving the fruits, vegetables and protein sources.
 There are only a few research that analyzing the multivitamin supplement only without analyzing the
specific vitamin B3 supplement.
 Cannot compare either the vitamin B3 supplement have same or less or more amount of doses compared
to nature products which it can lead in unhealthy supplement consume.
02. Unhealthy Consumption of Overdose B3 Supplement

 Everyone needs a certain amount of niacin -- from food or supplements -- for the body to function
normally.
 This amount is called follower the RDA (Recommended Daily Allowance).
 Vitamin B3 supplement sometimes provided higher dosage from the dietary recommended and consumer
still consume because there a lot of benefits from vitamin B3 especially in skin brightening.
 Overdose of nicotinic acid can lead to high blood pressure, abdominal pain, impaired vision, and liver
damage
 Overdose of niacinamide lead to side effects (redness and warmth in the face and neck).
SIGNIFICANCE
OF STUDY
 • Food industries responsible for high vitamin B3 food
• In the early 1900s, pellagra was prevalent
production
in the Southern Unites States due to the
• Determining the amount of vitamin B3 in natural food will help
low availability of corn, at the time the
the food production to produce the process food for consumer
main dietary source of niacin
who cannot take directly from natural products.
(Rajakumar,2000).
• This can lead the industries to produce the nutrients food
• Pellagra includes the triad of dermatitis,
according RDA.
dementia, and diarrhea and can result in
death.
• Every day people need to
• Overdose in consuming vitamin B3 also
give risk to consumer such as liver consume vitamin B3 by eating
PHARMACEUTICAL
PHARMACEUTICAL
damage. INDUSTRIES
INDUSTRIES food or any supplement

• Analyzing the amount of vitamin B3 in • Consuming vitamin B3 are

food sources and niacin supplement help different followed the gender and
INDIVIDUAL
the pharmaceutical industries to produce AND SOCIETY the age.
the supplement with the correct amount of • Analyzing the amount of vitamin
vitamin B3 follow the RDA and help the B3 in food sources and
consumer to take the ideal amount of supplement will help individual
vitamin B3 and society to take the right
amount of vitamin B3 in their
daily life,
RESEARCH
GAP
Most of the previous research only focusing to identify the concentration or the
amount of vitamin B3 consists in animal and plant based. These research lead in
build the vitamin B3 profile to Dietary Intake by natural food only.

In this research, there are different from the previous research in term of :
1) Determining the amount of vitamin B3 in niacin supplement and animal and
plant based food.
2) Analyzing the sample in HPLC – Fluorescence Detector (for animal and
plant food based-10 types)
Analyzing the sample in HPLC – PDA Detector (four types of supplement)
RESEARCH
OBJECTIVES
 RESEARH AIM :
To determine the amount of vitamin B3 in animal, plant based food and B3
supplement by using HPLC method

RESEARH OBJECTIVES :
 To analyze the amount of vitamin B3 in the animal, plant based food with B3
supplement by using HPLC method
 To compare the amount of vitamin B3 in the samples followed the Recommended
Dietary Allowance (RDA)
 To propose the type of B3 natural food and B3 supplement to consumer intake
LITERATURE
REVIEW
VITAMIN B3
 Vitamin B3 (niacin) is an important water-soluble vitamin found as nicotinic acid (C₆H₅O₂) and nicotinamide (C₆H₆O₂) in foods
(Ndaw.et.,Al,2002).

 Vitamin B3 performs an important metabolic process in living cells as a precursor of NAD+/NADH and NADP+/NADPH.

 Vitamin B3 in the form of the dinucleotides plays a central role in energy metabolism such as in oxidative phosphorylation and
protein, fat and carbohydrate metabolism in the body. It is responsible for neural and enzymatic functions and is actively involved
in preventing many pathological processes (Pollak.et.,Al,2007)

 Nutritional deficiency of Vitamin B3 causes pellagra and is associated with low NAD+ levels (Belenky.et.,Al, 2007)

 Lack of Vitamin B3 : fatigue, constipation, diarrhea, depression, headache, circulatory problems, pellagra and many more
 Overdose of Vitamin B3: itchy skin, eye problems, liver damage, lower bloods pressure, insulin resistance and many more.
VITAMIN B3
 The recommended daily allowance (RDA) for niacin, revised in 1998 is based on the prevention of deficiency
and given by the Food and Nutrition Board (FNB) of the US Institute of Medicine (Lule et al., 2015)
B3 SUPPLEMENT
 For those taking supplements, the fiftieth percentile of supplemental niacin intake was 20 mg for
men and 30 mg for women.
 In the 1986 National Health Interview Survey, 26 percent of all adults reported use of supplements
containing niacin (Moss.et.Al.,1989)
 Supplements containing up to about 400 mg of niacin are available without a prescription

 Disadvantages of Taking High Dose of B3 Supplement.

 Vasodilatory Effects (Flushing) : Case reports and clinical trials have reported flushing effects at
oral doses of 30 to 1000 mg/day within 30 minutes to 6 weeks of the initial dose
(McKenny.et.Al.,1994). This flushing covers a burning, tingling and itching sensation as well as a
reddened flush primarily on the face, arms and chest.

 Hepatotoxicity : In the most severe cases, patients develop liver dysfunction and fulminant
hepatitis and may progress to stage 3 and 4 encephalopathy requiring liver transplantation.  These
effects are typically associated with high doses (3 to 9 g/day of niacin) used to treat patients with
hypercholesterolemia for periods of months to years (Clementz and Holmes, 1987).
 HPLC METHOD
 Since 2010, High Performance Liquid Chromatography has become the most common method for
determination of vitamins.
 HPLC technique is used for the separation of vitamins in various matrices due to its high selectivity
and sensitivity (Schmidt.et.Al., 2017)
 Classical reversed-phase HPLC with ultraviolet (UV), photodiode array (PDA) and fluorescence
detector (Hasan.et.,Al, 2013) is still widely used for the routine quantification of vitamins in different
type of samples (Zhang.et.Al., 2018).
 HPLC METHOD
HPLC - FLD
• FL detection is typically three times of magnitude more sensitive than UV detector. HPLC-FLD are used routinely
assay in low ng/mL range and even in pg/mL concentration of analytes (Crotti.et.,Al, 2017).
• Vitamin B3 are water-soluble vitamins which can fluoresce. Separation of vitamin B3 in animal and plant based
food gives good results using HPLC-FLD.

HPLC-PDA
• Each photodiode gives a specific response to radiation with a certain wavelength so that electromagnetic radiation
with a broad range of wavelengths (UV-Visible) can be received simultaneously.
• The advantage of HPLC-PDA is the ability to choose the best wavelength for analysis (Synder.et.Al.,2010)
• HPLC-PDA has been used in the simultaneously determination of water-soluble vitamins (ascorbic acid, nicotinic
acid, nicotinamide, pantothenic acid, thiamine, folic acid, and syanocobalamin)
METHODOLOG
Y

FOR ANIMAL &
PLANT BASED
FOOD
by HPLC
Post-column UV derivatization system by
HPLC (high-performance liquid
chromatography) was used to determine
the presence and concentrations of
nicotinic acid and nicotinamide
(Catak,2019).
Reagents:
The vitamin standard (nicotinic acid and nicotinamide),
Teflon tube, UVA lamp, sodium hydroxide, hydrochloric acid,
hydrogen peroxide, copper sulfate, trichloroacetic acid and
potassium dihydrogen phosphate.
Materials :
Samples of chicken, fish, beef, mutton, egg, oat, chickpea,
eggplant, tomato and mushroom.
Instrumentation:
HPLC instrument with 3D pump and fluorescence detector.
Colum (5µm, 4.6 x 150mm)
Flowrate : 1mL/min
The column oven temperature maintained at 25ºC
Injection volume : 20µL
Analysis time : 40 min
 STANDARD PREPARATION
Prepares standard
Prepare working
stock solution of
standards in five
nicotinic acid and Each standard is
concentration levels
nicotinamide freshly prepared daily
were from stock
(100µg/mL) in 0.1 N
solution
HCl solution

SAMPLE PREPARATION
Add 2 mL of 20% tricycloacetic
acid (C2HCl3O2) solution to the
Place homogenized 1–10 g of the solution, proteins were
Add 60 mL of 0.1 N HCI and Take the samples from the
samples (based on the estimated precipitated, the volume was
autoclave at 121°C for 30 autoclaves and cool down to
amount of niacin it may contain) completed with 0.1 N HCI, filtered
minutes room temperature.
in 100 mL erlene. through filter paper. The final
solution filtered through 0.45 μm
filter into HPLC vials.
 HPLC Determination
• Connect between the analytical column and the fluorescence detector.
• Prepare the mobile phase daily and protect from light.
• Prepare the mobile phase by mixing 500 mL of deionized water and 9.5 g of potassium dihydrogen
phosphate (KH2PO4).
• Then, dissolve 7.5 mL of hydrogen peroxide (H2O2) and 2 mL of copper (II) sulfate solution (CuSO4) (0.12
copper (II) in 100 mL of deionized water) is added and the volume was completed with 1 L of deionized
water.
• Finally, filter the mobile phase through a 0.22 μm filter. The fluorescence detector set to excitation
wavelength of 322 nm and emission wavelength of 380 nm, respectively.
• Use an column (5 μm, 4.6 × 150 mm,) and flow rate of 1 mL/min. The injection volume was set at 20 μL,
and separation was completed in 40 min.
FOR NIACINE
SUPPLEMENT
by HPLC
A robust chromatographic method for the
separation of eight water-soluble B vitamins
in vitamin supplement tablets using HPLC
with photodiode array (PDA) (Andrew et al.,
2009)
Reagents:
The vitamin B standard, sodium hydroxide, hydrochloric acid,
water
Materials :
Two types of multivitamin supplement (brand A and B)
Two types of niacin supplement (brand C and D)
 STANDARD PREPARATION
1. Prepare two set of standard, working standard 1 (WS1) and working standard 2 (WS2)
Working standard 1 (WS1)
i. Weigh out 80-320 µg/mL of each vitamins depending on the vitamin’s relative absorptivity.
Working standard 2 (WS2)
ii. Weigh individually selected amounts for each vitamin, depending on its expected concentration
range in typical dietary supplements

For both WS1 and WS2

iii. Weigh out the vitamins and transfer into individual 50 mL volumetric flask and add 30 mL of water
iv. Add 0.1 N NaOH to the flask contains vitamins B9,B2 and B7 to improve solubility
v. Sovicate all flask in five minutes and then fill to the mark with water
vi. Equal volumes of the eight respective stock standard are add together to produce each of the two
WS solution.
vii. For WS1, the resulting concentration for the eight vitamins are between 10-40µg/mL
viii. For WS2, the vitamins ranged in concentration from 0.9 to 525 µg/mL depending on the vitamin
 SAMPLE PREPARATION
1. Purchase multivitamin and niacin supplement (brand A,B,C and D ) from a local store
2. For each sample, transfer a tablet into an individual 50 mL volumetric flask and add 30 mL of
water.
3. Stopper the flask and place on a heater / stirrer and heat at 40ºC while stir for 15 min.
4. After the tablet completely dissolve, sonicate each solution for 10 minutes.
5. Fill to the mark with HPLC- grade water
6. Filter all the standard and samples through 0.45µm filters prior to injection

INSTRUMENTATION
Colum: (2.7 µm, 3.0 x 100 mm)
Flowrate : 0.6 mL/min
The column oven temperature maintained at 40ºC
Injection volume : 3µL
Analysis time : 10 min
Wavelength : 214 nm
Detector : PDA (Photodiode-Array Detection)
Detection : 214 nm (other B vitamins), 267 nm (B1 and B2)
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Andrew, P. Z., Wilhad, M. R., & Inc, P. (2009). Analysis of BVitamins.
https://www.perkinelmer.com/lab-solutions/resources/docs/APP_Analysis-of-Water-Soluble-B-
Vitamins-by-HPLC-PDA.pdf
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