ANALISIS VITAMIN

Devinisi Vitamin
• Vitamin adalah senyawa organik dengan molekul
kecil yang dibutuhkan oleh tubuh dalam jumlah yang
relatif sedikit dan berguna untuk mempertahankan
kesehatan
• Vitamin tidak dapat disintesis dalam tubuh kecuali
vitamin K, sehingga harus tersedia dalam makanan
yang dikonsumsi
• Provitamin: senyawa yang bukan vitamin akan tetapi
dapat diubah menjadi vitamin di dalam tubuh. Contoh
provitamin A dan provitamin D
 Kelarutan Vitamin
• Vitamin larut air
– Vitamin B kompleks (B1, B2, B6, asam pantetonat,
niasin, biotin, B12, dan asam folat)
– Vitamin C
• Vitamin yang larut dalam minyak
– Vitamin A
– Provitamin A (-karoten)
– Vitamin D
– Vitamin E
– Vitamin K
 Analisis Vitamin
• Pada umumnya vitamin dalam makanan sulit
ditentukan kadarnya
– Secara kimia memiliki mikronutrien esensial yang
heterogen
– Berada dalam jumlah yang sangat kecil
– Sering tidak stabil selama analisis
• Bioassays menggunakan manusia dan hewan (tidak
mudah dilakukan, mahal, akurat)
• Microbiological assays (menggunakan bakteri
protozoa dan jamur, mudah, lebih murah, kurang
akurat)
 Analisis Vitamin
• Physico-chemical assays
– titrimetric / spectrophotometric
– fluorimetry
– chromatographic
– Enzymatic
 Paling mahal, paling akurat, tetapi sering tidak ada metode
alternatif lain,
 pilihan metode tergantung keakuratan yang diinginkan, dan
biaya
 Diperlukan pengambilan sampel yang tepat dan diperlukan
ekstraksi
 Stabil selama analisis, sensitif terhadap cahaya, pH, oksigen
dan panas
 Diperlukan tindakan pencegahan untuk melindungi terhadap
kerusakan selama analisis
 Vitamin A (Retinol)

CH3 CH3
H3C CH3
OH

Retinol
CH3
  - Carotene and Retinol
CH 3
CH 3 CH 3 CH 3
H3 C

CH 3
CH 3 CH 3 CH 3
CH 3

Oxidation

CH 3 CH 3 O
H3 C CH 3
C H

CH 3 Retainal
- 2H

CH 3 CH 3
H3 C CH 3
CH 2 OH

CH 3 Retinol (Vitamin A)
Vitamin A Activity Determnination in Foods

Vitamin A Activity = -Carotene + Retinol

Vitamin A and  - Carotene Determination
Food
KOH (Alcoholic)
Saponification

3 hrs. at room temperature

Ether for Extraction
Extract (retinol and -carotene)

HPLC
 Vitamin A

• Column: Nova-Pack C18; 150 x 3.9mm

• Eluent : 95:5 Methanol: CH3CN
• Detection wavelength: 285 nm
Thiamin (Vitamine B1) Determination

Pyrimidine Thiazole
H3C N NH2 S CH2CH2OH Mono-, pyro-,
N N or triphosphate.
CH2 CH3
Protein

K 3Fe(CN) 6
Oxidation

H3C N N S CH2CH 2OH

N N
CH2 CH3
measure the absorbance at 435 nm
THIOCHROME (Fluorescent)
 Example of fluorimetric analysis of vitamins

• Fluorometric thiamin (vitamin B1) determination

– sample and standards dissolved in HCl
– enzymatic hydrolysis of phosphate esters of thiamin
– sample ‘clean up’ using ion exchange cartridge
– converte thiamin to thiachrome using potassium
ferrocyanide
– immiscible isobutyl alcohol added, shaken and left
to separate into two layers
– isobutyl alcohol upper layer now containing
thiochrome decanted off into fluorescence tube
– fluorescence measured (at 365-435nm)
compared to standard solutions 12
Thiamin Determination in Flour

1. 5 g flour
2. 75 ml 0.1 N HCl
3. Make volume to 100 ml
o
4. Digest at 100 C for 30 min
5. Centrifuge and filter
 Thiamine

• Column : 250 x 4.6 mm, Spherisorb

• Eluent : 90:10 chloroform:methanol
• Wavelength: 435 nm
 Water-Soluble Vitamins
2
Pyridoxin

Riboflavin
3

Niacin Thiamin
Inject 4
1

Column: u Bondapak C18

Solvent: MeOH
Sample: Water-Soluble Vitamins
0 5 10 15 20
Riboflavin (Vitamine B2) Determination

Flavinmononucleotide(FMN)
CH2 OH
Flavoadeninedinucleotide (FAD)
HOCH Flavoprotein
HOCH
HOCH
HCH
CH 3
N N O
8 9 1
7 2
6 10 3
5 4
CH 3 N
O

6,7 Dimethyl-9-D-1-Ribitylisoalloxazine
 Sample Preparation

Autoclave in 0.1 N HCl for 30 min

1. Adjust pH to isoelectric point to precipitate
proteins
2. Filter
3. Dilute to 100 ml in volumetric flask
 Riboflavin Analysis by HPLC

• Column: 250 x 4.6 mm 5µm; Spherisorb

• Eluent : 50:50 methanol : water
• Wavelength: 510 nm
 Niacin (Vitamine B3)

O O
C OH C NH2

N N

Ribose Ribose

Nicotinamideadeninedincleotide (NAD)
 Niacin Determination
1. Digestion an Hydrolysis
Autoclave
Sample + Ca(OH) 2 2 Hours

2. Precipitated Protein with (NH4)2SO4

3. Centrifuge and Filter
4. Rupture (pecahnya) of Pyridine Ring with CNBr
(Cyanogen Bromide)
5. Color Formation with Sulfanilic Acid
6. Determine the Absorption at 470 nm
7. Plot the Standard Curve of Niacin vs. Concentration
 Niacine Determination

O O

OH + CNBr C OH REARRANGEMENT
1 C REARRANGED
RING IS OPENED DERIVATIVE
N N
Br CN

O
RERRANGED +
2 DERIVATIVE
H2 N S OH
O
O
C-OH O
R-N-CH=CH-CH=C-CH=N S OH
O
Niacine Standard Plot

mg Niacin
 Vitamin C
Ascorbic Acid Dehydroascorbic Acid

CH2 OH CH2 OH
HOCH O HOCH O
-2 H
O O
+2 H

HO OH O O

HSCH2CH(SH)CH2OH : 2, 3-dithiol-Propanol
Reducing agent and converts dehydroascorbic acid to ascorbic acid
 Titration Method for Reduced Vitamin C

1. Extract with metaphosphoric acid (HPO3) in HOAC.

2. Titrate with 2, 6-dichloroindophenol (blue).

At end point, rose pink (2, 6-dichloroindophenol in acidic

condition).
 Example of a titrimetric analysis of vitamins
• 2, 6-Dichloroindophenol (DCPIP or DPIP) titrimetric
method for ascorbic acid
• Sample homogenised in metaphosphoric acetic acid
solution
• Filtered and diluted
• Standard solutions of ascorbic acid prepared
• Standards and samples titrated to pink endpoint with
dichloroindophenol dye
– mg ascorbic acid/ml sample = C x V x (DF / WT)
C = mg ascorbic acid/mL dye
V = ml of dye used for titration of diluted sample
DF = dilution factor
WT = sample wgt, g

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 Oxidation and Reduction of Ascorbic Acid
2,6-dichloroindophenol
HO OH Cl
+ O N OH
H
H O H 2C O O Cl

Blue Color

Cl
O O
HO NH OH
+
H
O Cl
H O H 2C O

Pink color in acidic condition

Extra oxidized indophenol form in the absence of ascorbic acidic gives pink color
at end point
 Calculation for Reduced Vitamin C

F V
mg Ascorbic Acid/g = (A-B) x x
E X

A= ml of 2,6-dichloroindophenol for sample titration

B= ml of 2,6-dichloroindophenol for blank
F= mg of ascorbic acid equivalent to 1 ml of indophenol standard solution
V= Initial assay solution volume
E= Number of grams
X= Volume sample aliquot titrated
 Vitamin E (Tocopherols & Tocotrienols)

• Vitamin E is present in food as eight compounds (---

and -) tocopherols and tocotrienols

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 Structure of vitamin E; Tocopherols & Tocotrienols

RI

RII

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 Vitamin E Determination
• Method
– saponify
• mix with 6% (wt/vol) pyrogallol in ethanol, heat at
70°C & sonicate
• add 60% KOH solution digest at 70°C & sonicate
– extract with hexane
– filter & inject into normal phase HPLC
– use florescence detector at 290 and 330 nm
– quantify by external standard from peak area by linear
regression

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HPLC Chromatogram of Vit E

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