Determination of Vitamin A (Retinol), Vitamin E ( ( ) - Tocopherol) and SS-Carotene in Foodstuffs by HPLC-UV

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Vitamin A, vitamin E, ß-carotene
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Determination of vitamin A (retinol),

vitamin E ((±)- -tocopherol) and ß-carotene
in foodstuffs by HPLC-UV
Reasons for amendment of original German version:

V2: Summary of methods N03_01, N03_04, N03_08
V3 (11.07.08): Changes to parameters
V4 (21.01.10): New calibration, changes in processing
Modifications of norm method explained
Name Signature Date

First version written by: Melanie
Mongili
Translation by: easytrans24

Bei den Mühren 69a 11/2009
D-20457 Hamburg
verified (translation): Melanie
Mongili
verified (professional): Katharina
Lorenz
verified (norm conformity): Nadine
Wollnitz
approved: Matthias
Lieske
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1 Area of application
This method describes a procedure for the determination of vitamin A (retinol), vitamin E
(d,l -tocopherol) and ß-carotene in food products.
Vitamin A, E and ß-carotene are categorized as fat-soluble vitamins. They are essential,
as is the case for all vitamins, i.e., they cannot be synthesized by the body, but must be
taken up with food and are therefore added to baby food.
2 Principle of the method

All three vitamins are determined with the aid of saponification. The material under
investigation is saponified with aqueous ethanolic potassium hydroxide solution and the
vitamins thus released are extracted using petroleum ether. After concentration of the
extract, the residue is dissolved in methanol and the contents of vitamin A, E and
ß-carotene are determined using HPLC-UV/VIS.
This method is based on the norm methods DIN EN 12823-1, DIN EN 12823-2 and DIN
EN 12822. The following modifications are applied:
All 3 norm methods are summarized to one single method. The 3 vitamins are
determined from one sample preparation.
For 13-cis retinol, -carotine und - tocopherol no calibration is performed
No correction factor for HPLC is derived from purity assessment.
Saponification is performed by 3 h incubation at 50 °C in a water bath and
incubation at room temperature over night.
The extraction is performed just once and with 150 ml petrol ether
The neutralisation is performed just once and with approx. 400 ml water
A defined aliquot of the extract is evaporated on a drying device at 48 °C with
nitrogen
Only a single determination is performed. The determination is only repeated if there
are deviations from the expected value or mistakes during sample preparation are
suspected.
3 Definitions and abbreviations
Definitions
The vitamin A content is the content of all-trans retinol and 13-cis-retinol determined using
the procedure described here.
Conversion factors for the calculation of international units (IU) in units of weight:
1 IU vitamin A = 0.300 µg all-trans retinol
0.344 µg all-trans retinol acetate
0.550 µg all-trans retinol palmitate
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1 µg retinol equivalent = 1.00 µg retinol

3.333 IU vitamin A,
The vitamin E content is the content of d,l- -tocopherol determined using the procedure
described here.
Conversion factors for the calculation of international units (IU) in units of weight:
1 mg d,l- -tocopherol = 0.74 mg -tocopherol equivalent
= 1.10 IU vitamin E
The ß-carotene content is the content of ß-carotene determined using the procedure
described here which is employed for the calculation of the vitamin A equivalent. 6 µg
ß-carotene correspond to 1 µg retinol.
Abbreviations
HPLC high performance liquid chromatography
BHT butylhydroxytoluene
4 Equipment
Laboratory mill
Analytical scales, precision ± 0.1mg
Brown glass flat-bottomed flask, 250 ml
Stir bar
Magnetic stirrer
Dispenser (50 ml, 100 ml)
Graduated cylinder
Water bath with reflux condenser
Conical separating funnel, brown glass, 500 ml, with PTFE stopcock
Mechanical shaker
Volumetric flask, narrow neck, 50 ml, 100 ml
Glass funnel
Phase separation folding filters
Pipette
Brown glass vials, 6 ml
Drying device with nitrogen (evaporation system)
Brown glass vials, 1 ml
Vial clamp
HPLC equipment composed of a pump, injector with sample loop (20-100 µl), UV-VIS
detector, analytical software
HPLC columns: Vitamin A/E: e.g. LiChrosorb RP18, 7 µm (250 x 4.0 mm)
ß-carotene: e.g. Spherisorb ODS 2, 5 µm, (100 mm x 4.6 mm)
e.g. Vydac 201TP54 (RP18), 5 µm (250 x 4.6 mm)
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UV-VIS spectrophotometer suitable for the measurement of the absorption capacity at

predetermined wavelengths with suitable quartz cuvettes, e.g. 1 cm layer thickness
5 Chemicals
5.1 Reagents
Purified water
L-ascorbic acid
Ethanol, denatured, ~ 95 %
Ethanol, absolute, = 99 %
Potassium hydroxide p.a.
Nitrogen, oxygen-free, = 99.1 %
Petroleum ether (boiling range 40-60 °C)
Methanol for the liquid chromatography
Dichloromethane for the liquid chromatography
Acetonitrile for the liquid chromatography
Ammonium acetate
Triethylamine
Buthylhydroxytoluene
5.2 Solutions
All solutions and eluents for HPLC are made up using purified water ( 18M DI).
Potassium hydroxide solution (60 %)

600 g potassium hydroxide are dissolved in 1.0 l water.
5.3 Standards
Vitamin A standard:
Retinol, e.g. supplied by Sigma (purity > 95 %)
Retinol palmitate, e.g. supplied by Sigma (purity ~ 1,800,000 USP units/g)
Vitamin E standard:
d,l -tocoperol, e.g. supplied by Sigma (purity > 96 %)
ß-carotene standard:
ß-carotene, e.g. supplied by Sigma (purity > 95%)
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6 HPLC systems
The vitamins are separated under suitable HPLC conditions. For example, the following
have proven reliable:
HPLC equipment for the determination of vitamin A and E
Injection volume: 50 µl
Column: LiChrosorb RP18, 7 µm (250 x 4.0 mm)
Eluent: methanol/water (98/2, v/v)
Flow rate: 0 min 0.8 ml
5 min 1.0 ml
8.5 - 14 min 1.5 ml
14.5 min 0.8 ml
UV detector: 0 8.5 min 326 nm
8.5 15 min 292 nm
Run time: 15 min
HPLC equipment for the determination of vitamin ß-carotene
Injection volume: 20 µl
Column: Spherisorb ODS 2, 5 µm, (100 mm x 4.6 mm)
Vydac 201TP54 (RP18), 5 µm (250 x 4.6 mm)
Eluent: acetonitrile/ methanolic ammonium acetate solution/ dichloro-
methane (75/20/5, v/v) with the addition of 0.08 g BHT+ 0.085 g
triethylamine (per 100 ml eluent)
Flow rate: 1.5 ml (isocratic)
UV detector: 450 nm (Wolfram lamp)
Run time: 19 min
7 Calibration
Two different calibrations are carried out for the determination of the vitamin A content,
one using retinol and one using retinol palmitate as the standard. This is required, as
lower contents of vitamin A are otherwise determined for retinol palmitate, probably
because saponification is incomplete due to the high stability of retinol palmitate. A retinol
palmitate standard is saponified for the calibration, in order to compensate for these
"losses" during saponification.
7.1 Calibration for the determination of retinol, retinol acetate and

d,l- -tocopherol
Comment: Based on the differing quality of the standards supplied by different

manufacturers and on our experience, the calibration standards for retinol
and vitamin E are obtained exclusively from Sigma. The standards should
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always exhibit a purity of 95 % (for retinol) or 96 % (for vitamin E).

Due to stability problems, the purity of the standards must be checked prior
to their use for calibration purposes, even though a manufacturer's certificate
is available (see 7.1.1 and 7.1.2).
Vitamin A and vitamin E are measured simultaneously on one column. A standard mixture
of the two standards is used for the calibration. The stock solutions and the solution for
the purity assessment must be made up separately as the extinctions of the two
substances overlap in photometric measurements (see 7.1.3).
7.1.1 Assessment of concentration and purity pursuant to the European Standard

DIN EN 12823-1 (retinol)
Concentration and purity are checked against ethanol using spectrophotometric

measurements on a retinol standard solution of a defined concentration and subsequent
calculation of mass concentration.
There is a form that provides the exact details of how to produce the solution for
measurement for this assessment (see Appendix 1). This form documents the initial
weight, the dilution and the values measured with the photometer. It is filed in the
corresponding folder together with the documents on calibration.
The retinol standard A2 is used for the assessment of concentration and purity (see 7.1.3
Calibration).
To this end, the absorption capacity of the retinol standard solution A2 is measured
against ethanol in a quartz cuvette with a layer thickness of 1 cm at the absorption
maximum of 325 326 nm for retinol using a suitable spectrophotometer.
The mass concentration of all-trans retinol in micrograms per millilitre is calculated using
the following equation:
A 10 4
[ g / ml ]
1830
Where:
A Absorption maximum at 325 to 326 nm
1830 E11cm
%
value for retinol in ethanol
As an additional safeguard, the absorption capacity of the standard solution is measured

against ethanol at 300 nm, 325 nm, 350 nm and 370 nm.
The following ratio is determined for each wavelength:
E/E325 (or 326nm)

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The standard can be used for calibration purposes if the ratio for retinol does not exceed
0.602 (at 300 nm), 0.452 (at 350 nm) and 0.093 (at 370 nm). If the contents deviate from
the data provided in the certificate, the concentrations of the standard solutions are
calculated based on the contents measured photometrically.

DIN EN 12822 (d,l- -tocopherol)
Concentration and purity are checked against methanol using spectrophotometric

measurements on a d,l- -tocopherol standard solution of a defined concentration and
subsequent calculation of mass concentration.
In order to assess concentration and purity, 1.0 ml of the stock solution is pipetted into a
100 ml volumetric flask, evaporated under nitrogen and dissolved in 100 ml methanol. The
concentration is approx. 8 mg/100 ml.
To this end, the absorption capacity of the d,l- -tocopherol standard solution is measured
against methanol in a quartz cuvette with a layer thickness of 1 cm at the absorption
maximum of 292 nm for d,l- -tocopherol using a suitable spectrophotometer.
The mass concentration, , of d,l- -tocopherol in micrograms per millilitre is calculated

using the following equation:
A 10 4
[ g / ml ]
76
Where:
A Absorption maximum at 292 nm
76 E11cm
%
value for d,l- -tocopherol in methanol
In addition, the absorption capacity should be measured at 255 nm (minimum). At this

wavelength, the extinction should be between 6 and 8; otherwise the vitamin has
decomposed and the standard cannot be used.
If the contents deviate from the data provided in the certificate, the concentrations of the
standard solutions are calculated based on the contents measured photometrically.
7.1.3 Production of the calibration solutions
There is a form that provides the exact details on the concentrations of the individual
solutions for the production of the calibration solutions (see Appendix 1). This form
documents the initial weights and the dilution steps. It is filed in the corresponding folder
together with the chromatograms and the calibration curve.
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Retinol stock solution:

About 80 mg retinol are weighed into a 100 ml volumetric flask (not brown glass) at a
precision of ± 0.1 mg, dissolved in ethanol and the solution is made up to the mark. Add a
few ml ethyl acetate if the standard does not dissolve. The stock solution contains about
80 mg/100 ml retinol.
Vitamin E stock solution:

About 800 mg d,l- -tocopherol are weighed into a 100 ml volumetric flask (not brown
glass) at a precision of ± 0.1 mg, dissolved in ethanol and the solution is made up to the
mark.
Attention: Brown glass volumetric flasks should not be used in the production of the stock
solutions as it is not possible to see whether the standard is fully dissolved.
Comment: The stock solution for vitamin A is diluted again to 1/200 in ethanol, the stock
solution for vitamin E to 1/100 in methanol (after evaporating off the ethanol) and
spectrophotometrically assessed for concentration and purity, as described under points
7.1.1 and 7.1.2.
Whether or not the standard can be used as a calibration standard can only be
determined based on the values obtained from the photometric determination of purity.
This means that the dilutions in the following are only produced, or calculations of their
contents are made, if the results of the determination of purity confirm a purity of > 95 %.
Dilutions made from the stock solutions:
Standard mixture A/E 1:

5.0 ml of the retinol stock solution and 5.0 ml of the tocopherol stock solution are pipetted
into a 100 ml volumetric flask and the solution is made up to the mark with ethanol. The
standard solution contains about 4 mg/100 ml retinol and 40 mg/100 ml tocopherol.

10.0 ml of the standard mixture A/E 1 are pipetted into a 100 ml volumetric flask and the
solution is made up to the mark with ethanol. The standard mixture A/E 2 contains about
0.4 mg/100 ml retinol and 4 mg/100 ml tocopherol.
Calibration solutions and working standard (dilution from standard mixture A/E 2):

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0.08 mg/100 ml retinol and 0.8 mg/100 ml tocopherol.
Standard mixture A/E 6 (working standard):

solution is made up to the mark with ethanol. The standard solution A6 contains about
This solution is used as a working standard for regular checks on the HPLC equipment.

The standard solutions A/E 3 to A/E 7 are used for the calibration. The solutions are
analysed in triplicate. A calibration curve is produced with the aid of the software and a
corresponding response factor is calculated.
The chromatograms thus obtained and the calibration curves are filed together with the
form in the "calibration" folder.
The working standard (standard mixture A/E 6) is decanted into vials that are sealed and
stored in the refrigerator at 8 °C.
The standard mixture A/E 2 can also be stored in the refrigerator and has a shelf life of up
to one year if protected from light. This solution can be diluted and used to make working
standard if the working standard is used up prematurely.
A new calibration is required once the working standard has been used up or its
concentration deviates from the nominal concentration by more than 5%; however, it
should be conducted once a year.
7.2 Calibration for the determination of retinol palmitate

manufacturers and on our experience, the calibration standard for retinol
palmitate is obtained exclusively from Sigma. The standard should always
exhibit a purity of ~ 1,800,000 USP units/ g. Due to stability problems, the
purity of the standard must be checked prior to its use for calibration
purposes, even though a manufacturer's certificate is available (see 7.2.1).
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DIN EN 12823-1 (retinol palmitate)
Concentration and purity are checked against isopropanol using spectrophotometric

measurements on a retinol palmitate standard solution of a defined concentration and
The retinol palmitate standard P3 is used for the assessment of concentration and purity
(see 7.2.2 Calibration).
To this end, the absorption capacity of the retinol palmitate standard solution P3 is
measured against isopropanol in a quartz cuvette with a layer thickness of 1 cm at the
absorption maximum of 325 326 nm for retinol using a suitable spectrophotometer.
The mass concentration, , of retinol palmitate in IU per gram is calculated using the
following equation:
A 200 1900
[ IU / g ]
E
Where:
1900 E11cm
%
value for retinol palmitate in isopropanol
200 Dilution factor
E Initial weight [g]

against isopropanol at 300 nm, 325 nm, 350 nm and 370 nm.
E/E325 (or 326nm)
The standard can be used for calibration purposes if the ratio for retinol palmitate does not
exceed 0.602 (at 300 nm), 0.452 (at 350 nm) and 0.093 (at 370 nm). If the contents
deviate from the data provided in the certificate, the concentrations of the standard
solutions are calculated based on the contents measured photometrically.
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documents the initial weights and the dilution steps. It is filed in the corresponding folder
together with the chromatograms and the calibration curve.
Retinol palmitate stock solution:

About 150 mg retinol palmitate are weighed into a 100 ml volumetric flask (not brown
glass) at a precision of ± 0.1 mg, dissolved in isopropanol and the solution is made up to
the mark.
Attention: Brown glass volumetric flasks should not be used in the production of the stock
solution as it is not possible to see whether the standard is fully dissolved.
Comment: The stock solution is diluted again to 1/200 in isopropanol and
spectrophotometrically assessed for concentration and purity, as described under point
7.2.1.
Whether or not the standard can be used as a calibration standard and can only be
determined based on the values obtained from the photometric determination of purity.
This means that the dilutions in the following are only produced, or calculations of their
contents are made, if the results of the determination of purity confirm a purity of > 80 %.
Dilutions made from the stock solutions:
Standard solution P 1:
5.0 ml of the retinol palmitate stock solution are pipetted into a 100 ml volumetric flask and
the solution is made up to the mark with isopropanol. The standard solution contains
about 4 mg/100 ml retinol.
Standard solution P 2:
20.0 ml of the standard solution P 1 are pipetted into a 100 ml volumetric flask and the
solution is made up to the mark with isopropanol. The standard solution P 2 contains
about 0.8 mg/100 ml retinol.
Standard solution PV 1
The standard P 2 is processed like a sample for the production of the calibration solutions.
To this end, 5 ml of the standard solution P 2 are used for saponification and then
extracted with 150 ml petroleum ether (see sample processing 8.3 8.4). This is how the
standard solution PV 1 (standard solution after saponification) is obtained, with contents of
~ 30 µg/100 ml.
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Calibration solutions (dilutions made from standard solution PV 1):
Standard PV 2:
8.0 ml of the standard solution PV 1 are pipetted into a brown glass vial and the solvent is
evaporated off under nitrogen. The residue is then taken up in 1.0 ml methanol and
transferred to an HPLC vial. The standard PV 2 contains approx. 0.2 mg/100 ml retinol.
Standard PV 3:
Standard PV 4:
Standard PV 5:
Standard PV 6:
transferred to an HPLC vial. The standard PV 6 contains approx. 0.006 mg/100 ml
Retinol.
The standard solutions PV 2 to PV 6 are used for the calibration. The solutions are
analysed in triplicate. A calibration curve is produced with the aid of the software and a
The chromatograms thus obtained and the calibration curve are filed together with the
form in the calibration folder.
None of the retinol palmitate standard solutions can be stored as the standard solution
PV 1, in particular, is not stable. The standard mixture A/E 6 from the calibration with
retinol is used as the working standard in daily checks on HPLC measurements (see
7.1.3).
A new calibration should be carried out once a year.
7.3 Calibration for the determination of ß-carotene

manufacturers and on our experience, the calibration standard for ß-carotene
is obtained exclusively from Sigma . The standard should always exhibit a
purity of 95 %. In addition to the availability of the manufacturer's
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certificate, purity must be assessed prior to use of the standard for the
calibration (7.3.1).

DIN EN 12823-1
Concentration and purity are checked against n-hexane using spectrophotometric

measurements on a ß-carotene standard solution of a defined concentration and
measurement for this assessment (see Appendix 3).
This form documents the initial weight, the dilution and the values measured with the
photometer. It is filed in the corresponding folder together with the documents on
calibration.
In order to assess concentration and purity, 10.0 ml of stock solution I is pipetted into a
100 ml volumetric flask and the solution is made up to the mark with 100 ml n-hexane.
The concentration is approx. 0.7 mg/100 ml. (see also 7.3.2 Calibration).
To this end, the absorption capacity of the ß-carotene solution is measured against
n-hexane in a quartz cuvette with a layer thickness of 1 cm at the absorption maximum of
453 455 nm for ß-carotene using a suitable spectrophotometer (e.g. RPM002).
The mass concentration, , of ß-carotene in micrograms per millilitre is calculated using
the following equation:
A 10 4
[ g / ml ]
2592
Where:
2592 E11cm
%
value for ß-carotene in n-hexane

against n-hexane at 453 nm, 455 nm, 340 nm and 483 nm.
E/E453 (or 455nm)
The standard can be used for calibration purposes if the ratio for ß-carotene is > 15 (at
340 nm) and between 1.14 1.18 (at 483 nm). If the contents deviate from the data
provided in the certificate, the concentrations of the standard solutions are calculated
based on the contents measured photometrically.
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provides an exact documentation of the initial weights and the dilution steps. It is filed in
the corresponding folder together with the chromatograms and the calibration curve.
ß-carotene stock solution I:

About 70 mg ß-carotene are weighed into a 100 ml volumetric flask (not brown glass) at a
precision of ± 0.1 mg, dissolved in n-hexane/ dichloromethane (80/20, v/v) and the
solution is made up to the mark. The solution is then diluted 1/10 with n-hexane. The
stock solution contains about 7 mg/100 ml.
Attention: Brown glass volumetric flasks should not be used here as it is not possible to
see whether the standard is fully dissolved. All further dilutions are carried out in a brown
glass volumetric flask.
Comment: The stock solution I is diluted again to 1/10 in n-hexane and spectrophoto-
metrically assessed for concentration and purity, as described under point 7.3.1. Whether
or not the standard can be used as a calibration standard and can only be determined
based on the values obtained from the photometric determination of purity. This means
that the dilutions in the following are only produced, or calculations of their contents are
made, if the results of the determination of purity confirm a purity of > 95%.
ß-carotene stock solution II:

10.0 ml of stock solution I are pipetted into a 100 ml volumetric flask and the solvent is
evaporated off under nitrogen. The residue is dissolved in 100 ml eluent (6.2). The stock
solution II contains about 0.7 mg/100 ml.
Calibration solutions and working standard (dilutions made from stock solution II):
ß-carotene standard solution C1:

10.0 ml of stock solution II are pipetted into a 100 ml volumetric flask and the solution is
made up to the mark with eluent. The standard solution C1 contains about
0.07 mg/100 ml.
0.035 mg/100 ml.
ß-carotene standard solution C3 (working standard):

0.02 mg/100 ml.
This solution is used as a working standard for regular checks on the HPLC equipment.
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1.0 ml of stock solution II is pipetted into a 100 ml volumetric flask and the solution is
0.007 mg/100 ml.
The standard solutions C 1 to C 4 are used for the calibration. The solutions are analysed
in triplicate. A calibration curve is produced with the aid of the software and a
The chromatograms thus obtained and the calibration curve are filed together with the
form in the calibration folder.
The working standard (standard solution C3) is decanted into vials that are sealed and
stored in the freezer at -4 °C.
Stock solution I is highly unstable and cannot be stored in the refrigerator. A new
calibration must be conducted if the working standard is used up prematurely.
A new calibration is otherwise only required if the working standard has been used up or
its concentration deviates from the nominal concentration by more than 5 %. However, it
should be conducted once a year.
8 Conduct
General note: Vitamin A, E and ß-carotene are sensitive to UV radiation and oxidizing
agents (such as, e.g. oxygen). It is therefore important to protect the samples from
exposure to UV light (e.g. through the use of brown glass or aluminium foil) and oxygen
(by working under nitrogen, especially during the saponification) as much as is possible.
8.1 Preparation / processing

The sample material will need to be chopped up and homogenized by milling if it is a
cereal.
All other samples will generally not require milling unless they are inhomogeneous or if
they contain coarse-grained material.
8.2. Weighing out samples

All samples are weighed out into a 250 ml brown glass flat-bottomed flask.
The initial weights should never be below 0.5 g or above 10 g for solid samples. Following
extraction, the sample must be diluted accordingly if vitamin contents are high (e.g. for
vitamin mixtures).
The initial weight is generally tailored to the vitamin A content and is selected such that
the vitamin A content in the initial weight is at least 0.01 mg, but no more than 0.05 mg.
If only vitamin E is of interest in the sample, then the vitamin E content in the initial weight
should be at least 0.1 mg, but no more than 1.0 mg.
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If only vitamin ß-carotene is of interest in the sample, then the ß-carotene content in the
initial weight should be at least 0.005 mg, but no more than 0.05 mg.
The weight table in the following is used for guidance in the selection of the initial weight:
Vitamin A vitamin content [mg/100g] initial weight [g]
solid samples ~0.100 0.700 5.0
vitamin mixture/effervescent tablet >1.0 600.0 0.5 1.0*
vitamin content [mg/100g] initial weight [g]
liquid samples ~0.05 0.1 20.0
>0.1 10.0
creams (e.g. Forticream) 0.1 0.2 10.0
Vitamin E vitamin content [mg/100g] initial weight [g]

solid samples ~ 2.0 20.0 5.0
vitamin mixture/effervescent tablet >20 0.5 1.0*
>5.0 10.0
Vitamin ß-carotene vitamin content [mg/100g] initial weight [g]

solid samples ~ 0.05 1,0 5.0
vitamin mixture/effervescent
tablet >50 0.5 1.0*
>0.25 10.0
* Samples must be further diluted
8.3 Saponification
The samples are initially suspended in 20 to 40 ml of water and left to stand for a short
while.
Attention: Care must be taken to ensure that no lumps are formed; the sample must be
fully wetted with water.
While swirling the flask, add 100 ml ethanol, about 1 g sodium ascorbate or L-ascorbic
acid and 25 ml potassium hydroxide solution (60%) to the suspended sample. The flat-
bottomed flasks are then sealed with a glass stopper and shaken vigorously.
The reaction mixture is now heated to boiling point under reflux in a water bath that has
been pre-heated to 80 - 90°C and maintained at boiling point for 30 min under a slow flow
of nitrogen.
Alternatively, the samples can also be heated for 3 hours in a water bath at 50 °C and
then left to stand overnight after the saponification is complete. In this case, the samples
are sealed with a glass stopper.
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Attention: Ensure that the ground glass joint of the flat-bottomed flask is thoroughly
rinsed with water before it is fitted to the reflux condenser, as it will otherwise not be
possible to remove the flask from the reflux condenser after saponification. However, if too
much water is added, then saponification will no longer be complete.
After saponification, rinse the condenser with a little water and cool the contents of the
flask to about 15°C.
Attention: If oil drops are still visible on the surface of the saponified mixture after
saponification, then add more potassium hydroxide solution and extend the duration of
saponification.
8.4 Extraction
The saponified solution is transferred quantitatively to a separating funnel through multiple

rinsing with water. In order to avoid the formation of emulsions, sufficient water is added to
the saponified sample solution to ensure that the alcohol/water ratio in the resulting
solution is about 1:1. Following this, 150.0 ml petroleum ether is added and the vitamins
are extracted by shaking on a mechanical shaker for approx. 10 minutes.
Attention: The quantity of petroleum ether that is added must be measured accurately.
The aqueous phase is allowed to drain away and discarded after phase separation. Add a
little ethanol to dissolve the emulsion if the aqueous and the petroleum ether phases do
not separate.
Attention: The two phases must separate before the aqueous phase is allowed to drain
away.
The extraction solution is now washed neutrally with purified water through multiple
shaking and the washing water is allowed to drain away after the phases have separated.
The extraction solution is filtered through a phase separation filter into a 50 ml volumetric
flask to remove any potential suspended water drops.
8.5 Concentration
For vitamin A and E and for carotene, 3.0 ml each of the extraction solution are pipetted
into a brown glass vial and the solution is evaporated to dryness under nitrogen at 45 °C
in the evaporation system.
Attention: Vitamin mixtures and more highly concentrated samples (see 8.2 Weighing out
samples) must be diluted accordingly prior to evaporation to dryness. After evaporation to
dryness, each residue is taken up in 1.0 ml methanol for the determination of vitamin A
and E and ß-carotene. The solutions are transferred to HPLC vials and can be used for
the determination.
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8.6 Determination
Vitamin A and E:
Vitamin A and vitamin E are measured simultaneously on one column. To this end, 50 µl
of the sample and standard solutions (working standard A/E 6) are injected onto the HPLC
column and eluted using a methanol/water mixture (98+2, v/v). A flow-rate gradient is
used to ensure that the two substances elute at different retention times and to largely
eliminate interfering matrix peaks (see 6. HPLC). Furthermore, the wavelength is changed
from 326 nm (for retinol) to 292 nm (for tocopherol) after 8.5 min.
Comment: The HPLC separation conditions must be selected such that the isomers of
all-trans retinol and 13-cis-retinol are combined in one peak.
Vitamin ß-carotene:
20 µl of the sample and standard solution (working standard C3) are injected onto the
HPLC column and eluted isocratically at a flow rate of 1.5 ml/min using a mixture of
dichloromethane/ methanolic ammonium acetate solution/ acetonitrile (20/5/75, v/v) with
the addition of 0.08 g BHT and 0.085 g triethylamine per 100 ml. Determination is carried
out using VIS detection at 450 nm.
Comment: The conditions must be selected such that -carotene is separated from
ß-carotene.
9 Analysis
Vitamin A
The isomers of all-cis retinol and all-trans retinol are combined in one peak and their sum
is the content of vitamin A.
Comment: All samples that contain retinol palmitate are analysed using the retinol
palmitate calibration (see 7.2). All other samples containing retinol or retinol acetate are
analysed using the retinol calibration (see 7.1).
Vitamin E:
At higher contents, the peak for -tocopherol exhibits a small shoulder that does not need
to be separated off.
Vitamin ß-carotene
Two separate peaks are obtained for -carotene and ß-carotene. The peak for ß-carotene
exhibits a small shoulder, the area of which must be included for the determination of
content. -carotene elutes approx. 1 minute before ß-carotene and appears as a double
peak. If -carotene is present in the sample, then this peak is separated from the
ß-carotene peak and the contents are given separately. However, the value for -carotene
is only given as a guideline, as no calibration is conducted with -carotene.
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10 Calculation
The contents of vitamin A, E and ß-carotene are calculated using the external standard
method. A response factor is calculated based on the peak areas of the standard and this
is then used to calculate the vitamin content in the sample. The contents of vitamin A, E
and ß-carotene are given in mg/100g sample.
Calculation of the response factor from the standard chromatogram:
c( Std .) mg / 100 ml
Re sponsefactor
Area
Calculation of the vitamin content based on the response factor:
Area(Pr) Re sponsefactor
c Vita min mg / 100 g 100
W [ g]
Area (Sa ) c (Std .)[mg ] 150ml 1ml

c Vita min mg / 100 g 100 f
Area (Std ) 100ml E [ g ] 3ml
Where:
c (vitamin): Vitamin concentration [mg/100g]
Area (Std.): Standard peak area
Area (Sa) Sample peak area
W: Initial weight [g]
c (Std): Standard concentration [mg/100ml]
150 ml: Quantity of petroleum ether added
3 ml: Quantity evaporated off
1 ml: Quantity of MeOH or iso-octane added
100: Factor for the calculation per 100 g sample
f: Dilution factor dependent on dilution
11 Validation
11.1 Validation data for vitamin A
The current method essentially corresponds to the European Standard prEn 12823-1,
Determination of Vitamin A by high performance liquid chromatography Part 1:
Measurements of all-trans retinol and 13-cis retinol (Final draft, September 1999) .
The following characteristic procedural data were determined to assess and validate the
method at CLF:
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11.1.1 Linearity
Linearity was assessed with the aid of standard solutions at different concentrations. Two
separate calibration curves were produced. To this end, 7 different concentrations ranging
between ~ 8 µg/100ml and ~ 200 µg/100ml were measured for retinol. For retinol
palmitate, 6 different concentrations ranging between ~ 40 µg/100ml and ~ 200 µg/100ml
were measured.
The following correlation coefficients (R²) were calculated:
Retinol Retinol palmitate

Correlation coefficient (R²) 0.9999 0.9992
11.1.2 Precision
The precision was assessed with repeat analysis (6-fold) of the 3 most frequently
occurring matrices: infant formula, milk cereal and PKU. For the infant formula, repeated
precision (several determinations, one laboratory, different days, different analysts) was
assessed in addition to laboratory precision (repeat determination, one laboratory, one
day, one analyst).
The expanded uncertainty (UE) was then calculated from the standard deviation (s), which
permits a statement to be made on the confidence interval for the method.
Vitamin A
x (n=5) [mg/100g] 0.357
repeated precision s [mg/100g] 0.0106
infant formula CV [%] 3.0
U [mg/100g] 0.030
x (n=6) [mg/100g] 0.352
laboratory precision s [mg/100g] 0.0207
U [mg/100g] 0.048
x (n=6) [mg/100g] 1.071
PKU CV [%] 14.0
U [mg/100g] 0.386
x (n=6) [mg/100g] 0.358
milk cereal CV [%] 16.9
U [mg/100g] 0.156
11.1.3 Accuracy
The accuracy was assessed by spiking an infant formula with standard solutions. The
sample was initially analysed without spiking and then twice each with spiking in the lower
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and upper working ranges. In addition, a reagent blank was measured. Following this, the
recovery rate was determined for the added quantity of standard.
Vitamin A
Recovery - mean value (x) [% ] 84.6
11.1.4 Limit of determination

The limit of determination was determined based on the signal/noise ratio. Standard
solutions of different concentrations were injected to determine the limit of determination.
The concentration at which the peak area was still at least 10 times higher than the noise
was defined as the limit of determination.
Vitamin A
Limit of determination [mg/100 g] 0.02
11.1.5 Reference material

Within the framework of routine analysis, the current method is assessed through regular
analysis of internal reference material.
The internal reference material is always analysed together with the routine samples as a
control sample and the results are regularly entered into quality control charts.
11.1.6 Inter-laboratory comparisons

A further assessment of the method occurs through regular participation in inter-laboratory
comparisons.
11.2 Validation data beta-carotene

The current method essentially corresponds to the European Standard DIN EN 12823-2,
Measurement of ß-carotene (July 2000) .
The following characteristic procedural data, listed under points 1-4, were determined to
assess and validate the method at CLF:
11.2.1 Linearity
Linearity was assessed with the aid of standard solutions at different concentrations. To
this end, a calibration curve was produced using 6 different concentrations ranging
between ~ 30 µg/100ml and 190 µg/100ml.
Based on this, the following correlation coefficient (r) was calculated:
Beta-carotene
Correlation coefficient (R²) 0.9999
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11.2.2 Precision
Reproducibility was determined for beta-carotene. To this end, infant formula was
analysed by different people over a lengthy time period.
The expanded uncertainty (UE) was then calculated from the standard deviation (s), which
permits a statement to be made on the confidence interval for the method.
Beta-carotene
x (n=5) [mg/100g] 0.150
U [mg/100g] 0.025
11.2.3 Accuracy
Beta-carotene

Beta-carotene

control sample and the results are regularly entered into quality control records.

comparisons.
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11.3 Validation data Vitamin E

The current method essentially corresponds to the European Standard prEn 12822,
Determination of Vitamin E by high performance liquid chromatography Measurements
of -, -, - and -tocopherol (Final draft, September 1999) .
The following characteristic procedural data, listed under points 1-4, were determined to
assess and validate the method at CLF.
(If not otherwise noted, the data provided under vitamin E refer to -tocopherol).
11.3.1 Linearity
Linearity was assessed with the aid of standard solutions at different concentrations. To
this end, a calibration curve was produced using 7 different concentrations ranging
between ~ 5 µg/100ml and 180 µg/100ml.
Based on this, the following correlation coefficient (r) was calculated:
Vitamin E
Correlation coefficient (r) 0.9994
11.3.2 Precision
The precision was assessed with repeat analysis (6-fold) of the 3 most frequently
occurring matrices: infant formula, milk cereal and PKU. For the infant formula, repeated
precision (several determinations, one laboratory, different days, different analysts) was
assessed in addition to laboratory precision (repeat determination, one laboratory, one
day, one analyst).
The expanded uncertainty (UE) was then calculated from the standard deviation (s),
which permits a statement to be made on the confidence interval for the method.
Vitamin E
x (n=5) [mg/100g] 6.00
U [mg/100g] 0.34
x (n=6) [mg/100g] 6.18
U [mg/100g] 0.90
x (n=6) [mg/100g] 25.30
PKU CV [%] 3.0
U [mg/100g] 5.86
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x (n=6) [mg/100g] 3.03

milk cereal CV [%] 3.4
U [mg/100g] 0.27
11.3.3 Accuracy
Vitamin E

Vitamin E

control sample and the results are regularly entered into quality control records.

comparisons.
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9 References
1. European Standard prEn 12823-1
Measurements of all-trans retinol and 13-cis retinol, Final draft, September 1999
2. European Standard DIN EN 12823-2

Measurement of ß-carotene (July 2000)
3. European Standard prEn 12822

Determination of Vitamin E by high performance liquid chromatography
Measurements of -, -, - and -tocopherol, Final draft, September 1999
4. Official collection of methods for sampling and examining foods pursuant to § 64
of the Foods and Other Commodities Act (LMBG)
Determination of vitamin A in dietary food products L49.00-3, May 1985;
Determination of vitamin A in food products by HPLC Part 1: Measurements of all-
trans retinol and 13-cis retinol L00.00-63/1; Part 2: Measurement of ß-carotene
L00.00-63/2, July 2001; Determination of vitamin E in food products by HPLC,
L00.00-62, July 2001
5. VA504_06_V01 Method validation
10 Attachments
Attachement 1 Form for the calibration and purity assessment of retinol and
d,l- -tocopherol
Attachement 2 Form for the calibration and purity assessment of retinol palmitate
Attachement 3 Form for the calibration and purity assessment of ß-carotene
Attachement 4 Chromatogram of a retinol and tocopherol standard
Attachement 5 Chromatogram of a ß-carotene standard

Determination of Vitamin A (Retinol), Vitamin E ( ( ) - Tocopherol) and SS-Carotene in Foodstuffs by HPLC-UV

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Determination of Vitamin A (Retinol), Vitamin E ( ( ) - Tocopherol) and SS-Carotene in Foodstuffs by HPLC-UV

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Vitamin A, vitamin E, ß-carotene