SPSFAM-INGR-02

Based from Call for Methods 03-09-2012

Laboratory
Instructions

VITAMINS A and E by UPLC-UV or FLD

1 SCOPE OF APPLICATION

Description of a method for the quantitative determination of vitamins A (retinol) and E (alpha-
tocopherol) in infant formula, infant and breakfast cereals and beverages by UPLC.

The same extracts can be used to analyse vitamin D (full procedure described elsewhere).

The method has been validated in-house for infant formula. The method is similar to the AOAC and
CEN procedures.

2 DEFINITIONS AND ABBREVIATIONS

2.1 Definitions
Vitamin A Sum of all-trans retinol, 13-cis retinol and retinol esters
Vitamin E Sum of alpha-tocopherol and tocopherol esters

2.2 Abbreviations
UPLC Ultra performance LC RE Retinol Equivalent
UV Ultraviolet TE Tocopherol equivalent
FL Fluorescence IU International Unit
BHT Butylhydroxytoluene
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3 PRINCIPLE OF METHOD

• Alcoholic saponification with potassium hydroxide in the presence of antioxidants

• Solid Phase Extraction using diatomaceous earth cartridge. Elution with hexane
• Evaporation of an aliquot to dryness
• Dilution in n-hexane

Vitamin A analysis Vitamin E analysis Vitamin D preparative

chromatography
• HILIC-UPLC • HILIC-UPLC
• UV detection • FL detection • NP-HPLC
• Fraction collection
• Evaporation
• Dilution in appropriate solvent

Vitamin D analysis
• RP-HPLC
• UV detection

4 SAFETY PRECAUTIONS

Potassium hydroxide is extremely corrosive; avoid any contact with eyes and skin. Wear laboratory
safety goggles. Work in a fume hood when working with solvents. Refer to MSDS for specific
information.

5 CHEMICALS AND MATERIALS

Commercial references are only a guideline. Use equivalent chemicals or materials when listed
items are not locally available.
Numbers in the margin refer to CAS numbers for the chemical.

5.1 Chemicals
Before using chemicals, refer to adequate manuals or safety data sheets approved by your local
authorities and ensure that the safety guidelines are applied.

CAS number - Chemical or reagent, purity (e.g. supplier, article number and website)
64-17-5 Absolute ethanol, GR, ACS, ISO ( e.g. Merck,100983)
67-63-0 - 2-propanol
110-54-3 - n-hexane, for HPLC LiChrosolv (e.g. Merck 104391)
1310-58-3 - Potassium hydroxide pellets, for analysis (e.g. Merck 105033)
68-26-8 - All-trans retinol (vitamin A) cryst.,(e.g. Fluka nb.95144)
27610-45-3 - Sodium sulphide hydrated.(e.g. Merck 106638)
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CAS number - Chemical or reagent, purity (e.g. supplier, article number and website)
151-21-3 - Sodium dodecyl sulphate (e.g. Fluka nb..71727)
7757-82-6 - Sodium sulphate anhydrous, ACS,ISO (e.g.Merck 106649)
10191-41-0 - dl-alpha tocopherol, for biochem., 98-102% (e.g.108283)
207605-40-1 - Sodium 1-pentanesulfonate, (e.g. Fluka 76955)
134-03-2 - Sodium ascorbate (e.g. Merck 500076)
128-37-0 - Butylhydroxytoluene,(2,6-Di-tert-butyl-4-methylphenol ( e.g.Merck 822021
9001-19-8 - Takadiastase (e.g. Fluka nb.86247) or amylase

5.2 Materials
- Standard laboratory amber glassware,class A
- Membrane filter (e.g.Millipore,Millex,-GP 0.22 µm)
- Disposable plastic syringes, 2mL
- Cromabond adapter (e.g.Macherey-Nagel 730566)
Chromabond XTR cartridges 70 mL/14500 mg (Macherey–Nagel 730507) or
-
equivalent

- Chromabond columns empty 70 mL (e.g.Macherey-Nagel 730158)

- Filling material Chromabond XTR Btl 14.5g (e.g.Macherey-Nagel 730585)

- Polytron homogeniser
Water bath, with magnetic stirrers, equipped with Allihn condenser.,
-
(e.g.Labotech DWB 16 or equivalent) or magnetic stirrer
Laboratory oven, 40°C±5°C
- Rotary evaporator

5.3 Special equipment and instrumentation

5.3.1 Ultra performance Liquid Chromatograph (Vitamins A & E)
UPLC system equipped with a binary gradient pump and hexane kit, a sample injector equipped
with a 10µL injection loop, UV/DAD detector, a FL detector and data software.
5.3.2 UPLC column (Vitamins A & E)
Acquity UPLC BEH HILIC 1.7 µm, 2.1 x 100mm (Waters, 186003461) or equivalent.

6 PREPARATION OF REAGENTS

Volumes of glassware are purely indicative and may be modified as long as the proportion of
reagents is maintained.
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6.1 n-hexane containing 0.05 mg/mL BHT

Into a 1000-mL volumetric flask introduce 50 mg BHT, dissolve, and make up to the mark with
n-hexane.

This solution can be stored 6 months at room temperature.

6.2 Mobile phase A: 1 % (v/v) 2-propanol in n-hexane

Into a 1000-mL volumetric flask pipette 10 mL 2-propanol. Make up to the mark with degassed
n-hexane.

This solution can be stored 2 months at room temperature.

6.3 Mobile phase B: n-hexane

6.4 All-trans retinol standard solutions

6.4.1 All-trans retinol stock solution, about 150 µg/mL
Into a 100-mL amber glass volumetric flask weigh 15 mg ± 5 mg all-trans retinol cryst. Dissolve
and make up to the mark with ethanol.

Note: The concentration of this solution must be determined by spectrophotometry (6.6.2) each
day of use. The calculated concentration should be at least 80% of the theoretical, otherwise
prepare freshly the stock solution.

This solution is stable for at least 2 weeks at -20 °C.

Note: If all-trans retinol standard is not available, it is possible to use saponified retinyl acetate
(see Annex 1 for standard preparation procedure)
6.4.2 All-trans retinol intermediate solution, about 15 µg/mL
Into a 50-mL amber glass volumetric flask pipette 5 mL of stock solution (6.5.1). Make up to the
mark with n-hexane.

This solution must be prepared fresh daily.

6.4.3 All-trans retinol working solutions, about 3.0, 1.5 and 0.6 µg/mL
Into a 25 mL amber glass volumetric flask pipette 5 mL of intermediate solution (6.3.3). Make up to
the mark with n-hexane. This solution contains about 3.0 µg/mL.

Into two different 10-mL amber glass volumetric flasks, pipette 6.0 mL and 2.0 mL of this solution.
Make up to the mark with n-hexane. These solutions contain respectively about 1.5 and 0.6 µg/mL.
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These solutions must be prepared fresh daily.

6.5 Vitamin E standard solutions

6.5.1 dl-alpha-tocopherol stock solution, about 0.5 mg/mL
Into a 100-mL amber glass volumetric flask weigh 50 mg ± 5 mg dl-alpha-tocopherol. Dissolve and
make up to the mark with ethanol.

Note: The actual concentration of this solution must be determined by spectrophotometry (6.6.2)
each day of use. The calculated concentration should be at least 85% of the theoretical, otherwise
prepare freshly the stock solution.

This solution is stable for at least 1 month at -20 °C.

6.5.2 dl-alpha tocopherol intermediate solution, about 25 µg/mL
Into a 50-mL amber glass volumetric flask, pipette 2.5 mL dl-alpha tocopherol stock solution (6.6.1).
Make up to volume with hexane. This solution contains about 25 µg/mL.
This solution must be prepared fresh daily.
6.5.3 dl-alpha tocopherol working solutions, about 10, 7.5, 5 and 2.5 µg/mL
Into four separate 10-mL amber glass volumetric flasks pipette 4.0 mL, 3.0 mL, 2.0 mL and 1.0 mL
of the intermediate solution (6.6.2). Make up to the mark with n-hexane. These solutions contain
respectively about 10, 7.5, 5 and 2.5 µg/mL.
These solutions must be prepared fresh daily.

6.6 Determination of the concentration of the stock solutions by spectrometry

6.6.1 All-trans retinol stock solution
Into a 100-mL amber glass volumetric flask pipette 2 mL of all-trans retinol stock solution (6.3.1)
and make up to the mark with ethanol. This solution contains about 3 µg/mL of all-trans retinol.

Measure the absorbance (A) at 326 nm against ethanol. Determine the all-trans retinol
concentration according to the following formula:

A 326 nm
Concentration of stock solution (µg/mL) = ∗ 150
0.549

Where:
A326 nm = measured absorbance at 326 nm
0.549 = theoretical absorbance of a all-trans retinol solution in ethanol at 3 µg/mL ( E 11%cm = 1830 )

6.6.2 dl-alpha tocopherol stock solution

Into a 50-mL amber glass volumetric glass flask pipette 5 mL of dl-alpha tocopherol stock solution
(6.4.1) and make up to the mark with ethanol. This solution contains about 0.05 mg/mL.
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Measure the absorbance (A) at 292 nm against ethanol. Determine the dl-alpha tocopherol
concentration according to the following formula.

A 292 nm
Concentration of stock solution (mg/mL) = ∗ 0 .5
0.379

Where:
A292 nm = measured absorbance at 292 nm
0.379 = theoretical absorbance of a alpha-tocopherol solution in ethanol at 0.05 mg/mL
( E11%cm = 75.8 )

7 PREPARATION OF TEST SAMPLES

7.1 Test sample preparation

7.1.1 Solid products (e.g. infant formulas powder, infant and breakfast cereals, powder
beverages
In a 250 mL beaker weigh 50.0 g ± 1.0 g of powder sample and record the mass to 0.1 g. Add
100 g of water at 40°C ± 5°C and mix with a glass rod or Polytron until the suspension is
homogeneous.

If the sample contains starch, 50 mg ± 10 mg Takadiastase or amylase can be added to facilitate

handling. Mix well and let stand for a few minutes.

Proceed as described under 8.1.

7.1.2 Liquid products (e.g. RTD formulas, liquid milk)

Bring to room temperature the whole laboratory sample (original container or at least 75 g. Mix well.
If the sample contains starch, 50 mg ± 10 mg Takadiastase or amylase can be added to facilitate
handling. Mix well and let stand for 15 min at 40 °C ± 5 ° C.

Proceed as described under 8.1.

Avoid any fat separation: mix well.

8 PREPARATION OF TEST PORTIONS & SOLUTIONS

QS samples (certified reference materials, in-house reference samples or spiked samples) must be
regularly included and analysed in duplicate.
If necessary, different sized glassware may be substituted for specific volumes listed during the
preparation of test solutions as long as the proper dilutions ratios are maintained.
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8.1 Saponification
In a 250-mL brown glass flat bottomed flask with a ground-glass neck weigh 30.0 g ± 0.1 g of the
sample suspension.(7.1.1) (which corresponds to 10.0 g dry test portion, m) or 30.0 ± 0.1 g of
liquid sample.

Add 7 g potassium hydroxide puriss. Mix to dissolve. Add 50 ml absolute ethanol and the
antioxidant mix (1 g sodium sulphide, 1 g sodium ascorbate). Add a magnetic stirring rod.

Note: 1 g sodium ascorbate can be replaced by about 0,5 g pyrogallol. Hydroquinone (0.5 g) can
also be used as antioxidant instead of sodium sulphide and sodium ascorbate mixture.

Proceed as per 8.1.1 or 8.1.2

8.1.1 Hot saponification
Mount on the flask an adapter for gas introduction and an Allihn condenser. Introduce a slight
nitrogen stream and heat for 30 min at 85 °C + 3 °C while stirring in a water bath provided with
magnetic stirrers.
8.1.2 Overnight saponification
Introduce a slight nitrogen stream for about 30 sec. in order to replace oxygen. Stopper the flask.
Place the flask on a magnetic stirrer overnight at room temperature (25 °C + 5).

Note: During saponification ensure thorough stirring of the reaction medium

8.2 Extraction

Cool the flask to room temperature and transfer quantitatively into a 100-ml amber glass volumetric
flask. Add 2 g sodium 1-pentanesulfonate and make up to the mark with water. Shake well for
1 min.

Note: Sodium 1-pentanesulphonate is expensive and can be replaced by 1.5 g sodium dodecyl
sulphate which is much cheaper. Sodium dodecyl sulphate is less soluble. In principle it is easier to
use as aqueous solution (1.5 g sodium dodecyl sulphate in 3.5 ml water).

Prepare the cartridge by fitting a needle to the Luer connection at the lower end of the cartridge.
Fix the latter by means of a clamp.

Note: Too coarse granulometry of the filling material will reduce the extraction recovery - particle
size should not be larger than 0.4 mm diameter.

Pipette 20 ml of saponified solution on the top of the cartridge. The solution must be completely
retained by the packing. If not, start again with 15 ml and with another cartridge.

Note: It is also possible to mix the Cromabond XTR filling material in a beaker with 20 ml of the
hydrolysate with a glass rod and then transfer it into an empty cartridge. This improves the
dispersion and absorption of the liquid throughout the filling material. In this case, it is not
necessary to wait 15 min before starting the elution step.
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Wait 15 min, then elute with 100 ml n-hexane with added BHT (6.1). Collect the eluate into a
250 ml pointed-bottom amber glass flask. Stop the elution not later than 30 min after all the
n-hexane has been absorbed by the cartridge filling material (packing).

Remark: In some cases the saponified sample solution is not completely retained by the extraction
cartridge even when using 15 ml instead of 20 ml. The problem may be solved by using a smaller
quantity of potassium hydroxide during saponification, e.g. 4 g instead of 7 g or by adding 1 g
potassium dihydrogen phosphate to the 20 ml saponified sample solution before loading the
extraction cartridge.

8.3 Evaporation
Evaporate the solvent under reduced pressure; apply a nitrogen stream to evaporate the last few
milliliters.

Transfer the residue quantitatively into a 5 ml amber glass volumetric flask by means of small
portions of n-hexane. Make up to the mark with n-hexane.

Filter through 0.45 µm membrane filters. This solution is ready to be injected in the UPLC system.

Perform further dilutions in n-hexane to be within the calibration ranges ((retinol 0.6-3.0 µg/mL,
alpha tocopherol 2.5-10 µg/mL) if needed.

9 INSTRUMENTAL CONDITIONS

9.1 Chromatographic conditions

Start up the UPLC system with the following conditions:

9.1.1 UPLC conditions for vitamin A analysis

Analytical column : Acquity UPLC BEH HILIC 1.7µm 2.1x100mm

Column temperature : 25 °C
Mobile phase : mobile phase A: 1 % 2-propanol in n-hexane ()
Injection volume : 4 µL
Injection mode : Partial loop with needle overfill (5 µL loop)
UV detection : 326 nm
Data rate : pts/sec

9.1.2 UPLC conditions for vitamin E analysis

Analytical column : Acquity UPLC BEH HILIC 1.7µm 2.1x100mm

Column temperature : 25 °C
Mobile phase : mobile phase A: 1% 2-propanol in hexane ()
mobile phase B: hexane ()
Injection volume : 1 µL
Injection mode : Partial loop with needle overfill (5 µL loop)
Fluorescence detection : Excitation wavelength: 290 nm
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Emission wavelength: 326 nm

Data rate : 2 points/s
Flow rate : 1.2 mL/min

Gradient program: Time Mobile phase A Mobile phase B Curve

[min] [%] [%]
0 20 80
1.0 50 50
1.5 20 80

9.2 Instrument check test

Allow the chromatographic system to equilibrate for at least 15 minutes (UPLC) or 1 hour (HPLC)
before injecting standards and samples. Make sure the system pressure is stable and there are no
leaks. Before starting a series of analyses, inject the lowest point of the calibration curves at least 3
times and ensure the stability of the system, repeatable response and retention time. The
coefficients of variation should not be higher than 2% for retention time and peak response.

10 OPERATING PROCEDURE & DETERMINATION

10.1 Vitamins A & E

10.1.1 Sequence set up
Inject in duplicate, 4 or 1 µL (vitamins A and E respectively) of each of the working standard
solutions (6.4.3 and 6.5.3).

Sequentially inject 4 or 1 µL of the reagent blank and the test solutions. Include a working standard
or a QC sample every 6-8 samples to monitor system stability.
10.1.2 Calibration
Calculate average of peak area (or height) and standard deviation in the series of analysis.

Construct a calibration curve by plotting the peak area (or height) of each vitamin (all-trans retinol
and alpha-tocopherol) in each of the working standard solutions against concentration in
micrograms per millilitre. Calculate the slope (S) and the intercept (I) by linear regression.
10.1.3 Identification
Identify the all-trans retinol, 13-cis retinol and the alpha tocopherol peaks on the sample
chromatogram by comparison with the retention time of the corresponding peak in the standard
solutions (see example chromatograms in Enclosure).
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11 CALCULATIONS AND EXPRESSION OF RESULTS

11.1.1 All-trans retinol

Calculate the mass fraction, w, of all-trans retinol, in micrograms per 100 grams of sample, by
using the following equation:
(A - I) × V0 × V2
w all-trans retinol = s × 100
S × m × V1
Where:
m = mass of the test portion, in g (10.0 for powders and 30.0 for liquids)
As = area (or height) of the all-trans retinol peak in the sample chromatogram
S = slope of the calibration curve
I = intercept of the calibration curve
V0 = volume in which the saponified solution has been diluted, in mL (100.0)
V1 = aliquot of the saponified solution introduced in the cartridge, in mL (20.0)
V2 = final volume of the test solution, in mL (5.0)
11.1.2 13-cis retinol
Calculate the mass fraction, w, of 13-cis retinol, in micrograms per 100 grams of sample, by using
the following equation:
(A - I) × V0 × V2 × 1830
w 13-cis retinol = s × 100
S × m × V1 × 1680
Where:
m = mass of the test portion, in g (10.0 for powders and 30.0 for liquids)
As = area (or height) of the 13-cis retinol peak in the sample chromatogram
S = slope of the all-trans retinol calibration curve
I = intercept of the all-trans retinol calibration curve
V0 = volume in which the saponified solution has been diluted, in mL (100.0)
V1 = aliquot of the saponified solution introduced in the cartridge, in mL (20.0)
V2 = final volume of the test solution, in mL (5.0)
1830 = Absorbance coefficient E (1 cm, 1 %) of all-trans retinol
1680 = Absorbance coefficient E (1 cm, 1%) of 13-cis retinol
11.1.3 Vitamin E (alpha tocopherol)
Calculate the mass fraction, w, of alpha tocopherol, in milligrams per 100 grams of sample, by
using the following equation:
(A - I) × V0 × V2 × 100
w alpha tocopherol = s
S × m × V1 × 1000
Where:
m = mass of the test portion, in g (10.0 for powders and 30.0 for liquids)
As = area (or height) of the alpha tocopherol peak in the sample chromatogram
S = slope of the calibration curve
I = intercept of the calibration curve
V0 = volume in which the saponified solution has been diluted, in mL (100.0)
V1 = aliquot of the saponified solution introduced in the cartridge, in mL (20.0)
V2 = final volume of the test solution, in mL (5.0)
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11.2 Expression of results

11.2.1 Vitamin A
Express vitamin A as the sum of all-trans retinol and 13-cis retinol in Retinol Equivalents or
International Units.

Vitamin A (RE/100 g) = w all-trans retinol + 0.75 × w 13-cis retinol

11.2.2 Vitamin E
Express vitamin E in alpha-tocopherol equivalents using the appropriate factor according to
product composition:

1 alpha-TE = 1 mg d-alpha tocopherol

= 1.35 mg dl-alpha tocopherol

12 INTERNAL CONTROL PLAN

12.1 QC samples
QC samples (certified, in-house reference samples or spiked samples) must be regularly included
and analysed in duplicate.

12.2 Spiking experiments

Verify recovery rate by spiking samples. Calculate recovery rate (Rec) on the spiked samples
using the following equation:
Cs - C n
Rec = x 100
Ca
Where:
Cs = concentration of vitamin in the spiked test portion
Cn = concentration of vitamin in the non spiked test portion
Ca = concentration of vitamin A added to the test portion

Recovery rate should be higher than 85 %.

13 VALIDATION DATA

13.1 LoQ

Using a 10-g test portion, vitamins A and E can be accurately quantified (LoQ) at 30 RE/100 g and
< 0.45 TE/100 g.
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13.2 Precision
Repeatability and intermediate reproducibility (within laboratory variability) were estimated by
replicate analysis (nr) on different days (nd) using HPLC or UPLC.

Milk based infant formula Repeatability Intermediate reproducibility

Analyte Unit nd * nr Median SD(r) CV(r) r r% SD(iR) CV(iR) iR iR%

[%] [%] [%] [%]

Vitamin A HPLC RE/100 g 6*2 511 8.9 1.7 25 4.8 22.1 4.3 61 12.0

UPLC RE/100 g 7*2 475 6.3 1.3 17 3.7 22.7 4.8 63 13.2

Vitamin E HPLC TE/100 g 6*2 6.07 0.09 1.6 0.26 4.3 0.29 4.8 0.82 13.4

UPLC TE/100 g 7*2 6.17 0.16 2.6 0.44 7.1 0.19 3.1 0.54 8.7

Infant cereal Repeatability Intermediate reproducibility

Analyte Unit nd * nr Median SD(r) CV(r) r r% SD(iR) CV(iR) iR iR%

[%] [%] [%] [%]

Vitamin A HPLC RE/100 g 6*2 374 4.7 1.3 13 3.5 61.2 16.4 170 45.4

UPLC RE/100 g 7*2 404 6.4 1.6 18 4.4 69.5 17.2 193 47.6

Vitamin E HPLC TE/100 g 6*2 5.58 0.08 1.4 0.22 3.9 0.18 3.2 0.49 8.7

UPLC TE/100 g 7*2 5.29 0.14 2.6 0.38 7.1 0.20 3.7 0.55 10.4

Cocoa – malt beverage Repeatability Intermediate reproducibility

Analyte Unit nd * nr Median SD(r) CV(r) r r% SD(iR) CV(iR) iR iR%

[%] [%] [%] [%]

Vitamin A HPLC RE/100 g 6*2 604 28.3 4.7 78 13.0 35.9 5.9 99 16.5

UPLC RE/100 g 7*2 567 33.6 5.9 93 16.4 37.3 6.6 103 18.2

Vitamin E HPLC TE/100 g 6*2 0.33 0.01 2.0 0.02 5.5 0.02 5.5 0.05 15.3

UPLC TE/100 g 7*2 0.40 0.02 3.3 0.04 9.1 0.02 4.5 0.05 12.4

*This product has not been fortified with Vitamin E, the amount found corresponds to natural content from fat contribution. The values
are close to the LoQ and prove that vitamin E can be accurately quantified at this low level.

13.3 Trueness/Recovery
13.3.1 Analysis of internal reference samples
Samples with consensus values obtained from internal proficiency testing were analysed.
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Milk based infant formula Recovery

Analyte Unit nd * nr Consensus n Uncertainty of Median of Rec SD(Rec)

value consensus value results [%]
Vitamin A UPLC RE/100 g 7*2 474 41 13 475 100.2 0.033
Vitamin E UPLC TE/100 g 7*2 6.34 22 0.16 6.17 97.2 0.026
*Uncertainty of reference value calculated as SDref=1.2533*SDR/sqrt (n), where SDR is the standard deviation of reproducibility
obtained during proficiency testing and n is the number of participating laboratories.

Infant cereal Recovery

Analyte Unit nd * nr Consensus n Uncertainty of Median of Rec SD(Rec)

value consensus value results [%]
Vitamin A UPLC RE/100 g 7*2 365 20 22 404 110.8 0.099
Vitamin E UPLC TE/100 g 7*2 5.66 13 0.41 5.29 93.4 0.069
*Uncertainty of reference value calculated as SDref=1.2533*SDR/sqrt (n), where SDR is the standard deviation of reproducibility
obtained during proficiency testing and n is the number of participating laboratories.

13.3.2 Spiking experiments

Recoveries on spiking experiments were found to be in the range 93-103% for retinol and 95% for
alpha tocopherol in infant formula, infant cereals and enteral feeding products.

13.4 Measurement Uncertainty

Measurement uncertainty is estimated using the simplified approach based on existing validation
data proposed by Bartwick & Ellison (2000), mainly precision and trueness studies, which, if
properly planned to cover as many of the uncertainty sources previously identified as possible,
provide the necessary data required to calculate measurement uncertainty. Precision and trueness
contributions are combined together as followed to obtain the overall uncertainty :

Standard uncertainty : u = Median * CV(iR)2 + RSD(Re c )corrected

2

U = 2 * u (which gives a level of confidence o

95%)
Expanded uncertainty :

Milk based infant formula

Analyte Unit Median CV(iR) RSD(Rec)

Vitamin A UPLC RE/100 g 475 4.8 0.033

Vitamin E UPLC TE/100 g 6.17 3.1 0.026

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Infant cereal Standard Expanded

uncertainty uncertainty

Analyte Unit Median CV(iR) RSD(Rec) u %u U %U

Vitamin A UPLC RE/100 g 404 17.2 0.099 78 19.4 157 38.7

Vitamin E UPLC TE/100 g 5.29 3.7 0.069 0.44 8.3 0.87 16.5

14 REFERENCES

EN 12823-1 Foodstuffs – Determination of vitamin A by high performance liquid

chromatography – Part 1: Measurement of all-trans-retinol and 13-cis
retinol
EN 12822 Foodstuffs – Determination of vitamin E by high performance liquid
chromatography – Measurement of alpha, beta, gamma and delta
tocopherols
AOAC 992.04 Vitamin A (retinol isomers) in milk and milk-based infant formula
AOAC 992.06 Vitamin A (retinol) in milk-based infant formula
AOAC 992.03 Vitamin E activity (all-rac-alpha tocopherol) in milk-based infant formula
AOAC 2001.13 Vitamin A (retinol) in foods
Barwick VJ, Ellison SLR (2000) Development and harmonization of measurement uncertainty principles.
Part d : Protocol for uncertainty evaluation from validation data. LGC,
Teddington, UK.
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Annex 1: Preparation of all-trans retinol standard from all-trans retinyl acetate.

Into a 25 mL tube with screw cap weigh 20 mg all-trans retinyl acetate. Add 5 mL ethanol. Mix until
complete dissolution. Add 2 mL or 50 % KOH aqueous solution and 0.1 g sodium ascorbate.

Heat in a water bath at 85 °C for 30 minutes. Cool down to room temperature.

Add 5 mL water and 10 mL n-hexane. Stopper the tube and shake vigorously for about 30 sec.

Let stand to allow the phases to separate. Remove the lower aqueous layer with a pasteur pipette.

Add 10 mL water. Stopper the tube and shake vigorously.

Let stand to allow the phases to separate. Remove the lower layer with a pasteur pipette.

Add about 0.5 g anhydrous sodium sulphate. Swirl and filter into a 100 mL amber glass volumetric
flask. Rinse the tube with two portions of about 10 mL n-hexane. Make up to the mark with
n-hexane.
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Annex 2: Example chromatograms

a) dl-alpha tocopherol working solution, about 7.5 µg/mL (6.5.3) b) milk based infant formula powder, 6.2 TE/100 g

alpha-tocopherol - 1.014
100.00 100.00

95.00 95.00

90.00 90.00

85.00 85.00

alpha-tocopherol - 1.026
80.00 80.00

75.00 75.00

70.00 70.00

65.00 65.00

60.00 60.00

55.00 55.00

50.00 50.00
EU

EU
45.00 45.00

40.00 40.00

35.00 35.00

30.00 30.00

25.00 25.00

20.00 20.00

15.00 15.00

10.00 10.00

1.089
5.00 5.00

0.00 0.00

-5.00 -5.00
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00
Minutes Minutes

b) all-trans retinol working solution, about 1.8 µg/mL (6.4.3). b) milk based infant formula powder, 475 RE/100 g

0.034 0.034

all-trans-retinol - 1.350
all-trans-retinol - 1.346

0.032 0.032

0.030 0.030

0.028 0.028

0.026 0.026

0.024 0.024

0.022 0.022

0.020 0.020

0.018 0.018
AU

AU

0.016 0.016

0.014 0.014

0.012 0.012
13-cis-retinol - 1.058

0.010 0.010

0.008 0.008
13-cis-retinol - 1.054

0.006 0.006

0.004 0.004
1.289
1.137

0.002 0.002

0.000 0.000

-0.002 -0.002
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00
Minutes Minutes