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AOAC Official Method 2012.

10
Simultaneous Determination of 13-cis and all-trans
Vitamin A Palmitate (Retinyl Palmitate),
Vitamin A Acetate (Retinyl Acetate),
and Total Vitamin E (dl--Tocopherol
and dl--Tocopherol Acetate)
in Infant Formula and Adult Nutritionals
Normal-Phase HPLC
First Action 2012

[Applicable to the concurrent quantitative analysis of total


vitamin E (dl--tocopherol and dl--tocopherol acetate), vitamin A
palmitate, and vitamin A acetate (cis- and trans-isomers) present
in milk- and soy-based infant formula and adult nutritionals and
formulas containing hydrolyzed protein. Vitamin A is defined
as 13-cis and all-trans retinol (CAS No. 68-26-8), retinyl esters
(retinyl palmitate; CAS No. 79-81-2), and retinyl acetate (CAS
No, 127-47-9). The determination of vitamin E focuses on d-tocopherol (CAS No. 59-02-9), all-racemic -tocopherol (CAS
No. 1406-18-4), and their esters. -Tocopherol and esters can be
reported separately.]
The analytical range of the method is as follows:
Vitamin A, retinyl palmitate.2450 g/100 g reconstituted.
Vitamin A acetate.2450 g/100 g reconstituted.
dl--Tocopherol acetate.0.029.4 mg/100 g reconstituted.
dl--Tocopherol.0.038.0 mg/100 g reconstituted.
Caution: Correct personal and environmental safety standards
shall be used while performing this analytical method.
Laboratory personnel handling solvents, acids, and
reagents should be knowledgeable of their potential
hazards. Consult the Material Safety Data Sheets
(MSDSs) for information on the hazards and take proper
precautions. Transfer solvents and acids inside efficient
fume hoods and extractors. Ensure all glassware is free
from chipping and hairline cracks.
A. Principle

This procedure utilizes the proteolytic enzyme papain to hydrolyze


the hydrophilic protein coating of fat micelles in milk- or soybased infant formulations in an aqueous solution. The hydrophobic
contents of the micelles are then extracted quantitatively into isooctane in a single extraction and chromatographed by normalphase HPLC using a Zorbax NH2 analytical column. The analytes
are eluted with a gradient and dl--tocopherol and dl--tocopherol
acetate quantified using fluorescence detection, excitation/emission,
280/310 nm. Vitamin A palmitate (cis and trans) and vitamin A
acetate (cis and trans) are quantified using UV detection. In order
to account for the different biopotency values of the isomers, if
required, all analytes are quantified in IUs and converted to g or
mg/100 g reconstituted final product following summation of the
isomers.
B. Apparatus

(a) HPLC system.Contains pump, autosampler, and


programmable UV and fluorescence detectors (FLD), controlled by
applicable software, Agilent (Santa Clara, CA) 1200, or equivalent.
A Zorbax NH2, 150 4.6 mm id, 5 m particle size column
(Agilent), or equivalent with equal performance was used.
(b) Water bath.Capable of 37 2C.

(c) Centrifuge.With adapters for 50 mL centrifuge tubes


capable of 4000 rpm.
(d) Laboratory mechanical test tube shaker (optional).
(e) UV-Vis spectrophotometer.With 1 cm quartz cells.
(f) Standard laboratory glassware.
(g) Vials. 2 mL amber fitted with PTFE liners (Agilent).
(h) Duran bottles.1 and 2 L, for the mobile phase (Wertheim/
Main, Germany).
(i) Disposable centrifuge tubes.50 mL Falcon tubes, or
equivalent (Fisher, Pittsburgh, PA).
(j) Disposable Pasteur pipets.
C. Standards

(a) Vitamin A palmitate.Reference standard, Sigma (St. Louis,


MO) Cat. No. R3375, or equivalent.
(b) Vitamin A acetate.Reference standard, Sigma Cat. No.
46958, or equivalent.
(c) dl--Tocopherol acetate.Reference standard, Sigma Cat.
No. T3376, or equivalent.
(d) dl--Tocopherol.Reference standard, Sigma Cat. No.
95240, or equivalent.
D. Chemicals and Reagents

(a) Deionized water.>18 M resistance (EMD Millipore,


Billerica, MA, or equivalent).
(b) Methyl-t-butyl ether.HPLC grade (also known as tertbutyl methyl ether).
(c) Hexane, ethanol, methanol, and iso-octane (2,2,4trimethylpentane).HPLC grade.
(d) Papain (from Carica Papain), 3 units/mg.Sigma Cat. No.
76220, or equivalent.
(e) Hydroquinone.Sigma H9003, or equivalent.
(f) Anhydrous sodium acetate.BDH (Visela, CA) 10236, or
equivalent.
E. Solutions

(a) 2% Papain solution.Dissolve 100 mg hydroquinone and


4 g sodium acetate in approximately 80 mL water in a 100 mL
volumetric flask. Adjust the pH to 5.0 with dilute hydrochloric acid.
Add 2 g papain and make up to volume. Prepare fresh on day of
use.
(b) Glacial acetic acid.Reagent grade.
(c) Dilute hydrochloric acid.100 mL of 37% HCl diluted to
200 mL with distilled water.
(d) Acidified methanol.Add 20 mL acetic acid to 1 L methanol
and mix. Prepare fresh on day of use.
(e) Mobile phase A.Hexane, filtered, and deaerated for 10 min
in an ultrasonic bath.
(f) Mobile phase B.Hexanemethyl-t-butyl ether (75 + 25,
v/v). Add 3 mL methanol, filter, and deaerate for 10 min in an
ultrasonic bath.
F. Calibration Standards

Note: Class A certified glassware is recommended for the


preparation of stock reference standards.
(a) Vitamin A palmitate stock standard (P1).Weigh (to
0.01 mg) approximately 70 mg retinyl palmitate into a 50 mL
volumetric flask. Dissolve in and dilute to volume with iso-octane.
(b) Vitamin A acetate stock standard (A1).Weigh (to 0.01 mg)
approximately 35 mg retinol acetate into a 50 mL volumetric flask.
Dissolve in and dilute to volume with ethanol.
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Table 2012.10.
Time, min
0.0

Pump gradient elution cycle

Flow, mL/min % Mobile phase A

% Mobile phase B

1.5

95

3.0

1.5

95

12.0

1.5

95

14.0

1.5

95

15.0

1.5

95

20.0

1.5

95

(c) dl--Tocopherol acetate stock standard (E1).Weigh (to


0.01 mg) approximately 180 mg dl--tocopherol acetate into a
50 mL Class A certified volumetric flask. Dissolve in and dilute to
volume with iso-octane.
(d) dl--Tocopherol stock solution (E2).Weigh (to 0.01 mg)
100 mg -tocopherol into a 50 mL Class A certified volumetric
flask. Dissolve in and dilute to volume with iso-octane.
(e) Combined working standard (S3).Transfer by pipet 4 mL
Pl, 4 mL Al, 7 mL El, and 20 mL E2 into a 50 mL volumetric flask.
Dilute to volume with iso-octane, S2. Transfer by pipet 4 mL S2
into a 50 mL volumetric flask and dilute to volume with iso-octane.
Store in a refrigerator for up to 7 days.
(f) Level 1 calibration standard.Prepare as follows: Into a 50
mL volumetric flask, transfer by pipet 0.5 mL S3, and dilute to
volume with iso-octane. Transfer 2 mL into a 10 mL volumetric
flask and dilute to volume with iso-octane.
Level 2 calibration standard.Prepare as follows: Into a 50 mL
volumetric flask, transfer by pipet 8 mL S3, and dilute to volume
with iso-octane.
Level 3 calibration standard.Prepare as follows: Use S3
solution.
The standard curve calibration standards for levels 1 and 2
should be prepared daily.
G. Concentration of Stock Standards

(a) Vitamin A palmitate.(1) Pipet 3 mL stock solution P1 into


a l00 mL volumetric flask and make up to volume with iso-octane.

Pipet 3 mL this solution into a l00 mL volumetric flask and dilute


to volume with iso-octane.
(2) Determine the absorption at 325 nm, zeroed against
iso-octane in a 1 cm quartz cell.
(3) Repeat the reading twice, rinsing the sample cuvet with the
solution before each reading. Calculate the average absorbance
reading. The potency of the vitamin A palmitate stock solution is
then calculated as follows:
IU/mL=

Abs
975

100 100

10000 1.817
3
3

where Abs = average absorbance reading, determined above;


975 = extinction coefficient of retinyl palmitate at 325 nm;
10 000 = conversion of percent to g/mL; and 1.817 = conversion
from g to IU for retinyl palmitate.
(b) Vitamin A acetate.(1) Pipet 3 mL stock solution A1 into
a l00 mL volumetric flask and make up to volume with iso-octane.
Pipet 3 mL this solution into a l00 mL volumetric flask and dilute
to volume with iso-octane.
(2) Determine the absorption at 325 nm, zeroed against
iso-octane, in a 1 cm quartz cell.
(3) Repeat the reading twice, rinsing the sample cuvet with the
solution before each reading. Calculate the average absorbance
reading. The potency of the vitamin A acetate stock solution is then
calculated as follows:
IU / mL

Abs 100 100


u
u
u 10000 u 2.904
1560
3
3

where Abs = average absorbance reading, determined above;


1560 = extinction coefficient of retinyl acetate at 325 nm;
10 000 = conversion of percent to g/mL; and 2.904 = conversion
from g to IU for retinyl acetate.
(c) dl--Tocopherol acetate.(1) Pipet 3 mL stock solution E1
into a l00 mL volumetric flask and make up to volume with isooctane.
(2) Determine the absorption at 284 nm, zeroed against
iso-octane, in a 1 cm quartz cell.
(3) Repeat the reading twice, rinsing the sample cuvet with the
solution before each reading. Calculate the average absorbance
reading. The potency of the dl--tocopherol acetate stock solution
is then calculated as follows:

Figure 2012.10A. Vitamin A palmitate and vitamin A acetate calibration standard.


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Figure 2012.10B. Vitamin A palmitate sample chromatograms.

43.6

100
10
3

where Abs = average absorbance reading, determined above;


43.6 = extinction coefficient of tocopheryl acetate at 284 nm; and
10 = conversion of percent to mg/mL. (Note: For dl--tocopherol
acetate, 1 mg = 1 IU.)
(d) dl--Tocopherol.(1) Pipet 3 mL stock solution E2 into a
l00 mL volumetric flask and make up to volume with iso-octane.
(2) Determine the absorption at 292 nm, zeroed against
iso-octane in a 1 cm quartz cell.
(3) Repeat the reading twice, rinsing the sample cuvet with the
solution before each reading. Calculate the average absorbance
reading. The potency of the -tocopherol stock solution is then
calculated as follows:
/=

100

10 1.1
75.8
3

where Abs = average absorbance reading, determined above; 75.8


= extinction coefficient of -tocopherol at 292 nm; 10 = conversion

of percent to mg/mL; and 1.1 = conversion from mg to IU for dl-tocopherol.


(e) Calculation of working standard concentrations.The
concentration of each vitamin in the working standards is calculated
from the stock concentration using the appropriate dilution factor,
as shown (IU/mL), Section F. Refer to the example calculations
below.
(1) Level 1 calibration standard.
Stock IU / mL u

Vitamin A Palmitate ( IU / mL)

Vitamin A Acetate ( IU / mL)

Stock IU / mL u

DL  D Tocopherol Acetate ( IU / mL)

DL  D Tocopherol ( IU / mL)

4
4 0.5 2
u u
u
50 50 50 10

4
4 0.5 2
u
u
u
50 50 50 10

Stock IU / mL u

Stock IU / mL u

7
4 0.5 2
u
u
u
50 50 50 10

20 4 0.5 2
u
u
u
50 50 50 10

Figure 2012.10C. Vitamin A acetate sample chromatograms.


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Figure 2012.10D. -Tocopherol acetate and -tocopherol calibration standard.


H. Sample Preparation

(a) For powder samples, transfer 25 g, accurately weighed,


into a 250 mL volumetric flask. Dissolve using distilled water
(approximately 40C), cool and make up to 250 mL with distilled
water. Transfer 5 mL reconstituted sample to a 50 mL screw top
centrifuge tube.
(b) For ready-to-feed samples or concentrated liquid products,
transfer 5.0 mL thoroughly agitated sample directly to a 50 mL
screw top centrifuge tube. Liquid samples should be analyzed from
a freshly opened container, stored refrigerated for no more than
48 h, and never analyzed from a frozen sample.
(c) Add 5 mL 2% papain solution.
(d) Mix to disperse each sample, cap, and place the tubes in
a 37 2C water bath for 2025 min. Remove the samples from

the bath and cool. Place in a freezer for approximately 5 min or


refrigerate for approximately 20 min.
(e) Add approximately 20 mL acidified methanol to each sample
tube and mix.
(f) Accurately pipet 10.0 mL iso-octane into each sample tube.
Close tightly to avoid leakage and shake the tube for 10 min,
preferably with a mechanical shaker.
(g) Centrifuge for 10 min at 4000 rpm to obtain a clear isooctane layer. Remove enough iso-octane from the centrifuge tube
to fill an injection vial. This extract is ready for LC analysis.
(h) Typically, a 50 L injection volume is used for the standards
and sample extracts, but this can be varied (20100 L) to suit
sensitivity.

Figure 2012.10E. -Tocopherol acetate and -tocopherol sample chromatogram.


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I. HPLC Analysis

(a) Use the pump gradient elution cycle (Table 2012.10).


(Note: The gradient parameters can be altered as required to
maximize the analytical separation and avoid interferences.)
(b) Set the detectors to the following wavelengths: UV detector,
325 nm for vitamin A palmitate and vitamin A acetate. FLD
(excitation/emission) 280/310 nm for dl--tocopherol acetate and
dl--tocopherol (Figures 2012.10AD).
J. System Suitability

The following system suitability and standard checks are to be


met when running this method.
(a) Linearity.The coefficient of determination, R2, of each
calibration curve shall be 0.995.
(b) Standard injection precision.When a stable baseline
is obtained, inject a medium reference standard three times in
succession and determine the RSD. The RSD determined must be
2%.
(c) Standard response accuracy.Determine the slope change
between successive calibrations and the difference must be 3%.
The appropriate number of samples between successive calibrations
is typically 68.
(d) Tailing/asymmetry factor.The target limits of the tailing
factor for meeting system suitability are 0.81.2. The system should
be closely monitored if outside the target limits, and action taken if
the value is outside 0.51.5 (e.g., column cleaning, reconditioning,
or replacement).
K. Calculations of Sample Concentrations

(a) Powder samples.


( IU/100 g)
=

(b) Liquid samples.


. (IU/100 g reconstituted Final Product) =
=

(Area/Height of Sample Peak C)


10 100

where C = y-intercept, Y = dilution volume of test portion,


10 = volume (mL) of iso-octane, 100 = conversion to per 100 g,
W = sample amount in mL for liquid ready-to-feed and concentrates,
and D = density of liquid product. Total vitamin A is the sum
of the trans vitamin A concentration and the 13-cis vitamin A
concentration in IUs.
One IU is equal to 0.30 g all-trans retinol. Retinyl palmitate
or retinyl acetate in IU/100 g reconstituted final product can
be converted to g/100 g reconstituted final product in retinol
equivalents (REs) by multiplying by 0.30.
One RE is defined as 1 g retinol.
Total vitamin E (sum of dl--tocopherol and dl--tocopherol
acetate) in IU/100 g reconstituted final product can be converted
to mg/100 g reconstituted final product in -TEs by multiplying
by 0.671.
References: J. AOAC Int. 96, 1073(2013)
DOI: 10.5740/jaoacint.13-103
AOAC SMPR 2011.003
J. AOAC Int. 95, 291(2012)
DOI: 10.5740/jaoac.int.11-0439
AOAC SMPR 2011.010
J. AOAC Int. 96, 485(2013)
DOI: 10.5740/jaoac.int.SMPR2011.010

(Area/Height of Sample Peak C)

10 100

225

where C = y-intercept; Y = dilution volume of test portion;


10 = volume (mL) of iso-octane; 100 = conversion to per 100 g; W
= sample amount in g; and 225 = weight of dilution water.

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