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SM
AOAC® Official Method xxx

Spectrophotometric Method (iCheck® CHROMA)

for determination of Vitamin A in Vegetable Oil

For approval by: STAKEHOLDER PANEL ON STRATEGIC FOOD ANALYTICAL METHODS

(SPSFAM)

Submission date: 07.06.2012

Applicability:

Quantitative measurement of vitamin A is defined as 13-cis and all-trans retinol, retinyl esters
(retinyl palmitate, and retinyl acetate) in food ingredients such as vegetable oil at a concentration
range of 2.5 – 30 ppm. The upper limit can be increased by adjusting the dilution level.

Caution statement:

No special precautions needed as the method is developed for safe use inside as well as outside a
laboratory setting.

Interlaboratory study result table:

Unavailable at this point in time.

Principle:

Vegetable oil is directly injected into the sealed glass vial containing antimony trichloride (SbCl3)
and chloroform (CHCl3). The determination of vitamin A in the oil is based on the reaction of SbCl3
and the double bonds of the retinol molecule in CHCl3 that forms anhydroretinylic and retinylic
cation yielding transient brilliant blue color. The absorption of the solution is immediately measured
in the iCheck® CHROMA (BioAnalyt, Germany) photometer. The device automatically calculates
and displays the concentration of vitamin A as retinyl equivalents/RE (retinol and retinyl esters) in
mg/kg oil.

Apparatus and Materials:

iCheck CHROMA – portable photometer

designed specifically for determination of
vitamin A in vegetable oil. The optical design
has been developed for the iEx™ ELAN
reagent vial and cuvette. The unit is
equipped with the latest light emitting diode
(LED) technology. Modern LEDs emit high
and extremely stable light energy together
with very low energy consumption at no heat
development. Therefore, user calibration is
not required for the anticipated shelf life of
the iCheck™. Based on the half life of
30,000 hours of the LED, approximately 10
million measurements can be conducted. iCheck™ software has programmed algorithm that
converts the absorption units of the blue-color reaction of several automatic measurements into
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RE/kg. For quality control purposes the device conducts and auto-control to verify the emitter and
receptor performance. A vitamin A standard to control the device is not needed.

iEx™ ELAN - disposable reaction vial and cuvette used for the measurement with iCheck™. iEx™
ELAN contains 2 mL of mixture of solvents containing essentially SbCl3 and CHCl3 developed to
determine Vitamin A in the sample. BioAnalyt guarantees that each vial contains the mixture of
reagents safely and permanently crimped (shelf life 12 months).

Reagents:

iEx™ ELAN - disposable reaction vial. No additional chemicals are required.

Preparation of Test Sample:

For sample preparation the oil sample may be warmed up to 30° C in order to avoid any
precipitate. No additional preparation required.

Procedure and Determination:

Oil is drawn up with the applicator (syringe) at a usual volume of 500 µl. The complete volume is
injected slowly into the sealed glass vial iEx™ ELAN through the red rubber septum. Sealed vial
prevents toxic exposure to reagents. Briefly invert the vial and immediately measure the absorption
of the concentration-dependent blue colour of the solution at 610 nm with iCheck® CHROMA
photometer. The measurement is accompanied by the device displaying the instructions for the
investigator of each single step. The concentration of vitamin A is displayed as retinyl equivalents
(= retinol and retinyl esters) in mg/kg oil.

Reference:

Int. J. Vitam. Nutr. Res., 81 (5), 2011, p.335-342.

Method Performance Requirements

AOAC requirements iCheck® CHROMA

Analytical range 100 ppm – 100% 2.5 -30 ppm [100%*]

0.01% ≤ 4% 1.62%
Repeatability
1% ≤ 2% N/A
(RSDr)
100% ≤ 1% N/A

Recovery 90-110% 99-101%

0.01% ≤ 8% 3.92%
Reproducibility N/A
1% ≤ 4%
(RSDR)
100% ≤ 2% N/A

*100% can be reached by adjusting the dilution.

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INTERNAL VALIDATION OF iCheck® CHROMA

Working and linear range

Based on the injection of 500µl sample the linear range was determined. Palm oil fortified with
retinyl palmitate was gradually diluted with five equivalent concentrations (0, 20, 40, 60, 80 and
100%) of non fortified palm oil and measured in triplicates. The linear range is 2.5 - 30 mg/kg.

Figure 1. Linearity of fortified palm oil spiked with increasing amounts of non fortified palm oil. Each sample was measured
in triplicates. Each dot represents one measurement.

LOD & LOQ

Limits of detection and quantitation were assumed to be interchangeable for this method and
determined by functional sensitivity (Clin. Chem. 44, p. 2217). Unfortified palm oil was increasingly
fortified with retinyl palmitate, and measured, until a coefficient of variation (CV) consistently below
20% was reached. Because of the algorithms programmed in the portable device, a separate
definition of LOD and LOQ is not possible. The threshold where the portable device can reliably
distinguish the signal from the background noise (<20% CV) is 2.5 mg RE/kg and is representative
of LOD.

Repeatability

Intra-assay (precision within series) and day-to-day precision was determined by measuring 3
fortified palm oil samples at three different days by one person. Palm oil fortified with retinyl
palmitate was mixed with non-fortified palm oil in three concentrations (20, 40 and 80%) and
measured ten times:
Fortified Expected Expected
Non-fortified
Concentration concentration in concentration
palm oil palm oil
% (mg RE/kg)
Low 20% 80% 0.00065 6.50

Medium 40% 60% 0.0013 13.0

High 80% 20% 0.0025 25.0

The intra-assay and day-to-day precision show that all concentration levels (Low, Medium, High)
comply with the expected concentrations. The repeatability relative standard deviation (%RSDr)
among the results of the three days ranges between 0.21 and 3.34%. The intra-assay %RSDr was
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7.14%, 5.86% and 3.24% for the Low, Medium and High concentration on Day 1 (10
measurements by one person / the same day).
Vitamin A [mg RE / kg]
Oil
Sample RSDr
Day 1 Day 2 Day 3 MW SDr
(%)
Mean 6.53 6.58 6.18 6.43 ±0.21 3.34
Low SDr ±0.47 ±0.70 ±0.38
RSDr (%) 7.14 10.60 6.20
Mean 13.09 13.05 13.10 13.08 ±0.03 0.21
Medium SDr ±0.77 ±0.38 ±0.50
RSDr (%) 5.86 2.92 3.78
Mean 25.24 24.71 24.45 24.80 ±0.04 1.62
High SDr ±0.82 ±1.29 ±0.42
RSDr (%) 3.24 5.24 1.73

Reproducibility

Inter-person precision was determined by measuring 3 fortified palm oil samples at three different
days by three persons. Palm oil fortified with retinyl palmitate was mixed with non-fortified palm oil
in three concentrations (20, 40 and 80%) and measured in triplicates.

The inter-person precision shows that all three measured concentrations (Low, Medium, High)
comply with the expected concentrations. The % reproducibility relative standard deviation
(%RSDR) coefficient of variation among the results of the three days ranges between 1.07 and
3.92%.
Vitamin A [mg RE / kg]
Oil RSDR
Person 1 Person 2 Person 3 MW
Sample (%)
Mean 6.58 6.72 6.67 6.66 1.07
Low
RSDR (%) 10.60 3.08 2.81
Mean 13.05 13.67 14.11 13.61 3.92
Medium
RSDR (%) 2.92 2.38 2.35
Mean 24.71 25.43 25.82 25.32 2.24
High
RSDR (%) 5.24 6.87 1.22

Recovery

Recovery was calculated as the percentage of expected/spiked concentration of vitamin A in oil

recovered using iCheck® CHROMA and ranged between 98.9% and 100.6%.

Expected Measured
Concentration concentration concentration Recovery (%)
(mg RE/kg) (mg RE/kg)
Low 6.50 6.43 98.9

Medium 13.0 13.08 100.6

High 25.0 24.80 99.2

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EXTERNAL VALIDATION OF iCheck® CHROMA

iCheck® CHROMA method performance internally was further validated against external well-
established HPLC reference method performed by Potsdam University (Germany), and against
external use by technicians with limited training in a low-resource setting in West Africa (Helen
Keller International, Abidjan, Côte d’Ivoire).

Validation of internally performed iCheck® CHROMA method against externally performed

HPLC method

To validate the iCheck® CHROMA device against the HPLC reference method, 200 samples
between 0 and 16 mg RE/kg as previously determined by HPLC in Côte d’Ivoire were randomly
selected from 2500 samples collected in a oil fortification household coverage survey in Côte
d’Ivoire. The vitamin A concentrations of the selected oil samples ranged from 0-16 mg RE/kg. All
oil samples were collected in November 2009 and stored in cool, dark, conditions (+4°C) before
and after analysis in-country, and during shipment of the aliquots to Germany. Upon arrival in
Germany, the oil aliquots were split again, and: a) after warming to 30°C for 5 minutes and mixing,
analyzed using the iCheck® CHROMA by experienced technicians involved in the development of
the device, and b) after warming to 30°C for 5 minutes and mixing, analyzed at the University of
Potsdam by HPLC as described below. All technicians were blinded to the results of the other
analyses until results were merged in a database.

All reagents purchased were of HPLC-grade: Methanol, ethyl-acetate, iso-propanol (Sigma,

Deisenhofen, Germany). Oil samples were warmed to 30°C and mixed thoroughly. Samples were
diluted in isopropanol at a ratio of 1:10 (oil: isopropanol; v:v) and the diluted oil sample (20 µL) was
injected without prior saponification and analysed on a reverse-phase column (Repro Sil 70 C18
(5µm; 200x3) Dr. Maisch GmbH, Germany). The solvent system consisted of methanol (A) and
ethyl acetate (B) which were used as the eluent at a flow rate of 0.5 mL/min (HPLC pump
Shimadzu LC 20 AD, Duisburg, Germany). Each analytical run consisted of the following gradient
profile: 3.0 min 0 % B; 3.1 min 50% B; 10.0 min 50% B, 10.1 min 0% B, 15.0 min rinsing time. The
column temperature was 40°C. Absorption of vitamin A esters (palmitate) were identified by
comparison of retention times to the external standard (retinyl palmitate) by use of a photodiode
array detector at a wavelength of 325 nm (PDA Shimadzu SPD-M 20A, Duisburg, Germany). The
exact concentration of the external standard - retinyl palmitate - was measured on a weekly basis
using a UV-VIS spectrophotometer (Specord 205, Analytik Jena AG, Germany). The detection limit
for retinyl pamitate is 50 µg RE/kg.

Data processing and statistics were conducted using Excel version 2007. Besides plotting the two
data sets and calculating the Spearman coefficient and the regression equation, the Bland and
Altman plot was used. The absolute differences of the values in the samples analyzed by the two
methods were calculated. Limits of agreement (LOA) were calculated using:
∆ - 2s = LOAlow
∆ + 2s = LOAhigh
where ∆ is the mean of the difference between the two methods, and s is the standard deviation
(SD) of this difference.

Of the 200 samples collected, sufficient volume for both analyses was available in 189 samples.
The results of this comparison are presented graphically in Figure 2. The equation of the
correlation is y = 0.83x + 0.08, with y being the reference HPLC method. The corresponding
2
Spearman coefficient for the relationship is R = 0.92. The limits of agreement are as follows:
LOAlow is -1.24 mg RE/kg
LOAhigh 2.53 mg RE/kg
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Figure 1 shows the regression (A) and Bland and Altman plot (B) of the comparison between the iCheck™-iEx and the
reference method (HPLC). The dashed line in A is the line of identity, whereas in B they represent the ± 2 SD.

The Bland-Altman plot (Figure 2) indicates

indicates that there are data points that are outside the 2
standard deviation lines and although tests for identifying outliers exist, no such outliers were
excluded.

Among the samples analyzed, using a target range to define an “adequate fortification level” of 6.4
– 9.6 mg RE/kg (which represents the legally stated fortification concentration in the Ivorian
fortification standards, i.e. 8 mg RE retinol
retinol palmitate/kg oil, plus a fortification precision of ± 20%),
the difference in the percentage of oil samples
samples that were categorized as adequately fortified by the
HPLC reference method and the portable device was 0.6%.

Analysis of oil samples from Côte D’Ivoire using the portable device and by the reference HPLC
method yielded good correlation and agreement, with the portable device measuring consistently
0.6mg RE/kg higher than the reference method. As AOL show, 97.5% of the results obtained by
iCheck® CHROMA do not differ more than 2.8 mg RE/kg (-1.24 to 1.53 mf RE/kg) from the
reference method.

Validation of internally against externally performed iCheck® CHROMA method

Two local technicians in Côte d’Ivoire underwent a brief training module in Côte d’Ivoire, and were
then asked to analyze the samples on-site and then ship those samples to Germany to be
analyzed internally by BioAnalyt. The training included the information on the test, the technology,
and the operation of the instrument and the testing of 10 samples by each technician. Technicians
were blinded to the HPLC results previously obtained with the same samples.

Comparison between the iCheck® CHROMA conducted externally in the field and internally in the
Bioanalyt laboratory: For this comparison, all 200 collected samples contained sufficient amounts
(over 3 mL) and could be measured both by the technicians in Côte d’Ivoire and in Germany. The
results of this comparison are presented graphically in Figure 2. The equation of the correlation is y
= 0.93x + 0.08, with y being the portable device method conducted in the field; the corresponding
2
Spearman coefficient is R =0.94. The LOAlow is -0.60 mg RE/kg and the LOAhigh 1.99 mg RE/kg.
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Figure 2 shows the regression (A) and Bland and Altman plot (B) of the comparison between the iCheck™-iEx conducted in
the field and in the laboratory. The dashed line in A is the line of identity, whereas in B they represent the ± 2 SD.

The comparison between the analysis of the same oil samples by the iCheck® CHROMA used in
the field by technicians with only brief training, and expert technicians in the BioAnalyt laboratory
gave good agreement, proving the device to be easy to use and to deliver reliable results. This is
an important aspect, as HPLC-based or other spectrophotometric methods require a high level of
expertise and a well-established laboratory infrastructure (including high equipment and
maintenance cost).
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Int. J. Vitam. Nutr. Res., 81 (5), 2011, 335 –342 335

Original Communication

Quantification of Vitamin A in
Palm Oil Using a Fast and
Simple Portable Device: Method
Validation and Comparison to
High-Performance Liquid
Chromatography
Fabian Rohner1, Simone K. Frey2, Ralf Mothes2, Andrea Hurtienne 3, Simone
Hartong1, Patrice Emery Bosso4, Mai Bui5, Florian J. Schweigert2,3, and
Christine Northrop-Clewes1
1
GAIN – Global Alliance for Improved Nutrition, Geneva, Switzerland
2
BioAnalyt GmbH, Teltow, Germany
3
University of Potsdam, Potsdam-Rehbrücke, Germany
4
Helen Keller International, Abidjan, Côte d’Ivoire
5
Swiss Vitamin Institute, Epalinges, Switzerland

Received: May 8, 2011; Accepted: September 25, 2011

Abstract: Vitamin A deficiency continues to be a global public health problem. Fortification of oil with
vitamin A is considered a cost-effective, feasible strategy to prevent this problem but quality control
poses a challenge to program implementation. To overcome this, we have validated a newly developed
device that quantitatively measures the content of retinyl palmitate in refined palm oil, is simple to use,
and yields immediate results.
Linearity of analysis ranged from 2.5 – 30 mg retinol equivalents (RE)/ kg of palm oil, with 2.5 mg RE/
kg being the determination limit; inter- and intra-assay precision ranged from 1.4 – 7.1 %. Comparison
with a high-performance liquid chromatography method showed high agreement between the methods
(R2 = 0.92; Limits of Agreement: –1.24 mg to 2.53 mg RE/kg), and further comparisons illustrate that
the new device is useful in low-resource settings. This device offers a field- and user-friendly solution
to quantifying the vitamin A content in refined palm oil.

Key words: Vitamin A, retinyl palmitate, fortification, monitoring, rapid test kit, palm oil

DOI 10.1024/0300 – 9831/a000081 Int. J. Vitam. Nutr. Res. 81 (5) © 2011 Hans Huber Publishers, Hogrefe AG, Bern

336 F. Rohner et al.: Quantification of Vitamin A in Palm Oil
Introduction
Data collected by the World Health Organization
(WHO) between 1995 – 2005 estimates that 190 million
pre-school children in 122 countries and 19.1 million
pregnant women in 88 countries have vitamin A de-
ficiency (VAD). The estimates of VAD are based on
the specified population groups having plasma retinol
concentrations < 0.7μmol/L. Low vitamin A intake
during nutritionally demanding periods in life greatly
increases the risks of adverse health consequences,
and VAD is therefore considered an important de-
terminant of child survival and is of public health
concern [1].
Of the most commonly used strategies to help re-
duce VAD (dietary diversity; food fortification; vita-
min A supplementation through capsules; other public
health measures such as breastfeeding promotion),
the fortification approach is thought to be the most
sustainable and cost-efficient way to reduce VAD [2].
Among the most commonly fortified food vehicles
are vegetable oils, sugar, and cereal flours. Vegetable
oils, in particular palm, peanut, and cottonseed oils,
are widely consumed in many African and Asian
countries, where the prevalence of VAD is of public
health concern [1]. Oil presents an ideal matrix for
the addition of fat-soluble vitamin A, and methods
of fortifying vegetable oil are fairly simple and easy
to implement at low cost. Furthermore, oil stabilizes
the fortificant, retinyl acetate or retinyl palmitate, and
delays oxidation of the vitamin [3].
For a successful and sustainable fortification pro-
gram, ongoing quality assurance monitoring and
regulatory quality control enforcement is required to
ensure that appropriately fortified oil is produced and
marketed, and that vitamin A retains its stability over
time. In addition, a population-based assessment of
the coverage of adequately fortified oil and its impact
on the vitamin A status of the population is needed to
inform and guide program implementation. Current
semi-quantitative and quantitative analysis methods
for assessment of retinyl palmitate in oil are techni-
cally quite demanding and can be expensive; e. g. the
qualitative method based on the formation of anhy-
droretinol [4], the quantitative spectrophotometric [5],
or high-performance liquid chromatography (HPLC)
methods [6]. Qualitative methods do not measure the
adequacy of fortification; rather they provide informa-
tion on presence or absence of a fortificant, which is
often insufficient information [7].
To ensure adequate fortification levels, simple-to-
use quantitative methods that yield immediate and re-
liable results are needed at the points of production for
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regulatory monitoring and for program evaluation at
the household level. BioAnalyt GmbH has developed
a simple portable device to rapidly quantify vitamin A
concentration in fortified refined palm oil (iCheck™-
iEx , hereafter referred to as “portable device”).
The objectives of the present study were 1) to vali-
date the portable device used by well-trained techni-
cians against an HPLC reference method, and 2) to
evaluate the field-friendliness and robustness of the
portable device through its use by technicians with lim-
ited training in a low-resource setting in West Africa.
Material and methods
Portable device
Method
The portable device determines the concentration of
vitamin A (retinol, retinyl acetate, retinyl palmitate)
in palm oil based on a color reaction described by
Carr and Price [8]. Antimony trichloride (SbCl3) in
chloroform and the double bonds of the retinol mol-
ecule react in the formation of an anhydroretinylic
and a retinylic cation, yielding a brilliant blue color.
The absorption of the blue color is dependent on
the concentration of the solution and is measured
at 610 nm in the spectrophotometric measuring unit.
Results are displayed and saved in the measuring
unit, and can be downloaded via a universal serial
bus (USB) as a text file (.txt) to a computer. The
device can be battery-operated or be connected to
the mains (100 – 240 V). The device’s software has a
programmed algorithm that converts the absorption
units of the blue-color reaction of several measure-
ments into mg RE/ kg.
Material
The portable device consists of two units, the measur-
ing unit (iCheck™ CHROMA) and the disposable
reagent vial (iEx™ ELAN), in which the reaction
is performed (Figure 1). The disposable reagent vi-
als contain 2 mL of a mixture of reagents, which are
needed for completion of the Carr-Price reaction [8];
no additional reagents are required. Both the measur-
ing unit and reagent vials are commercially available
(www.bioanalyt.com). Currently, shelf life at 25 °C
has been verified for 12 months. For quality control
purposes the device conducts an auto-control to verify

F. Rohner et al.: Quantification of Vitamin A in Palm Oil
Figure 1: Photograph of the prototype of the portable de-
vice that has been validated as described in this paper. To
conduct the analysis, 500 μL of the oil sample is injected
into the reagent vial using a syringe; the vial is inverted and
inserted into the device for measurement. As an indication
of size, a match has been placed in the picture.
the emitter and receptor are working correctly. A vi-
tamin A standard to control the device is not needed.
Working conditions
The reagent vials and measuring unit were stored at
room temperature (20 – 30 °C) prior to analysis. With-
out any further preparation, palm oil samples (500
μL) were drawn using a syringe, included as part of
the portable device test kit, and the 500 μL of oil were
injected slowly through the red rubber septum into the
reagent vial. After inverting the vial once (max. 1 sec-
ond), it was placed immediately into the photometric
measuring chamber of the measuring unit. The device
displays instructions to the analyst at each step of the
analysis. After five minutes, the photometer displays
the calculated vitamin A concentration in mg RE/kg.
During the production process, the measuring unit
was calibrated and good quality control procedures
were used at all stages. Because the measuring unit
uses a stable light source (LED), user calibration is
not required for the anticipated shelf life of the por-
table device. Based on the half-life of the light source
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337
(30,000 hours), approximately 10 million measure-
ments can be conducted.
Reference method
Method
To conduct the reference method, a gradient reverse-
phase, HPLC system was used; the method is based
on that described by Gimeno and colleagues [9] for
the determination of vitamin E and was adopted for
vitamin A.
Material
All reagents purchased were of HPLC-grade: Metha-
nol, ethyl acetate, and isopropanol (Sigma, Deisen-
hofen, Germany).
Working conditions
Oil samples were warmed to 30 °C and mixed thor-
oughly. Samples were diluted in isopropanol at a ratio
of 1:10 (oil:isopropanol; v:v) and the diluted oil sample
(20 μL) was injected without prior saponification and
analyzed on a reverse-phase column [Repro Sil 70
C18 (5μm; 200 × 3) Dr. Maisch GmbH, Germany].
The solvent system consisted of methanol (A) and
ethyl acetate (B), which were used as the eluent at a
flow rate of 0.5 mL/minute (HPLC pump Shimadzu
LC 20 AD, Duisburg, Germany). Each analytical
run consisted of the following gradient profile: 0 – 3
min – 0 %B; 3 – 3.1 min –50 %B; 3.1 – 10 min – 50 %B;
10 – 10.1 min – 0 %B; and 15.0 min rinsing time. The
column temperature was 40 °C. Absorption of vitamin
A esters (palmitate) were identified by comparison
of retention times to the external standard (retinyl
palmitate) by use of a photodiode array detector at a
wavelength of 325 nm (PDA Shimadzu SPD-M 20 A,
Duisburg, Germany). The exact concentration of the
external standard – retinyl palmitate – was measured
on a weekly basis using a UV-VIS spectrophotometer
(Specord 205, Analytik Jena AG, Germany). The de-
tection limit for retinyl palmitate is 50 μg RE/kg. To
ensure validity of the results obtained by this reference
method, the results of a subset (n = 30) of fortified palm
oil samples (range: 0 – 30 mg RE/kg) were compared
with those obtained by a third laboratory, the Swiss
Vitamin Institute (Epalinges, Switzerland; external
validation), using saponification, followed by retinol

338 F. Rohner et al.: Quantification of Vitamin A in Palm Oil
analysis using an HPLC method [10]. Agreement be-
tween the HPLC reference method and the external
validation was very good [HPLC = 0.9485*(external
validation) + 0.3092 mg RE/kg; R2 = 0.98].
Method validation of the portable device
a) Linearity of the portable device was determined
by the injection of a 500-μL sample of fortified palm
oil (fortificant: retinyl palmitate) with the following
calculated concentrations: 2.8, 5.6, 11.2, 16.8, 22.4, and
28 mg RE/kg; each sample was measured in triplicate.
b) Limits of detection and quantitation were assumed
to be interchangeable for this method and determined
by functional sensitivity [11]: unfortified palm oil was
increasingly fortified with retinyl palmitate, and mea-
sured, until a coefficient of variation (CV) consistently
below 20 % was reached.
c) Intra-assay precision: Palm oil fortified with retinyl
palmitate was prepared to achieve three concentra-
tions of 6.5, 13.0, and 25.0 mg RE/kg, respectively. The
same technician measured each of the three samples
ten times on the same day, and the CV of the results
was calculated.
d) The inter-assay precision was determined by one
person measuring the same three concentrations of
fortified palm oil in triplicate on three different days,
and the CV of the results was calculated. Samples were
stored in the dark and at room temperature.
e) The inter-person precision was determined by three
people measuring the same three concentrations of
fortified palm oil in triplicate, over three days, and
the CV was calculated.
f) Comparison of the portable device to the reference
method: To validate the portable device against the
HPLC reference method, 200 samples between 0 and
16 mg RE/kg, as previously determined by HPLC in
Côte d’Ivoire, were randomly selected from 2500 re-
fined palm oil samples collected in an oil fortification
household coverage survey in Côte d’Ivoire. The vita-
min A concentrations of the selected oil samples ranged
from 0 – 16 mg RE/kg. All oil samples were collected
in November 2009 and stored in cool, dark, conditions
(+4 °C) before and after analysis in-country, and during
shipment of the aliquots to Germany. Upon arrival in
Germany, the oil aliquots were split again, and: a) after
warming to 30 °C for 5 minutes and mixing, analyzed
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using the portable device by experienced technicians
involved in the development of the device, and b) after
warming to 30 °C for 5 minutes and mixing, analyzed
at the University of Potsdam by HPLC as described
above. All technicians were blinded to the results of the
other analyses until results were merged in a database.
g) Comparison of the use of the portable device in
the field and in the laboratory: prior to shipment of
aliquots to Germany, two local technicians in Côte
d’Ivoire underwent a one-hour training module in Côte
d’Ivoire. The training included information on the test,
the technology and operation of the instrument, and
the testing of 10 samples by each technician. After-
wards they were asked to analyze the same samples
on-site as described in section f) above. Technicians
were blinded to the HPLC results previously obtained.
Statistical analysis
For the laboratory validation of the method, standard
protocols were followed, unless otherwise described.
For the field validation, there were two levels of com-
parison: a) the comparison of the results obtained by
the portable device (performed in Germany) com-
pared to the HPLC reference method, and b) the
comparison of the results obtained using the portable
device by local technicians in-country in a hot climate
with those obtained by well-trained technicians in Ger-
many. Data processing and statistics were conducted
using Excel version 2007. Besides plotting the two data
sets and calculating the Spearman coefficient and the
regression equation, the Bland and Altman plot was
used [12]. The absolute differences of the values in the
samples analyzed by the two methods were calculated.
Limits of agreement (LOA) were calculated using
Δ – 2 s = LOAlow
Δ + 2 s = LOAhigh
where Δ is the mean of the difference between the
two methods, and s is the standard deviation (SD) of
this difference.
Results
Method validation of the portable device
a) Linearity: The linear range of the portable device
was determined to be 2.5 – 30 mg RE/kg; over this
range, R2 was 0.996 and the regression equation was
y = 0.9916x – 0.1182.
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F. Rohner et al.: Quantification of Vitamin A in Palm Oil 339

Figure 2: Regression (A) and Bland and Altman plot (B) of the comparison between HPLC and the iCheck™-iEx con-
ducted in the laboratory (above), and between the iCheck™-iEx conducted in the field and in the laboratory. The dashed
line in A is the line of identity, whereas in B the dashed lines represent the ± 2 SD.

b) Limits of detection and quantitation: Because of the
algorithms programmed in the portable device, a sepa-
rate definition of limit of detection and quantitation is
not possible. The threshold where the portable device
can reliably distinguish the signal from the background
noise (< 20 % CV) is 2.5 mg RE/kg.
c) Intra-assay precision: the CV of the 10 measure-
ments conducted within one day by one technician for
the three concentrations of retinyl palmitate (6.5, 13.0,
and 25.0 mg RE/kg) were 7.14 %, 5.86 %, and 3.24 %
respectively, and the mean of the measurements were
close to the expected concentrations: 6.5, 13.1, and
25.2 mg RE/kg, respectively. This corresponds to re-
covery rates of 100.4, 100.7, and 100.9 % for each of
the three levels, respectively (recovery rate: measured
value/expected value * 100).
d) Inter-assay precision: The CV for the results of
one person measuring 3 fortified palm oil samples
over three days were 8.82, 1.35, and 4.00 % for the
three concentrations (6.5, 13.0, and 25.0 mg RE/kg),
respectively. The mean concentration of each fortified
sample was 6.4, 13.1 and 25.3 mg RE/kg, again close
to the expected concentrations (see above). This cor-
responds to recovery rates of 98.6, 100.9 and 101.2 %
for each of the three levels, respectively.

Int. J. Vitam. Nutr. Res. 81 (5) © 2011 Hans Huber Publishers, Hogrefe AG, Bern

340 F. Rohner et al.: Quantification of Vitamin A in Palm Oil
e) The inter-person precision, obtained by calculating
the CV from three technicians measuring the three
concentrations of fortified palm oil in triplicate, over
three days were 4.53, 3.82, and 4.79 % respectively;
the respective mean concentrations obtained were 6.6,
13.6 and 25.5 mg RE/kg. This corresponds to recovery
rates of 100.7, 104.7 and 102.0 % for each of the three
levels, respectively.
f) Comparison between the portable device and the
reference method: Of the 200 samples collected, suf-
ficient volume for both analyses was available in 189
samples. The results of this comparison are presented
graphically in Figure 2. The equation of the correlation
is y = 0.83x + 0.08, with y being the reference HPLC
method. The corresponding Spearman coefficient for
the relationship is R2 = 0.92. The limits of agreement
are as follows: LOAlow is -1.24 mg RE/kg and the LOA-
high 2.53 mg RE/kg.
The Bland-Altman plot (Figure 2) indicates that
there are data points that are outside the 2 SD line
and although tests for identifying outliers exist, no
such outliers were excluded.
Among the samples analyzed, using a target
range to define an “adequate fortification level” of
6.4 – 9.6 mg RE/kg (which represents the legally stated
fortification concentration in the Ivorian fortification
standards; i. e. 8 mg RE retinyl palmitate/kg oil, plus
a fortification precision of ± 20 %), the difference in
the percentage of oil samples that were categorized as
adequately fortified by the HPLC reference method
and the portable device was 0.6 %.
g) Comparison between the portable device method
conducted in the field and in the laboratory: For this
comparison, all 200 collected samples contained suffi-
cient amounts (over 3 mL) and could be measured both
by the technicians in Côte d’Ivoire and in Germany.
The results of this comparison are presented graphi-
cally in Figure 3. The equation of the correlation is y =
0.93x + 0.08, with y being the portable device method
conducted in the field; the corresponding Spearman
coefficient is R2 = 0.94. The LOAlow is 0.60 mg RE/kg
and the LOAhigh 1.99 mg RE/kg.
Discussion
Not having field-friendly and cost-effective tools to
monitor micronutrient levels in fortified foods at the
production site, at national borders, and in popula-
tions is a common problem among food laboratories,
Int. J. Vitam. Nutr. Res. 81 (5) © 2011 Hans Huber Publishers, Hogrefe AG, Bern
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governmental food control authorities, non-govern-
ment organizations (NGOs), and development or-
ganizations. To fulfill the requirements of users in
low-resource settings, micronutrient assays should be
inexpensive, accurate, reliable, rugged, and well suited
to the context of the developing world.
To our knowledge, this is the first time a simple,
quantitative, portable field-friendly device has been
validated for the quantitative analysis of retinyl palmi-
tate in palm oil. In this paper, the recently developed
portable device has been described. In addition to
standard method validation tests, our objective was
to validate the portable device against an HPLC ref-
erence method and to evaluate the field-friendliness
and robustness of the portable device by testing the
potential difference in analytical performance by com-
paring its use by an experienced laboratory technician
to that of technicians only briefly trained.
Analysis of oil samples from Côte D’Ivoire using the
portable device and by the reference HPLC method
yielded good correlation and agreement, with the
portable device measuring consistently 0.6 mg RE/
kg higher than the reference method. As the Limits
of Agreement show, 97.5 % of the results obtained
by the device do not differ more than 2.8 mg RE/kg
(–1.24 to 1.53 mg RE/kg) from the reference method.
Using a target range for defining an adequate fortifica-
tion level of 6.4 – 9.6 mg RE/kg, the portable device
showed 27.2 % of oil samples were adequately forti-
fied, compared to 27.8 % by HPLC.
The comparison between the analysis of the same
oil samples by the portable device used in the field
by technicians with only brief training, and expert
technicians in the BioAnalyt laboratory, showed good
agreement, proving the device to be simple to use, but
still able to deliver valid results. This is an important
aspect, as HPLC-based or other spectrophotometric
methods require a high level of expertise and a well-
established laboratory infrastructure (including high
equipment and maintenance cost).
The linear range of the portable device was deter-
mined to be 2.5 – 30 mg RE/kg palm oil and according
to recently published guidelines by WHO/FAO, the
levels of vitamin A currently added to oil are in the
range of 5 – 15 mg/kg [13], a range for which the device
shows high linearity. Hence, the portable device is able
to deliver useful results as to whether oil samples are
adequately fortified or not.
The validation of the instrument showed that intra-
assay precision was between 3 and 7 % and the inter-
assay precision between 1 and 9 %. The inter-person
precision was also acceptable at between 4 and 5 %.
With the variations being below 11.3 %, the precision

F. Rohner et al.: Quantification of Vitamin A in Palm Oil
can be judged satisfactorily for field applications [14].
A limitation of the portable device to date is that the
device has only been validated for palm oil. In many
countries, the use of other types of oil or mixtures
of palm oil with other types of vegetable oils is com-
mon. Although a laboratory validation of other oils
such as sunflower, rapeseed, and peanut oil (data not
presented) has been performed and holds promise, a
rigorous and systematic validation will need to take
place to state the device can be used for all types of
vegetable oil.
When compared to other methods based on the
Carr-Price principle, the use of hazardous reagents
(e. g., antimony trichloride) is considerably reduced
in volume in this system, but still, these reagents are
necessary and hence, an appropriate waste manage-
ment needs to be implemented.
In conclusion, the portable device presented in this
paper offers a viable solution to the current problem
of lack of quantitative and field- and user-friendly
analytical methods to analyze vitamin A content in
refined palm oil. Further research and use in the field
is warranted to expand its use for other vegetable oils.
Abbreviations and nomenclature
CV Coefficient of variation
FAO Food and Agriculture Organization
HPLC High-performance liquid chromatography
LOA Limit of agreement
RE Retinol equivalents (or: μg retinol/g, equal
to 3.3 IU of vitamin A)
VAD Vitamin A deficiency
WHO World Health Organization
Acknowledgments
The authors thank Helen Keller International, Côte
d’Ivoire for allowing additional testing on samples
collected for a household coverage survey. We also
thank the National Health Institute of Côte d’Ivoire
(Institut National de Santé Publique) who provid-
ed technicians to conduct the field analysis on the
portable device, as well as colleagues at the Global
Alliance for Improved Nutrition (GAIN) for their
support in the logistics.
Int. J. Vitam. Nutr. Res. 81 (5) © 2011 Hans Huber Publishers, Hogrefe AG, Bern
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341
Author disclosures
FR, AH, PEB, MB, and CC declare no conflict of
interest; SKF, RM, and FJS are employees of Bio-
Analyt GmbH, the holder of patents related to the
device presented in this manuscript. The latter con-
ducted the basic laboratory validation of the method
and were involved in planning the field study in Côte
d’Ivoire. However, SKF, RM, and FJS were not in-
volved in the analysis using the portable device in
Côte d’Ivoire or using the reference method. SKF,
RM, and FJS were involved in conducting the parallel
analysis using the portable device in Germany, but
they were blinded to the results from the reference
method until the results in the databases were locked.
SKF, RM, and FJS were not involved in any of the
statistical analysis.
References
1. WHO (2009) Global prevalence of vitamin A defici-
ency in populations at risk 1995 – 2005: WHO global
database on vitamin A deficiency. Geneva.
2. Edejer, T.T., Aikins, M., Black, R., Wolfson, L., Hu-
tubessy, R. and Evans, D.B. (2005) Cost effectiveness
analysis of strategies for child health in developing
countries. BMJ 331, 1177.
3. Dary, O. and Mora, J.O. (2002) Food fortification to
reduce vitamin A deficiency: International Vitamin A
Consultative Group recommendations. J. Nutr. 132,
2927S-2933S.
4. Bayfield, R.F. (1971) Colorimetric determination
of vitamin A with trichloroacetic acid. Anal. Bio-
chem. 39, 282 – 287.
5. Kamangar, T. and Fawzi, A.B. (1978) Spectrophoto-
metric determination of vitamin A in oils and fats. J.
Assoc. Off. Anal. Chem. 61, 753 – 755.
6. Bui, M.H. (1994) Simple determination of retinol,
[alpha]-tocopherol and carotenoids (lutein, all-trans-
lycopene, [alpha]- and [beta]-carotenes) in human
plasma by isocratic liquid chromatography. Journal
of Chromatography B: Biomedical Sciences and Ap-
plications 654, 129 – 133.
7. Pandav, C.S., Arora, N.K., Krishnan, A., Sankar, R.,
Pandav, S. and Karmarkar, M.G. (2000) Validation of
spot-testing kits to determine iodine content in salt.
Bull. World Health Organ. 78, 975 – 980.
8. Carr, F.H. and Price, E.A. (1926) Colour Reactions
Attributed to Vitamin A. Biochem. J. 20, 497 – 501.
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342 F. Rohner et al.: Quantification of Vitamin A in Palm Oil

9. Gimeno, E., Castellote, A.I., Lamuela-Raventos,
R.M., de la Torre, M.C. and Lopez-Sabater, M.C.
(2000) Rapid determination of vitamin E in vegetable
oils by reversed-phase high-performance liquid chro-
matography. J. Chromatogr. A 881, 251 – 254.
10. European Committee for Standardization (2000)
Determination of vitamin A in foodstuffs by high
performance liquid chromatography – Part 1: Measu-
rement of all-trans-retinol and 13-cis-retinol: EN
12823 – 1 – 2000.
13. Bland, J.M. and Altman, D.G. (1986) Statistical me-
thods for assessing agreement between two methods
of clinical measurement. Lancet 1, 307 – 310.
14. Fraser, C.G., Hyltoft Petersen, P., Libeer, J.C. and
Ricos, C. (1997) Proposals for setting generally appli-
cable quality goals solely based on biology. Ann. Clin.
Biochem. 34 ( Pt 1), 8 – 12.
Fabian Rohner

11. Schambeck, C.M., Bedel, A. and Keller, F. (1998) GAIN – Global Alliance for Improved Nutrition
Limit of quantification (functional sensitivity) of the PO Box 55
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Switzerland
12. Allen, L.H., de Benoist, B., Dary, O. and Hurrell, Tel.: +41 (0)22 749 18 05
R.F. (2006) Guidelines on food fortification with mi- Fax: +41 (0)22 749 18 51
cronutrients. World Health Organization, Geneva. frohner@gainhealth.org

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TM
Instruction Guide
Read all of the procedures before starting. Be sure to study the
separate iEx™ TECH Syringe Use & Mixing guide first.

1.1 1.2 1.1 – BLANK MEASUREMENT

Place your iCheck™ CHROMA on a stable flat surface
and switch on. Take a new vial and place it in the
measurement chamber. Press the measurement key
for the blank measurement, wait some seconds.
1.2 – TAKE UP 0.5 mL SAMPLE
Use the green syringe without a needle attached and
take up at least 0.5 mL of an oil sample. Clean the end
of the syringe with a tissue and attach a needle. Turn
the syringe in order to release air bubbles. Adjust the
volume of the sample to exact 0.5 mL by ejecting
excessive volume out, into the tissue.

1.3 1.4 1.3 – INJECT THE SAMPLE.

Now slowly inject the sample into the iEx™ ELAN vial
which has been used for blank measurement. Take
special care to inject the sample along the side of the
glass, and above the clear liquid in order to produce
two distinct liquids.
1.4 - If you have injected the sample correctly, the
contents of your vial should look very similar to the
image at left. Note: A very small amount of blue liquid
may appear where the sample has met the clear liquid.

1.5 1.6 1.5 – PRESS MEASUREMENT

Now that your sample is ready, press the
measurement button on the iCheck™ CHROMA and
follow the procedures as indicated by the count-down
on the display.
1.6 – INVERT THE VIAL.
At ZERO as indicated on the display of the iCheck™
CHROMA, invert the vial overhead twice and insert the
vial into the iCheck™ within 2 seconds. Note: The
content of the vial will instantly turn into a bluish
colour, if the sample contains vitamin A.

1.7 1.8 1.7 – INSERT THE VIAL INTO THE iCHECKTM.

Insert the vial into your iCheck™ CHROMA device as
long as the display indicates “Insert vial”.

1.8 - Your iCheck™ CHROMA will now analyse your

sample. This will take approx. 30 seconds. The result is
displayed in mg RE/kg.

Note: For detailed instructions on using the iCheck™

please refer to the iCheck™ CHROMA User Manual.