DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO.

2, 2012 1

INFANT FORMULA AND ADULT NUTRITIONALS

Determination of Vitamin A (Retinol) in Infant Formula and

Adult Nutritionals by Liquid Chromatography:
First Action 2011.15
JONATHAN W. DEVRIES, KARLENE R. SILVERA, and ELLIOT MCSHERRY
Medallion Laboratories, 9000 Plymouth Ave North, Minneapolis, MN 55427
DAWN DOWELL
AOAC INTERNATIONAL, 481 N. Frederick Ave, Gaithersburg, MD 20877

O
During the “Standards Development and International n March 28, 2011, the AOAC Board of Directors
Harmonization: AOAC INTERNATIONAL Mid- approved an alternative path to achieve Official First
Year Meeting,” held on June 29, 2011, an Expert Action status for methods selected and reviewed
Review Panel (ERP) reviewed the method for the using the AOAC volunteer consensus standards development
“Determination of Vitamins A (Retinol) and E (alpha- processes. Under this path, an Expert Review Panel (ERP)
Tocopherol) in Foods by Liquid Chromatography: vetted by the Official Methods Board (OMB) reviewed the
Collaborative Study,” published by Jonathan W. method for the “Determination of Vitamins A (Retinol) and
DeVries and Karlene R. Silvera in J. AOAC Int. in
E (alpha-Tocopherol) in Foods by Liquid Chromatography:
2002. After evaluation of the original validation data,
Collaborative Study” (1). After evaluation of the original
an ERP agreed in June 2011 that the method meets
validation data, an ERP agreed in June 2011 that the method
standard method performance requirements (SMPRs)
for vitamin A, as articulated by the Stakeholder Panel meets standard method performance requirements (SMPRs)
on Infant Formula and Adult Nutritionals. The ERP for vitamin A, as articulated by the Stakeholder Panel on Infant
granted the method First Action status, applicable Formula and Adult Nutritionals, and approved this method as
to determining vitamin A in ready-to-eat infant and Official First Action status for vitamin A in infant formula and
adult nutritional formula. In an effort to achieve Final adult nutritionals. Methods approved First Action under the
Action status, it was recommended that additional alternative path will remain First Action for a period of about
information be generated for different types of infant 2 years. During this time, methods will be used in laboratories,
and adult nutritional formula matrixes at varied generating additional information. ERPs will monitor the
concentration levels as indicated in the vitamin methods’ performance, and after about 2 years, will determine
A (retinol) SMPR. Existing AOAC LC methods are whether the methods should be recommended to the OMB for
suited for specific vitamin A analytical applications. Final Action (2).
The original method differs from existing methods For First Action adoption, all methods were reviewed by
in that it can be used to assay samples in all nine
primary, as well as secondary, expert reviewers. Panel members
sectors of the food matrix. One sector of the food
summarized their reviews; the advantages and disadvantages of
matrix was powdered infant formula and gave
each method were then discussed thoroughly by the entire panel,
support for the First Action approval for vitamin
A in infant and adult nutritional formula. In this stakeholders, and observers present. Methods were evaluated
method, standards and test samples are saponified for completeness of validation and likelihood of meeting the
in basic ethanol–water solution, neutralized, and SMPRs (applicability to intended use, clarity of the method
diluted, converting fats to fatty acids and retinol description, ruggedness, reproducibility, recovery, analytical
esters to retinol. Retinol is quantitated by an LC range, and LOQ).
method, using UV detection at 313 or 328 nm for Based on the data presented, the ERP agreed that the method
retinol. Vitamin concentration is calculated by for determining vitamin A (retinol), in addition to being granted
comparison of the peak heights or peak areas of Official First Action status, should undergo additional evaluation
retinol in test samples with those of standards. consisting of data collected from multiple laboratories on a series
of infant formula matrixes shared amongst the laboratories and
Submitted for publication October 28, 2011.
The method was approved by the Expert Review Panel on Infant available through AOAC INTERNATIONAL. The protocol
Formula and Adult Nutritionals as First Action. See “Standards News,” for data collection should be guided by the ERP in conjunction
(2011) Inside Laboratory Management, July/August issue. with the OMB. With vitamin A being essential for many health-
The AOAC Stakeholder Panel on Infant Formula and Adult
Nutritionals (SPIFAN) invites method users to provide feedback on related functions, it is important for analytical laboratories to
the First Action methods. Feedback from method users will help verify have accurate methods available to determine the concentration
that the methods are fit for purpose and are critical to gaining global of vitamin A in infant formula and adult nutritionals. The
recognition and acceptance of the methods. Comments can be sent
directly to the corresponding author. method provides a straightforward, modern way to achieve
Corresponding author’s e-mail: [email protected] these results using a combination of traditional chemistry and
DOI: 10.5740/jaoacint.CS2011_15 LC technology.
2 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, 2012

AOAC Official Method 2011.15 B. Apparatus and Materials

Vitamin A (Retinol) in Infant Formula (a) HPLC system.—(1) Pump.—High-pressure pump
and Adult Nutritionals operating continuously at 1.0 to 2.0 mL/min with a flow
Liquid Chromatography precision of ±1% or better.
First Action 2011 (b) Injector.—Manual injector or autosampling injector
(Applicable for the determination of retinol from 7 to with a 20 μL fixed loop having a typical sampling precision of
383 g/100 g in ready-to-feed infant formula and adult ±0.25% or better.
nutritionals.) (c) Chromatography columns.—Reversed-phase C18, 10 μm
Caution: Potassium hydroxide is extremely caustic and can (4.6  250 mm), capable of separating cis and trans isomers of
retinol with a resolution of 1.5 or greater. Cis-retinol typically
cause severe burns. Protect skin and eyes and wear
elutes prior to trans-retinol on columns providing effective
all personal protective equipment while performing separation.
method, which involves the use of flammable liquids (d) Detector.—Photometric detector monitoring absorbance
and toxic chemicals. Perform saponification behind at 328 nm. (Alternatively a wavelength of 313 nm can be used.)
a barrier when using hot water, steam, or an electric (e) Recorder, integrator, or data collection system.—
heating mantle. Use an effective fume removal Compatible with detector used.
(f) Erlenmeyer flasks.—Low-actinic, 125 mL, with neck
device to remove flammable and toxic vapors/fumes
adapted for connecting reflux condenser.
produced during operation of the method. Leave
(g) Hot plate.—With sufficient heating surface area to handle
ample headroom in flask and add boiling chips before multiple reflux apparatus setups preferred.
heating a flammable liquid. (h) Reflux condensers.—With adapters (if necessary) to
attach 125 mL low-actinic Erlenmeyer flasks and nitrogen lines.
See Tables 2011.15A and B for the results of the interlaboratory
(i) Volumetric flasks.—Low-actinic, 100 and 10 mL.
study supporting acceptance of the method.
(j) Nitrogen blanket apparatus.—Supply of nitrogen gas
A. Principle with appropriate tubing and connectors to provide a constant
nitrogen atmosphere blanket in the reflux apparatus during
Standards and test samples are saponified in basic ethanol–
saponification.
water solution, neutralized, and diluted, converting fats to fatty
acids and retinol esters to retinol. Retinol is quantitated by an C. Reagents
LC method, using UV detection at 313 or 328 nm. Vitamin (a)(1) Certified vitamin A acetate concentrate.—Equivalent
concentrations are calculated by comparison of peak heights or to ca 30 mg retinol/g oil. Content certified by U.S. Pharmacopeia
peak areas of vitamins in test samples with those of standards. (USP; Rockville, MD; www.usp.org); or (2) Retinyl palmitate,

Table 2011.15A. Interlaboratory study results for the determination of vitamin A by LC

Matrix
sector No. Matrix Labsa(b) Mean, μg/100 g Rec., % SR RSDR, % R HorRat
1 Margarine/butter (50/50 mix) 12(0) 857.08 68.36 7.98 191.41 0.69
1 Margarine/butter (50/50 mix; spiked) 10(2) 1457.20 106.6 89.90 6.17 251.72 0.58
2 Chicken gravy (canned; spiked) 9(1) 131.13 97.1 19.17 14.62 53.68 0.95
3 Cheese sauce (spiked) 11(0) 271.45 103.7 34.96 12.88 97.89 0.94
4 Whole egg powder 10(0) 161.98 62.48 38.57 174.94 2.59
4 Whole egg powder (spiked) 12(0) 426.12 91.7 205.71 48.28 575.99 3.75
5 Multigrain cereal 12(0) 634.38 114.85 18.10 321.58 1.49
5 Corn cereal 12(1) 945.52 86.78 9.18 242.98 0.80
5 Corn cereal (spiked) 12(1) 1395.91 108.5 76.11 5.45 213.11 0.51
6 Infant formula (powdered) 12(0) 584.15 95.98 16.43 268.74 1.34
6 NIST SRM 1846 12(0) 464.28 79.5 49.06 10.57 137.37 0.83
7 Dried nonfat milk 12(0) 817.27 102.94 12.60 288.23 1.08
7 Dried nonfat milk (spiked) 12(0) 1708.25 125.5 195.68 11.46 547.90 1.10
8 Cottage cheese 8(1) 46.02 8.19 17.80 22.93 0.99
8 Cottage cheese (spiked) 10(2) 411.03 98.9 69.29 16.86 194.01 1.30
9 Canned tuna in oil (spiked) 11(1) 262.36 89.5 56.11 21.39 157.11 1.55
Avg. 100.1 ± 13.2
a(b)
Number of laboratories: a = number of laboratories retained after outliers were removed, and b = number of outlier laboratories.
 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, 2012 3

all-trans.—Fluka Chemical Co. (Ronkonkoma, NY, +1-800-

358-5287). Request Certificate of Lot Analysis when ordering.

HorRat

1.74
0.67
0.66
0.90

1.10
1.42

1.21
If manufacturer’s certification is unavailable, or purity of
standards needs to be verified, test vitamin A palmitate purity as
follows: Dissolve 50 mg (record to the nearest 0.1 mg) retinol
palmitate standard in 2-propanol (UV-spectroscopy grade) in a

102.42
314.22
239.37
244.66

171.44
296.32

126.20
500 mL flask and dilute to volume. Dilute 10 mL of this solution
R

to 100 mL with 2-propanol (final concentration is ca 10 mg/L).

Measure maximum absorbance obtained at 325–328 nm using
1 cm path length cell and 2-propanol as a blank. Inject 10 L
300.41
135.32
185.44

169.12
244.78

109.59
62.13
of solution onto an LC system with a reversed-phase column,
r

a mobile phase of methanol–heptane (93 + 7), and detection at

the wavelength of the absorbance maximum. Record the peak
area of the all-trans retinyl palmitate (PArp) and the total of all
Interlaboratory study results for the determination of vitamin A by LC (Youden pair statistical treatment)
RSDR, %

26.52
10.44

14.04
17.37

16.74

peak areas present other than the solvent front (PAt). Calculate
7.04
7.29

the purity of the retinol palmitate as follows:

6
Percent purity = (PArp/PAt) × (ABS  5  10 )/(960  W)
105.83
112.22

36.58
85.49
87.38

61.23
45.07

Number of laboratories: a = number of laboratories retained after outliers were removed, and b = number of outlier laboratories.
SR

where ABS = absorbance maximum; 960 = absorbance of pure

retinol palmitate (1% solution in 1 cm cell); W = weight of
6
test portion in mg; and 5  10 = combined dilution factors,
RSDr, %

16.09
13.85
14.35

14.54
6.73
4.12
7.91

conversion to 1% equivalent solution, and conversion to %.

Store retinol palmitate standard at 0–4°C to allow for easier
handling while weighing.
(b) Acetic acid.—Glacial.
107.29

22.19
48.33
66.23
87.42

39.14
60.4

(c) Methanol.—HPLC grade.

sr

(d) Ethanol.—95%.
(e) Tetrahydrofuran (THF).
Mean, μg/100 g

(f) Hexane.
137.94
1595.1

837.18

436.18
609.27

269.25
1173.3

(g) Pyrogallol (pyrogallic acid).—Crystals, Sigma-Aldrich,

(www.sigmaaldrich.com) CAS 87-66-1 or equivalent.
(h) Vitamin A mobile phase.—Combine 860 mL methanol
(HPLC grade) and 140 mL water. Mix well. Stir overnight to
degas or mechanically degas prior to use.
Labsa(b)
10(2)
10(2)
12(0)

10(2)
12(0)

11(1)
8(1)

(i) THF–ethanol (50+50).—Combine 500 mL THF and

500 mL 95% ethanol. Mix well.
(j) Potassium hydroxide solution, 50%.—Slowly add 500 g
Dried nonfat milk (spiked) and margarine/butter (50/50 mix; spiked)

KOH pellets to 500 mL water contained in a 2 L thick-wall

Erlenmeyer flask. (Caution: Solution gives off substantial heat
while KOH is dissolving; add KOH in 100 g portions while
flask is being cooled with cold water. Swirl flask gently to aid in
Chicken gravy (canned; spiked) and whole egg powder
Canned tuna in oil (spiked) and cheese sauce (spiked)

dissolution of KOH. Store in glass container with cork stopper.)

Margarine/butter (50/50 mix) and dried nonfat milk

(k) Vitamin A working standard (approximately 15 μg/mL).—

Multigrain cereal and infant formula (powdered)
Cottage cheese (spiked) and NIST SRM 1846

(1) Using USP standard.—Weigh 50 mg vitamin A acetate

concentrate into a 100 mL low-actinic volumetric flask. Record
weight to nearest 0.1 mg. Record concentration in mg/g per
Corn cereal and corn cereal (spiked)

USP certification. Add small amount of acetone (<3 mL) to aid

dissolution. Dilute to volume with 95% ethanol. Store at 4°C in
dark. Solution is stable for 2 weeks.
(2) Using retinyl palmitate.—Weigh 55 mg retinyl palmitate
into a 100 mL low-actinic volumetric flask. Record weight to
Table 2011.15B.

nearest 0.1 mg. Record purity per supplier certification or purity

test. Add pea-sized piece of pyrogallic acid, approximately
Youden pairs

50 mg. Dissolve and dilute to volume with hexane. Pipet 5 mL

of solution to second 100 mL low-actinic flask and dilute to
volume with 95% alcohol. Store at 4°C in dark. Solution is
a(b)

stable for 2 weeks.

4 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, 2012

D. Extraction and Saponification peak heights of 50–90% of full scale at the high standard. Repeat
injection of standard until peak height(s) are reproducible.
Turn on hot plate to preheat. Start and adjust cooling water
Inject test solutions. Intersperse with standard solution
flow to precool reflux condensers.
injections after every nine tests. [If retinol in test exceeds the
Prepare high standard (approximately 750 ng/mL) by
peak height of the high standard by more than 25%, dilute
pipeting 5 mL vitamin A working standard into a 125 mL low-
test solutions using a solution of 10 mL 50% KOH, 40 mL of
actinic Erlenmeyer flask. Add 35 mL of 95% ethanol. Proceed
95% ethanol, 10 mL glacial acetic acid, and 40 mL THF–95%
to addition of pyrogallic acid.
ethanol (50 + 50).]
Prepare intermediate standard (approximately 300 ng/mL) by
pipeting 2 mL vitamin A working solution into a second 125 mL F. Calculations
low-actinic Erlenmeyer flask. Add 38 mL of 95% ethanol. Calculate μg/g vitamin A (as retinol) as follows: Measure the
Proceed to addition of pyrogallic acid. peak heights or areas of the standards.
Prepare low standard (approximately 75 ng/mL) by pipeting (1) Using USP standard.—Determine the response factor for
0.5 mL vitamin A working standard into a third 125 mL low- vitamin A (RFA) using the following calculation:
actinic Erlenmeyer flask. Add 39.5 mL of 95% ethanol. Proceed
to addition of pyrogallic acid. RFA = mgstd  mLstd  concnstd/PkHTstd  10 000
Grind solids to pass a 40 mesh sieve. Blend liquid or wet
materials to homogeneity and store ≤4°C in the dark. where PkHTstd = peak height or area of standard from
To prepare low-fat (<40% fat) test samples, weigh enough chromatogram; mLstd = mL of working standard used in
test sample (≤5 g) to give approximately 50 μg vitamin A into a procedure; concnstd = concentration of USP vitamin A (as
125 mL low-actinic Erlenmeyer flask. For test samples high in retinol) per USP certification (mg/g); mgstd = mg of USP
sugar, add 3 mL water and disperse the test portion as a slurry. standard weighed in reagents section; 10 000 = combined
Add 40 mL of 95% ethanol. dilution factors for vitamin A standard.
To prepare high-fat test samples, weigh test sample (≤2 g) to (2) Using retinyl palmitate.—Determine RFA using the
give approximately 50 μg vitamin A into a 125 mL low-actinic following calculation:
Erlenmeyer flask. Add 40 mL of 95% ethanol.
Add a pea-sized piece (approximately 50 mg) of pyrogallic RFA = mgstd  mLstd  puritystd  0.5458/PkHTstd  200
acid (antioxidant) to each standard and test flask. Add a glass
bead or boiling stone to promote even boiling. where puritystd = percent purity certified by supplier or
Swirl all flasks to ensure that all materials are thoroughly determined, divided by 100; mgstd = mg of retinyl palmitate
dispersed in the solution. weighed; PkHTstd = peak height or area of standard from
Turn on N flow and ensure N atmosphere for all flasks before chromatogram; mLstd = mL of working standard used in
and while refluxing. procedure; 0.5458 = ratio of retinol to retinyl palmitate
Pipet 10 mL of 50% KOH solution into each flask and molecular weights; and 200 = combined dilution factors/
immediately place flask on hot plate under reflux condenser. conversion from mg to mg.
Swirl. The RFA values of the low, medium, and high standards should
Reflux 45 min. Swirl flasks every 10 min. agree with each other within 3% relative since the detector
Remove reflux flasks from hot plate, stopper with corks, and response should be linear across this concentration range. Use
quickly cool flasks to room temperature using cold water or ice an average of RFA values calculated from high, medium, and
water. low standards for test sample quantitation.
Pipet 10 mL glacial acetic acid into each flask to neutralize the Measure the peak heights or areas corresponding to retinol
KOH. Mix well and let flasks cool again to room temperature. (vitamin A) in the test sample extracts. The 13-cis isomer of
Quantitatively transfer solution in each flask to a 100 mL retinol (eluting immediately preceding the all-trans-isomer)
low-actinic volumetric flask using THF–95% ethanol (50 + 50). might be present in some test samples. Measure the 13-cis peak
Dilute to volume with the same solvent mixture. also.
Stopper and invert volumetric flask 10 times. Multiply the height or area of the 13-cis retinol peak by 1.08
Allow flasks to set for at least 1 h at room temperature and (to compensate for difference in absorbance compared to the
preferably overnight in refrigerator to precipitate fatty acid salts trans-isomer).
formed during saponification. In some cases, centrifugation Add the corrected peak height or area for the 13-cis isomer to
may reduce settling time. that of the all-trans isomer to give total test sample peak height
or area. Calculate the concentration of vitamin A (in μg/100 g as
E. Determination
retinol) using the following equation:
Start HPLC system(s) and allow to warm up and equilibrate
for a minimum of 30 min with mobile phase flowing at flow rate Vitamin A, μg/100 g (as retinol) =
of 1.0 mL/min. RFA  PkHTSPLE  10 000/W
Inject vitamin A standards that have been taken through
saponification onto HPLC system. Adjust mobile phase to where RFA = response factor for vitamin A; PkHTSPLE = total
achieve a resolution of 1.5 or better for cis and trans forms. test sample peak height or area of all-trans and 13-cis retinol;
All-trans-retinol should elute in approximately 9 min (the cis- 10 000 = dilution volume of test portion, mL  conversion to
isomer will elute just prior to the all-trans-isomer. Inject high, 100 g portion; and W = weight of test portion, g.
medium, and low standards. Adjust detector sensitivity to give References: J. AOAC Int. 85, 424(2002); in press(2012)
 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, 2012 5

Table 1. Samples selected for collaborative study

Matrix sector Test samples Protein, % Fat, % Carbohydrate, %
1 Margarine/butter (50/50 mix) 0 100 0
2 Chicken gravy (canned) 6 42 52
3 Cheese sauce 19 60 21
4 Whole egg powder 42 48 10
5 Multigrain cereal 17 6 77
5 Corn cereal 9 0 91
6 Infant formula (powdered) 24 17 59
6 NIST SRM 1846 24 17 59
6 Baked beans with franks (60/40, w/w) 23 26 51
7 Dried milk 41 1 58
7 Full fat soy flour 42 23 35
8 Cottage cheese 66 20 14
9 Canned tuna in oil 86 14 0

Results and Discussion protein content based on satisfactory results from test samples
in adjacent sectors 7 and 8 that have similar protein content.
The original study data presented in Tables 2011.15A and B In the original study, Youden pair statistics was used since the
collected for vitamins A and E in nine food matrixes. The data method was applicable to all foods and a similar variance was
were presented for review by the ERP to determine the method’s expected. This approach was applied to the data to calculate the
appropriateness for use to determine the vitamin A content in variability between laboratories for this method. Youden pairs
infant and adult nutritional formula. The study data presented were established based on samples in which the analyte levels
was reported from 13 laboratories, with one laboratory report were closest. As shown in Table 2011.15B, the 14 test samples
showing a systematic high bias in relation to that of the other were combined to form seven pairs. The results show that the
laboratories. The cause of this bias was determined to be a within-laboratory variability for each sample pair is less than
result of not properly following the protocol. Therefore, the the variability between laboratories.
results were omitted from the calculations. Tabulated results are The recovery of the method was assessed by spiking
found in Table 2011.15A for vitamin A from the remaining 12 eight samples with a vitamin concentration range. National
laboratories. The test samples for chicken gravy, baked beans Institute of Standards and Technology (NIST) Standard
Reference Material (SRM) 1846 was also included in the sample
with franks, cheese sauce, and tuna (canned) did not provide
set. This approach allowed a recovery sample for each sector of
data sufficient for statistical review. In addition, soy flour (full
the food analysis triangle. As shown in Tables 2011.15A and
fat) does not have a measurable quantity of retinol because it is
B, the average recovery for the method was 100 ± 13%. This
plant-based. The decision to include it in the original study was
is in line with the expected recovery variability based on the
made based on theoretical data for content of vitamin A. Because
expected reproducibility RSD (RSDR) of approximately 13%
the typical operating LOD for retinol on LC is 15 μg/100 g
(based on HorRat values in Tables 2011.15A and B).
for most laboratories, it is possible that the level of retinol is
Accuracy of the method was determined by including a
<15 μg/100 g, or that the detectors used by the laboratories were
powdered, milk-based infant formula (NIST SRM 1846).
not tuned adequately to a sufficiently low S/N to detect the low This sample was included as an unknown sample. The NIST
levels of retinol present. Because of the expected low levels of Certificate of Analysis gives the noncertified vitamin A content
retinol, one additional test sample was included for each of the of NIST SRM 1846 as 584 ± 68 μg/100 g, 95% uncertainty
five food sectors to obtain valid data. range. The reported study results showed a recovery of 79.5%,
It was decided to apply the Dixon test to the individual with the results being 464 ± 31 mg/100 g, 95% uncertainty
test samples that had sufficient data points to obtain between- range. SRM 1846 packets were obtained approximately 6
laboratory statistical data based on similar analyte level. As seen months prior to the study beginning. The packets were stored
in Table 2011.15A, the results are good except over sector 4 unopened in a dark cabinet in an office. The temperature ranged
(see Table 1), which is represented by whole egg samples (fat, from 20 to 25°C (68–77°F), as suggested by the instructions
33–67%; protein, 33–67%; carbohydrates, 0–33%). Based received with SRM 1846.
on the HorRat results, the RSD at various concentrations is Because the cis-retinol proved to be somewhat elevated
in agreement relative to the analyte levels. The analytical from what might be expected from the SRM, it was decided
variability of the whole egg sample cannot be specifically to investigate possible loss of retinol during storage of the
attributed to the particular fat, carbohydrate, or protein ratio. materials. As part of the investigation, new packets of SRM 1846
Possible explanations include difficulty in digesting the matrix were purchased and analyzed in a comparison study of the
and extract, or homogeneity was not achieved for the whole previously procured SRM packets used in the collaborative
egg powder sample. It is plausible to exclude issues involving study (stored an additional 8 months, for a total of 14 months).
 6

Table 2. Collaborative study data for vitamin A

Laboratory
Matrix Spike level,
sector Vitamin A samples μg/100 g 1 2 3 4 5 6 7 8 9 10 11 12
1 Margarine/butter (50/50 mix) 874 806 732 871 860 960 820 934 832 948 781 867
1 Margarine/butter (50/50 mix; spiked) 563 1470 1283 969a 1480 1480 1570 1390 1492 1347 1530 4278a 1530
2 Chicken gravy (canned) 10
a
2 Chicken gravy (canned; spiked) 135 140 92 127 158 140 140.2 113 141 342 129
3 Cheese sauce 39 64 80 29.5 35
3 Cheese sauce (spiked) 214 270 258 304 296 300 250 222 227 263 338 258
4 Whole egg powder 176 67 144 188 160 186.8 118 191 293 96
4 Whole egg powder (spiked) 288 467 155 680 433 430 180 337.4 479 881 506 282 283
5 Corn cereal 968 1008 766 970 950 100a 847.7 894 1043 1063 910 981
5 Corn cereal (spiked) 415 1430 1369 1316 1340 1390 1380 1279 1455 1465 1551 359a 1380
5 Multigrain cereal 599 571 500 717 620 630 627.6 841 727 778 561 441
6 Baked beans with franks (60/40, w/w) 2 395 40
6 Infant formula (powdered) 620 575 604 608 350 660 593.8 635 563 737 475 589
6 NIST SRM 1846 584 499 453 362 457 450 450 468.3 400 480 547 508 497
7 Nonfat dry milk 896 822 658 725 750 890 780.2 691 914 1005 794 882
7 Nonfat dry milk (spiked) 710 1720 1738 1924 1750 1520 1800 1625 1748 1825 1994 1245 1610
7 Full fat soy flour 15 40 363.8 208
DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, 2012

8 Cottage cheese 36 39 84a 57.7 50 50 38.19 55 42

a a
8 Cottage cheese (spiked) 369 407 404 459 354 270 1430 392.3 491 391 512 838 430
9 Canned tuna in oil
9 Canned tuna in oil (spiked) 293 287 163 227 263 530a 260 284 266 338 351 187 260
a
Dixon outlier.
 DEVRIES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 2, 2012 7

A testing schedule was set for the newly obtained SRM 1846 during weekends. It is possible that the cabinet that housed
at about 2 weeks, 5 weeks, and 6 months after receipt. Results the SRM sample was exposed to periods of high temperature
obtained for the stored NIST sample were 80.7, 78.6, and 81.1%, during SRM sample storage, as the cabinet was against the wall
respectively (average 80.1%), compared to those of the newly
adjacent to the food production facility. Thus, it is possible the
obtained NIST samples. The average concentration of the stored
NIST samples was 465 mg/100 g; the newly purchased sample SRM sample was exposed to degradation temperature prior to
average was 580 mg/100 g. The cause for the level reduction of being included in the collaborative study.
vitamin A in the collaborative study sample of SRM cannot be Collaborative study data for vitamins A and E were received
determined. However, the levels found during this investigation and reviewed during the AOAC Official MethodsSM approval
are consistent with the comparative results obtained during the process. From the data reported, vitamin A was shown to
collaborative study.
perform as expected (Table 2).
One possible reason for the change in the SRM could be
attributed to office environment. The office that was used
to store the SRM prior to the collaborative study is climate- References
controlled. However, the area of the building adjacent to the
office contains a food production pilot plant faculty. This area (1) DeVries, J., & Silvera, K. (2002) J. AOAC Int. 85, 424–434
was periodically heated above 100°F for pest control purposes (2) Sullivan, D. (2012) J. AOAC Int. 95, in press