FOOD COMPOSITION AND ADDITIVES

Extension of Dry Ash Atomic Absorption and Spectrophotometric

Methods to Determination of Minerals and Phosphorus in
Soy-Based, Whey-Based, and Enteral Formulae (Modification of
AOAC Official Methods 985.35 and 986.24): Collaborative Study
COOK: JOURNAL OF AOAC INTERNATIONAL VOL. 80, NO. 4, 1997
KATHLEEN K. COOK
U.S. Food and Drug Administration, Office of Food Labeling, Division of Science and Applied Technology, 200 C St, SW,
Washington, DC 20204
Collaborators: R. Allen; A. Choudhry; R. Fleener; F.E. Greene; C. Johnson; N. Miller-Ihli; R. Powell; W.-L. Yip

Eight laboratories participated in a collaborative

study of AOAC Official Method 985.35, Minerals in
Ready-to-Feed Milk-Based Infant Formula and Pet
Foods, Atomic Absorption Spectrophotometric
Method; and 7 laboratories participated in a study
of AOAC Official Method 986.24, Phosphorus in
Milk-Based Infant Formula, Spectrophotometric
Method, to extend these methods to infant formulae (other than milk-based) and enteral products.
Three ready-to-feed soy-based formulae and 2 soybased powder formulae were chosen to represent
the plant matrix. A whey-based formula and a casein-based enteral formula were also included in
the study. Soy formulae containing nearly identical
concentrations of particular elements were matched,
and an application of the Youden closely matched
pair approach was used to estimate repeatability
parameters. Average reproducibility values were as
follows: calcium, 9.3%
%; copper, 9.7%
%; iron, 5.5%
%; potassium, 4.0%
%; magnesium, 5.2%
%; manganese,
10.6%
%; sodium, 4.7%
%; phosphorus, 10.5%
%; and zinc,
7.3%
%. At similar analyte concentrations, the betweenlaboratory variabilities compared well with those reported for the official methods. Most repeatability
and reproducibility parameters compared well with
the original collaborative study. AOAC Official Methods 985.35 and 986.24 have been modified to extend their applicability to infant formulae (other
than milk-based) and enteral products.

Submitted for publication October 28, 1996.

The recommendation was approved by the Methods Committee on
Food Nutrition, and was adopted by the Official Methods Board of the
Association. See Official Methods Board Actions (1997) J. AOAC Int. 80,
35A, and Official Methods Board Actions (1997) Inside Laboratory
Management, March issue.

collaborative study was performed to confirm that the

dry ash atomic absorption method for determination of
Ca, Mg, Zn, Cu, Fe, Mn, K, and Na in ready-to-feed
milk-based infant formula, AOAC Official Method 985.35,
and the spectrophotometric method for phosphorus in milkbased infant formula, AOAC Official Method 986.24, could be
extended to determination of these minerals in all types of infant formula and enteral products.

Collaborative Study
In the original collaborative studies of methods of analysis
for infant formula, 3 liquid ready-to-feed (RTF) milk-based
formulae were analyzed and the same analysis was repeated on
a different day (1, 2). For the study reported here, 3 RTF formulae and 2 soy-based powders were selected to represent a
plant source matrix, which was not included in the original
study. A whey-based powder and a casein-based enteral formula were also included to represent other matrixes derived
from a milk base. Eight collaborators, including the author,
used Method 985.35 (3), and 7 collaborators, including the
author, used Method 986.24 (4) to analyze soy, whey, and enteral formulae. In addition, each collaborator was asked to add
a spiking solution to one of the following: the enteral formula,
an RTF soy-based formula, a soy powder formula, or the whey
powder formula.
The protocol of the International Union of Pure and Applied
Chemistry (IUPAC; 5) was used to interpret study results. This
protocol uses sequential application of the Cochran and
Grubbs tests to the data to identify outliers. The same protocol
was used to determine recovery. Results (Tables 111) show
that Methods 985.35 and 986.24 are reliable and applicable to
other formulae.

Powders
The contents of 6 cans from the same lot of a particular
brand of powder formula were composited by adding half of
their 12 to 16 oz. contents to a large beaker. After thorough

mixing, these composites were apportioned into screw-top

plastic jars for shipment to collaborators. For the replicate portion to be spiked, 6 g of either soy powder or whey powder was
weighed into a screw-top plastic jar. The material was to be
added to the ashing vessel with the spiking solution. The minimum weight specified for use for powders was 1.5 g.

985.35 Minerals in Infant Formula, Enteral

Products, and Pet Foods, Atomic Absorption
Spectrophotometric Method
Final Action 1985
Final Action 1988
Revised First Action 1997

Liquids
Collaborators analyzed 4 liquid formulae: 8 fl. oz. cans and
bottles of the same lot, with labels removed, representing 2 of
the RTF soy products and the single enteral formula, and a 32 fl.
oz. can with the label printed on the can representing the third
RTF soy formula. No attempt was made to eliminate can-to-can
variation within lots because of the instability of the product
once opened. Collaborators analyzing a spiked liquid were
given directions to weigh a 30 g portion of the 32 fl. oz. RTF
soy formula or the enteral formula into the ashing vessel and to
add the accompanying spiking solution. The only formula identified for the collaborators was the 32 fl. oz. can with the label
printed directly on the can.

Spiking Solution
A spiking solution containing all 9 minerals in a plastic
screw-top vial was included. Collaborators were directed to
add this solution to the designated formula to be spiked and
then to rinse the vial 3 times, collecting the rinses in the ashing
vessel. Because P was included in the same spiking solution and
only one portion was to be spiked, collaborators were required to
use the same preparation step for P and the other minerals.
An in-house study was conducted to investigate whether results for phosphorus would be compromised by this procedure.
No differences were found between results obtained from using
the extracts from the atomic absorption method for minerals
and the results of the official phosphorus method in the inhouse study. The results are summarized in Table 12.

Within-Laboratory Variability
Liquid RTF soy formulae and soy powders with nearly identical concentrations of particular elements were used to estimate within-laboratory variability by mechanical application
of the Youden closely matched pair approach.

Blank Determination
To identify problems due to contamination, collaborators
were asked to run 2 blanks through each method.

Concentration Ranges
Estimates of concentration ranges for all minerals in the
products were supplied to each collaborator. However, actual
concentrations found for some elements were outside these
ranges. As a consequence, one collaborator reported 2 determinations for elements found to be outside the ranges supplied.
The first reported values were used in this report.

(Applicable to Ca, Mg, Fe, Zn, Cu, Mn, Na, and K.)
Caution: See Appendix B: safety notes on safe handling of
acids. Dispose of waste solvents in an appropriate manner compatible with applicable environmental rules and regulations.
Method Performance:
See Table 985.35A for method performance data.

A. Principle
Organic matrix is destroyed by dry ashing in muffle furnace.
Remaining ash is dissolved in diluted acid and analyte is determined by atomic absorption spectrophotometry (AAS).

B. Apparatus
(a) Glassware.Thoroughly clean all glassware by soaking overnight in 20% HNO3. Rinse all glassware 3 with distilled-deionized or 18 M resistance H2O.
(b) Evaporation dish.100 mL unetched Vycor (or Pt),
flat-bottom, with pour spout; capable of withstanding temperatures up to 600C.
(c) Atomic absorption spectrophotometer.Equipment
should be well maintained with good response per unit concentration, for example, 0.200 abs or above 4 mg/L Cu.
(d) Furnace.With pyrometer to control temperature
range of 250600 10C.

C. Reagents
(a) Water.Distilled, deionized, or 18 M resistance for
preparation of standard or sample solutions.
(b) Standard stock solutions.Commercially prepared,
certified AA standards, or prepared in laboratory by
Method 969.23A(c) (see 35.1.21) for Na, 969.23A(d) (see
2.6.01) for K, 965.09B(a) (see 2.6.01) for Ca, 965.09B(b) (see
2.6.01) for Cu, 965.09B(c) (see 2.6.01) for Fe, 965.09B(e) (see
2.6.01) for Mg, 965.09B(f) (see 2.6.01) for Mn, and
965.09B(g) (see 2.6.01) for Zn.
(c) Nitric acid.Unless specified otherwise, use redistilled
or ultrapure.
(d) Lanthanum oxide.La2O3, 99.99%; AAS quality.
(e) Lanthanum chloride solution.LaCl3, 1% (w/v).
Weigh 11.7 g ( 100 mg) La2O3 and transfer to 1 L volumetric
flask. Add enough H2O to wet powder and then slowly add
50 mL concentrated HCl (Caution: exothermic reaction). Let
powder dissolve and then dilute to volume with H2O and mix.
Lanthanum chloride solution is stable up to 6 months when
stored at room temperature.
(f) Cesium chloride solution.CsCl, 10% (w/v). Weigh
12.7 g (100 mg) CsCl and transfer to 100 mL volumetric flask.
Dilute to volume with H2O and mix. Make fresh every 6 months.
(g) Filter pulp.Analyzed ash-free.

D. Ashing Procedure
Note: For liquid formulaes, shake container before weighing.
Place composite portion in previously cleaned Vycor evaporating dish (which may contain 5 g filter pulp for ease of handling). Exact amount of composite required will depend on
concentration of minerals present. (For powders, take 1.5 g.)
In general, 25 mL will be adequate. If some minerals, in particular Fe, Cu, or Mn, are at very low levels, a larger portion
(50 mL) may be necessary.
Dry portion in 100C oven overnight or in microwave oven
(programmed over ca 30 min). When dry, heat on hot plate until
smoking ceases, and then place dish in 525C furnace (carefully avoiding ignition) for minimum time necessary to obtain
ash that is white and free from C, normally 35 h, but 8 h.
Remove dish from furnace and let cool. Ash should be white
and free from C. If ash contains C particles (i.e., it is gray), wet
with H2O and add 0.53 mL HNO3. Dry on hot plate or steam
bath and return dish to 525C furnace 12 h.
Dissolve ash in 5 mL 1N HNO3, warming on steam bath or
hot plate 23 min to aid solution. Add solution to 50 mL volumetric flask and repeat with 2 additional portions of 1N HNO3.
Dilute to 50 mL with 1N HNO3. (Note: Additional dilutions
may be necessary to bring concentrations within the linear
range of instrument.)

E. Determination
Add LaCl3 solution to final dilution of each standard and test
solution to make 0.1% (w/v) La for determination of Ca and
Mg only. Add CsCl solution to final dilution of each standard
and test solution to make 0.5% (w/v) Cs (0.04M) for determination of Na and K only.
Prepare blanks representing all reagents and glassware, and
carry through entire procedure.
Prepare calibration curve (concentration vs absorbance) for
each mineral to be determined, using wavelength and flame
specified in Table 985.35B. Optimize flame parameters in accordance with instrument manufacturers instructions. Prepare
solutions for calibration of instrument to cover linear range of
calibration curve. See instrument instruction manual.
Assay samples in similar manner. Determine concentration
of each mineral from its calibration curve, and calculate concentration in test sample, taking into account test portion size
and dilutions.
Ref.: J. Assoc. Off. Anal. Chem. 68, 514(1985); J. AOAC Int.
80, 834844(1997)
986.24 Phosphorus in Infant Formula and Enteral
Products, Spectrophotometric Method
First Action 1986
Final Action 1988
Revised First Action 1997

Method Performance:
See Table 986.24 for method performance data.

A. Principle
Phosphorus is determined by spectrophotometry on ashed
test portion by complexing with molybdovanadate reagent.

B. Apparatus
(a) Spectrophotometer.Capable of operation at 400 nm.
(b) Muffle furnace.Equipped with pyrometer and controller.
(c) Ashing dishes.Silica or porcelain.

C. Reagents
(a) Hydrochloric acid solution.(1 + 3, v/v). Add 250 mL
HCl to 750 mL H2O.
(b) Molybdovanadate reagent.Dissolve 20 g ammonium molybdate in 200 mL hot H2O and cool. Dissolve 1.0 g
ammonium metavanadate in 125 mL hot H2O, cool, and add
160 mL HCl. Gradually add, with stirring, molybdate solution
to vanadate solution and dilute with H2O to 1.0 L.
(c) Phosphorus standard solutions.(1) Stock standard
solution.2 mg P/mL. Weigh 8.7874 g KH2PO4 previously
dried 2 h at 105C. Quantitatively transfer to 1 L volumetric
flask and add ca 750 mL H2O to dissolve. Dilute to volume
with H2O. Store in refrigerator. (2) Working standard solution.0.1 mg P/mL. Dilute 50 mL stock standard solution
with H2O to 1 L. Store in refrigerator. Prepare fresh on day of
analysis.

D. Preparation of Test Solution

Note: For liquid formulae, shake container before weighing.
Accurately weigh amount of test portion to contain ca
4.0 mg P into ashing dish and evaporate to dryness on hot plate
or steam bath. Ignite in muffle furnace at maximum temperature of 600C until free of C (34 h). Cool; add 40 mL HCl
solution, C(a), and several drops of HNO3; and bring to boil on
hot plate. Cool, transfer quantitatively to 100 mL volumetric
flask, and dilute to volume with H2O.

E. Determination
Transfer portions of 0.0, 5.0, 8.0, 10.0, and 15.0 mL working
standard solution to respective 100 mL volumetric flasks.
These represent 0.0, 0.5, 0.8, 1.0, and 1.5 mg P. Pipet 20.0 mL
test solution into each 100 mL volumetric flask. To each standard and test flask, add 20.0 mL molybdovanadate reagent, dilute to volume with H2O, and mix well. Let flasks stand 10 min
for complete color development.
Determine absorbance of standards and sample in 1 cm cells
at maximum near 400 nm. Use 0.0 mg standard to zero spectrophotometer. Use linear regression of standard absorbance vs
mg P of standards to determine mg P for each sample.

F. Calculations
Caution: See Appendix B: safety notes on safe handling of
acids. Dispose of waste solvents in an appropriate manner compatible with applicable environmental rules and regulations.

Calculate content of phosphorus in test sample as follows:

P, mg/L = mg P 5000 test sample density/g sample

Test sample density should be ca 1.03 g/mL for RTF formula.

Ref.: J. Assoc. Off. Anal. Chem. 69, 777(1986); J. AOAC Int.
80, 834844(1997)
Results and Discussion
Results for Ca, Mg, Zn, Cu, Fe, Mn, K, Na, and P are summarized in Tables 19, respectively. RTF liquid soy formulae
and soy powders with similar concentrations of analyte were
pooled together as Youden closely matched pairs for estimates of within-laboratory variability (RSDr). Despite a different technique in estimating repeatability and reproducibility
(RSDR) from that used in the original study, estimates of these
parameters can be compared. Use of results from different-day
analysis on the same RTF milk-based formulae in the original
study produced estimates of repeatability and reproducibility
close to those determined in this study. Averaged estimates of
repeatability and reproducibility from the original collaborative
study are listed with the averaged estimates from this study in
Table 10. Exceptions are for Ca and P, which had twice the repeatability and reproducibility estimates as in the original
study.
Calcium results with the highest variation had the poorest
HORRAT ratios (ratio of relative standard deviation among
laboratories/relative standard deviation expected, extrapolated
from previous studies for analytes at those particular levels).
The resulting relative standard deviations estimating betweenlaboratory variation were quite high, and for 2 test samples the
HORRAT ratio was more than twice what would be expected
at that concentration of analyte. However, no outlier among the
Ca results was identified by the Cochran and Grubbs tests for
outliers.
The sum ranking test proposed by Youden and Steiner (6)
identifies laboratories with consistently high or consistently
low results for a significant number of the test samples analyzed. With the sum ranking test applied, 2 laboratories would
rank significantly low and one laboratory would rank significantly high. Eliminating the high ranking laboratory results and
the low ranking laboratory results for each test sample (see bottom of Table 1) results in relative standard deviations for Ca
between laboratories that are closer to the average reproducibility estimate of the original study as listed in Table 10.
One collaborative study for Ca, Mg, and P in cheese (7)
identified the potential for Ca contamination as a leading factor
in between-laboratory variation. In this study, results from
2 laboratories ranked consistently low compared with those reported by other laboratories. No blanks with significant levels
of Ca were reported, indicating that Ca contamination was not
a problem.
One of the low-ranking laboratories weighed a larger
amount of test portion and exceeded the 50 g limit recommended by the method for sample 6. The other low-ranking
laboratory used different amounts of sample for determining
minerals by Method 986.24. This method specifies that liquid
formula must be adequately mixed before each weighing to
prevent calcium phosphate from settling. The poorest RSDR for
Ca was for the 32 fl. oz. can, sample 6. This variation would be

expected for a sample in which 4 times the amount of calcium

phosphate could settle to the bottom of the container. The poor
RSDR also may be due to greater can-to-can variation. This is
indicated by the high RSDR for P for sample 6, which increases
to 11% from <4% for other test samples.
The IUPAC protocol was used to identify outliers. Although
more than the required 8 laboratories began the collaborative
study, only 8 laboratories, including the authors, completed
the study. In Table 9, which shows results for determination of
P, only 7 laboratories reported results. For whey powder sample 3, removal of the Grubbs outlier would have increased the
outlier removal rate above the standard 2/9 laboratories. In accordance with harmonization guidelines, the outlier was included in the reproducibility estimate. Table 9 also shows estimates without the outlier.
Recoveries from spiked sample are listed in Table 11. Most
recoveries are within the 90110% range, and results indicate
the method is accurate for soy-based, whey-based, and enteral
formulae. For all elements excluding Ca, the HORRAT ratio
was <1 for most test samples, indicating that between-laboratory variation was slightly less than what would be expected for
analytes at these concentrations. As shown in Table 10, reproducibilty estimates are close to those of the original study. In
the case of liquid formula, it may be essential to include directions to shake the container before weighing to ensure complete
mixing, as stated in AOAC Official Method 984.27, Calcium,
Copper, Iron, Magnesium, Manganese, Phosphorus, Potassium, Sodium, and Zinc in Infant Formula, Inductively Coupled Plasma Emission Spectroscopic Method.
Recommendation
On the basis of the results of this study, it is recommended
that AOAC Official Methods 985.35 and 986.24 be extended
to include all types of infant formulae and enteral products.
Acknowledgments
I thank the following collaborators:
Robert Allen, Covance Laboratories, Madison, WI
Anjum Choudhry, U.S. Food and Drug Administration,
Atlanta Center for Nutrient Analysis, Atlanta, GA
Randy Fleener, Silliker Laboratories of Illinois, Chicago
Heights, IL
Nancy Miller-Ihli and F. Ella Greene, U.S. Department of
Agriculture, Nutrient Composition Laboratory, Beltsville, MD
Carol Johnson, Lancaster Laboratories, Lancaster, PA
Rochelle Powell, Woodson-Tenent Laboratories, Inc.,
Memphis, TN
Wai-Leung Yip, U.S. Food and Drug Administration,
North Eastern Regional Laboratory, Brooklyn, NY
I also thank Richard H. Albert and Richard F. Newell, Division of Mathematics, Office of Scientific Support, U.S. Food
and Drug Administration, for statistical analysis of the data. I
also acknowledge the support of Martin P. Bueno, former General Referee for Infant Formula and Medical Diets.

References
(1) Tanner, J.T., & Barnett, S.A. (1985) J. Assoc. Off. Anal.
Chem. 68, 514522
(2) Tanner, J.T., & Barnett, S.A. (1986) J. Assoc. Off. Anal.
Chem. 69, 777785
(3) Official Methods of Analysis (1990) 15th Ed., AOAC, Arlington, VA, sec. 985.35

(4) Official Methods of Analysis (1990) 15th Ed., AOAC, Arlington, VA, sec. 986.24
(5) Horwitz, W. (1988) Pure Appl. Chem. 60, 855864
(6) Youden, W.J., & Steiner, E.H. (1975) Statistical Manual of
the AOAC, AOAC, Arlington, VA
(7) Pollman, R.M. (1991) J. Assoc. Off. Anal. Chem. 74, 2731

Table 985.35A. Method performance for determination of minerals in infant formula by atomic absorption
spectrophotometrya
Infant formula

Mean, g/g

sr

RSDr, %

sR

RSDR, %

rb

Rc

Calcium
Enteral 7
d
RTF soy 2
e
RTF soy (4 and 6)
e
Soy powder (1 and 5)
Whey powder

660
808
660
5270
3510

36
320

70
81
73
482
194

5.4
6.1

11
10
11
9.2
5.5

100.8
896.0

196.0
226.8
204.4
1349.6
543.2

Magnesium
Enteral 7
e,f
RTF soy (2 and 6)
RTF soy 4
e
Soy powder (1 and 5)
Whey powder 3

208
69.9
101
454
367

3.1
15.8

12.7
3.7
3.8
24.1
17.6

4.4
3.5

6.8
5.3
3.8
5.3
4.8

8.7
44.2

35.6
10.4
10.6
67.5
49.3

Zinc
Enteral 7
RTF soy 2
e
RTF soy (4 and 6)
e
Soy powder (1 and 5)
Whey powder 3

11.0
7.50
6.45
44.3
48.4

0.43
0.87

0.89
0.52
0.51
3.2
3.2

6.7
2.0

8.0
6.9
7.9
7.3
6.6

1.20
2.44

2.49
1.46
1.43
8.96
8.96

Copper
Enteral 7
e
RTF soy (2 and 4)
RTF soy 6
e
Soy powder (1 and 5)
Whey powder 3

1.02
0.80
0.56
3.90
4.76

0.056
0.19

0.12
0.106
0.06
0.30
0.23

7.0
4.8

12
13
11
7.6
4.8

0.157
0.53

0.34
0.30
0.17
0.84
0.64

Iron
Enteral 7
e
RTF soy (2 and 4)
RTF soy 6
e
Soy powder (1 and 5)
Whey powder 3

10.1
12.8
13.5
89.6
95

0.53
5.6

0.6
0.53
0.56
7.6
4.5

4.1
6.2

5.9
4.1
4.1
8.4
4.8

1.48
15.7

1.68
1.48
1.57
21.28
12.6

Manganese
Enteral 7
e
RTF soy (2 and 6)
RFT soy 4
e
Soy powder (1 and 5)
Whey powder 3

1.67
0.305
0.408
2.87
0.87

0.015
0.32

0.10
0.026
0.056
0.40
0.093

4.8
11.0

6.1
8
14
14
11

0.042
0.896

0.28
0.073
0.157
1.12
0.26

Potassium
Enteral 7
RTF soy 2
e,f
RTF soy (4 and 6)
e
Soy powder (1 and 5)
Whey powder 3

1340
1050
840
6390
5080

14
128

85
28
18
356
153

2.0
2.0

6.8
2.7
2.1
5.6
3.0

39.2
358.4

238
78.4
50.4
996.8
428.4

Table 985.35A. (continued)

Infant formula

Mean, g/g

sr

sR

RSDr, %

RSDR, %

rb

Rc

Sodium
Enteral 7
RTF soy 2
e,f
RTF soy (4 and 6)
e
Soy powder (1 and 5)
Whey powder 3
a
b
c
d
e
f

544
312
238
2090
1290

13.7
73

20
9.4
13.7
90
87

5.8
3.5

3.7
3.0
5.8
4.3
6.7

38.36
204.4

Based on collaborative study results of modified method. Data received from 8 laboratories were used in statistical analysis.
r = 2.8 sr.
R = 2.8 sR.
RTF = ready-to-feed.
Closely matched (Youden) pairs.
Based on results from 7 laboratories (excluding Grubbs outliers).

56
26.32
38.36
252
243.6

Table 985.35B. Wavelength and flame parameters for AAS determination of minerals in infant formula
Element
Ca (CAS-7440-70-2)
Cu (CAS-7440-50-8)
Fe (CAS-7439-89-6)
K (CAS-7440-09-7)
Na (CAS-7440-23-5)
Mg (CAS-7439-95-4)
Mn (CAS-7439-96-5)
Zn (CAS-7440-66-6)

Wavelength, nm

Instrument setting
UV/vis range

Flame type

422.7
324.7
248.3
766.5 or 769.9
589.0
285.2
279.5
213.9

211-vis
325-UV
248-vis
383-vis
295-vis
285-UV
279-UV
214-UV

Reducing air-C2H2
Oxidizing air-C2H2
Oxidizing air-C2H2
Oxidizing air-C2H2
Oxidizing air-C2H2
Oxidizing air-C2H2
Oxidizing air-C2H2
Oxidizing air-C2H2

Table 986.24. Methods performance for determination of phosphorus in infant formula by spectrophotometrya
Infant formula
Enteral 7
d
e
RTF soy (2 and 4)
RTF soy
e
Soy powder (1 and 5)
f
Whey powder 3
a
b
c
d
e
f

Mean, g/g
541
526
447
3908
2350

sr

15.6
94

sR
18.8
19.3
49
133
739

RSDr, %

3.0
2.4

RSDR, %
3.5
3.7
11
3.4
31

rb

43.7
263.2

Based on collaborative study results of modified method. Data received from 7 laboratories were used in statistical analysis.
r = 2.8 sr.
R = 2.8 sR.
RTF = ready-to-feed.
Closely matched (Youden) pairs.
Calculated including Grubbs outlier.

Rc
52.6
54.0
137.2
372.4
2069.2

Table 1. Results for determination of calcium (g/g) by AOAC Official Method 985.35 in soy, whey, and enteral
formulae
Youden pair

Youden pair

Enteral
sample 7

RTF soy
sample 2

RTF soy
sample 4a

RTF soy
sample 6a

Soy powder
sample 1b

Soy powder
sample 5b

Whey powder
sample 3

1
2
3
4
5
6
c
7
8

656
628
545
679
641
708
789
634

685
863
750
846
786
829
947
755

577
703
644
705
693
745
758
658

519
741
524
620
605
681
736
650

4080
5070
4860
5500
5060
5710
5490
4860

5400
5520
4700
6080
5440
5700
5860
4990

3380
3410
3370
3520
3500
3620
3940
3370

Mean
sr
RSDr, %
sR
RSDR, %
c
RSDR, %

660

808

Laboratory
c

a
b
c

70
11
8.7

81
10
6.0

Youden closely matched pairs.

Youden closely matched pairs.
If sum ranking outlier were identified and excluded.

660a
36a
5.4a
73a
11a
8.7

5270b
320b
6.1b
482b
9.2b
8.1

3510

194
5.5
2.9

Table 2. Results for determination of magnesium (

g/g) by AOAC Official Method 985.35 in soy, whey, and enteral
formulae
Youden pair
Enteral
sample 7

RTF soy
sample 2a

1
2
3
4
5
6
7
8

215
202
194
207
202
236
204
204

62.0
58.8
57.0
61.5
56.2
60.0
71.8
60.5

Mean
sr
RSDr, %
sR
RSDR, %

208

Laboratory

a
b
c
d
e

12.7
6.8

Youden closely matched pairs.

Youden closely matched pairs.
Grubbs outlier.
7 laboratories.
Grubbs outlier included.

Youden pair

RTF soy
sample 6a
c

122c
76.3
80.0
80.0
77.4
78.4
81.0
79.0

69.9a,d, 72.6e
3.1a,d, 11e
4.4a,d, 15e
3.7a,d, 12e
5.3a,d, 16e

RTF soy
sample 4

Soy powder
sample 1b

Soy powder
sample 5b

Whey powder
sample 3

99
94
101
104
101
105
106
101

518
493
520
531
519
580
570
545

364
342
378
383
368
368
400
390

360
340
370
363
353
381
398
372

101

3.8
3.8

454b
15.8b
b
3.5b
24.1b
5.3b

367

17.6
4.8

Table 3. Results for determination of zinc (

g/g) by AOAC Official Method 985.35 in soy, whey, and enteral formulae
Youden pair

Youden pair

Enteral
sample 7

RTF soy
sample 2

RTF soy
sample 4a

RTF soy
sample 6a

Soy powder
sample 1b

Soy powder
sample 5b

Whey powder
sample 3

1
2
3
4
5
6
7
8

12.8
10.2
11.0
9.8
11.0
10.9
11.4
11.1

7.40
7.46
8.00
6.67
7.00
8.31
7.50
7.65

5.92
5.96
7.00
5.63
6.00
6.13
6.70
6.45

7.70
6.81
7.00
5.96
6.00
6.46
6.70
6.72

47.6
40.1
41.0
38.8
43.0
39.8
46.1
45.3

50.7
42.9
44.0
42.7
45.0
44.5
47.5
50.2

53.2
47.3
45.0
43.5
48.0
47.5
51.5
50.7

Mean
sr
RSDr, %
sR
RSDR, %

11.0

7.50

Laboratory

a
b

0.89
8.0

Youden closely matched pairs.

Youden closely matched pairs.

0.52
6.9

6.45a
0.43a
6.7a
0.51a
7.9a

44.3b
0.87b
2.0b
3.2b
7.3b

48.4

3.2
6.6

Table 4. Results for determination of copper (

g/g) by AOAC Official Method 985.35 in soy, whey, and enteral
formulae
Youden pair

Youden pair

Enteral
sample 7

RTF soy
sample 2a

RTF soy
sample 4a

RTF soy
sample 6

Soy powder
sample 1b

Soy powder
sample 5b

Whey powder
sample 3

1
2
3
4
5
6
7
8

1.17
1.10
1.00
0.79
1.10
1.04
0.94
1.02

1.04
0.78
1.00
0.80
0.80
0.87
0.83
0.90

0.85
0.53
0.80
0.62
0.80
0.72
0.66
0.83

0.65
0.56
0.60
0.48
0.60
0.51
0.52
0.52

4.16
3.33
3.70
3.42
4.00
3.59
3.60
3.17

4.73
3.95
4.20
4.28
4.00
4.22
4.06
3.99

5.11
4.46
4.80
4.81
5.00
4.75
4.46
4.65

Mean
sr
RSDr, %
sR
RSDR, %

1.02

Laboratory

a
b

0.12
12

Youden closely matched pairs.

Youden closely matched pairs.

0.80a
0.056a
7.0a
0.106a
13a

0.56

0.06
11

3.90b
0.19b
4.8b
0.30b
7.6b

4.76

0.23
4.8

Table 5. Results for determination of iron (

g/g) by AOAC Official Method 985.35 in soy, whey, and enteral formulae
Youden pair

Youden pair

Enteral
sample 7

RTF soy
sample 2a

RTF soy
sample 4a

RTF soy
sample 6

Soy powder
sample 1b

Soy powder
sample 5b

Whey powder
sample 3

1
2
3
4
5
6
7
8

10.4
9.8
9.0
9.7
11.0
10.2
10.6
10.1

13.4
12.7
12.0
13.2
12.0
12.8
13.4
13.0

12.2
12.4
13.0
12.9
13.0
13.3
13.1
12.9

13.7
13.9
13.0
13.1
13.0
13.7
14.6
13.2

105
90
89
96
102
98
100
99

78
82
64
76
85
86
90
93

100
91
87
93
99
96
98
98

Mean
sr
RSDr, %
sR
RSDR, %

10.1

Laboratory

a
b

0.6
5.9

Youden closely matched pairs.

Youden closely matched pairs.

12.8a
0.53a
4.1a
0.53a
4.1a

13.5

0.56
4.1

89.6b
5.6b
6.2b
7.6b
8.4b

95

4.5
4.8

Table 6. Results for determination of manganese (

g/g) by AOAC Official Method 985.35 in soy, whey, and enteral
formulae
Youden pair

Youden pair

Enteral
sample 7

RTF soy
sample 2a

RTF soy
sample 6a

RTF soy
sample 4

Soy powder
sample 1b

Soy powder
sample 5b

Whey powder
sample 3

1
2
3
4
5
6
7
8

1.60
1.66
1.70
1.56
1.80
1.79
1.73
1.53

0.327
0.309
0.300
0.330
0.300
0.328
0.360
0.260

0.286
0.305
0.300
0.290
0.300
0.320
0.310
0.250

0.357
0.370
0.400
0.410
0.500
0.454
0.440
0.330

2.37
2.83
1.60
2.44
2.80
2.82
2.82
2.08

3.16
3.05
3.40
3.24
3.70
3.53
3.53
2.59

0.83
0.91
1.00
0.77
0.90
0.84
0.98
0.74

Mean
sr
RSDr, %
sR
RSDR, %

1.67

Laboratory

a
b

0.10
6.1

Youden closely matched pairs.

Youden closely matched pairs.

0.305a
0.015a
4.8a
0.026a
8a

0.408

0.056
14

2.87b
0.32b
11.0b
0.40b
14b

0.87

0.093
11

Table 7. Results for determination of potassium (

g/g) by AOAC Official Method 985.35 in soy, whey, and enteral
formulae
Youden pair

Youden pair

Enteral sample 7

RTF soy
sample 2

RTF soy
sample 4a

RTF soy
sample 6a

Soy powder
sample 1b

Soy powder
sample 5b

Whey powder
sample 3

1
2
3
4
5
6
7
8

1290
1430
1450
1260
1430
1220
1260
1350

1070
1055
1090
1060
1060
1060
1010
1010

861
838
c
968c
864
887
844
876
870

795
829
848
820
842
803
834
800

6410
6081
6050
6730
6900
5800
6640
6200

6340
6550
6220
6630
6950
5860
6600
6290

4870
5010
4970
4980
5230
5160
5330
5120

Mean
sr
RSDr, %
sR
RSDR, %

1340

1050

Laboratory

a
b
c
d
e

85
6.8

Youden closely matched pairs.

Youden closely matched pairs.
Grubbs outlier.
7 laboratories.
Grubbs outlier included.

28
2.7

840a,d, 849a,e
14a,d, 23a,e
2.0a,d, 2.7a,e
18a,d, 32a,e
2.1a,d, 3.8a,e

6390b
128b
2.0b
356b
5.6b

5080

153
3.0

Table 8. Results for determination of sodium (

g/g) by AOAC Official Method 985.35 in soy, whey, and enteral
formulae
Youden pair
Laboratory
1
2
3
4
5
6
7
8
Mean
sr
RSDr, %
sR
RSDR, %
a
b
c
d
e

Youden pair

Enteral
sample 7

RTF soy
sample 2

RTF soy
sample 4a

RTF soy
sample 6a

Soy powder
sample 1b

Soy powder
sample 5b

Whey powder
sample 3

552
551
c
726c
515
554
545
520
572

317
291
323
317
312
311
312
309

224
247
242
232
237
222
231
262

233
221
263
223
237
241
252
238

1950
1640
1770
1720
1770
1680
1730
1790

2440
2280
2310
2470
2540
2430
2430
2480

1280
1154
1340
1250
1270
1240
1420
1390

544d, 567e

312

20d, 67c
3.7d, 12e

Youden closely matched pairs.

Youden closely matched pairs.
Grubbs outlier.
7 laboratories.
Grubbs outlier included.

9.4
3.0

238a
13.7a
5.8a
13.7a
5.8a

2090b
73b
3.5b
90b
4.3b

1290

87
6.7

Table 9. Results for determination of phosphorus (

g/g) by AOAC Official Method 986.24 in soy, whey, and enteral
formulae
Youden pair
Laboratory
1
2
3
4
5
6
7
8
e

Mean
sre
e
RSDr, %
sRe
e
RSDR, %
a
b
c
d
e
f

Youden pair

Enteral
sample 7

RTF soy
sample 2a

RTF soy
sample 4a

RTF soy
sample 6

Soy powder
sample 1b

Soy powder
sample 5b

Whey powder
sample 3

541
538
527
536
580
d d

522
544

491
552
518
528
510

510
516

504
538
528
539
570

525
538

364
488
507
415
480

433
443

4160
3853
3920
3870
4000

4010
4135

4130
3810
3740
3810
3800

3680
3790

2450
2006
c
3980c
1900
2000

2040
2090

541

18.8
3.5

526a
15.6a
3.0a
19.3a
3.7a

447

Youden closely matched pairs.

Youden closely matched pairs.
Grubbs outlier.
, laboratory 6 did not assay for phosphorus.
7 laboratories.
If Grubbs outlier excluded (exclusion would exceed 2/9 IUPAC Guidelines).

49
11

3908b
94b
2.4b
133b
3.4b

2350, 2081f

739, 191f
31, 9f

Table 10. Average repeatability estimates (RSDr)

and reproducibility estimates (RSDR) of original
collaborative study and extension study
Estimate

Original study

Extension study

Calcium
RSDr
RSDR

2.9
5.4

5.8
9.3

Magnesium
RSDr
RSDR

2.3
7.1

3.9
5.2

Zinc
RSDr
RSDR

3.3
5.3

4.4
7.3

Copper
RSDr
RSDR

3.2
11.4

5.9
9.7

Iron
RSDr
RSDR

3.1
16.8

5.2
5.5

Manganese
RSDr
RSDR

6.2
13

6.9
10.4

Potassium
RSDr
RSDR

3.4
6.4

2.0
4.0

Sodium
RSDr
RSDR

3.9
9.2

4.6
4.7

Phosphorus
RSDr
RSDR

1.1
2.0

2.7
10.5

Table 11. Amount of spike and recoveries for spiked replicates

Mineral

Enteral

RTF soy

Whey powder

Soy powder

Ca
Spike, mg/g
Recovery, %

0.36
105

0.6
90, 129

4
113, 113, 98a

4.4
95, 127a

Spike, g/g
Recovery, %

119
93

64
84, 99

300
113, 111, 104a

500
106, 109a

Spike, g/g
Recovery, %

7.5
105, 100

2040
90, 119, 103a

42
122, 115a

Spike, g/g
Recovery, %

0.6
90

0.8
95, 90

24
68, 123, 90a

4
104, 86a

Spike, g/g
Recovery, %

5
89

15
81

50
125, 103, 96a

80
104, 91a

Spike, g/g
Recovery, %

0.8
118

0.2
87, 86

1
119, 105, 101a

2
98, 87a

Mg

Zn
b

Cu

Fe

Mn

K
Spike, mg/g
Recovery, %

0.75
96

0.6
86, 100

5
101, 104, 103a

6
98, 125a

Spike, mg/g
Recovery, %

0.8
88, 110

13
97, 75, 106a

3
98, 95a

0.5
101a

0.3
93, 100

13
108, 101, 102a

4
96a

Na

P
Spike, mg/g
Recovery, %
a
b

In-house determination.
, not added to spiking solution.

Table 12. Comparison of phosphorus results

determined by AOAC Official Method 985.35, atomic
absorption, and by AOAC Official Method 986.24 for
phosphorus
Formula
Soy powder 1
RTF soy 2
Whey powder 3
RTF soy 4
Soy powder 5
RTF soy 6
Enteral 7

Method 985.35
4062 53 (n = 6)
529
2060
540
3810 20 (n = 6)
453
538

Method 986.24
4110
517
2103
536
3730
448
545

4160
516
2070
540
3850
438
544