2011 14

AOAC Official Method 2011.
14
Calcium, Copper, Iron, Magnesium, Manganese,
Potassium, Phosphorus, Sodium, and Zinc
in Fortified Food Products
Microwave Digestion and
Inductively Coupled Plasma-Optical Emission Spectrometry
First Action 2011
Final Action 2013
(Applicable to analysis of calcium, copper, iron, potassium,

magnesium, manganese, phosphorus, sodium, and zinc in fortified
food products.)
Upper limits (mg/kg): Ca (24 000); Cu (210); Fe (850);
K (32 000); Mg (7500); Mn (20); Na (16 000); P (16 000); Zn (320).
Limit of quantitation (LOQ; mg/kg): Ca (150); Cu (2); Fe (10);
K (200); Mg (50); Mn (0.05); Na (100); P (100); Zn (5).
See Tables 2011.14AI for the results of the interlaboratory
study supporting acceptance of the method. [Note: Results shown
in Tables 2011.14AI are slightly different from those shown in
J. AOAC Int. 92, 1484(2009) due to classical statistics applied
instead of robust statistics applied in the tables included in J. AOAC
Int. 95, 177(2012).]
Digestion
A. Principle
Test portion is heated at 200C either with nitric acid in a closedvessel microwave digestion system (MDC) or with a combination
of hydrogen peroxide, nitric acid, and hydrochloric acid in an openvessel microwave digestion system (MDO).
B. Apparatus
Microwave.____Commercial MDC or MDO designed for

laboratory use at 200 20C, up to 600 psi, and controlled
temperature or pressure ramping capability. It is recommended
that vessel design be selected that will withstand the maximum
possible pressure (600 psi) since organic residues of rich-fat or
rich-carbohydrate samples, if not given sufficient time to predigest,
will generate significant pressure during digestion.
Table 2011.14A.
C. Reagents
(a) High-grade water, (18 M).For slurry preparation and/

or dilution.
(b) Nitric acid (HNO3), 65% (w/v).Trace metal grade
throughout.
(c) Hydrochloric acid (HCl), 37% (w/v).Trace metal grade
throughout.
(d) Hydrogen peroxide (H2O2), 97% (w/v).Trace metal grade
throughout.
D. Determination
[Caution: Before using chemicals, refer to the supplier guide

for chemical safety and/or other adequate manuals or safety data
sheets approved by local authorities. Use fume hood and wear full
personal laboratory protective clothing, gloves, and appropriate
eye protection (safety glasses) when using glassware and preparing
standards or test portions with acid solutions.]
(a) Sample preparation.(1) Test sample preparation.
Homogenize a representative sample by grinding as finely as
possible and/or by preparing a slurry with H2O. For example:
Infant cereals and fortified milk powders, preheat water at 50C.
Prepare the slurry by weighing 10.0 0.1 g test sample and place
into a 100 mL Erlenmeyer flask; add 90.0 0.1 g H2O. Mix well
with stopper.
(2) Test portion preparation.Accurately weigh 0.50 0.01 g
test portion or sample mass on a dry weight basis in the prepared
slurry to MDC vessel (1.00 0.01 g into a 100 mL volumetric flask
for MDO). [Note: An optimal analytical test portion mass of 0.5 g
(1.0 g for MDO) is based on an empirical maximum energy release
by the food of 3 kcal and 90110% recovery.]
Line the MDC vessel walls or Pasteur pipet with weighing paper
during sample transfer to keep sample from adhering to sides of
vessel or use a Pasteur pipet to transfer liquid samples. (Weigh fluid
samples or test portion from slurry test sample directly after mixing.)
(Note: Remove weighing paper from sample prior to next step.)
Carefully add 5.0 0.1 mL HNO3 into MDC/MDO vessel (and
then 5 mL H2O2 only into MDO vessel).
Loosely cap MDC vessel without sealing.
Interlaboratory study results for calcium

Mean, mg/kg
Rec., %
Sra
RSDrb, %
SRc, ng/g
RSDRd, %
HorRat
Chocolate milk powder
9732.17
99
152.10
1.56
264.03
2.71
0.68
NSg
Dietetic milk powder 2
5678.58
98
75.93
1.34
184.11
3.24
0.74
NS
Infant cereal
5983.54
100
210.58
3.52
251.67
4.21
0.97
NS
Peanut butter NIST SRM 2387
409.90
(411)h
100
13.37
3.26
28.20
6.88
1.06
NS
Wheat gluten NIST RM 8418
367.56
(369)h
100
3.51
0.95
12.90
3.51
0.53
2, 4C
Matrix
Sr = Standard deviation for repeatability (within laboratory).
RSDr = Relative standard deviation for repeatability.
SR = Standard deviation for reproducibility (among laboratories).
RSDR = Relative standard deviation for reproducibility.
Reporting laboratories minus outliers.
C = Cochran outlier; SG = Single Grubbs outlier; DG = Double Grubbs outlier.
NS = Not significant.
Value in parentheses = Certified or reference values.
No. of labse Outlier labsf
2013 AOAC INTERNATIONAL
Table 2011.14B.
Interlaboratory study results for copper
Matrix
Mean, mg/kg
Sra
RSDrb, %
SRc, ng/g
RSDRd, %
HorRat
0.09
1.57
0.29
4.97
0.41
NSg
Rec., %
5.92
NA
6.19
101
0.19
3.06
0.38
6.12
0.50
NS
Infant cereal
2.01
NA
0.10
4.98
0.24
11.72
0.81
NS
4.98
(4.93)i
101
0.19
3.84
0.35
7.08
0.56
NS
5.64
(5.94)i
95
0.13
2.27
0.27
4.77
0.39
NS
NA = Not available.
Predigest for at least 10 min at room temperature or until

vigorous foaming subsides.
Close MDC vessels and distribute onto microwave carousel to
ensure uniform microwave power application on all samples.
(3) Food-grade salt sample preparation.Weigh 0.20 0.01 g
food-grade salt into a 100 mL volumetric flask. Add deionized
water and 10 mL HNO3. Dissolve salt and dilute to volume with
deionized water.
(b) Test portion digestion.[Caution: Application of
microwave digestion systems involves hot pressurized acid
solutions and concentrated acids. Use fume hood and wear full
personal laboratory protective clothing, gloves and appropriate eye
protection (safety glasses) when using glassware and preparing
standards or samples with acid solutions. Follow manufacturers
directions for safety risk and safety environment of microwave
Table 2011.14C.
systems. Never remove hot vessels from microwave; wait until

they are near room temperature. Keep microwave door closed
while vessels are hot. The door is the primary safety device if a
vessel vents.]
(1) Sample digestion.With power setting appropriate to MDC
(maximum power of 1600 W) and MDO models (maximum power
of 600 W), and number of vessels used, heat MDO vessels at 200
20C for 20 min or ramp MDC temperature from ambient to 200
20C in 15 min and hold at 200C for 25 min. (Note: Yellow vapors
will be emitted during the hydrolysis in MDO vessels.)
Carefully remove the MDO vessels. Allow the vessels to cool
down to room temperature.
Add 5 mL HCl 35% (w/v) into MDO vessels and heat MDO
vessels at 200 20C for 20 min.
Interlaboratory study results for iron

Mean, mg/kg
Rec., %
Sra
RSDrb, %
SRc, ng/g
RSDRd, %
HorRat
164.77
97
6.72
4.08
11.25
6.83
0.92
NSg
83.11
102
2.51
0.02
4.62
5.56
0.68
NS
Infant cereal
75.18
94
1.36
1.81
3.32
4.42
0.53
NS
15.65
(16.4)h
95
0.65
4.17
1.95
12.45
1.18
6C
49.29
(54.3)h
91
0.93
1.89
1.54
3.12
0.35
2C
Matrix
Table 2011.14D.
Interlaboratory study results for potassium

Mean, mg/kg
Rec., %
Sra
RSDrb, %
SRc, ng/g
RSDRd, %
HorRat
8996.74
99
135.82
1.51
208.98
2.32
0.57
NSg
6443.79
100
65.17
1.01
130.04
2.02
0.47
NS
Matrix
Infant cereal
6438.19
99
119.83
1.86
135.78
2.11
0.49
6SG
6200.24
(6070)h
102
148.84
2.40
187.59
3.03
0.70
NS
411.08
(472)h
87
10.19
2.48
31.31
7.62
1.18
NS

a
Cool vessels to room temperature before venting (MDC vessels).

Transfer the MDC digests to 50 mL (100 mL for MDO) volumetric
flasks. Dilute to volume with H2O and mix. (Note: A digestion is judged
complete when clear to yellow analytical solutions are produced.)
Filter the digested solution using an ashless filter paper for turbid
samples containing fat. Discard the first 20 mL filtrate and collect
the remaining filtrate for analysis. [Note: Membrane disc filters (0.45
m) are not recommended as they are generally not metal-free.]
Transfer to polyethylene containers within 2 h.
Dilute the samples that are found to be above the standard curve
range or have total content of minerals higher than 1000 mg/L
with H2O. [Note: Final dilutions require the addition of appropriate
amounts of HNO3 for MDC digests (or appropriate amounts of
combined acids for MDO) to maintain the proportion of 10% (v/v)
HNO3 (15%, v/v, combined acids and hydrogen peroxide) in the
final solution to be analyzed.]
Table 2011.14E.
(2) Digestion vessel decontamination.Decontaminate the

used MDC vessels with 5 mL HNO3 according to a program close
to that in b(1).
Wash the MDO glass volumetric flasks in a laboratory washing
machine or soak overnight in a water bath with HNO3 (30%, v/v).
Rinse with H2O to avoid or to minimize risk of iron contamination.
Dry MDC vessels at 60C in drying oven for 1 h.
Detection
E. Principle
Digested test solution or an appropriate dilution is presented

to the inductively coupled plasma-optical emission spectrometry
(ICP-OES) instrument. The instrument is calibrated with acid(s)
matched standard calibrant solutions. An ionization buffer (cesium)
and internal standard(s) (chromium, indium, strontium, and
yttrium) are used with or without appropriate in-line addition to
Interlaboratory study results for magnesium

Mean, mg/kg
Rec., %
Sra
RSDrb, %
SRc, ng/g
RSDRd, %
HorRat
1746.74
98
38.46
2.20
51.03
2.92
0.56
2SG
439.31
99
5.57
1.27
13.37
3.04
0.48
NSg
Matrix
Infant cereal
859.48
98
33.62
3.91
35.38
4.12
0.71
2, 6DG
1714.02
(1680)h
102
56.83
3.32
73.68
4.30
0.82
2SG
482.05
(510)h
95
8.52
1.77
16.53
3.43
0.54
NS
Table 2011.14F.
Interlaboratory study results for manganese
Matrix
Mean, mg/kg
Rec., %
h
Sra
RSDrb, %
SRc, ng/g
RSDRd, %
HorRat
0.14
1.88
0.67
8.82
0.75
NSg
7.54
NA
0.40
99
0.01
2.56
0.0197
4.89
0.27
2C
Infant cereal
16.74
NA
1.14
6.79
1.44
8.61
0.82
NS
15.85
(16.0)i
99
0.45
2.86
1.32
8.31
0.79
NS
12.87
(14.3)1
90
0.23
1.75
1.13
8.75
0.80
NS
NA = Not available.
minimize easily ionizable element (EIE) effects and to correct for

physical and chemical interferences.
F. Instrumentation
(Note: ICP-OES equipments must be used by qualified laboratory

personnel who are familiar with the operation and maintenance of
analytical systems and must follow manufacturers directions for
safety risk, safety environment, and use of instruments.)
(a) ICP-OES spectrometer.Instrument with axial, radial,
or dual view grating configurations and autosampler, capable of
determining multiple wavelengths for each element of interest
with the required sensitivity. A 3-channel peristaltic pump with
or without appropriate in-line addition system (e.g., T connector)
are linked between the peristaltic pump and nebulizer to avoid
having to manually add ionization buffer and internal standards
Table 2011.14G.
to each sample solution. A thermostated cyclonic spray chamber

equipped with a micro-concentric nebulizer or other components
designed to optimize aerosol and maximize precision was used.
Follow manufacturers recommendations. Select peristaltic pump
rotation speed, sample, and internal standard pump tubes of similar
size to maximize mixing accuracy while maintaining the required
detection levels.
Ionization buffer (cesium chloride) is combined with the internal
standard solution to compensate EIEs effects (e.g., K, Na, and Ca)
in the plasma since certain food materials can contain substantial
concentrations of these elements. This provides a significant source
of electrons in the plasma. The presence of ionization buffer in
all samples and standards will minimize the effects of varying
concentrations of EIEs in the sample. The solution presented to
the nebulizer contains a maximum of 5000 mg/kg cesium for high-
Interlaboratory study results for sodium

Mean, mg/kg
Rec., %
Sra
RSDrb, %
SRc, ng/g
RSDRd, %
HorRat
1927.84
97
43.30
2.25
51.07
2.65
0.52
NSg
1769.19
96
18.64
1.05
33.39
1.89
0.36
NS
Matrix
Infant cereal
992.41
95
37.67
3.80
39.72
4.00
0.71
6SG
4924.12
(4890)h
101
158.61
3.22
158.61
3.22
0.72
NS
1346.82
(1420)h
95
19.56
1.45
26.85
1.99
0.37
6SG
Table 2011.14H.
Interlaboratory study results for phosphorus

Mean, mg/kg
Rec., %
Sra
RSDrb, %
SRc, ng/g
RSDRd, %
HorRat
4138.12
101
84.53
2.04
115.22
2.78
0.61
NSg
3729.50
100
46.37
1.24
101.72
2.73
0.59
NS
Infant cereal
4341.81
101
60.56
1.39
111.79
2.57
0.57
NS
3444.06
(3378)h
102
98.10
2.85
111.97
3.25
0.69
NS
2050.02
(2190)h
94
37.86
1.85
67.15
3.28
0.65
NS
Matrix
salted food raw materials (e.g., culinary products or tastemakers)

and a minimum of 500 mg/kg cesium (for main food samples);
20 mg/kg indium and 5 mg/kg strontium, yttrium, and chromium;
less than half of each element concentration of the higher working
standard Std6 and less than 0.5 g/kg total dissolved minerals.
Power settings and nebulizer gas flow must be optimized to ensure
that the Mg280.271:Mg285.213 ratio (Mermet principle of robust
plasma) demonstrates robust operating conditions in accordance
with the ratio established by the instrument manufacturer. At least
three replicate readings of the same sample are performed, with
relatively long integration times to minimize noise.
(b) ICP wavelengths.____A number of recommended and
alternative wavelengths may be used for the nine elements to
be determined and internal standards. As a minimum, select one
recommended and one alternative wavelength for each element
Table 2011.14I.
corrected by one recommended wavelength for appropriate internal

standard. All responses for both recommended and alternative
wavelengths for each element are corrected using only one internal
standard line. The following is a list of wavelengths for each
element (and its appropriate internal standard) in priority order that
have been found acceptable for main foodstuffs: Wavelength (nm):
Ca: 317.933 (In: 303.936); Cu: 324.754 (In: 303.936); Fe: 259.94
(Sr: 338.071); K: 766.491 (Sr: 460.733); Mg: 285.213 (In: 303.936);
Mn: 257.610 (Sr: 338.071); Na: 589.592 (Sr: 460.733); P: 213.618
(In: 303.936); Zn: 213.857 (Sr: 338.071).
Other wavelengths that are acceptable for both elements and
internal standards could be used as confirmatory analytical lines
or alternative wavelengths as certain recommended lines may not
be available on some ICP-OES systems: Wavelength (nm): Ca:
317.933 (Y: 371.028); Cu: 324.754 (Y: 371.028), 327.395 (In:
Interlaboratory study results for zinc
Matrix
Mean, mg/kg
Rec., %
h
Sra
RSDrb, %
SRc, ng/g
RSDRd, %
HorRat
18.06
NA
0.396
2.19
0.95
5.24
0.51
5SG
53.65
105
1.43
2.66
2.36
4.40
0.50
NS
Infant cereal
36.51
NA
0.92
2.25
1.27
3.44
0.37
NS
26.64
(26.3)i
101
1.06
3.96
1.78
6.69
0.69
NS
49.30
(53.8)i
92
0.96
1.95
3.19
6.46
0.73
NS
NA = Not available.
303.936 or Y: 371.028); Fe: 259.94 (Y: 371.028 or Cr: 283.563);

Mg: 285.213 (Y: 371.028), 279.028 (In: 303.936); Mn: 257.610 (Sr:
460.733 or Y: 371.028); P: 178.222 (Sr: 460.733 or Y: 371.028);
Zn: 213.857 (Sr: 460.733 or Y: 371.028).
Other lines of appropriate sensitivity, free of interferences or
corrected for interferences in specific matrixes, may be just as
acceptable.
Table 2011.14J. Suggested concentration of the six standard

solutions, mg/kg
Element
Std0
Std1
Std2
Std3
Std4
Std5
Std6
Ca
7.5
15
30
75
150
225
Cu
0.05
0.1
0.2
0.5
1.0
1.5
Fe
0.25
0.5
1.0
2.5
7.5
G. Reagents
10
20
40
100
200
300
(a) Ionization buffer/internal standard solution.Weigh 1.27 g

cesium chloride into a 1000 mL acid-washed volumetric flask
[Note: This cesium 0.1% (w/v) solution was tested as the minimal
recommended concentration required for element analysis in most
food matrixes. Cs solution 1% (w/v) is recommended if an element
is present at low concentration in high-salted food raw materials,
e.g., culinary products or tastemakers, or if it is analyzed as an
impurity in food-grade salts.] Add 40 mL indium 1000 mg/kg and
10 mL each of strontium, yttrium, and chromium 1000 mg/kg
stock standard solutions, as internal standards. Add 10 mL HNO3.
Dilute to volume with H2O, mix, and transfer to an acid-washed
polyethylene bottle. (Note: Reagent concentrations assume the use
of same pump tubing internal diameter for both internal standard/
ionization buffer and sample pump tubes using automatic addition.)
(b) Stock standard solution.Working standards can be prepared
from ICP-grade individual element 1000 mg/kg (e.g., for copper,
iron, manganese, and zinc) and 10 000 mg/kg (e.g., for calcium,
magnesium, phosphorus, potassium, and sodium) commercial
stock standard solutions. However, it is also acceptable to use
commercially prepared, custom blended stock standard mixtures
containing all of the nine elements at appropriate concentrations. A
number of companies provide this stock standard service.
(c) Intermediate stock solution.(Suggested composition of the
intermediate stock standard solution, in mg/kg: Ca = 1500; Cu =
10; Fe = 50; K = 2000; Mg = 500; Mn = 0.25; Na = 1000; P =
1000; Zn = 20.) Add into a 500 mL volumetric flask, 75 mL calcium
10 000 mg/kg, 5 mL copper 1000 mg/kg, 25 mL iron 1000 mg/kg,
100 mL potassium, 25 mL magnesium 10 000 mg/kg, 0.125 mL
manganese 1000 mg/kg, 50 mL sodium 10 000 mg/kg, 50 mL
phosphorus 10 000 mg/kg, and 10 mL zinc 1000 mg/kg. Add 10 mL
HNO3 and dilute to volume with H2O.
(d) Working standard solutions.Standards prepared from
intermediate stock standard solution are designed to have the same
acid concentration as digested test solutions (i.e., 10%, v/v, HNO3)
for MDC or 15% (v/v) for MDO using combined acids (HNO3,
H2O2, and HCl).
(1) Std6.Pipet 15.0 mL intermediate stock standard solution
into a 100 mL acid-washed volumetric flask. Add 10 mL HNO3
(MDC) or 15 mL combined acids (MDO), dilute to volume with
H2O, mix, and transfer to acid-washed polyethylene bottle.
(2) Std5.Pipet 10 mL intermediate stock standard solution
Mg
2.5
10
25
50
75
Mn
Na
10
20
50
100
150
10
20
50
100
150
Zn
0.1
0.2
0.4
1.0
2.0
3.0
0.00125 0.0025 0.005 0.0125 0.025 0.0375

(7) Blank.Add 10 mL HNO3 (MDC) or 15 mL combined
acids (MDO) into a 100 mL acid-washed volumetric flask, dilute to
volume with H2O, mix, and transfer to acid-washed polyethylene
bottle. All calibration solutions when made are stable for 1 week in
glass volumetric flasks.
(e) Sampler wash solution, 10% HNO3 (v/v).Dilute 100 mL
trace metal-grade HNO3 to 1000 mL with H2O.
H. Determination
Make a calibration curve using either weighted linear or

quadratic regression with correlation coefficients of at least
0.9999 from seven standards prepared from intermediate standard
solution, including a blank (Std0) and six suggested concentrations
of the standard solution (Std1Std6) shown in Table 2011.14J and
expressed in mg/kg.
Analyze test solutions using an ICP-OES instrument calibrated with
the working standard solutions. Insert a working standard or other
suitable quality control solution every 10 test portions to monitor for
instrument drift. The inclusion of a digestion blank, a sample duplicate,
and known reference materials is highly encouraged.
I. Calculations
The concentration (C) of each element, in mg/kg, is calculated

as follows:
C
aVF
m
where C = concentration in the test portion sample (mg/kg); a

= concentration (mg/L) of the element in the digest solution as
obtained from instrument; V = volume (mL) of the test solution
after being made up (i.e., 50 mL for MDC and 100 mL for MDO);
F = dilution factor of the test solution; m = weight of the test
portion (g).
Reference: J. AOAC Int. 95, 177(2012)

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AOAC Official Method 2011.

(Applicable to analysis of calcium, copper, iron, potassium,

Microwave.____Commercial MDC or MDO designed for

(a) High-grade water, (18 M).For slurry preparation and/

[Caution: Before using chemicals, refer to the supplier guide

Interlaboratory study results for calcium

Chocolate milk powder

Dietetic milk powder 2

Peanut butter NIST SRM 2387

Wheat gluten NIST RM 8418

Sr = Standard deviation for repeatability (within laboratory).

RSDr = Relative standard deviation for repeatability.

SR = Standard deviation for reproducibility (among laboratories).

RSDR = Relative standard deviation for reproducibility.

Reporting laboratories minus outliers.

C = Cochran outlier; SG = Single Grubbs outlier; DG = Double Grubbs outlier.

Value in parentheses = Certified or reference values.

No. of labse Outlier labsf

2013 AOAC INTERNATIONAL

Interlaboratory study results for copper

No. of labse Outlier labsf

Chocolate milk powder

Dietetic milk powder 2

Peanut butter NIST SRM 2387

Wheat gluten NIST RM 8418

Sr = Standard deviation for repeatability (within laboratory).

RSDr = Relative standard deviation for repeatability.

SR = Standard deviation for reproducibility (among laboratories).

RSDR = Relative standard deviation for reproducibility.

Reporting laboratories minus outliers.

C = Cochran outlier; SG = Single Grubbs outlier; DG = Double Grubbs outlier.

Value in parentheses = Certified or reference values.

Predigest for at least 10 min at room temperature or until

systems. Never remove hot vessels from microwave; wait until

Interlaboratory study results for iron

Chocolate milk powder

Dietetic milk powder 2

Peanut butter NIST SRM 2387

Wheat gluten NIST RM 8418

Sr = Standard deviation for repeatability (within laboratory).

RSDr = Relative standard deviation for repeatability.

SR = Standard deviation for reproducibility (among laboratories).

RSDR = Relative standard deviation for reproducibility.

Reporting laboratories minus outliers.

C = Cochran outlier; SG = Single Grubbs outlier; DG = Double Grubbs outlier.

Value in parentheses = Certified or reference values.

No. of labse Outlier labsf

2013 AOAC INTERNATIONAL

Interlaboratory study results for potassium

Chocolate milk powder

Dietetic milk powder 2

No. of labse Outlier labsf

Peanut butter NIST SRM 2387

Wheat gluten NIST RM 8418

Sr = Standard deviation for repeatability (within laboratory).

RSDr = Relative standard deviation for repeatability.

SR = Standard deviation for reproducibility (among laboratories).

RSDR = Relative standard deviation for reproducibility.

Reporting laboratories minus outliers.

C = Cochran outlier; SG = Single Grubbs outlier; DG = Double Grubbs outlier.

Value in parentheses = Certified or reference values.

Cool vessels to room temperature before venting (MDC vessels).

(2) Digestion vessel decontamination.Decontaminate the