Improvement of AOAC Official Method 98427 For The

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Improvement of AOAC Ofﬁcial Method 984.27 for the Determination of Nine

Nutritional Elements in Food Products by Inductively Coupled Plasma-Atomic
Emission Spectroscopy After Micr...
Article in Journal of AOAC International · September 2009

DOI: 10.1093/jaoac/92.5.1484
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1484 POITEVIN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 5, 2009
FOOD COMPOSITION AND ADDITIVES
Improvement of AOAC Official Method 984.27 for the

Determination of Nine Nutritional Elements in Food Products by
Inductively Coupled Plasma-Atomic Emission Spectroscopy After
Microwave Digestion: Single-Laboratory Validation and Ring Trial
ERIC POITEVIN, MARINE NICOLAS, LAETITIA GRAVELEAU, JANIQUE RICHOZ, DANIEL ANDREY, and FLORENCE MONARD
Downloaded from https://academic.oup.com/jaoac/article-abstract/92/5/1484/5656012 by guest on 18 February 2020

Nestlé Research Center, Vers-Chez-Les-Blanc, CH-1000 Lausanne 26, Switzerland
Collaborators: A. Abrahamson; A. Baillon; J. Barrios; S. Berger; R. Berrocal; R. Bos; L. Brullebaut; C. Caseiro; L.F. Choo;
G. Cole; G. Daix; C. Dekussche; G.-S. Dhillon; A. Fortineau; C. Gaudin; M.J. Gonzales; R. Leal; R.O. Mabiog; T. Noorlos;
R. Reba; C. Senechal
R
A single-laboratory validation (SLV) and a ring trial obust and efficient methods with well-characterized
(RT) were undertaken to determine nine nutritional reference materials are needed by food-testing and
elements in food products by inductively coupled nutrition laboratories to facilitate compliance with
plasma-atomic emission spectroscopy in order to nutritional labeling laws and claim requirements, to provide
improve and update AOAC Official Method 984.27. traceability for food exports needed for acceptance in many
The improvements involved optimized microwave foreign markets, and to improve the accuracy of nutrition
digestion, selected analytical lines, internal information that is provided to assist consumers in making
standardization, and ion buffering. Simultaneous sound dietary choices (1, 2). Inductively coupled plasma-
determination of nine elements (calcium, copper, atomic emission spectroscopy (ICP-AES) is one of the most
iron, potassium, magnesium, manganese, sodium, commonly used techniques within the food industry for
phosphorus, and zinc) was made in food products. accurate and cost-efficient routine analyses of nutritional
Sample digestion was performed through wet minerals in food products, plants, pet food, raw materials, and
digestion of food samples by microwave feeding stuffs (3–15).
technology with either closed or open vessel AOAC Method 984.27 was validated 25 years ago via a
systems. Validation was performed to characterize collaborative study in which acid digestion with
the method for selectivity, sensitivity, linearity, HNO3/HClO4 and ICP-AES analysis with radial grating
accuracy, precision, recovery, ruggedness, and configuration were used to determine nine nutritional
uncertainty. The robustness and efficiency of this minerals in infant formula (16). Standard procedures for the
method was proved through a successful internal decomposition of food samples usually involve dry-ashing in
RT using experienced food industry laboratories. a muffle furnace or wet digestion with combined
Performance characteristics are reported for acids (6, 10, 17). Compared to the classical digestion
13 certified and in-house reference materials, procedures, microwave digestion has some significant
populating the AOAC triangle food sectors, which advantages with respect to speed of analysis, analyte loss,
fulfilled AOAC criteria and recommendations for sample contamination, and safe acid handling during sample
accuracy (trueness, recovery, and z-scores) and preparation (8). The use of microwave digestion before
precision (repeatability and reproducibility RSD ICP-AES analysis provides an accurate method for the
and HorRat values) regarding SLV and RT. This determination of nutritional minerals targeted for labeling of
multielemental method is cost-efficient, food products (5).
time-saving, accurate, and fit-for-purpose
The objective of this study is to improve AOAC Method
according to ISO 17025 Norm and AOAC
984.27 in terms of sample preparation time and throughput
acceptability criteria, and is proposed as an
using microwave digestion, and analytical performance using
improved version of AOAC Official Method 984.27
the latest generation of ICP-AES equipment under robust
for fortified food products, including infant
conditions (18), internal standardization (19, 20), and ion
formula.
buffer (21) to correct for physical and chemical interferences,
to compensate for matrix effects (22–25) induced by the
complexity of the food samples, and to improve short-term
Received April 30, 2009. Accepted by SG June 19, 2009. accuracy (repeatability) and long-term stability (reproducibility
Corresponding author’s e-mail: eric.poitevin@rdls.nestle.com and calibration curve validity in a long analysis batch.
POITEVIN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 5, 2009 1485
Table 1. Microwave digestion systems (MD1, MD2, MD3, MD4) and ICP-AES equipment used for single-laboratory
validation and ring trial
Test codea Digestion modeb Digestion equipment Nebulizer/chamber ICP equipment Grating configuration
SLV MDC CEM Mars Xpress Concentric/cyclonic Varian Vista Pro AX Axial view
MD1 MDC CEM Mars Xpress Concentric/cyclonic Varian Vista Pro AX Axial view
MD2 MDO CEM MDS 2000 Concentric/cyclonic Varian Vista Pro AX Axial view
MD3 MDC Milestone MLS Ethos Concentric/cyclonic Spectro Ciros Radial view
MD4 MDC Milestone MLS 1200 Concentric/cyclonic Perkin Elmer Optima 5300 DV Dual view
RT MDC CEM Mars Xpress Concentric/cyclonic Perkin Elmer Optima 5300 DV Dual view
RT MDC CEM Mars 5 Concentric/cyclonic Perkin Elmer Optima 2000 DV Dual view

RT MDC CEM Mars Xpress Concentric/cyclonic Varian 720 ES Axial view
RT MDC CEM Mars Xpress Crossflow/scott Perkin Elmer Optima 3300 RL Radial view
RT MDO CEM MDS 2000 Crossflow/scott Perkin Elmer Optima 4300 DV Dual view
RT MDC CEM Mars Xpress Concentric/cyclonic Varian 720 ES Axial view
a
SLV = Single-laboratory validation; MD = ruggedness test using different microwave digestion systems and ICP-AES equipment; RT = ring
trial.
b
MDC = Closed-vessel microwave digestion system; MDO = open-vessel microwave digestion system.
The validation of this method involved a single-laboratory including ruggedness test MD1 (Table 1). Other microwave
validation (SLV), a ruggedness study, and a ring trial (RT) in systems from CEM and Milestone (Shelton, CT) suppliers
accordance with ISO 17025 and AOAC guidelines (26): were used for ruggedness tests MD2, MD3, and MD4 and for
(a) SLV.—Performed using ICP-AES with axial grating the collaborative study RT (Table 1).
after a closed-vessel microwave digestion, on 10 in-house and (b) Open-vessel microwave digestion system.—A CEM
certified reference materials. MDS 2000 was used for test MD2 and by one laboratory for
(b) Ruggedness study.—Performed in parallel, analyzing the RT.
with and without ionization buffer, the spiking elements in (c) ICP-AES spectrometers.—A Varian Vista-Pro axial
eight food-grade salts and testing six in-house and certified ICP-AES instrument with SPS-5 autosampler (Varian Inc.,
reference materials in repeatability and reproducibility Mulgrave, Victoria, Australia) was used for the SLV,
conditions with different ICP-AES equipment (axial, radial, including tests MD1 and MD2. Other ICP-AES spectrometers
and dual-view grating configurations) after open- and from Perkin Elmer (Norwalk, CT), Varian, and Spectro
closed-vessel microwave-based digestions. (Kleve, Germany) were used for the MD3 and MD4 tests and
(c) RT.—Involving nine laboratories was organized to for the RT.
analyze five in-house and certified reference materials. (d) Balance.—A balance (AT 200, Mettler-Toledo, Inc.,
Thirteen well-characterized reference materials were Greifensee, Switzerland) with readability of 0.1 mg (SLV, tests
selected to populate the AOAC triangle, in which foods could MD1 and MD2) or equivalent (tests MD3, MD4, and RT).
be categorized into nine sectors based on their fat, protein, and (e) Sub-boiling quartz distillation apparatus for acids.—A
carbohxdrate contents (27). All the results obtained on Kürner Analysentechnik (Rosenheim, Germany) distillator
reference materials have been treated with robust (SLV, MD1, and MD2) for purifying acids or equivalent (tests
statistics (28, 29) to verify that the updated procedure is MD3, MD4, and RT).
fit-for-purpose in terms of specificity, sensitivity, linearity, (f) Fumehood.—A fumehood acid-resistant was used
trueness, and precision; and can be proposed as an improved (SLV, tests MD1 and MD2) or equivalent (tests MD3, MD4,
version of official AOAC Method 984.27 regarding microwave and RT).
digestion, internal standardization, and ion buffering. (g) Drying oven.—A Heraeus drying oven 6120T
(Heraeus, Hanau, Germany) was used for the SLV, and tests
Experimental MD1 and MD2, and similar equipment was used for tests
Apparatus MD3, MD4, and RT.
(h) Mixer or grinder for homogenization.—A Retsch
(a) Closed-vessel microwave digestion systems.—A CEM (Haan, Germany) mixer GM200 was used for the SLV, tests
Mars Xpress (CEM, Matthews, NC) was used for the SLV, MD1 and MD2, or equivalent (tests MD3, MD4, and RT).
Table 2. Raw materials, certified, and in-house reference materials used for SLV and RT
Material type Sample reference AOAC food triangle sectora Test code
Sterilized cream LGC RM 7104 1 SLV

Baking chocolate NIST SRM 2384 2 SLV
Peanut butter NIST SRM 2387 3 RT
Whole egg powder NIST RM 8415 4 SLV
Chocolate milk powder CMP 5 SLVb - RT
Infant cereals IC 5 SLVb - RT
Baby food composite NIST SRM 2383 5 SLV

Corn bran NIST RM 8433 5 SLVb
Dietetic milk powder 1 DMP1 6 SLVb
Dietetic milk powder 2 DMP2 6 RT
Pet food PET 6 SLVb
Nonfat milk powder NIST SRM 1549 7 SLVb
Wheat gluten NIST RM 8418 9 RT
c
Calcium carbonate CAS No. 471-34-1 NA SLV
Calcium phosphate tribasic CAS No. 7758-84-4 NA SLV
Calcium citrate tribasic CAS No. 5785-44-4 NA SLV
Potassium chloride CAS No. 7447-40-7 NA SLV
Magnesium carbonate CAS No. 546-93-0 NA SLV
Magnesium chloride CAS No. 7791-18-6 NA SLV
Manganese sulfate CAS No. 10034-96-5 NA SLV
Sodium chloride CAS No. 7647-14-5 NA SLV
a
Sector number (1 to 9) of the food triangle (27).
b
SLV, including ruggedness tests (MD).
c
NA = Not applicable.
Materials (Table 2) were used for the SLV, ruggedness tests MD1,
MD2, MD3, and MD4 and internal RT, including dairy-based
(a) Volumetric flasks.—Glass, 50, 100, 500, 1000 mL products, milk, and cereals-based products, chocolate and
(SLV, tests MD1 and MD2) or equivalent (tests MD3, MD4, dietetic milk powders, refrigerated meals, pet food, and raw
and RT). materials, such as whole egg powder, wheat gluten, corn bran,
(b) Erlenmeyer flask for slurry preparation.—Glass, and added food-grade salts (calcium carbonate, calcium
100 mL (SLV, tests MD1 and MD2) or equivalent (tests MD3, phosphate tribasic, calcium citrate tribasic, potassium
MD4, and RT). chloride, magnesium carbonate, magnesium chloride,
(c) Sample tubes.—Polyethylene or polystyrene, tubes 15 manganese sulfate, and sodium chloride).
or 50 mL with caps (Falcon) for ICP analysis (SLV, tests
MD1 and MD2) or equivalent (tests MD3, MD4, and RT). Chemicals and Reference Solutions
(d) Filters.—Ashless filter papers, quantitative (SLV,
tests MD1 and MD2), Whatman 589/1 (diameter 125 mm (a) High grade water, H2O (18 MW).—MilliQ™ Plus
and/or 90 mm; Schleicher & Schuell, Whatman, Dassel, system (Millipore, Bedford, MA).
Germany), or equivalent (tests MD3, MD4, and RT). (b) Nitric acid (HNO3).—65% (w/v), Suprapure.
(e) Pipets.—Macropipet, variable volume 0.5–5.0 mL; (c) Nitric acid (HNO3).—65% (w/v), analytical grade.
micropipet, variable volume 50–200 mL and 200–1000 mL; (d) Hydrochloric acid.—37% (w/v), analytical grade.
electronic digital pipet 50–1000 mL (SLV, tests MD1 and (e) Hydrogen peroxide.—97% (w/v), analytical grade.
MD2), or equivalent (tests MD3, MD4, and RT). (f) Multistandard.—CPI International (Santa Rosa, CA).
(f) Samples.—Certified and standard reference materials Composition of the CPI standard solution, in mg/L: Ca =
from National Institute of Standards and Technology (NIST; 7500; Cu = 50; Fe = 250; K = 10 000; Mg = 2500; Mn = 1.25;
Gaithersburg, MD) and National Metrology Institute (LGC Na = 5000; P = 5000; Zn = 100.
standards, Teddington, UK), in-house reference available (g) Calcium standard solution.—10 000 mg/L (Merck,
samples (from internal proficiency tests), and raw materials Darmstadt, Germany).
Table 3. Open-vessel microwave digestion program HNO3 (SLV, MD1) or 15 mL combined acids (MD2), dilute
(CEM MDS 2000) used for MD2 ruggedness test to volume with H2O, mix, and transfer to acid-washed
No. of samples Hold time, min Hold time, min
polyethylene bottle. (5) Std2.—Pipet 0.2 mL stock standard
to digest Powera (step 1) (step 2) solution into a 100 mL acid-washed volumetric flask. Add
10 mL analytical grade HNO3 (SLV, MD1) or 15 mL
5 23 20 10 combined acids (MD2), dilute to volume with H2O, mix, and
transfer to acid-washed polyethylene bottle. (6) Std1.—Pipet
6 27 20 10
0.1 mL stock standard solution into a 100 mL acid-washed
7 31 20 10
volumetric flask. Add 10 mL analytical grade HNO3 (SLV,
8 35 20 10 MD1) or 15 mL combined acids (MD2), dilute to volume with
9 39 20 10 H2O, mix, and transfer to acid-washed polyethylene bottle.
10 43 20 10 (7) Blank.—Add 10 mL analytical grade HNO3 (SLV, MD1)

11 47 20 10 or 15 mL combined acids (MD2) into a 100 mL acid-washed
volumetric flask, dilute to volume with H2O, mix, and transfer
12 51 20 10
to acid-washed polyethylene bottle. All calibration solutions
a
Power expressed in % of maximum power (600 watts for CEM are stable for 1 week in glass volumetric flasks.
MDS 2000 system). (c) Ionization buffer/internal standard solution for
automatic addition.—Weigh 12.67 g cesium chloride into a
1000 mL acid-washed volumetric flask [Note: Ruggedness
(h) Potassium standard solution.—10 000 mg/L (Merck). tests.—Weigh 1.27 g cesium chloride. Cesium 0.1% (w/v)
(i) Sodium standard solution.—10 000 mg/L (Merck). solution was tested as the minimal recommended
(j) Magnesium standard solution.—10 000 mg/L (Merck). concentration required for element analysis in most food
(k) Phosphorus standard solution.—10 000 mg/L (Merck). matrixes. Cs solution 1% (w/v) is recommended if an element
(l) Iron standard solution.—1000 mg/L (Merck). is present at low concentration in high-salted food raw
(m) Zinc standard solution—1000 mg/L (Merck). materials (e.g., culinary products or tastemakers) or if it is
(n) Copper standard solution.—1000 mg/L (Merck). analyzed as an impurity in food-grade salts. Add 40 mL
(o) Manganese standard solution.—1000 mg/L (Merck). indium 1000 mg/L and 10 mL each of strontium, yttrium, and
(p) Indium standard solution.—1000 mg/L (Merck). chromium 1000 mg/L stock solutions, as internal standards.
(q) Chromium standard solution.—1000 mg/L (Merck). Add 10 mL analytical grade HNO3. Dilute to volume with
(r) Strontium standard solution.—1000 mg/L (Merck). H2O, mix, and transfer to an acid-washed polyethylene bottle.
(s) Yttrium standard solution.—1000 mg/L (Merck). (Note: Reagent concentrations assume the use of same pump
(t) Cesium chloride.—Analytical grade (Merck). tubing internal diameter for both internal standard/ionization
Preparation of Reagents and Standard Solutions

(a) Stock standard solution.—Working standards were
Table 4. ICP-AES conditions (Varian Vista AX PRO) for
prepared from a CPI multistandard commercial stock standard
SLV, MD1, and MD2 ruggedness tests
solution.
(b) Working standard solutions.—Standards prepared Instrument Varian Vista Pro AX
from stock standard solution are designed to have the same
acid concentration as digested test solutions (i.e., 10% v/v RF generator 40 MHz
using sub-boiled or analytical grade HNO3 (SLV and MD1) RF power 1100 W
or 15% v/v (MD2) using combined acids (HNO3, H2O2, and Torch quartz injector id 2.5 mm
HCl). (1) Std6.—Pipet 3.2 mL stock standard solution into a
Argon flow rate Plasma 18 L/min
100 mL acid-washed volumetric flask. Add 10 mL analytical
grade HNO3 (SLV, MD1) or 15 mL combined acids (MD2), Auxiliary 2.25 L/min
dilute to volume with H2O, mix, and transfer to acid-washed Nebulizer 0.8 L/min
polyethylene bottle. (2) Std5.—Pipet 1.6 mL stock standard Cyclonic thermostated (water
solution into a 100 mL acid-washed volumetric flask. Add Spray chamber cooled)
10 mL analytical grade HNO3 (SLV, MD1) or 15 mL Nebulizer Microconcentric
combined acids (MD2), dilute to volume with H2O, mix, and Pump Peristaltic, 3 channels
transfer to acid-washed polyethylene bottle. (3) Std4.—Pipet Pump tubing id 0.64 mm
0.8 mL stock standard solution into a 100 mL acid-washed
Sample flow rate 0.75 mL/min
volumetric flask. Add 10 mL analytical grade HNO3 (SLV,
MD1) or 15 mL combined acids (MD2), dilute to volume with Polychromator Echelle
H2O, mix, and transfer to acid-washed polyethylene bottle. Resolution 6.9 pm at Mo 202.032 nm
(4) Std3.—Pipet 0.4 mL stock standard solution into a 100 mL Detector Segmented CCD
acid-washed volumetric flask. Add 10 mL analytical grade
Table 5. Recommended and alternate ICP analytical sealing, and predigest at room temperature until vigorous
lines used for SLV and RT foaming subsides. Close vessels and place in CEM Mars
Element lines, nm Internal standard lines, nm Xpress microwave or in other closed-vessel microwave
systems.
Ca IIa 317.933 In I 303.936
(c) Test portion preparation for open-vessel microwave
b digestion (MD2 ruggedness test).—Accurately weigh 1.0 ±
Ca II 317.933 Y II 371.028
a
0.01 g test portion (or sample mass in the slurry) into a 100 mL
Cu I 324.754 In I 303.936 volumetric flask. Carefully add 5 mL HNO3 and then 5 mL
b
Cu I 324.754 Y II 371.028 H2O2. Allow the sample to stand for at least 10 min at room
b
Cu I 327.395 In I 303.936 temperature. Distribute the volumetric flasks onto the
Cu I
b
327.395 Y II 371.028 open-vessel microwave carousel to ensure homogeneous
a microwave power application on all samples.

Fe II 259.94 Sr II 338.071
Fe II
b
259.94 Y II 371.028
(d) Sample digestion for closed-vessel microwave
b digestion (SLV, MD1, MD3, and MD4 ruggedness
Fe II 259.94 Cr II 283.563
a
tests).—Digest sample according to a ramp program as
KI 766.491 Sr I 460.733 follows: Conditions: Stage 1.—Power, expressed as
a
Mg I 285.213 In I 303.936 maximum power (1600 watts for CEM Mars Xpress system)
b
Mg I 285.213 Y II 371.028 = 75%; ramp time, 5 min; 120°C; hold time, 5 min.
Mg II
a
279.078 In I 303.936 Stage 2.—Power, 75%; ramp time, 5 min; 200°C; hold time,
Mn II
b
257.61 Sr II 338.071
25 min. Then cool vessels, vent, and transfer digests to 50 mL
b volumetric flasks, dilute to volume with H2O, and mix. A
Mn II 257.61 Sr I 460.733
b
digestion is judged complete when clear-to-yellow analytical
Mn II 257.61 Y II 371.028 solutions are produced. Filter the digested solution using an
a
Na I 589.592 Sr I 460.733 ashless filter paper for turbid samples containing fat,
a
PI 213.618 In I 303.936 discarding the first 20 mL of filtrate and collecting the
PI
b
178.222 Sr I 460.733 remaining filtrate for analysis. [Note: Membrane disc filters
PI
b
178.222 Y II 371.028
(0.45 mm) are not recommended as they are generally not
a metal free.] Transfer to polyethylene containers within 2 h.
Zn I 213.857 Sr II 338.071
b
Dilute with H2O the samples that are found to be above the
Zn I 213.857 Sr I 460.733 standard curve range, or have total content of minerals higher
b
Zn I 213.857 Y II 371.028 than 1000 mg/L. Final dilutions require addition of
a
appropriate amounts of HNO3 to maintain the proportion of
Recommended lines for ICP-AES analysis.
b
10% (v/v) HNO3 in the final solution to be analyzed.
Alternate confirmatory lines for ICP-AES analysis.
(e) Sample digestion for open-vessel microwave digestion
(MD2 ruggedness test).—Digest sample according to an
appropriate power program close to that described in Table 3
buffer and sample pump tubes (i.e., orange/white, 0.64 mm id) for a CEM MDS 2000 system depending on the number of
using automatic addition. distributed flasks. This first step requires 20 min heating time.
Sample Preparation and Digestion Yellow vapors will be emitted during the hydrolysis.
Carefully remove the flasks from the microwave system.
(a) Test sample preparation.—Homogenize the test Allow the flasks to cool to room temperature and add 5 mL
sample (e.g., serving size or declared labeled size) by either HCl 35% (w/v). Heat the flasks for an additional 10 min using
grinding as fine as possible using a mixer to obtain a fine the same program in the microwave system. Allow the flasks
powder and/or by preparing a slurry with H2O: weigh 10 to cool to room temperature. Transfer digests to 100 mL
± 0.1 g test sample, and put into a 100 mL Erlenmeyer flask; volumetric flasks, dilute to volume with H2O, and mix. Filter
add 90 ± 0.1 g H2O, and mix well with stopper (Note: Water and prepare the digested solution before analysis as described
can be preheated at 50°C for a better homogenization of for closed-vessel microwave digestion in (d) but maintain the
samples, e.g., infant cereals and fortified milk powders). proportion of 15% (v/v) combined acids in the final solution
(b) Test portion preparation for closed-vessel microwave to be analyzed.
digestion (SLV, MD1, MD3, and MD4 ruggedness (f) Digestion vessel decontamination for closed-vessel
tests).—Accurately weigh 0.50 ± 0.01 g test portion (or microwave digestion (SLV, MD1, MD3, and MD4 ruggedness
sample mass in the prepared slurry) into digestion vessel. Use tests).—Conditions: Step 1.—Power, express in % of
either weighing paper insert to line the vessel walls or Pasteur maximum power (1600 watts for CEM Mars Xpress system)
pipet during sample transfer to keep sample from adhering to = 75%; ramp time, 5 min; 180°C; hold time, 5 min.
sides of vessel. Fluid samples or test portion from slurry test Decontaminate used digestion vessels with 5 mL HNO3 for a
sample may be weighed directly after mixing. Add 5.0 ± CEM Mars Xpress system and rinse with H2O. Dry vessels at
0.1 mL analytical grade HNO3, loosely cap vessels without 60°C in drying oven for 1 h.
Table 6. Recovery and SD values found for nine elements using recommended lines after optimization of CEM Mars
Xpress microwave digestion of two Standard Reference Materials (NIST SRM 2384, NIST SRM 2383) and one
Reference Material (NIST RM 8415)
Concentration, mg/kg
Baking chocolate Whole egg powder Baby food composite

Element (NIST SRM 2384) (NIST RM 8415) (NIST SRM 2383)
Calcium Certifieda 840 ± 74 2480 ± 190 853 ± 28

Found (recovery, %)c
b
793 ± 100 (94) 2627 ± 358 (106) 868 ± 12 (102)
Copper Certifieda 23.2 ± 1.2 2.70 ± 0.35 1.42 ± 0.12
Foundb (recovery, %)c 24.1 ± 0.2 (104) 2.90 ± 0.05 (107) 1.77 ± 0.22 (125)

Iron Certifieda 132.0 ± 1.1 112 ± 16 8.44 ± 0.44
Foundb (recovery, %)c 129.6 ± 1.2 (98) 105 ± 0.3 (94) 10.41 ± 1.11 (123)
Potassium Certifieda 8200 ± 500 3190 ± 370 3600 ± 100
b
7750 ± 100 (95) 3125 ± 27 (98) 3793 ± 33 (105)
Magnesium Certifieda 2570 ± 150 305 ± 27 248 ± 5
b
2567 ± 34 (100) 292 ± 1 (96) 245 ± 3 (99)
Manganese Certifieda 20.3 ± 1.3 1.78 ± 0.38 1.39 ± 0.11
b
19.9 ± 0.1 (98) 1.62 ± 0.01 (91) 1.42 ± 0.002 (102)
Sodium Certifieda 40 ± 4 3770 ± 340 390 ± 28
b
NDd 3550 ± 222 (94) 378 ± 7 (97)
Phosphorus Certifieda 3330 ± 210 10010 ± 320 948 ± 33
b
3301 ± 48 (99) 11643 ± 295 (116) 962 ± 10 (101)
Zinc Certifieda 36.6 ± 1.7 67.5 ± 7.6 10.5 ± 0.3
b
35 ± 0.5 (96) 60.9 ± 0.1 (90) 10.6 ± 0.03 (101)
a
Uncertainty expressed either as a 95% confidence interval or as an interval based on the entire range of accepted results for a single
determination.
b
SD from average of triplicates analysis.
c
Found value/certified value ratio expressed in %.
d
ND = Not determined (<LOQ).
(g) Digestion vessel decontamination for open-vessel A 3-channel peristaltic pump (Gilson, Middleton, WI) and a T
microwave digestion (MD2 ruggedness test).—The glass connector (id 1.5 mm/Socochim, Lausanne, Switzerland)
volumetric flasks can be directly used after being washed in a linked between the peristaltic pump and nebulizer were used
laboratory washing machine or soaked overnight in a water to avoid having to manually add ionization buffer and IS to
bath with HNO3 30% (v/v) and rinsed with H2O to avoid or each sample solution. A thermostatted (15°C) cyclonic spray
minimize risk of iron contamination. chamber (Jacketed ‘Althea’ Cyclonic Glass Spray Chamber,
(h) Spiked food grade salt preparation (SLV).—Weigh water-cooled, EPOND SA, Vevey, Switzerland) equipped
0.2 ± 0.01 g food-grade salt into a 100 mL volumetric flask. with a micro-concentric nebulizer [C-Type ‘Caliber’ standard
Add deionized water and 10 mL HNO3. Add 0.8 mL stock Concentric Glass Nebulizer with ‘NebLink’ sample capillary
standard solution corresponding to standard Std4 [prepared tube and argon connector (EPOND)] were used to obtain the
from CPI commercial solution (Ca, 60 mg/L; Cu, 0.4 mg/L; best method performance. Sample and IS pump tubes (e.g.,
Fe, 2 mg/L; K, 80 mg/L; Mg, 20 mg/L; Mn, 0.01 mg/L; Na, orange/white, 0.64 mm id Socochim), and peristaltic pump
40 mg/L; P, 40 mg/L; Zn, 0.8 mg/L)]. Dissolve salt and dilute rotation speed (15 rpm), were selected to keep sample and IS
to volume with deionized water. pump tubes of similar size to maximize mixing accuracy,
ICP Analysis while maintaining the required detection levels. Ionization
buffer (cesium chloride) was combined with IS solution to
(a) ICP-AES spectrometer (SLV-MD1 and MD2 compensate for easy ionizable element (EIE) effects (e.g., K,
ruggedness tests).—A Varian Vista-Pro axial ICP-AES Na, and Ca) in the plasma, because certain food materials can
instrument with autosampler SP5 was used during SLV, using contain substantial concentrations of these elements that
the operating conditions shown in Table 4. The recommended provide a significant source of electrons in the plasma. The
and alternative analytical lines (nm) used for elements to be presence of ionization buffer in all samples and standards will
determined and internal standards (IS) are shown in Table 5. minimize the effects of varying concentrations of EIEs in the
Table 7. Regression coefficients obtained from well as sample and IS pump tubes and peristaltic pump
weighed linear and nonlinear regression for rotation speed used were selected according to manufacturer’s
recommended and alternate lines recommendations and were adapted to optimize aerosol and
Calibration curvea Concn range, mg/L maximize precision, and to demonstrate robust operating
conditions. They were capable of determining at least the
Element line R² nonlinearb R² linearc Low High recommended multiple lines for each element of interest
(Table 5). Combined ionization buffer/IS solution was
Ca IId 0.99997 0.99980 0 240 prepared such that after mixing digest sample and
d
Cu I 0.99999 1.00000 0 2.1 IS/ionization buffer solutions using the instrument’s
Fe II
d
0.99997 0.99999 0 8.5 peristaltic pump, the combined solution presented to the
KI
d
0.99924 0.99326 0 320
nebulizer contained at least 500 mg/L Cs; 20 mg/L indium;
5 mg/L strontium, yttrium, and chromium; and less than half

d
Mg I 0.99999 0.99891 0 75
d
of each element concentration of the higher working standard
Mn II 1.00000 0.99998 0 0.04 and <.5 g/L total dissolved minerals. All sample instrument
d
Na I 0.99996 0.99666 0 160 responses of recommended and alternate lines for each
d
PI 0.99998 0.99997 0 160 element are corrected using one IS line (Table 5).
Zn I
d
0.99999 0.99998 0 3.2 (c) Quantification.—The concentration (C) of each
Ca II
e
0.99994 0.99993 0 240
element, in mg/kg, was calculated as follows:
e
Cu I 1.00000 0.99995 0 2.1 a ´ V´ F
e C=
Cu I 0.99999 0.99999 0 2.1 m
e
Cu I 0.99999 0.99998 0 2.1
Fe II
e
0.99996 0.99997 0 8.5
where C = concentration in the test portion sample (mg/kg); a
e = concentration (mg/L) of the element in the digest solution; V
Fe II 0.99999 0.99998 0 8.5
e
= volume (mL) of the test solution after being made up [i.e.,
Mg I 0.99997 0.99929 0 80 50 mL for closed vessel microwave digestion (MDC) for SLV
e
Mg II 1.00000 0.99999 0 80 and 100 mL for open-vessel microwave digestion (MDO)
e
Mn II 1.00000 0.99997 0 0.04 used for test MD2]; F = dilution factor of the test solution; m =
Mn II
e
1.00000 0.99998 0 0.04 weight of the test portion (g).
e
PI 0.99999 0.99967 0 160 Statisic Calculations
e
PI 1.00000 0.99993 0 160
e
All data used for selectivity, accuracy and precision
Zn I 0.99999 0.99991 0 3.2
performance tests were treated using robust statistics (29)
e
Zn I 1.00000 1.00000 0 3.2 based in the concept of Rousseeuw and Croux (28). The
a
presence of some suspect values (outliers) can strongly distort
Calibration with six standard solutions and a blank.
b
classical estimations. However, results must not be eliminated
Weighed second degree quadratic regression.
c
Weighed linear regression without forcing zero.
without a valid justification. For that reason, robust statistics,
d
Recommended lines for ICP-AES analysis. which provide good estimations even without the elimination
e
Alternate confirmatory lines for ICP-AES analysis. of suspect values, have been used in the SLV and RT. These
robust estimations are insensitive to extreme values and
depend only slightly on data distribution. It is then neither
necessary to test for outliers nor to exclude suspect values.
sample. Power settings and nebulizer gas flow were optimized The median has been used as a robust estimation of the central
to ensure that the Mg 280.271:Mg 285.213 ratio demonstrates value. The robust standard deviation SD(Sn) drawn from the
robust operating conditions (18) in accordance with the ratio algorithm Sn of Rousseeuw and Croux (28) has been used as a
established by the instrument manufacturer. Five replicate robust estimation of spread. This algorithm uses median
readings of the same sample were performed, with relatively absolute distances between all the couples we can set up with
long integration times to minimize noise. The solution the data:
presented to the nebulizer contains at the most 5000 mg/L
cesium (Note: 500 mg/L Cs for ruggedness tests MD1, MD2); Sn = medi {medj *xi – xj *}
20 mg/L indium; and 5 mg/L strontium, yttrium, and SD(Sn) = 1.1926 ´ Sn
chromium; less than half of each element concentration of the
higher working standard Std6 and <0.5 g/L total dissolved Statistical comparative t-test is the robust adaptation of the
minerals. All responses for both recommended and alternative classical t-test by replacing the mean with the median, and the
lines for each element are corrected using one IS line SD with the robust SD(Sn). Robust t-test was used for
(Table 5). selectivity (median comparison) and accuracy was evaluated
(b) ICP-AES spectrometers (MD3 and MD4 ruggedness to check if the recovery is significantly different from 100%
tests).—Operating conditions of both instruments (Table 1) as (at 95 and 99% confidence intervals).
Table 8. Estimated LOD and LOQ for the nine elements range of food matrixes is analyzed on a routine basis, and an
in food matrixes obtained from recommended and additional predigestion step is often used to allow various
alternate element lines matrixes to be digested concurrently. Thus, approach of a
Element LODa, mg/L LOQb, mg/L LOQc, mg/kg single optimized MDC program for duty vessels with only
HNO3 operating up to 200°C, which has been adapted on a
Calcium 0.5 1.50 150
CEM Mars Xpress system, was favored: an optimal analytical
test portion mass of 0.5 g [based on an empirical maximum
Copper 0.006 0.02 2
energy release by the food of 3 kcal (8)] was used, and
Iron 0.03 0.1 10 concurrent digestions of triplicates of three NIST-certified
Potassium 0.5 2 200 RMs (baking chocolate, NIST SRM 2384; whole egg powder,
Magnesium 0.2 0.5 50 NIST RM 8415; and Baby Food Composite, NIST SRM
Manganese 0.0002 0.0005 0.05 2383) were performed. Similar programs to that of CEM Mars

Sodium 0.4 1 100
Xpress system were adapted on other MDC systems used for
MD3 and MD4 ruggedness tests. An optimized program for
Phosphorus 0.4 1 100
MDO system CEM MDS 2000 (Table 3) was also developed
Zinc 0.02 0.05 5 in parallel, and used for all matrixes during ruggedness test
a
MD2 and by one volunteer laboratory during the RT.
Instrumental LOD calculated in solution.
b
Instrumental LOQ calculated in solution.
(c) Linearity.—A calibration curve was obtained from
c
LOQ estimated in food matrixes (dilution factor 100). seven standards, including a blank (Std0) and six
concentrations of the standard solution (Std1–Std6).
Weighted linear and quadratic regression analyses were
Recovery is judged satisfactory if it is not significantly performed and correlation coefficient (R2) values were
different from 100% at 95% confidence interval (P-value calculated.
>0.05) between the median concentration and the certified or (d) LOD and LOQ.—The instrumental detection (DL)
reference values (Table 2). Recovery is judged questionable if and quantification (QL) limits expressed in mg/L were
it is not significantly different from 100% at 99% confidence estimated using the SD approach by analyzing 10 replicates of
interval (0.01 < P-value < 0.05) between the median the low standard solution (Std1) according to the formula:
concentration and the certified or reference values (Table 2).
DL = 3 ´ SD of the mean of Std1 determinations (n = 10)
Recovery is judged unsatisfactory if it is significantly
QL = 10 ´ SD of the mean of Std1 determinations (n = 10)
different from 100% at 99% confidence interval (P-value
<0.01) between the median concentration and the certified or The DL and QL values of elements in real samples were
reference values (Table 2). hence estimated assuming a sample weight of 0.5 g and a
SLV dilution to 50 mL (i.e., a dilution factor of 100). Verification
of QL estimation was performed by checking satisfactory
(a) Study material.—Ten materials included six reference performance characteristics (accuracy and precision) for
materials (RM) of LGC Promochem LGC RM 7104 elements present in real matrixes at these estimated
(sterilized cream), of existing NIST Standard Reference concentrations, e.g., Ca, Cu, Fe, K, Na, P in corn bran (NIST
Material (SRM) NIST SRM 2384 (baking chocolate), NIST RM 8433), Mg, Zn in sterilized cream (LGC RM 7104), and
RM 8415 (whole egg powder), NIST SRM 2383 (baby food Mn in nonfat milk powder (NIST SRM 1549).
composite), NIST RM 8433 (corn bran), NIST SRM 1549 (e) Selectivity.—Element median recovery was calculated
(nonfat milk powder), and four Nestlé well-characterized using all the lines displayed in Table 5 from duplicates of eight
reference samples of infant cereals with milk powder, different solutions of spiked food grade salts (Table 2) with a
chocolate milk powder, dietetic milk powder (DMP1), and pet spiking concentration corresponding to Std4 prepared from
food (PET). The reference values of element concentrations in the CPI commercial solution. Robust t-test for comparison of
the four in-house reference materials and their associated median recovery and SD (i.e., median of averages of
reproducibility SD were obtained from Nestlé proficiency duplicates found per element using all analytical lines) of each
tests performed by a significant number of internal and spiked element in all food-grade salts with and without
external laboratories (between 12 and 45 laboratories, addition of ionization buffer Cs 1% (w/v) was performed.
depending on element and materials analyzed) using robust
(f) Accuracy.—Three replicate test portions (r = 3) were
statistics (28).
collected from each of seven (n = 7) different food samples
(b) Microwave digestion optimization.—Main reagents
for microwave-assisted digestion are HNO3 and HCl. H2O2 is populating four of the nine food triangle sectors, and then
generally used to remove any remaining organic residues of were digested and analyzed on 8 days (d = 8) corresponding to
rich-fat or rich-carbohydrate samples. MDC systems supplied nine series (i.e., 9 elements) of 168 data (n ´ r ´ d = 168).
by manufacturers typically apply to a single food matrix or Recovery median values (ratio of found and certified values)
minimize the analytical portion mass to account for the most and associated RSD were determined. Accuracy was
reactive food matrix. This approach is not efficient when a full statistically evaluated.
Table 9. Recovery values (%) and standard deviations (%) found using recommended lines for nine elements spiked
in eight food-grade salts
Spiked Calcium
element Calcium Calcium citrate phosphate Potassium Magnesium Magnesium Manganese
line carbonate tribasic tribasic chloride carbonate chloride sulfate Sodium chloride
Ca IIa NDc ND ND 102.8 ±1.9 98.5 ± 0.3 102.1 ± 0.7 90.2 ± 5.6 102.8 ± 6.7
b
Ca II ND ND ND 104.5 ± 2.1 100.6 ± 0.2 103.4 ± 0.6 98.2 ± 6.8 105.4 ± 6.7
a
Cu I 103.2 ± 0.5 100.4 ± 0.7 99.2 ± 1.2 105.0 ± 1.7 105.0 ± 0.4 105.1 ± 0.3 93.8 ± 5.6 105.3 ± 6.0
b
Cu I 106.8 ± 0.1 102.6 ± 0.5 102.5 ± 1.5 107.4 ± 1.9 107.0 ± 0.3 106.9 ± 0.3 102.3 ± 6.9 108.4 ± 6.0
b
Cu I 105.0 ± 0.6 102.4 ± 0.9 101.1 ± 1.5 106.9 ± 1.8 106.3 ± 0.5 107.0 ± 0.7 96.9 ± 5.6 106.6 ± 6.0

b
Cu I 108.7 ± 0.2 104.7 ± 0.7 104.5 ± 1.9 109.4 ± 2.0 108.0 ± 0.4 109.0 ± 0.7 105.5 ± 6.8 109.8 ± 6.0
a
Fe II 100.8 ± 0.5 96.6 ± 0.9 95.7 ± 2.2 104.5 ± 2.5 106.3 ± 1.7 104.8 ± 0.1 98.2 ± 6.2 105.3 ± 6.6
b
Fe II 99.6 ± 0.6 97.1 ± 0.9 96.0 ± 2.3 103.8 ± 2.5 102.5 ± 1.3 103.3 ± 0.1 96.9 ± 6.3 104.8 ± 6.4
b
Fe II 102.0 ± 0.3 99.0 ± 0.7 98.9 ± 2.2 104.5 ± 2.5 105.8 ± 1.2 104.6 ± 0.1 97.5 ± 5.9 105.7 ± 6.8
a
KI 105.3 ± 0.4 99.2 ± 0.9 98.1 ± 1.4 ND 109.1 ± 3.8 106.5 ± 0.8 102.5 ± 6.5 112.2 ± 7.7
a
Mg I 104.5 ± 0.1 101.3 ± 1.3 100.8 ± 2.5 107.4 ± 2.7 ND ND 94.3 ± 5.6 108.6 ± 7.4
b
Mg I 107.3 ± 0.3 102.7 ± 1.0 103.2 ± 2.9 109.0 ± 2.9 ND ND 102.3 ± 6.8 111.0 ± 7.4
b
Mg I 97.3 ± 0.6 96.4 ± 1.2 94.5 ± 2.1 101.2 ± 2.5 ND ND 88.4 ± 5.7 108.6 ± 7.4
a
Mn II 110.5 ± 8.9 98.0 ± 5.6 103.1 ± 1.1 110.2 ± 2.8 100.9 ± 18.7 103.9 ± 1.2 ND 107.8 ± 6.7
b
Mn II 107.7 ± 8.0 93.2 ± 5.6 106.1 ± 0.9 102.9 ± 2.4 100.4 ± 18.8 97.8 ± 0.6 ND 99.2 ± 6.2
b
Mn II 111.8 ± 8.0 99.6 ± 6.0 102.3 ± 1.2 109.0 ± 2.7 103.1 ± 17.0 102.1 ± 1.1 ND 106.7 ± 6.5
a
Na I 103.8 ± 1.0 99.7 ± 0.1 95.3 ± 0.6 106.5 ± 2.5 107.1 ± 1.3 107.9 ± 1.1 102.3 ± 7.1 ND
a
PI 100.7 ± 1.0 99.2 ± 1.3 ND 104.8 ± 1.9 99.2 ± 0.5 103.8 ± 1.0 90.5 ± 6.1 105.1 ± 6.8
b
PI 98.9 ± 1.0 96.2 ± 0.9 ND 102.2 ± 2.2 99.3 ± 1.0 104.1 ± 1.1 96.6 ± 6.8 101.6 ± 6.2
b
PI 105.6 ± 0.6 102.6 ± 1.0 ND 108.6 ± 2.2 102.0 ± 0.6 107.1 ± 0.9 100.2 ± 7.2 109.7 ± 6.6
a
Zn I 103.9 ± 0.3 101.6 ± 2.3 99.0 ± 1.3 107.4 ± 2.2 105.9 ± 0.3 115.0 ± 12.0 101.8 ± 6.9 108.3 ± 6.9
b
Zn I 97.0 ± 0.8 96.8 ± 2.4 91.3 ± 0.4 101.3 ± 2.2 98.6 ± 1.1 111.2 ± 11.3 97.5 ± 6.8 100.6 ± 6.5
b
Zn I 102.3 ± 0.3 101.8 ± 2.4 98.9 ± 1.4 106.2 ± 2.0 101.1 ± 0.5 113.0 ± 11.8 100.1 ± 7.0 107.4 ± 6.4
a
Recommended lines for ICP-AES analysis.
b
c
ND = Not determined.
(g) Precision.—Both repeatability standard deviation RSDiR , %

HorRat value =
(SDr; within day) and intermediate reproducibility standard 2C-0.1505 , %
deviation (SDiR, between day) were determined.
(h) Uncertainty.—Uncertainty (U) was estimated using
where RSDiR is the relative intermediate reproducibility
the simplified approach based on existing validation data
standard deviation of each element in each of the 10 tested,
proposed by Barwick and Ellison (30, 31), mainly from
certified or in-house reference matrixes; C is the relative
precision and trueness studies, which, if properly planned to
concentration (expressed as a dimensionless mass fraction) of
cover as many of the uncertainty sources previously identified
the analyte in question (valid for concentration ratio
as possible, provide the necessary data required to calculate
above 10–9).
measurement uncertainty. SDs of intermediate precision
The HorRat value is a simple performance parameter that
(SDiR) and recovery (SDrec) contributions are combined
reflects the acceptability of a chemical method of analysis
together as followed to obtain the overall uncertainty (U):
with respect to precision (32).
HorRat value of 1.0 is judged satisfactory with limits of
U = SDiR + SDrec
2 2
acceptability of 0.3–1.5. Consistent deviations from the ratio
on the low side (values <0.3) may indicate unreported
averaging or excellent training and experience; consistent
(i) HorRat values.—Horwitz ratio (HorRat value) was deviations on the high side (values >1.5) may indicate
calculated for SLV according to the equation: inhomogeneity of the test samples, need for further method
Table 10. Median recovery (%) and standard deviations cereals with milk powder, chocolate milk powder, and dietetic
(%) by element and for all elements in eight food-grade milk powder 2 (DMP2). The three in-house reference
salts using recommended lines materials were validated through Nestlé proficiency tests
Recovery, %b performed by a significant number of internal and external
laboratories (between 12 and 45 depending on element and
Spiked element Concn, mg/La Without Csc With Cs 1% w/vd materials analyzed). Their reference values and associated
SDR values were calculated using robust statistics (28).
Calcium 60 93 ± 4 101 ± 4 (c) Protocol.—Three lots of each NIST material were
Copper 0.4 92 ± 3 105 ± 4 simply remixed in an amber container and sample portions of
Iron 2 92 ± 3 101 ± 4 minimum 10 g were then extracted from random locations in
Potassium 80 117 ± 18 105 ± 5 the container using a small weighing spatula and transferred
into numbered amber PVC 100 mL boxes (Greiner Bio-one,

Magnesium 20 95 ± 5 102 ± 6
Frickenhausen, Germany) for distribution to participants.
Manganese 0.01 95 ± 7 105 ± 6
Sample portions of minimum 10 g were extracted from
Sodium 40 110 ± 14 103 ± 5 random locations in each original P-test container of the
Phosphorus 40 90 ± 7 102 ± 4 three in-house references using a small weighing spatula and
Zinc 0.8 91 ± 7 103 ± 6 transferred into numbered amber PVC 100 mL boxes (Greiner
All elements
e
0.01–60 94 ± 9 103 ± 5 Bio-one) for distribution to participants. One set of five
numbered samples, including five amber 100 mL flasks
a
Concentration of spiked element in salt solution. containing 10 g materials, was mailed to nine collaborators
b
Average of spiked element recoveries in all food-grade salts who volunteered to participate in this study.
solutions.
c
(d) Stock standard solution.—Prepare working standards
Recovery value without Cs ion buffer addition.
d
Recovery value with Cs ion buffer 1% (w/v) addition.
either from a multistandard commercial stock standard
e
Median of all spiked elements recoveries in all food-grade salts. solution (equivalent to that described for SLV, MD1, and
MD2 tests) or from an intermediate stock standard solution
previously prepared with ICP-grade individual element 1000
and 10 000 mg/L solutions.
optimization or training, operating below the limit of (e) Intermediate stock standard solution.—(Composition
determination, or an unsatisfactory method. It is now one of of the intermediate standard solution, in mg/L: Ca = 1500; Cu
the acceptability criteria for many of the recently adopted = 10; Fe = 50; K = 2000; Mg = 500; Mn = 0.25; Na = 1000; P =
chemical methods analysis of AOAC INTERNATIONAL, 1000; Zn = 20.) Add into a 500 mL volumetric flask, 75 mL
the European Union, and other European organizations calcium 10 000 mg/L, 5 mL copper 1000 mg/L, 25 mL iron
dealing with food analysis (33). 1000 mg/L, 100 mL potassium, 25 mL magnesium
(j) Ruggedness.—Three replicate test portions (r = 3) 10 000 mg/L, 0.125 mL manganese 1000 mg/L, 50 mL
were collected from each of the six (n = 6) different food sodium 10 000 mg/L, 50 mL phosphorus 10 000 mg/L, and
samples populating three of the nine food triangle sectors, and 10 mL zinc 1000 mg/L. Add 10 mL of analytical grade HNO3
then were digested using different closed-vessel (MD1, MD3, and dilute to volume with H2O.
and MD4) and open-vessel (MD2) microwave digestion (f) Working standard solutions.—Standards prepared
systems (Table 1). Triplicates were analyzed for 8 days (d = 8) from intermediate stock standard solution are designed to
corresponding to nine series (i.e., nine elements) of 144 data have the same acid concentration as digested test solutions.
(n ´ r ´ d = 144), and using different ICP equipment with the (1) Std6.—Pipet 15.0 mL intermediate standard solution into a
recommended lines (except the use of alternate 2MnII and 100 mL acid-washed volumetric flask. Add 10 mL analytical
2
Zn I alternate lines for the MD4 test) according to adapted grade HNO3, dilute to volume with H2O, mix, and transfer to
ICP operating conditions used for SLV with addition of ion acid-washed polypropylene bottle. (2) Std5.—Pipet 10.0 mL
buffer Cs 0.1% (w/v). Recovery median values, SDr (within intermediate standard solution into a 100 mL acid-washed
day), SDiR (between day), and HorRat values were volumetric flask. Add 10 mL analytical grade HNO3, dilute to
determined. Accuracy was statistically evaluated. volume with H2O, mix, and transfer to acid-washed
Ring Trial polypropylene bottle. (3) Std4.—Pipet 5.0 mL intermediate
standard solution into a 100 mL acid-washed volumetric flask.
(a) Design of the study.—Five materials that cover four of Add 10 mL analytical grade HNO3, dilute to volume with
the nine sectors of AOAC food triangle (Table 2) were H2O, mix, and transfer to acid-washed polypropylene bottle.
proposed for testing the proposed ICP-AES method for (4) Std3.—Pipet 2.0 mL intermediate standard solution into a
determining the nine elements of interest in food matrixes 100 mL acid-washed volumetric flask. Add 10 mL analytical
after microwave digestion through an internal RT. grade HNO3, dilute to volume with H2O, mix, and transfer to
(b) Study material.—Five materials were samples of acid-washed polypropylene bottle. (5) Std2.—Pipet 1.0 mL
existing NIST SRM 2387 (peanut butter) and NIST RM 8418 intermediate standard solution into a 100 mL acid-washed
(wheat gluten), and three in-house reference samples of infant volumetric flask. Add 10 mL analytical grade HNO3, dilute to
volume with H2O, mix and transfer to acid-washed where x is the found mean value of analyte concentration in
polypropylene bottle. (6) Std1.—Pipet 0.5 mL intermediate the test material calculated from the nine means reported by
standard solution into a 100 mL acid-washed volumetric flask. the nine laboratories; X is the certified (or reference) value of
Add 10 mL analytical grade HNO3, dilute to volume with the certified (or in-house) reference material; RSDR is the
H2O, mix, and transfer to acid-washed polypropylene bottle. relative reproducibility standard deviation.
All calibration solutions should be stable for 1 week. If z £ 2, the result is satisfactory; if 2 < z < 3, the result is
(g) Test portion preparation.—Three single test portions questionable; if z > 3, the result is unsatisfactory.
were first extracted using a small weighing spatula from each (n) HorRat values.—HorRat values for each element in
of the five amber PVC 100 mL boxes (Greiner Bio-one) each of the five matrixes were calculated as follows:
containing 10 g of test sample (Table 2), then weighed, and
finally prepared as described for SLV before MCD or MDO RSDR , %
HorRat value =
microwave digestions. 2C-0.1505 , %

(h) Sample digestion.—All 15 test portions (i.e.,
triplicates extracted from each of the five test samples) were where RSDR is the relative reproducibility standard deviation
digested according to MCD or MDO programs adapted or of each element in each of the five tested matrixes; C is the
similar to those described for SLV (Mars Xpress and MDS relative concentration (expressed as dimensionless mass
2000 systems, respectively). Information was provided to fraction) of the analyte in question (valid for concentration
collaborators concerning the maximum concentration for each ratio above 10–9).
element in the five delivered food matrixes in order to The original data developed from interlaboratory studies
pre-adapt the final dilution of digest test portions before were assigned a HorRat value of 1.0 with limits of
ICP-AES analysis. acceptability of 0.5–2.0. Consistent deviations from the ratio
(i) Detection.—Digested test solutions, or appropriate on the low side (values <0.5) may indicate unreported
dilutions were presented to the ICP-AES instrument averaging or excellent training and experience; consistent
calibrated as described for SLV with acid-matched standard deviations on the high side (values >2) may indicate
calibrant solutions. inhomogeneity of the test samples, need for further method
(j) Calculations.—Element concentrations in the five optimization or training, operating below the LOD, or an
food matrixes were calculated using the equation described unsatisfactory method.
for SLV in the ICP Analysis Section, part (c). (o) Horwitz equation.—Horwitz equation expressed using
(k) Statistical calculations.—Medians, SDr, and SDiR, log scale (log SDR = 0.8495 log C 1.6991) was compared with
repeatability (r) and reproducibility (R) limits, z-scores, and a similar equation found using the 45 SDR values and medians
HorRat values for nine elements in five blind samples of concentration (i.e., SDR and median values for nine
prepared in triplicate were determined by a statistical elements in five matrixes) obtained from the data treatment of
treatment of 45 data sets. the RT.
(l) Repeatability and reproducibility limits.— Results and Discussion
Repeatability limit r, which is the maximum tolerated relative
variation between two measurements taken by a single person SLV
or instrument on the same matrix and under the same (a) Closed-vessel microwave digestion optimization.—
conditions, is expressed as follows: Average values of each element concentration in whole egg
powder, baby food composite, and baking chocolate fell
2.772 ´ SDr within confidence intervals of reference value for the three
matrixes and are in agreement with AOAC recovery criteria,
where SDr is the repeatability standard deviation. except for iron and copper in baking chocolate, with recovery
Reproducibility limit R is the maximum variation between values of 123 and 125%, respectively, and for phosphorus in
two measurements taken by several persons in different whole egg powder, with a recovery value of 116% (Table 6).
laboratories, on different days, under different conditions, and Significant variation of results for iron and copper in baking
is expressed as follows: chocolate have previously been observed (34). The higher
value of recovery for phosphorus in whole egg powder is due
2.772 ´ SDR to the use of the optimized microwave program in CEM Mars
Xpress, whereas generally lower values are obtained with a
where SDR is the reproducibility standard deviation. program leading to incomplete digestion (6, 8). This single
(m) Z-score.—Evaluation of the whole laboratory optimized microwave digestion program on a CEM Mars
performance in term of accuracy was obtained based on the Xpress system is adapted to be applicable to various matrixes
calculation of the z-score. The z-score “z” is given by the covering the AOAC food triangle.
following equation: (b) Linearity.—The calibration curves constructed by
plotting element concentration versus peak ratio response
x-X
z= (element/IS) showed good linearity either in linear or in
RSDR weighted nonlinear regression (Table 7). Weighted nonlinear
Table 11. SLV study: accuracy, precision, and HorRat values found for calcium in seven certified and in-house reference materials using recommended and
alternate lines
Reference Median ± U,
Material description Line value, mg/kga,b mg/kgc Recovery, % P-valued SDr, mg/kge SDiR, mg/kgf RSDr, % RSDiR, % HorRatg
Sterilized cream Cah 845 ± 18 756 ± 132 90 ± 5 0.07 72 125 9.5 16.5 2.8
i
Ca 726 ± 132 86 ± 5 0.02 67 112 9.3 15.4 2.6
Chocolate milk powder Cah 9870 ± 124 10092 ± 343 102 ± 2 0.21 135 303 1.3 3.0 0.7
i
Ca 9882 ± 179 100 ± 1 0.93 117 133 1.2 1.3 0.3
Infant cereals Cah 6000 ± 44 6189 ± 249 103 ± 1 0.05 151 219 2.4 3.5 0.8
i
Ca 5983 ± 215 100 ± 1 0.82 160 204 2.7 3.4 0.8
Corn bran Cah 420 ± 19 435 ± 29 104 ± 5 0.60 6 20.0 1.4 4.5 0.7
Cai 424 ± 24 101 ± 5 0.97 6 14 1.4 3.3 0.5
Dietetic milk powder 1 Cah 3020 ± 15 3047 ± 56 101 ± 1 0.35 33 49 1.1 1.6 0.3
i
Ca 2987 ± 54 99 ± 1 0.11 22 49 0.7 1.7 0.4
Pet food Cah 6190 ± 140 6580 ± 317 106 ± 3 0.05 94 192 1.4 2.9 0.7
i
Ca 6410 ± 212 104 ± 3 0.18 92 150 1.4 2.3 0.5
Nonfat milk powder Cah 13 000 ± 250 13070 ± 988 101 ± 3 0.86 206 908 1.6 6.9 1.8
i
Ca 12910 ± 813 99 ± 3 0.80 222 735 1.7 5.7 1.5
a
SD expressed as half of stated uncertainty (NIST and LGC certified materials) given as 95% confidence interval.
b
SD expressed as a reproducibility SD (in-house reference materials) calculated according to robust statistics (29).
c
Uncertainty (U) estimated through simplified approach (30, 31).
d
Statistic evaluation of robust t-test: Satisfactory result (P > 0.05); questionable result (0.01<P <0.05); unsatisfactory result (P < 0.01).
e
SDr = SD of repeatability.
f
SDiR = SD of intermediate reproducibility.
g
HorRat = Ratio using RSDiR and Horwitz denominator according to ref. 32.
h
Recommended line for ICP-AES analysis.
i

Table 12. SLV study: accuracy, precision, and HorRat values found for copper in three certified and in-house reference materials using recommended and
alternate lines
Corn bran Cuh 2.47 ± 0.20 2.69 ± 0.24 109 ± 9 0.36 0.05 0.09 1.8 3.1 0.2
Cui 2.61 ± 0.24 106 ± 9 0.53 0.05 0.1 1.9 3.9 0.3
i
Cu 2.56 ± 0.26 103 ± 9 0.7 0.06 0.15 2.4 5.6 0.4
Cui 2.52 ± 0.25 102 ± 8 0.82 0.06 0.14 2.5 5.4 0.4
Dietetic milk powder 1 Cuh 7.55 ± 0.13 7.78 ± 0.21 103 ± 2 0.15 0.09 0.15 1.1 2 0.2
Cui 7.63 ± 0.23 101 ± 2 0.6 0.08 0.18 1 2.4 0.2
i
Cu 7.72 ± 0.25 102 ± 2 0.28 0.08 0.2 1 2.6 0.2
Cui 7.58 ± 0.21 100 ± 2 0.84 0.08 0.15 1 2 0.2
Nonfat milk powder Cuh 0.70 ± 0.05 0.75 ± 0.08 107 ± 8 0.44 0.06 0.06 8.3 8.5 0.5
Cui 0.78 ± 0.11 112 ± 9 0.22 0.06 0.09 8.3 11.4 0.7
Cui 0.78 ± 0.09 112 ± 8 0.21 0.05 0.07 7 8.5 0.5
Cui 0.79 ± 0.10 113 ± 9 0.17 0.05 0.08 6.8 10.2 0.6
a
b
c
d
Statistic evaluation of robust t-test: Satisfactory result (P > 0.05); questionable result (0.01 < P < 0.05); unsatisfactory result (P < 0.01).
e
f
g
h
i

Table 13. SLV study: accuracy, precision, and HorRat values found for iron in four certified and in-house reference materials using recommended and alternate
lines
Chocolate milk powder Feh 169.9 ± 2.4 166.9 ± 12.0 98 ± 2 0.33 13.2 11.7 7.9 7.0 1.0
i
Fe 163.1 ± 12.4 96 ± 2 0.10 13.1 12.2 7.9 7.3 0.8
Fei 166.6 ± 13.1 98 ± 2 0.35 13.3 12.7 8.0 7.6 1.2
h
Infant cereals Fe 80.4 ± 0.6 79.6 ± 1.3 99 ± 1 0.28 1.0 1.1 1.2 1.4 1.0
Fei 79.0 ± 1.2 98 ± 1 0.09 1.0 1.1 1.3 1.3 0.7
Fei 79.5 ± 1.3 99 ± 1 0.25 1.1 1.2 1.3 1.4 0.3
Corn bran Feh 14.8 ± 0.9 14.7 ± 1.3 99 ± 6 0.88 0.2 1.0 1.3 6.7 0.2
i
Fe 14.7 ± 1.2 99 ± 6 0.92 0.2 0.8 1.3 5.4 0.3
Fei 14.7 ± 1.1 99 ± 6 0.88 0.2 0.7 1.4 4.8 0.3
h
Dietetic milk powder 1 Fe 58.5 ± 0.4 59.3 ± 0.9 101 ± 1 0.11 0.8 0.8 1.3 1.3 0.2
Fei 59.1 ± 0.9 101 ± 1 0.24 0.7 0.8 1.1 1.4 0.3
Fei 59.1 ± 1.0 101 ± 1 0.26 0.7 0.9 1.1 1.5 0.4
a
b
c
d
e
f
g
h
i

regression was used during SLV, MD1, and MD2 tests, as the
best regression coefficients are obtained with R2 > 0.99995 for
HorRatg
2.0
1.1
1.7
1.4
0.6
1.5
all lines except for 1K766.491 (R2 = 0.99924) and for
Table 14. SLV study: accuracy, precision, and HorRat values found for potassium in six certified and in-house reference materials using recommended and
2
Ca317.933 (R2 = 0.99994) lines.
(c) LOD and LOQ.—The detection and quantification
limits of elements in real matrixes were estimated in Table 8
RSDiR, %
by affecting a dilution factor of 100 (i.e., corresponding to
4.6
7.1
8.6
2.8
5.6
10.5
0.5 g digest sample diluted to 50 mL solution after MDC and
before ICP-AES analysis) to the DL and QL, as satisfactory
trueness/RSDr and RSDiR values were found for elements
present in real matrixes at these concentrations (e.g., Ca, Cu,
RSDr, %
Fe, K, Na, P in corn bran; Mg, Zn in sterilized cream, and Mn

3.9
1.4
1.7
1.8
1.0
1.9
in nonfat milk powder).
(d) Selectivity.—Element mean recoveries and associated
SD values calculated from spiked element of interest in eight
SDiR, mg/kgf
different solutions of food-grade salts are displayed in
Table 9. Good recoveries ranging between 88 and 115% were
146
399
454
154
958
46
found in all food-grade salt solutions with SDs ranging
Statistic evaluation of robust t-test: Satisfactory result (P > 0.05); questionable result (0.01<P < 0.05); unsatisfactory result (P < 0.01).
between 0.1 and 18.8% for spiking that ranged between 0.01
and 60 mg/L, respectively. Comparative t-tests have shown
that recovery and SD values obtained from all averages of
SDr, mg/kge
element spiked duplicates in all food-grade salt solutions with
125
108
322
54
10
54
and without added ion buffer Cs 1% (w/v) are different at 95%
confidence with better recoveries and SD values (103 ± 5%)
using ion buffer Cs 1% (w/v; Table 10).
(e) Accuracy.—Statistic treatment of accuracy values
P-valued
0.02
0.03
0.66
0.70
0.88
0.77
(trueness/recovery) displayed in Tables 11–19 for seven
matrixes tested has shown global satisfactory results (i.e.,
P-value >0.05: recovery not significantly different from
100%, at 95% level of confidence interval). Questionable
Recovery, %
recoveries (i.e., 0.01 < P-value <0.05: recovery statistically

101 ± 1
101 ± 2
96 ± 2
99 ± 3
94 ± 7
120 ± 7
different from 100% at 95% level of confidence interval, but

not significantly different from 100% at 99% confidence
interval) were found for Ca and K in sterilized cream (86 ± 5%
and 120 ± 7%, respectively) using 2CaII and 1KI lines, for Mg
17008 ± 1342
Median ± U,
6423 ± 485
5600 ± 175
1395 ± 203
8726 ± 468
and Zn in dietetic milk powder 1 (98 ± 1% and 97 ± 1%,

531 ± 60
mg/kgc
respectively) using 3Mg, 1ZnI, and 3ZnI lines and for P in

infant cereals (104 ± 1%) using 1P line. AOAC requirements
for recovery are, however, globally fulfilled for all elements in

all matrixes (recovery range 79–120%) for concentrations
value, mg/kga,b
16900 ± 150
9090 ± 133
between 0.26 mg/kg (Mn) and 16 900 mg/kg (K). Only

1160 ± 50
6500 ± 50
5570 ± 25
Reference
566 ± 38
recovery values for 2CaII and 1KI lines in sterilized cream

showed unacceptable values by AOAC criteria, as previously
demonstrated width statistic recovery test at 95% confidence.
(f) Precision.—SDr and SDiR values, respectively,

displayed in Tables 13–21 for seven matrixes tested fulfill
Line
AOAC criteria regarding RSDr and RSDiR values for all

Kh
h
Kh
h
Kh
h
K
K
K
elements in all matrixes ranging between 0.6 and 16.8% and

between 0.9 and 23.3%, respectively, for concentrations

between 0.26 mg/kg (Mn) and 16 900 mg/kg (K).
Chocolate milk powder
Dietetic milk powder 1

Material description
(g) Uncertainty.—Overall uncertainty measurements

Nonfat milk powder
alternate lines
Sterilized cream
(±U) are displayed in Tables 11–19 with medians obtained

Infant cereals
through reproducibility conditions as combinations of

Corn bran
precision (SDiR) and trueness (SDrec) contributions.

Calculated derived relative U values range from 1 to 26% for
all elements in the seven tested matrixes.
a
h
c
f
Table 15. SLV study: accuracy, precision, and HorRat values found for magnesium in seven certified and in-house reference materials using recommended and
alternate lines
Material description Line value, mg/kga, b mg/kgc Recovery, % P-valued SDr, mg/kge SDiR, mg/kgf RSDr, % RSDiR, % HorRatg
Sterilized cream Mgh 84.4 ± 2.0 81.7 ± 4.5 97 ± 3 0.28 2.0 3.9 2.4 4.7 0.6
i
Mg 79.9 ± 4.2 95 ± 3 0.08 1.7 3.6 2.1 4.5 0.5
Mgi 82.7 ± 4.4 98 ± 3 0.48 2.3 3.8 2.7 4.6 0.6
h
Chocolate milk powder Mg 1779 ± 23 1771 ± 22 100 ± 1 0.82 23 32 1.3 1.8 0.3
Mgi 1757 ± 67 99 ± 2 0.48 22 59 1.3 3.4 0.7
Mgi 1744 ± 54 98 ± 2 0.22 21 47 1.2 2.7 0.5
Infant cereals Mgh 881 ± 8 911 ± 46 103 ± 2 0.10 25 43 2.7 4.7 0.8
i
Mg 902 ± 43 102 ± 2 0.22 21 40 2.3 4.5 0.8
Mgi 892 ± 48 101 ± 2 0.53 24 46 2.7 5.1 0.9
h
Corn bran Mg 818 ± 30 791 ± 45 97 ± 4 0.53 9 35 1.1 4.4 0.8
Mgi 775 ± 45 95 ± 4 0.25 8 33 1.0 4.2 0.7
i
Mg 785 ± 40 96 ± 4 0.41 9 26 1.1 3.3 0.6
Dietetic milk powder 1 Mgh 1200 ± 8 1206 ± 25 101 ± 1 0.88 8.0 23 0.7 1.9 0.3
Mgi 1180 ± 30 98 ± 1 0.06 7.0 28 0.6 2.3 0.4
Mgi 1181 ± 34 98 ± 1 0.03 9 29 0.8 2.5 0.5
Pet food Mgh 481 ± 2 483 ± 8 101 ± 1 0.31 7 8 1.4 1.6 0.3
Mgi 471 ± 12 98 ± 1 0.08 6 11 1.2 2.4 0.4
i
Mg 477 ± 12 99 ± 1 0.52 8 11 1.6 2.3 0.4
Nonfat milk powder Mgh 1200 ± 15 1206 ± 89 101 ± 3 0.87 18 83 1.5 6.9 1.3
i
Mg 1186 ± 70 99 ± 2 0.60 19 65 1.6 5.5 1.0
Mgi 1156 ± 87 96 ± 3 0.21 16 81 1.4 7.0 1.3
a
b
c
d
e
f
g
h
i

Table 16. SLV study: accuracy, precision, and HorRat values found for manganese in three certified and in-house reference materials using recommended and
alternate lines
Corn bran Mnh 2.55 ± 0.15 2.33 ± 0.18 91 ± 6 0.15 0.03 0.12 1.1 4.9 0.4
Mni 2.55 ± 0.25 100 ± 6 0.96 0.03 0.20 1.1 7.3 0.5
Mni 2.39 ± 0.17 94 ± 6 0.25 0.03 0.09 1.1 3.6 0.3
Dietetic milk powder 1 Mnh 12.7 ± 0.85 12.8 ± 1.1 102 ± 7 0.83 0.27 0.68 2.1 5.3 0.5
Mni 13.4 ± 1.3 105 ± 7 0.48 0.26 0.88 2.0 6.6 0.6
Mni 12.8 ± 1.1 101 ± 7 0.88 0.24 0.67 1.8 5.2 0.5
h
Nonfat milk powder Mn 0.26 ± 0.03 0.22 ± 0.03 85 ± 10 0.16 0.01 0.02 3.1 6.8 0.3
Mni 0.20 ± 0.03 79 ± 9 0.06 0.01 0.02 3.8 10.8 0.5
Mni 0.22 ± 0.03 85 ± 10 0.08 0.01 0.01 3.6 5.9 0.3
a
b
c
d
e
f
g
h
i

Table 17. SLV study: accuracy, precision, and HorRat values found for sodium in six certified and in-house reference materials using recommended and alternate
(h) HorRat values.—HorRat values never exceed 1.5
HorRatg
except for several analytical lines of Ca (2.8 and 2.6), K (2.0),
1.0
1.8
0.6
0.7
0.4
1.4
Na (1.8), P (1.6), and Zn (1.6, 1.7) in sterilized cream, of Ca
(1.8), P (1.9, 2.2, 1.6) in nonfat milk powder, and of K in
infant cereals (1.7). However, the reliability and applicability
of the method is proved, as AOAC criteria are fulfilled with no
RSDiR, %
values higher than 3.0. If several values are between 0.2 and
10.9
3.0
5.7
4.4
1.6
6.1
0.5, this can be explained by experience, training of analysts, a
good knowledge of tested matrixes, and careful application.
(i) Ruggedness.—Accuracy (SDrec) and intermediate
precision (SDiR) results are displayed in Tables 20–28 for the
RSDr, %
six matrixes tested. Global statistic recovery results were

3.4
1.6
3.2
1.5
0.8
1.3
satisfactory for all elements in all matrixes whatever the
digestion systems and ICP-AES equipments used.
Unsatisfactory statistic results (P <0.01) were found mainly
for the infant cereals matrix: Ca for MD1 and MD3, Fe for
SDiR, mg/kgf
MD4, K for MD1, Mg for MD1 and MD3, and Na for MD4.
297
59
60
19
79
6
Other unsatisfactory results concern Ca in corn bran for MD4,
Cu in nonfat milk powder for MD3, Fe in dietetic milk
powder 1 and in corn bran for MD4, K in dietetic milk
powder 1 for MD1, P in corn bran for MD4, and Zn in dietetic
SDr, mg/kge
milk powder for MD1. Unsatisfactory statistical recovery
6
32
34
40
65
2
results mainly concern infant cereals matrix due to relative
heterogeneity of the used batch and corn bran matrix due to
whole element concentration values close to QL. These
statistical unsatisfactory results have shown that general
P-valued
applicability of the method is limited by less analytical
0.13
0.37
0.89
0.40
0.20
0.61
experience and competency regarding sample preparation
(e.g., sample homogeneity and adapted digestion program)
and ICP analysis (adapted ion buffering and use of robust
conditions).
Recovery, %
However, AOAC requirements for recovery performance

110 ± 6
100 ± 2
98 ± 2
99 ± 4
99 ± 1
99 ± 1
are globally fulfilled for all elements in all matrixes between
74 and 118% for element concentrations from 0.26 mg/kg
(Mn) to 16 900 mg/kg (K). Only specific recovery values of
Ca (75%), Fe (72%), and P (68%) in corn bran for MD4; of K
Median ± U,
4910 ± 321
1950 ± 74
1054 ± 64
4890 ± 87
554 ± 67
424 ± 26
(81 and 82%) for MD1 and MD2, respectively; recovery

mg/kgc
values of Cu (67 and 74%) in nonfat milk powder and dietetic

milk powder for MD3 and MD4, respectively; and recovery
value of Ca (113%) in infant cereals for MD1 showed

unacceptable values regarding AOAC criteria, as already
value, mg/kga,b
1990 ± 42
1050 ± 14
4920 ± 30
4970 ± 50
Reference
505 ± 19
430 ± 16
demonstrated for statistic recovery test at 95% confidence.

AOAC requirements for RSDr and SDiR were fulfilled for
all elements in all matrixes between 0.6 and 18.9% and 0.9
and 23.3%, respectively, for concentrations from 0.26 mg/kg
(Mn) to 16 900 mg/kg (K).

These ruggedness tests have shown good robustness of this
Line
NAh
NAh
h
NAh
NAh
NAh
NA
method applied by different operators (at least two operators

per test of eight analytical series) after open- and closed-vessel
digestions on different ICP-AES equipments with axial,

radial, and dual view grating configurations using Cs 0.1%
(w/v) as minimal ionization buffer concentration.

Material description
Nonfat milk powder

Sterilized cream
Ring Trial
Infant cereals
Corn bran
(a) Calcium.—Table 29 presents estimated performance

lines
characteristics of the ICP-AES method for determination of

Ca, Cu, Fe, K, Mg, Mn, Na, P, and Zn. The RSDr of the
a
h
c
f
Table 18. SLV study: accuracy, precision, and HorRat values found for phosphorus in seven certified and in-house reference materials using recommended and
alternate lines
Reference Median ± U ,
Sterilized cream Ph 823 ± 22 807 ± 60 98 ± 3 0.56 32 53 4.0 6.6 1.1

Pi 821 ± 56 99 ± 3 0.95 36 49 4.4 6.0 1.0
i
P 783 ± 82 95 ± 4 0.26 32 76 4.1 9.6 1.6
Chocolate milk powder Ph 4080 ± 30 4110 ± 150 101 ± 1 0.60 67 138 1.6 3.4 0.7
i
P 4150 ± 88 102 ± 1 0.61 60 70 1.5 1.7 0.4
Pi 4050 ± 85 99 ± 1 0.59 60 70 1.6 1.8 0.4
Infant cereals Ph 4320 ± 21 4470 ± 165 104 ± 1 0.02 53 136 1.2 3.1 0.7
Pi 4510 ± 297 104 ± 2 0.10 77 279 1.7 6.2 1.4
Pi 4340 ± 116 100 ± 1 0.69 63 110 1.5 2.5 0.6
Corn bran Ph 171 ± 6 163 ± 18 95 ± 4 0.86 2 17 1.4 10.4 1.4
Pi 166 ± 9 97 ± 4 0.44 2 7 1.3 4.4 0.6
Pi 162 ± 8 95 ± 3 0.17 3 5 1.6 3.2 0.4
h
Dietetic milk powder 1 P 3080 ± 11 3100 ± 65 101 ± 1 0.27 28 58 0.9 1.9 0.4
Pi 3120 ± 68 101 ± 1 0.12 37 63 1.2 2.0 0.4
Pi 3080 ± 43 100 ± 1 0.65 21 39 0.7 1.3 0.3
Pet food Ph 4510 ± 40 4630 ± 122 103 ± 1 0.05 48 92 1.0 2.0 0.4
i
P 4620 ± 176 102 ± 2 0.16 63 72 1.5 1.7 0.4
Pi 4490 ± 88 100 ± 1 0.67 61.0 78 1.3 1.7 0.4
Nonfat milk powder Ph 10600 ± 100 10830 ± 845 102 ± 3 0.46 169 788 1.6 7.3 1.9
Pi 10840 ± 981 102 ± 3 0.50 200 922 1.8 8.5 2.2
Pi 10500 ± 707 99 ± 2 0.71 144 657 1.4 6.3 1.6
a
b
c
d
Statistic evaluation of robust t-test: Satisfactory result (P > 0.05); questionable result (0.01< P < 0.05); unsatisfactory result (P < 0.01).
e
f
g
h
i

Table 19. SLV study: accuracy, precision, and HorRat values found for zinc in four certified and in-house reference materials using recommended and alternate
lines
Sterilized cream Znh 3.1 ± 0.2 2.6 ± 0.6 83 ± 7 0.05 0.4 0.60 16.9 22.5 1.6
i
Zn 2.7 ± 0.6 88 ± 7 0.14 0.5 0.50 16.7 19.3 1.4
Zni 2.6 ± 0.7 82 ± 8 0.05 0.4 0.60 16.8 23.3 1.7
h
Corn bran Zn 18.6 ± 1.1 17.6 ± 1.3 95 ± 6 0.38 0.8 2.1 1.8 4.5 0.4
Zni 17.8 ± 1.4 96 ± 6 0.50 0.2 0.9 1.2 5.0 0.4
Zni 17.5 ± 1.3 94 ± 6 0.35 0.2 0.7 1.4 4.3 0.4
Dietetic milk powder 1 Znh 74.8 ± 0.6 72.8 ± 1.4 97± 1 0.02 0.5 0.6 0.6 0.9 0.1
Zni 73.9 ± 1.7 99 ± 1 0.24 0.8 1.6 1.1 2.1 0.3
Zni 72.7 ± 1.3 97 ± 1 0.01 0.5 0.5 0.7 0.7 0.1
h
Nonfat milk powder Zn 46.1 ± 1.1 45.6 ± 2.4 99 ± 3 0.68 0.8 2.1 1.8 4.5 0.5
Zni 44.1 ± 2.4 96 ± 3 0.14 0.8 2.1 1.8 4.7 0.5
Zni 45.2 ± 2.1 98 ± 3 0.51 0.8 1.8 1.7 3.9 0.4
a
b
c
d
e
f
g
h
i

Table 20. MD1–4 ruggedness tests: accuracy, precision, and HorRat values found for calcium in six certified and in-house reference materials using
recommended lines
Reference Median,
Material description b-test codea value, mg/kgb,c mg/kg Recovery, % P-valued SDr, mg/kge SDiR, mg/kgf RSDr, % RSDiR, % HorRatg
Chocolate milk powder MD1 9870 ± 124 9915 100 ± 1 0.92 91 171 0.9 1.7 0.3
MD2 9913 100 ± 2 0.93 97 250 1.0 2.5 0.4
MD3 9697 98 ± 1 0.20 155 250 1.6 2.6 0.5
MD4 10042 101 ± 2 0.36 68 211 0.7 2.1 0.4
Infant cereals MD1 6000 ± 44 6752 113 ± 3 0.003 337 526 5.0 7.7 0.8
MD2 6027 100 ± 1 0.80 111 270 1.8 4.4 0.5
MD3 5764 96 ± 1 0.01 89 148 1.5 2.6 0.3
MD4 5731 96 ± 2 0.03 158 277 2.8 4.8 0.3
Corn bran MD1 420 ± 19 374 89 ± 6 0.09 13 46 3.3 12.2 1.3
MD2 405 96 ± 6 0.55 7 44 1.6 11.0 1.2
MD3 411 98 ± 5 0.71 4 37 1.1 8.9 1.0
MD4 313 75 ± 5 0.002 9 51 2.8 16.1 1.7
Dietetic milk powder 1 MD1 3020 ± 15 3062 101 ± 1 0.07 33 43 1.1 1.4 0.2
MD2 3031 100 ± 1 0.77 26 95 0.9 3.1 0.5
MD3 2958 98 ± 2 0.23 30 130 1.0 4.4 0.7
MD4 2986 99 ± 1 0.21 43 66 1.4 2.2 0.3
Pet food MD1 6190 ± 140 6365 103 ± 2 0.29 83 144 1.3 2.3 0.4
MD2 6457 104 ± 3 0.15 80 226 1.2 3.5 0.6
MD3 6198 100 ± 3 0.96 151 251 2.4 4.1 0.7
MD4 6384 103 ± 3 0.25 176 210 2.7 3.0 0.5
Nonfat milk powder MD1 13000 ± 250 13012 100 ± 2 0.97 93 192 0.7 1.5 0.3
MD2 12931 100 ± 2 0.81 170 377 1.3 2.9 0.5
MD3 12612 97 ± 2 0.17 113 220 0.9 1.7 0.3
MD4 12845 99 ± 2 0.57 157 257 1.2 2.0 0.4
a
Ruggedness test using different microwave digestion systems and ICP-AES equipment (Table 1).
b
c
d
e
f
g

Table 21. MD1–4 ruggedness tests: accuracy, precision, and HorRat values found for copper in three certified and in-house reference materials using
recommended lines
Reference Median,
Corn bran MD1 2.47 ± 0.20 2.39 97 ± 9 0.74 0.06 0.32 2.7 13.1 0.7
MD2 2.39 97 ± 11 0.78 0.06 0.55 2.5 22.9 1.2
MD3 2.27 92 ± 8 0.32 0.07 0.17 3.0 7.5 0.4
MD4 2.47 100 ± 10 0.99 0.11 0.12 4.3 4.7 0.2
Dietetic milk powder 1 MD1 7.55 ± 0.13 7.73 102 ± 2 0.29 0.33 0.40 4.3 5.3 0.3
MD2 7.59 101 ± 2 0.83 0.06 0.35 0.8 4.6 0.3
MD3 7.61 101 ± 2 0.76 0.10 0.35 1.3 4.6 0.3
MD4 7.79 103 ± 2 0.20 0.30 0.38 3.9 5.0 0.3
Nonfat milk powder MD1 0.70 ± 0.05 0.52 74 ± 20 0.23 0.08 0.41 13.1 67.6 2.7
MD2 0.59 84 ± 21 0.46 0.04 0.39 6.0 67.0 2.8
MD3 0.47 67 ± 8 0.004 0.06 0.13 12.8 29.6 1.2
MD4 0.74 105 ± 27 0.85 0.13 0.52 18.9 73.6 3.1
a
b
c
d
e
f
g

Table 22. MD1–4 ruggedness tests: accuracy, precision, and HorRat values found for iron in four certified and in-house reference materials using recommended
lines
Reference Median,
Chocolate milk powder MD1 169.9 ± 2.4 167.7 99 ± 2 0.48 1.7 5.2 1.0 3.1 0.3
MD2 164.8 97 ± 2 0.10 1.4 3.9 0.8 2.4 0.2
MD3 173.6 102 ± 2 0.22 2.1 3.8 1.2 2.2 0.2
MD4 169.9 100 ± 4 0.99 9.3 20.9 5.4 12.2 1.2
Infant cereals MD1 80.4 ± 0.6 79.9 99 ± 2 0.72 3.0 3.8 3.7 4.7 0.4
MD2 77.8 97 ± 1 0.02 1.8 2.0 2.3 2.6 0.2
MD3 77.7 97 ± 2 0.05 1.3 2.7 1.7 3.5 0.3
MD4 72.8 91 ± 2 0.005 3.2 5.5 4.3 7.5 0.6
Corn bran MD1 14.8 ± 0.9 12.5 85 ± 7 0.05 0.3 1.8 2.6 13.8 0.9
MD2 13.9 94 ± 6 0.31 0.9 0.9 6.2 6.1 0.4
MD3 12.4 84 ± 6 0.03 0.4 1.3 2.8 10.7 0.7
MD4 10.7 72 ± 6 0.002 0.5 1.7 4.9 15.9 1.0
MD2 57.6 98 ± 1 0.24 0.7 1.8 1.2 3.1 0.3
MD3 57.8 99 ± 1 0.31 1.0 1.8 1.8 3.2 0.3
MD4 56.3 96 ± 1 0.10 0.9 1.6 1.6 2.9 0.2
a
b
c
d
Statistic evaluation of robust t-test: Satisfactory result (P > 0.05); questionable result (0.01 <P < 0.05); unsatisfactory result (P < 0.01).
e
f
g

Table 23. MD1–4 ruggedness tests: accuracy, precision, and HorRat values found for potassium in five certified and in-house reference materials using
recommended lines
Reference value,
Material description b-test codea mg/kgb,c Median, mg/kg Recovery, % P-valued SDr, mg/kge SDiR, mg/kgf RSDr, % RSDiR, % HorRatg
MD2 8915 98 ± 2 0.34 71 317 0.8 3.6 0.6
MD3 9025 99 ± 2 0.65 65 130 0.7 1.4 0.2
MD4 9031 99 ± 2 0.69 39 153 0.4 1.7 0.3
MD2 6450 99 ± 1 0.56 80 180 1.2 2.8 0.5
MD3 6360 98 ± 1 0.06 50 120 0.8 1.9 0.3
MD4 6260 96 ± 1 0.03 100 210 1.6 3.3 0.5
Corn bran MD1 566 ± 38 460 81 ± 7 0.02 15 59 3.3 12.6 1.4
MD2 463 82 ± 6 0.02 10 41 2.1 8.8 1.0
MD3 538 95 ± 7 0.48 7 35 1.4 6.3 0.7
MD4 561 99 ± 8 0.91 13 73 2.4 13.1 1.5
MD2 5633 101 ± 1 0.32 65 158 1.1 2.8 0.5
MD3 5561 100 ± 1 0.83 30 99 0.5 1.8 0.3
MD4 5640 101 ± 1 0.08 32 75 0.6 1.3 0.2
MD2 16230 96 ± 1 0.01 220 450 1.4 2.8 0.3
MD3 16580 98 ± 1 0.10 80 250 0.5 1.5 0.3
MD4 16730 99 ± 1 0.30 220 230 1.3 1.4 0.9
a
b
c
d
e
f
g

Table 24. MD1–4 ruggedness tests: accuracy, precision, and HorRat values found for magnesium in six certified and in-house reference materials using
recommended lines
Reference value,
Material description b-test codea mg/kgb,c Median, mg/kg Recovery, % P-valued SDr, mg/kge SDiR, mg/kgf RSDr, % RSDiR, % HorRatg
MD2 1780 100 ± 2 0.97 18 74 1.0 4.1 0.6
MD3 1789 101 ± 1 0.69 24 25 1.3 1.4 0.2
MD4 1770 100 ± 1 0.72 11 33 0.6 1.8 0.2
MD2 898 102 ± 2 0.29 17 37 1.9 4.1 0.5
MD3 922 105 ± 1 0.004 21 22 2.2 2.4 0.3
MD4 893 101 ± 1 0.33 19 26 2.1 2.9 0.4
Corn bran MD1 818 ± 30 778 95 ± 3 0.20 9 11 1.1 1.4 0.2
MD2 749 92 ± 3 0.04 14 16 1.8 2.1 0.3
MD3 778 95 ± 4 0.21 7 15 1.0 1.9 0.2
MD4 761 93 ± 3 0.08 5 9 0.7 1.2 0.1
MD2 1198 100 ± 1 0.79 8 44 0.7 3.7 0.5
MD3 1216 101 ± 1 0.21 5 17 0.4 1.4 0.2
MD4 1193 99 ± 1 0.40 12 24 1.0 2.0 0.3
Pet food MD1 48 ± 2 462 96 ± 3 0.17 7 8 1.5 1.8 0.2

MD2 474 99 ± 3 0.67 4 14 0.9 3.0 0.3
MD3 472 98 ± 3 0.55 6 12 1.3 2.5 0.3
MD4 456 95 ± 3 0.12 7 21 1.5 4.6 0.5
MD2 1194 100 ± 2 0.80 14 53 1.2 4.4 0.6
MD3 1185 99 ± 1 0.37 9 16 0.8 1.3 0.2
MD4 1172 98 ± 1 0.12 11 21 0.9 1.8 0.2
a
b
c
d
Statistic evaluation of robust t-test: Satisfactory result (P > 0.05); questionable result (0.01< P < 0.05); unsatisfactory result (P < 0.01).
e
f
g

Table 25. MD1–4 ruggedness tests: accuracy, precision, and HorRat values found for manganese in three certified and in-house reference materials using
recommended lines (MD1, MD2, MD3) and alternate 2MnII line (MD4)
Reference
Material description b-test codea value, mg/kgb,c Median, mg/kg Recovery, % P-valued SDr, mg/kge SDiR, mg/kgf RSDr, % RSDiR, % HorRatg
Corn bran MD1 2.55 ± 0.15 2.36 93 ± 6 0.22 0.03 0.05 1.1 2.1 0.1
MD2 2.27 89 ± 5 0.08 0.04 0.08 1.9 3.5 0.2
MD3 2.17 85 ± 5 0.02 0.04 0.08 2.0 3.7 0.2
MD4 2.37 93 ± 6 0.23 0.03 0.04 1.4 1.7 0.1
MD2 12.31 97 ± 7 0.65 0.12 0.22 0.9 1.7 0.1
MD3 11.71 92 ± 6 0.25 0.12 0.22 1.0 1.8 0.1
MD4 12.77 101 ± 7 0.93 0.16 0.14 1.2 1.1 0.1
MD2 0.23 90 ± 11 0.37 0.01 0.02 4.9 6.9 0.2
MD3 0.31 118 ± 14 0.25 0.02 0.03 6.7 9.7 0.4
MD4 0.21 82 ± 10 0.10 0.01 0.01 4.8 4.3 0.2
a
b
c
d
e
f
g

Table 26. MD1–4 ruggedness tests: accuracy, precision, and HorRat values found for sodium in five certified and in-house reference materials using
recommended lines
Reference
Material description b-test codea value, mg/kgb,c Median, mg/kg Recovery, % P-valued SDr, mg/kge SDiR, mg/kgf RSDr, % RSDiR, % HorRatg
MD2 2000 101 ± 3 0.86 42 99 2.1 4.9 0.7
MD3 1981 100 ± 2 0.85 29 68 1.5 3.4 0.5
MD4 1949 98 ± 2 0.36 14 34 0.7 1.7 0.2
MD2 1002 95 ± 2 0.04 21 42 2.1 4.2 0.5
MD3 970 92 ± 3 0.01 12 59 1.3 6.1 0.8
MD4 935 89 ± 3 0.002 23 63 2.5 6.8 0.8
Corn bran MD1 430 ± 16 395 92 ± 3 0.05 4 10 1.1 2.4 0.3
MD2 395 92 ± 4 0.08 4 27 1.1 6.8 0.7
MD3 391 91 ± 5 0.09 10 40 2.5 1.0 0.1
MD4 388 90 ± 4 0.03 4 19 1.0 5.0 0.5
MD2 4870 99 ± 1 0.31 40 110 0.8 2.4 0.4
MD3 4910 100 ± 1 0.81 32 122 0.6 2.5 0.4
MD4 5000 102 ± 1 0.09 39 77 0.8 1.5 0.2
MD2 4940 99 ± 1 0.60 62 106 1.3 2.2 0.4
MD3 4810 97 ± 1 0.02 66 98 1.4 2.0 0.3
MD4 4930 99 ± 1 0.51 75 110 1.5 2.2 0.4
a
b
c
d
e
f
g

Table 27. MD1–4 ruggedness tests: accuracy, precision, and HorRat values found for phosphorus in six certified and in-house reference materials using
recommended lines
Reference Median,
MD2 4150 102 ± 1 0.28 62 150 1.5 3.6 0.6
MD3 4140 101 ± 1 0.14 30 49 0.7 1.2 0.2
MD4 4170 102 ± 1 0.07 36 86 0.9 2.0 0.3
MD2 4508 104 ± 1 0.01 75 161 1.7 3.6 0.6
MD3 4384 102 ± 1 0.10 57 87 1.3 2.0 0.3
MD4 4321 100 ± 1 0.97 77 97 1.8 2.3 0.4
Corn bran MD1 171 ± 6 151 88 ± 6 0.06 4 23 2.7 15.1 1.4
MD2 163 95 ± 6 0.45 4 24 2.4 15 1.4
MD3 169 99 ± 4 0.30 2 9 1.0 5.3 0.5
MD4 116 68 ± 6 0.001 4 27 3.3 23.4 2.1
MD2 3170 103 ± 1 0.08 27 123 0.8 3.9 0.6
MD3 3133 102 ± 1 0.06 17 61 0.5 1.9 0.3
MD4 3111 101 ± 1 0.21 45 67 1.4 2.2 0.3
Pet food MD1 4510 ± 40 449 100 ± 1 0.60 28 49 0.6 1.1 0.2
MD2 464 103 ± 1 0.11 30 173 0.6 3.7 0.6
MD3 454 101 ± 1 0.56 36 78 0.8 1.7 0.3
MD4 453 101 ± 1 0.60 66 84 1.4 1.9 0.3
MD2 1088 103 ± 2 0.12 156 367 1.4 3.4 0.6
MD3 1044 99 ± 1 0.20 81 153 0.8 1.5 0.3
MD4 1040 98 ± 1 0.12 66 176 0.6 1.7 0.3
a
b
c
d
e
f
g

Table 28. MD1–4 ruggedness tests: accuracy, precision, and HorRat values found for zinc in three certified and in-house reference materials using recommended
lines (MD1, MD2, MD3) and alternate 2ZnI line (MD4 )
Reference Median,
Corn bran MD1 18.6 ± 1.1 17.6 95 ± 6 0.38 0.2 0.5 1.1 2.8 0.2
MD2 17.0 92 ± 5 0.17 0.2 0.3 1.4 1.7 0.1
MD3 17.5 94 ± 6 0.38 0.5 1.4 3.1 8.3 0.6
MD4 18.3 98 ± 7 0.83 0.8 2.4 4.5 13.6 0.9
MD2 72.6 97 ± 1 0.03 0.6 1.8 0.9 2.4 0.2
MD3 75.3 101 ± 3 0.78 1.0 5.0 1.3 6.7 0.6
MD4 76.3 102 ± 1 0.12 1.2 1.9 1.6 2.5 0.2
MD2 44.8 97 ± 2 0.28 0.7 0.9 1.6 2.0 0.2
MD3 45.1 98 ± 2 0.38 0.7 0.9 1.6 2.0 0.2
MD4 46.4 101 ± 3 0.83 0.9 2.1 1.9 4.7 0.4
a
b
c
d
e
f
g

method for the determination of Ca ranged between 1.1% acceptable precision for the determination of Mg in all
(dietetic milk powder) and 3.9% (infant cereals). The RSDR foodstuffs analyzed with Mg concentration above the
ranged between 2.9% (chocolate milk powder) and 5.0% estimated QL. The trueness as z-score (Table 30) is calculated
(peanut butter). The HorRat value should not exceed 1.5. to range from –1.8 (wheat gluten) to 0.6 (peanut butter). The
Table 29 shows that the present method has an acceptable results indicate that this method can determine, with sufficient
precision for the determination of Ca in all foodstuffs precision, from 1779 to 444 mg/kg.
analyzed with Ca concentration above the estimated QL. The (f) Manganese.—Table 29 presents estimated
trueness as z-score (Table 30) is calculated to range from –0.6 performance characteristics of the ICP-AES method for Mn.
(chocolate milk powder) to 0.2 (peanut butter). The results The RSDr of the method for the determination of Mn ranged
indicate that this method can determine, with sufficient between 1.4% (wheat gluten) and 7.2% (infant cereals). The
precision, from 9870 to 411 mg/kg. RSDR ranged between 6.5% (chocolate milk powder) and
(b) Copper.—Table 29 presents estimated performance

9.2% (infant cereals). The HorRat value should not exceed
characteristics of the ICP-AES method for Cu. The RSDr of 1.0. Table 29 shows that the present method has an acceptable
the method for the determination of Cu ranged between 1.8% precision for the determination of Mn in all foodstuffs
(wheat gluten) and 5.0% (infant cereals). The RSDR ranged analyzed with Mn concentration above the estimated QL. The
between 4.4% (wheat gluten) and 12.0% (infant cereals). The trueness as z-score (Table 30) is calculated to range from –1.1
HorRat value should not exceed 1.0. Table 29 shows that the (wheat gluten) to 0.1 (dietetic milk powder). The results
present method has an acceptable precision for the indicate that this method can determine, with sufficient
determination of Cu in all foodstuffs analyzed with Cu precision, from 16.0 to 0.405 mg/kg.
concentration above or close to the estimated QL. The
(g) Sodium.—Table 29 presents estimated performance
trueness, expressed as z-score (Table 30), is calculated to
characteristics of the ICP-AES method for Na. The RSDr of
range from –0.7 (wheat gluten) to 0.03 (peanut butter). The
the method for the determination of Na ranged between 1.2%
results indicate that this method can determine, with sufficient
(wheat gluten and dietetic milk powder) and 3.9% (infant
precision, from 6.09 to 1.94 mg/kg.
cereals). The RSDR ranged between 2.2% (chocolate milk
(c) Iron.—Table 29 presents estimated performance
powder) and 5.5% (infant cereals). The HorRat value should
characteristics of the ICP-AES method for Fe. The RSDr of
not exceed 1.0. Table 29 shows that the present method has
the method for the determination of Fe ranged between 1.5%
an acceptable precision for the determination of Na in all
(infant cereals) and 5.0% (peanut butter). The RSDR ranged
foodstuffs analyzed with Na concentration above the
between 4.1% (wheat gluten) and 14.9% (peanut butter). The
estimated QL. The trueness as z-score (Table 30) is calculated
HorRat value should not exceed 1.5. Table 29 shows that the
to range from –1.6 (dietetic milk powder and wheat gluten)
present method has an acceptable precision for the
to –0.03 (peanut butter). The results indicate that this
determination of Fe in all foodstuffs analyzed with Fe
method can determine, with sufficient precision, from 4890
concentration above the estimated QL. The trueness as z-score
to 1420 mg/kg.
(Table 30) is calculated to range from –2.2 (wheat gluten) to
0.2 (dietetic milk powder 2). The results indicate that this (h) Phosphorus.—Table 29 presents estimated
method can determine, with sufficient precision, from 169.9 performance characteristics of the ICP-AES method for P.
to 16.4 mg/kg. The RSDr of the method for the determination of P ranged
(d) Potassium.—Table 29 presents estimated between 1.2% (wheat gluten) and 2.0% (peanut butter). The
performance characteristics of the ICP-AES method for K. RSDR ranged between 2.1% (infant cereals) and 3.3% (wheat
The RSDr of the method for the determination of K ranged gluten). The HorRat value should not exceed 1.0. Table 29
between 0.9% (dietetic milk powder) and 1.6% (peanut shows that the present method has an acceptable precision for
butter). The RSDR ranged between 1.2% (dietetic milk the determination of P in all foodstuffs analyzed with
powder) and 7.8% (wheat gluten). The HorRat value should P concentration above the estimated QL. The trueness as
not exceed 1.5. Table 29 shows that the present method has an z-score (Table 30) is calculated to range from –2.0 (wheat
acceptable precision for the determination of K in all gluten) to 0.8 (peanut butter). The results indicate that this
foodstuffs analyzed with K concentration above the estimated method can determine, with sufficient precision, from 4320
QL. The trueness as z-score (Table 30) is calculated to range to 2190 mg/kg.
from –1.8 (wheat gluten) to 0.7 (peanut butter). The results (i) Zinc.—Table 29 presents estimated performance
indicate that this method can determine, with sufficient characteristics of the ICP-AES method for Zn. The RSDr of the
precision, from 9090 to 472 mg/kg method for the determination of Zn ranged between 1.4%
(e) Magnesium.—Table 29 presents estimated (wheat gluten) and 3.0% (infant cereals). The RSDR ranged
performance characteristics of the ICP-AES method for Mg. between 3.8% (infant cereals) and 6.0% (peanut butter). The
The RSDr of the method for the determination of Mg ranged HorRat value should not exceed 1.0. Table 29 shows that the
between 1.2% (dietetic milk powder) and 3.4% (infant present method has an acceptable precision for the
cereals). The RSDR ranged between 2.9% (dietetic milk determination of Zn in all foodstuffs analyzed with Zn
powder) and 5.4% (infant cereals). The HorRat value should concentration above the estimated QL. The trueness as z-score
not exceed 1.5. Table 29 shows that the present method has an (Table 30) is calculated to range from –1.7 (wheat gluten) to 1.1
Table 29. Estimated performance characteristics of the ICP-AES method for determination of nine elements (Ca, Cu, Fe, K, Mg, Mn, Na, P, and Zn)
Reference
Sample description value, mg/kga Median, mg/kg nb SDr, mg/kgc SDR, mg/kgd RSDr, % RSDR, % r, mg/kge R, mg/kgf HorRatg z-score
Calcium
Peanut butter 411 ± 18 416 9 14 21 3.3 5.0 39 57 0.8 0.2

Chocolate milk powder 9870 ± 248 9712 9 162 280 1.7 2.9 448 775 0.7 –0.6
Infant cereals 6000 ± 88 5972 9 234 290 3.9 4.9 649 803 1.1 –0.1
Dietetic milk powder 2 5780 ± 56 5696 9 61 218 1.1 3.8 170 606 0.9 –0.4
Wheat gluten 369 ± 35 365 9 5 15 1.4 4.0 15 41 0.6 –0.3
Copper
Peanut butter 4.93 ± 0.15 4.94 9 0.20 0.31 4.1 6.2 0.56 0.85 0.5 0.03
Chocolate milk powder NAh 5.92 9 0.11 0.41 1.9 6.9 0.31 1.13 0.6
Infant cereals NA 1.94 9 0.10 0.23 5.0 12 0.27 0.65 0.8
Dietetic milk powder 2 6.10 ± 0.20 6.09 9 0.12 0.40 2.0 6.5 0.33 1.10 0.5 –0.02
Wheat gluten 5.94 ± 0.72 5.77 9 0.10 0.26 1.8 4.4 0.29 0.71 0.4 –0.7
Iron
Peanut butter 16.4 ± 0.8 15.9 9 0.8 2.4 5.0 14.9 2.2 6.5 1.4 –0.2
Chocolate milk powder 169.9 ± 4.8 166.2 9 3.8 7.4 2.3 4.4 10.5 20.4 0.6 –0.5
Infant cereals 80.4 ± 1.2 74.8 9 1.2 3.9 1.5 5.2 3.2 10.8 0.6 –1.4
Dietetic milk powder 2 81.1 ± 1.0 81.9 9 2.2 3.6 2.7 4.3 6.0 9.9 0.5 0.2
Wheat gluten 54.3 ± 6.8 49.8 9 1.2 2.1 2.4 4.1 3.4 5.7 0.5 –2.2
Potassium
Peanut butter 6070 ± 200 6157 9 101 118 1.6 1.9 279 326 0.4 0.7
Infant cereals 6500 ± 100 6466 9 100 124 1.5 1.9 277 343 0.4 –0.3
Dietetic milk powder 2 6420 ± 52 6431 9 61 76 0.9 1.2 169 212 0.3 0.1
Wheat gluten 472 ± 61 413 9 6 32 1.5 7.8 17.1 89.3 1.2 –1.8
Magnesium
Peanut butter 1680 ± 70 1753 9 42 116 2.4 6.6 117 321 1.3 0.6
Chocolate milk powder 1779 ± 46 1751 9 33 54 1.9 3.1 91.7 151 0.6 –0.5
Infant cereals 881 ± 16 860 9 29 47 3.4 5.4 81 129 0.9 –0.5
Dietetic milk powder 2 444 ± 5 441 9 5.4 12.7 1.2 2.9 15.0 35.3 0.5 –0.2
Wheat gluten 510 ± 47 483 9 10 15 2.0 3.1 26 41 0.5 –1.8

Table 29. (continued)
Reference Median,
Sample description value, mg/kga mg/kg nb SDr, mg/kgc SDR, mg/kgd RSDr, % RSDR, % r, mg/kge R, mg/kgf HorRatg z-score
Manganese
Peanut butter 16.0 ± 0.6 16.0 9 0.4 1.3 2.4 7.8 1.1 3.5 0.7 0.0
Chocolate milk powder NA 7.53 9 0.15 0.49 2.1 6.5 0.43 1.36 0.6
Infant cereals NA 16.5 9 1.2 1.5 7.2 9.2 3.3 4.2 0.9
Wheat gluten 14.3 ± 0.8 13.0 9 0.2 1.2 1.4 9.1 0.5 3.3 0.8 –1.1
Sodium
Peanut butter 4890 ± 140 4886 9 129 122 2.6 2.5 358 337 0.6 0.03
Infant cereals 1050 ± 28 997 9 39 55 3.9 5.5 107 152 1.0 –1.0
Dietetic milk powder 2 1840 ± 20 1767 9 22 46 1.2 2.6 60 128 0.5 –1.6
Wheat gluten 1420 ± 110 1353 9 17 41 1.2 3.0 47 112 0.6 –1.6
Phosphorus
Peanut butter 3378 ± 92 3446 9 70 88 2.0 2.5 193 243 0.5 0.8
Chocolate milk powder 4080 ± 60 4127 9 70 123 1.7 3.0 194 341 0.7 0.4
Infant cereals 4320 ± 42 4302 9 58 91 1.3 2.1 160 253 0.5 –0.2
Dietetic milk powder 2 3720 ± 24 3760 9 52 122 1.4 3.2 144 338 0.7 0.3
Wheat gluten 2190 ± 150 2053 9 25 68 1.2 3.3 69 189 0.7 –2.0
Zinc
Peanut butter 26.3 ± 1.1 26.8 9 0.6 1.6 2.4 6.0 1.8 4.5 0.6 0.3
Chocolate milk powder NA 18.5 9 0.4 0.9 2.1 4.8 1.1 2.5 0.5
Infant cereals NA 36.7 9 1.1 1.4 3.0 3.8 3.1 3.8 0.4
Wheat gluten 53.8 ± 3.7 49.9 9 0.7 2.3 1.4 4.5 2.0 6.3 0.5 –1.7
a
Expanded uncertainty expressed as uncertainty with a coverage factor of 2.
b
n = Number of laboratories.
c
SDr = Repeatability SD.
d
SDR = Reproducibility SD.
e
r = Repeatability limit.
f
R = Reproducibility limit.
g
HorRat value as ratio using RSDR and Horwitz denominator according to ref. 32.
h
NA = Not available.

Table 30. Statistical analysis of nine elements determined in food products tested by ICP-AES
Statistic Ca Cu Fe K Mg Mn Na P Zn
Peanut butter
Found mean level, mg/kg 416 4.94 15.9 6157 1753 16.0 4886 3446 26.8
Certified value, mg/kg 411 4.93 16.4 6070 1680 16.0 4890 3378 26.3
SDR, mg/kg 21 0.31 2.4 118 116 1.3 122 88 1.6
z-score 0.2 0.03 –0.2 0.7 0.6 0 –0.03 0.8 0.3
Certified value, mg/kg 9870 NAa 169.9 9090 1779 NA 1990 4080 NA
SDR, mg/kg 280 0.41 7.4 166 54 0.49 42 123 0.9
z-score –0.6 –0.5 –0.5 –0.5 –1.3 0.4
Infant cereals
Certified value, mg/kg 6000 NA 80.4 6500 881 NA 1050 4320 NA
SDR, mg/kg 290 0.23 3.9 124 47 1.5 55 91 1.4
z-score –0.1 –1.4 –0.3 –0.5 –1 –0.2
Certified value, mg/kg 5780 6.1 81.1 6420 444 0.405 1840 3720 51
SDR, mg/kg 218 0.4 3.6 76 13 0.029 46 122 2.4
z-score –0.4 –0.02 0.2 0.1 –0.2 0.1 –1.6 0.3 1.1
Wheat gluten
Found mean level, mg/kg 365 5.77 49.8 413 483 13 1353 2053 49.9
Certified value, mg/kg 369 5.94 54.3 472 510 14.3 1420 2190 53.8
SDR, mg/kg 15 0.26 2.1 32 15 1.2 41 68 2.3
z-score –0.3 –0.7 –2.2 –1.8 –1.8 –1.1 –1.6 –2 –1.7
a
NA = Not available.


Figure 1. Ring trial: Comparison of Horwitz equation and experimental equation found for the ring trial.
(dietetic milk powder). The results indicate that this method can fulfilled AOAC criteria and recommendations in terms of
determine, with sufficient precision, from 53.6 to 18.5 mg/kg. accuracy (trueness, recovery, and z-scores) and precision
(j) Horwitz equation.—Comparison of the Horwitz (repeatability and reproducibility RSDs, HorRat values)
function with that obtained from 45 results (i.e., nine elements regarding SLV and collaborative study.
in five matrixes) of the collaborative study highlights The determination of nine nutritional minerals in fortified
excellent precision as shown in Figure 1. This graph shows food products by ICP-AES is fit-for-purpose according to ISO
that the trend of data from collaborative study follows the
17025 norm and AOAC acceptability criteria.
theoretical Horwitz function, as the slope is similar. The
intercept is significantly different, and hence, this consistent
Improvement of AOAC Official Method 984.27
and constant deviation from predicted HorRat value can be
explained by the excellent experience and training of analysts
(microwave digestion handling/ICP practice) in laboratories Main improvements compared to the AOAC Method
that were also informed of the maximum concentration of 984.27 are: (1) The use of microwave digestion systems with a
each element in the five matrixes tested. single acid (nitric acid) for an optimized sample preparation in
order to improve element recoveries from difficult matrixes
Conclusions and to increase sample throughput, favoring safety
Method Performance precautions and time-savings for operators in laboratories. It
is a significant improvement regarding long, time-consuming
The MDO and MDC procedures used for SLV and RT acid digestion with unsafe acid handling (HNO3/HClO4) used
were shown to be effective with acceptable agreement toward for AOAC Method 984.27. (2) The use of appropriate
certified and in-house reference values in terms of recovery analytical wavelengths for each element of interest and the
for most samples covering the nine sectors of the food automatic addition of a solution of appropriate IS and
triangle. ionization buffer to correct for physical and chemical
This ICP-AES analytical method was proven to be simple, interferences (i.e., to compensate for matrix effects induced
selective, accurate, and reliable for the determination of Ca, by the complexity of the food samples). The aim is to improve
Cu, Fe, K, Mg, Mn, P, Na, and Zn in most food matrixes.
short-term accuracy (repeatability) and long-term stability
Recommended and alternate analytical lines corrected by
(reproducibility and calibration curve validity in a long
appropriate IS and Cs ion buffer concentration ranging
analytical batch). Neither internal standardization nor ion
between 0.1 and 1% (w/v) were validated according the
buffer are used for AOAC Method 984.27. (3) The application
statistic treatment of selectivity, sensitivity, linearity,
accuracy, and precision through SLV using ICP-AES for all food matrixes covering all of the nine AOAC food
equipment with axially viewing plasma after MDC, and triangle sectors, including infant formula, which were the
through ruggedness tests using ICP-AES equipment with unique types of matrixes validated for AOAC Method 984.27.
radial and dual viewings of the plasma after MDC and MDO This fully validated multielemental method is
procedures. cost-efficient, time-saving, accurate, and fit-for-purpose. It is
Performance characteristics reported for 13 certified and proposed as an improved version of AOAC Official Method
in-house reference materials covering the triangle food sectors 984.27 for fortified food products, including infant formula.
Acknowledgments (12) CEN/TS 15621-Animal feeding stuffs (2007) Brussels,

Belgium
We would like to thank Florence Monard and Thierry (13) NF EN 1551-Animal feeding stuffs (2007) Brussels, Belgium
Delatour from the Nestlé Research Center for useful (14) International Organization for Standardization (2002) Animal
discussions and support given during the work. We are also and Vegetable Fats and Oils: Determination of Phosphorus
indebted to the following collaborators for their skilfull Content, Part 3, ISO 10540-3, Method using ICP-AES,
participation in ruggedness tests MD3, MD4, and the Geneva, Switzerland
collaborative study: (15) Benzo, Z., Marcano, E., Gomez, C., Ruiz, F., Salas, J.,
Susanna Berger and Rafael Berrocal, PTC, Konolfingen, Quintal, M., & Garaboto, A. (2002) J. AOAC Int. 85,
Switzerland 967–970
Ria Bos, Ton Noorlos, and Genevieve Daix, NQAC, (16) Suddendorf, R.F., & Cook, K.K. (1984) J. AOAC Int. 67,
Nunspeet, The Netherlands 985–992

Anne Baillon, Christine Senechal, Laure Brullebaut, and (17) Jorhem, L. (2000) J. AOAC Int. 83, 1204–1211
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(22) Todoli, J.L., Gras, L., Hernandis, V., & Mora, J. (2002) J.
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Improvement of AOAC Ofﬁcial Method 984.27 for the Determination of Nine

Article in Journal of AOAC International · September 2009

Eric Poitevin Daniel Andrey

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Marvin Joshua Gonzales

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FOOD COMPOSITION AND ADDITIVES

Improvement of AOAC Official Method 984.27 for the

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Sterilized cream LGC RM 7104 1 SLV

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Preparation of Reagents and Standard Solutions

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Baking chocolate Whole egg powder Baby food composite

Calcium Certifieda 840 ± 74 2480 ± 190 853 ± 28

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(g) Precision.—Both repeatability standard deviation RSDiR , %

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recoveries (i.e., 0.01 < P-value <0.05: recovery statistically

different from 100% at 95% level of confidence interval, but

and Zn in dietetic milk powder 1 (98 ± 1% and 97 ± 1%,

respectively) using 3Mg, 1ZnI, and 3ZnI lines and for P in

for recovery are, however, globally fulfilled for all elements in

between 0.26 mg/kg (Mn) and 16 900 mg/kg (K). Only

recovery values for 2CaII and 1KI lines in sterilized cream

(f) Precision.—SDr and SDiR values, respectively,

AOAC criteria regarding RSDr and RSDiR values for all

elements in all matrixes ranging between 0.6 and 16.8% and

between 0.9 and 23.3%, respectively, for concentrations

Dietetic milk powder 1

(g) Uncertainty.—Overall uncertainty measurements

(±U) are displayed in Tables 11–19 with medians obtained

through reproducibility conditions as combinations of

precision (SDiR) and trueness (SDrec) contributions.

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However, AOAC requirements for recovery performance

(81 and 82%) for MD1 and MD2, respectively; recovery

values of Cu (67 and 74%) in nonfat milk powder and dietetic

value of Ca (113%) in infant cereals for MD1 showed

demonstrated for statistic recovery test at 95% confidence.

(Mn) to 16 900 mg/kg (K).

method applied by different operators (at least two operators

digestions on different ICP-AES equipments with axial,

Dietetic milk powder 1

(w/v) as minimal ionization buffer concentration.

Nonfat milk powder

(a) Calcium.—Table 29 presents estimated performance

characteristics of the ICP-AES method for determination of

Sterilized cream Ph 823 ± 22 807 ± 60 98 ± 3 0.56 32 53 4.0 6.6 1.1