Improvement of AOAC Official Method 984.27 for The

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FOOD COMPOSITION AND ADDITIVES

Improvement of AOAC Official Method 984.27 for theDetermination of Nine Nutritional Elements in Food Products byInductively Coupled Plasma-Atomic Emission Spectroscopy After

Microwave Digestion: Single-Laboratory Validation and Ring Trial

ERIC POITEVIN, MARINE NICOLAS, LAETITIA GRAVELEAU, JANIQUE R ICHOZ, DANIEL ANDREY, and FLORENCE MONARD

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

A single-laboratory validation (SLV) and a ring trial

(RT) were undertaken to determine nine nutritional

elements in food products by inductively coupledplasma-atomic emission spectroscopy in order to

improve and update AOAC Official Method 984.27.

The improvements involved optimized microwave

digestion, selected analytical lines, internal

standardization, and ion buffering. Simultaneous

determination of nine elements (calcium, copper,

iron, potassium, magnesium, manganese, sodium,

phosphorus, and zinc) was made in food products.

Sample digestion was performed through wet

digestion of food samples by microwave

technology with either closed or open vessel

systems. Validation was performed to characterize

the method for selectivity, sensitivity, linearity,

accuracy, precision, recovery, ruggedness, and

uncertainty. The robustness and efficiency of this

method was proved through a successful internal

RT using experienced food industry laboratories.

Performance characteristics are reported for

13 certified and in-house reference materials,

populating the AOAC triangle food sectors, which

fulfilled AOAC criteria and recommendations for

accuracy (trueness, recovery, and z -scores) and

precision (repeatability and reproducibility RSD

and HorRat values) regarding SLV and RT. This

multielemental method is cost-efficient,time-saving, accurate, and fit-for-purpose

according to ISO 17025 Norm and AOAC

acceptability criteria, and is proposed as an

improved version of AOAC Official Method 984.27

for fortified food products, including infant

formula.

R

obust and efficient methods with well-characterized

reference materials are needed by food-testing and

nutrition laboratories to facilitate compliance with

nutritional labeling laws and claim requirements, to provide

traceability for food exports needed for acceptance in many

foreign markets, and to improve the accuracy of nutrition

information that is provided to assist consumers in making

sound dietary choices (1, 2). Inductively coupled plasma-

atomic emission spectroscopy (ICP-AES) is one of the most

commonly used techniques within the food industry for

accurate and cost-efficient routine analyses of nutritional

minerals in food products, plants, pet food, raw materials, and

feeding stuffs (3–15).

AOAC Method 984.27 was validated 25 years ago via a

collaborative study in which acid digestion withHNO3/HClO4 and ICP-AES analysis with radial grating

configuration were used to determine nine nutritional

minerals in infant formula (16). Standard procedures for the

decomposition of food samples usually involve dry-ashing in

a muffle furnace or wet digestion with combined

acids (6, 10, 17). Compared to the classical digestion

procedures, microwave digestion has some significant

advantages with respect to speed of analysis, analyte loss,

sample contamination, and safe acid handling during sample

preparation (8). The use of microwave digestion before

ICP-AES analysis provides an accurate method for the

determination of nutritional minerals targeted for labeling of

food products (5).

The objective of this study is to improve AOAC Method

984.27 in terms of sample preparation time and throughput

using microwave digestion, and analytical performance using

the latest generation of ICP-AES equipment under robust

conditions (18), internal standardization (19, 20), and ion

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

accuracy (repeatability) and long-term stability (reproducibility

and calibration curve validity in a long analysis batch.

1484 POITEVIN ET AL.: JOURNAL OF AOAC I NTERNATIONAL VOL. 92, NO. 5, 2009

Received April 30, 2009. Accepted by SG June 19, 2009.Corresponding author’s e-mail: [email protected]



The validation of this method involved a single-laboratory

validation (SLV), a ruggedness study, and a ring trial (RT) in

accordance with ISO 17025 and AOAC guidelines (26):

(a) SLV .—Performed using ICP-AES with axial grating

after a closed-vessel microwave digestion, on 10 in-house and

certified reference materials.

(b) Ruggedness study.—Performed in parallel, analyzing

with and without ionization buffer, the spiking elements ineight food-grade salts and testing six in-house and certified

reference materials in repeatability and reproducibility

conditions with different ICP-AES equipment (axial, radial,

and dual-view grating configurations) after open- and

closed-vessel microwave-based digestions.

(c) RT. —Involving nine laboratories was organized to

analyze five in-house and certified reference materials.

Thirteen well-characterized reference materials were

selected to populate the AOAC triangle, in which foods could

be categorized into nine sectors based on their fat, protein, and

carbohxdrate contents (27). All the results obtained on

reference materials have been treated with robust

statistics (28, 29) to verify that the updated procedure is

fit-for-purpose in terms of specificity, sensitivity, linearity,

trueness, and precision; and can be proposed as an improved

version of officialAOAC Method 984.27 regardingmicrowave

digestion, internal standardization, and ion buffering.

Experimental

Apparatus

(a) Closed-vessel microwave digestion systems.—A CEM

Mars Xpress (CEM, Matthews, NC) was used for the SLV,

including ruggedness test MD1 (Table 1). Other microwave

systems from CEM and Milestone (Shelton, CT) suppliers

were used for ruggedness tests MD2, MD3, and MD4 and for

the collaborative study RT (Table 1).

(b) Open-vessel microwave digestion system.—A CEM

MDS 2000 was used for test MD2 and by one laboratory for

the RT.

(c) ICP-AES spectrometers.—A Varian Vista-Pro axialICP-AES instrument with SPS-5 autosampler (Varian Inc.,

Mulgrave, Victoria, Australia) was used for the SLV,

including tests MD1 and MD2. Other ICP-AES spectrometers

from Perkin Elmer (Norwalk, CT), Varian, and Spectro

(Kleve, Germany) were used for the MD3 and MD4 tests and

for the RT.

(d) Balance.—A balance (AT 200, Mettler-Toledo, Inc.,

Greifensee, Switzerland) with readability of 0.1 mg (SLV, tests

MD1 and MD2) or equivalent (tests MD3, MD4, and RT).

(e) Sub-boiling quartz distillation apparatus for acids.—A

Kürner Analysentechnik (Rosenheim, Germany) distillator

(SLV, MD1, and MD2) for purifying acids or equivalent (tests

MD3, MD4, and RT).(f ) Fumehood .—A fumehood acid-resistant was used

(SLV, tests MD1 and MD2) or equivalent (tests MD3, MD4,

and RT).

(g) Drying oven.—A Heraeus drying oven 6120T

(Heraeus, Hanau, Germany) was used for the SLV, and tests

MD1 and MD2, and similar equipment was used for tests

MD3, MD4, and RT.

(h) Mixer or grinder for homogenization.—A Retsch

(Haan, Germany) mixer GM200 was used for the SLV, tests

MD1 and MD2, or equivalent (tests MD3, MD4, and RT).

POITEVIN ET AL.: JOURNAL OF AOAC I NTERNATIONAL 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 = ringtrial.

b MDC = Closed-vessel microwave digestion system; MDO = open-vessel microwave digestion system.



Materials

(a) Volumetric flasks.—Glass, 50, 100, 500, 1000 mL

(SLV, tests MD1 and MD2) or equivalent (tests MD3, MD4,

and RT).

(b) Erlenmeyer flask for slurry preparation.—Glass,

100mL (SLV, tests MD1 andMD2) or equivalent (tests MD3,

MD4, and RT).

(c) Sample tubes.—Polyethylene or polystyrene, tubes 15

or 50 mL with caps (Falcon) for ICP analysis (SLV, tests

MD1 and MD2) or equivalent (tests MD3, MD4, and RT).

(d) Filters.—Ashless filter papers, quantitative (SLV,

tests MD1 and MD2), Whatman 589/1 (diameter 125 mm

and/or 90 mm; Schleicher & Schuell, Whatman, Dassel,Germany), or equivalent (tests MD3, MD4, and RT).

(e) Pipets.—Macropipet, variable volume 0.5–5.0 mL;

micropipet, variable volume 50–200 L and 200–1000 L;

electronic digital pipet 50–1000 L (SLV, tests MD1 and

MD2), or equivalent (tests MD3, MD4, and RT).

(f ) Samples.—Certified and standard reference materials

from National Institute of Standards and Technology (NIST;

Gaithersburg, MD) and National Metrology Institute (LGC

standards, Teddington, UK), in-house reference available

samples (from internal proficiency tests), and raw materials

(Table 2) were used for the SLV, ruggedness tests MD1,MD2, MD3, and MD4 and internal RT, including dairy-based

products, milk, and cereals-based products, chocolate and

dietetic milk powders, refrigerated meals, pet food, and raw

materials, such as whole egg powder, wheat gluten, corn bran,

and added food-grade salts (calcium carbonate, calcium

phosphate tribasic, calcium citrate tribasic, potassium

chloride, magnesium carbonate, magnesium chloride,

manganese sulfate, and sodium chloride).

Chemicals and Reference Solutions

(a) High grade water, H 2O (18 M ).—MilliQ™ Plus

system (Millipore, Bedford, MA).(b) Nitric acid (HNO3 ).—65% (w/v), Suprapure.

(c) Nitric acid (HNO3 ).—65% (w/v), analytical grade.

(d) Hydrochloric acid .—37% (w/v), analytical grade.

(e) Hydrogen peroxide.— 97% (w/v), analytical grade.

(f ) Multistandard.— CPI International (Santa Rosa, CA).

Composition of the CPI standard solution, in mg/L: Ca =

7500; Cu = 50; Fe = 250; K = 10 000; Mg = 2500; Mn = 1.25;

Na = 5000; P = 5000; Zn = 100.

(g) Calcium standard solution.— 10 000 mg/L (Merck,

Darmstadt, Germany).


Table 2. Raw materials, certified, and in-house reference materials used for SLV and RT

Material type Sample reference AOAC food triangle sector a 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

Calcium carbonate CAS No. 471-34-1 NAc

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.



(h) Potassium standard solution.—10 000 mg/L (Merck).

(i) Sodium standard solution.—10 000 mg/L (Merck).

( j) Magnesiumstandard solution.—10 000 mg/L (Merck).

(k ) Phosphorus standard solution.—10 000 mg/L (Merck).

(l) Iron standard solution.—1000 mg/L (Merck).

(m) Zinc standard solution —1000 mg/L (Merck).

(n) Copper standard solution.—1000 mg/L (Merck).

(o) Manganese standard solution.—1000 mg/L (Merck).

(p) Indium standard solution.—1000 mg/L (Merck).

(q) Chromium standard solution.—1000 mg/L (Merck).

(r) Strontium standard solution.—1000 mg/L (Merck).

(s) Yttrium standard solution.—1000 mg/L (Merck).

(t) Cesium chloride.—Analytical grade (Merck).

Preparation of Reagents and Standard Solutions

(a) Stock standard solution.—Working standards were

prepared from a CPI multistandard commercial stock standard

solution.

(b) Working standard solutions.—Standards prepared

from stock standard solution are designed to have the same

acid concentration as digested test solutions (i.e., 10% v/v

using sub-boiled or analytical grade HNO3 (SLV and MD1)

or 15% v/v (MD2) using combined acids (HNO3, H2O2, and

HCl). (1) Std6 .—Pipet 3.2 mL stock standard solution into a

100 mL acid-washed volumetric flask. Add 10 mL analytical

grade HNO3 (SLV, MD1) or 15 mL combined acids (MD2),

dilute to volume with H2O, mix, and transfer to acid-washed

polyethylene bottle. (2) Std5.—Pipet 1.6 mL stock standard

solution into a 100 mL acid-washed volumetric flask. Add

10 mL analytical grade HNO3 (SLV, MD1) or 15 mL

combined acids (MD2), dilute to volume with H2O, mix, and

transfer to acid-washed polyethylene bottle. (3) Std4.—Pipet

0.8 mL stock standard solution into a 100 mL acid-washed

volumetric flask. Add 10 mL analytical grade HNO3 (SLV,

MD1) or 15 mL combined acids (MD2), dilute to volume with

H2O, mix, and transfer to acid-washed polyethylene bottle.

(4) Std3.—Pipet0.4 mL stock standard solution into a 100 mL

acid-washed volumetric flask. Add 10 mL analytical grade

HNO3 (SLV, MD1) or 15 mL combined acids (MD2), dilute

to volume with H2O, mix, and transfer to acid-washed

polyethylene bottle. (5) Std2.—Pipet 0.2 mL stock standard

solution into a 100 mL acid-washed volumetric flask. Add

10 mL analytical grade HNO3 (SLV, MD1) or 15 mL

combined acids (MD2), dilute to volume with H2O, mix, and

transfer to acid-washed polyethylene bottle. (6 ) Std1.—Pipet

0.1 mL stock standard solution into a 100 mL acid-washed

volumetric flask. Add 10 mL analytical grade HNO3 (SLV,MD1) or 15 mL combined acids (MD2), dilute to volume with

H2O, mix, and transfer to acid-washed polyethylene bottle.

(7 ) Blank .—Add 10 mL analytical grade HNO3 (SLV, MD1)

or 15 mL combined acids (MD2) into a 100 mL acid-washed

volumetric flask, dilute to volume with H2O, mix, and transfer

to acid-washed polyethylene bottle. All calibration solutions

are stable for 1 week in glass volumetric flasks.

(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

tests.—Weigh 1.27 g cesium chloride. Cesium 0.1% (w/v)

solution was tested as the minimal recommended

concentration required for element analysis in most food

matrixes. Cs solution 1% (w/v) is recommended if an element

is present at low concentration in high-salted food raw

materials (e.g., culinary products or tastemakers) or if it is

analyzed as an impurity in food-grade salts. Add 40 mL

indium 1000 mg/L and 10 mL each of strontium, yttrium, and

chromium 1000 mg/L stock solutions, as internal standards.

Add 10 mL analytical grade HNO3. Dilute to volume with

H2O, mix, and transfer to an acid-washed polyethylene bottle.

( Note: Reagent concentrations assume the use of same pump

tubing internal diameter for both internal standard/ionization


Table 3. Open-vessel microwave digestion program

(CEM MDS 2000) used for MD2 ruggedness test

No. of samplesto digest Power a

Hold time, min(step 1)

Hold time, min(step 2)

5 23 20 10

6 27 20 10

7 31 20 108 35 20 10

9 39 20 10

10 43 20 10

11 47 20 10

12 51 20 10

a Power expressed in % of maximum power (600 watts for CEMMDS 2000 system).

Table 4. ICP-AES conditions (Varian Vista AX PRO) for

SLV, MD1, and MD2 ruggedness tests

Instrument Varian Vista Pro AX

RF generator 40 MHz

RF power 1100 W

Torch quartz injector id 2.5 mm

Argon flow rate Plasma 18 L/min

Auxiliary 2.25 L/min

Nebulizer 0.8 L/min

Spray chamber

Cyclonic thermostated (water

cooled)

Nebulizer Microconcentric

Pump Peristaltic, 3 channels

Pump tubing id 0.64 mm

Sample flow rate 0.75 mL/min

Polychromator Echelle

Resolution 6.9 pm at Mo 202.032 nm

Detector Segmented CCD



buffer and sample pump tubes (i.e., orange/white, 0.64 mm id)

using automatic addition.

Sample Preparation and Digestion

(a) Test sample preparation.—Homogenize the test

sample (e.g., serving size or declared labeled size) by either

grinding as fine as possible using a mixer to obtain a fine

powder and/or by preparing a slurry with H2O: weigh 10

± 0.1 g test sample, and put into a 100 mL Erlenmeyer flask;

add 90 ± 0.1 g H2O, and mix well with stopper ( Note: Water

can be preheated at 50C for a better homogenization of samples, e.g., infant cereals and fortified milk powders).

(b) Test portion preparation for closed-vessel microwave

digestion (SLV, MD1, MD3, and MD4 ruggedness

tests).—Accurately weigh 0.50 ± 0.01 g test portion (or

sample mass in the prepared slurry) into digestion vessel. Use

either weighing paper insert to line the vessel walls or Pasteur

pipet during sample transfer to keep sample from adhering to

sides of vessel. Fluid samples or test portion from slurry test

sample may be weighed directly after mixing. Add 5.0 ±

0.1 mL analytical grade HNO3, loosely cap vessels without

sealing, and predigest at room temperature until vigorous

foaming subsides. Close vessels and place in CEM Mars

Xpress microwave or in other closed-vessel microwave

systems.

(c) Test portion preparation for open-vessel microwave

digestion (MD2 ruggedness test).—Accurately weigh 1.0 ±

0.01 g test portion (orsample mass in theslurry) into a 100mL

volumetric flask. Carefully add 5 mL HNO3 and then 5 mL

H2O2. Allow the sample to stand for at least 10 min at roomtemperature. Distribute the volumetric flasks onto the

open-vessel microwave carousel to ensure homogeneous

microwave power application on all samples.

(d) Sample digestion for closed-vessel microwave

digestion (SLV, MD1, MD3, and MD4 ruggedness

tests).—Digest sample according to a ramp program as

follows: Conditions: Stage 1.—Power, expressed as

maximum power (1600 watts for CEM Mars Xpress system)

= 75%; ramp time, 5 min; 120C; hold time, 5 min.

Stage 2.—Power, 75%; ramp time, 5 min; 200C; hold time,

25 min. Then cool vessels, vent, and transfer digests to 50 mL

volumetric flasks, dilute to volume with H2O, and mix. A

digestion is judged complete when clear-to-yellow analytical

solutions are produced. Filter the digested solution using an

ashless filter paper for turbid samples containing fat,

discarding the first 20 mL of filtrate and collecting the

remaining filtrate for analysis. [ Note: Membrane disc filters

(0.45 m) are not recommended as they are generally not

metal free.] Transfer to polyethylene containers within 2 h.

Dilute with H2O the samples that are found to be above the

standard curve range, or have total content of minerals higher

than 1000 mg/L. Final dilutions require addition of

appropriate amounts of HNO3 to maintain the proportion of

10% (v/v) HNO3 in the final solution to be analyzed.

(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

for a CEM MDS 2000 system depending on the number of

distributed flasks. This first step requires 20 min heating time.

Yellow vapors will be emitted during the hydrolysis.

Carefully remove the flasks from the microwave system.

Allow the flasks to cool to room temperature and add 5 mL

HCl 35% (w/v). Heat the flasks for an additional 10 min using

the same program in the microwave system. Allow the flasks

to cool to room temperature. Transfer digests to 100 mL

volumetric flasks, dilute to volume with H2O, and mix. Filter

and prepare the digested solution before analysis as described

for closed-vessel microwave digestion in (d) but maintain the proportion of 15% (v/v) combined acids in the final solution

to be analyzed.

(f ) Digestion vessel decontamination for closed-vessel

microwave digestion (SLV, MD1, MD3, and MD4 ruggedness

tests). —Conditions: Step 1.—Power, express in % of

maximum power (1600 watts for CEM Mars Xpress system)

= 75%; ramp time, 5 min; 180C; hold time, 5 min.

Decontaminate used digestion vessels with 5 mL HNO3 for a

CEM Mars Xpress system and rinse with H2O. Dry vessels at

60C in drying oven for 1 h.


Table 5. Recommended and alternate ICP analytical

lines used for SLV and RT

Element lines, nm Internal standard lines, nm

Ca IIa

317.933 In I 303.936

Ca IIb

317.933 Y II 371.028

Cu Ia

324.754 In I 303.936

Cu Ib 324.754 Y II 371.028

Cu Ib

327.395 In I 303.936

Cu Ib

327.395 Y II 371.028

Fe IIa

259.94 Sr II 338.071

Fe IIb

259.94 Y II 371.028

Fe IIb

259.94 Cr II 283.563

K Ia

766.491 Sr I 460.733

Mg Ia

285.213 In I 303.936

Mg Ib

285.213 Y II 371.028

Mg IIa

279.078 In I 303.936

Mn IIb

257.61 Sr II 338.071

Mn IIb 257.61 Sr I 460.733

Mn IIb

257.61 Y II 371.028

Na Ia

589.592 Sr I 460.733

P Ia

213.618 In I 303.936

P Ib

178.222 Sr I 460.733

P Ib

178.222 Y II 371.028

Zn Ia

213.857 Sr II 338.071

Zn Ib

213.857 Sr I 460.733

Zn Ib

213.857 Y II 371.028

a Recommended lines for ICP-AES analysis.b Alternate confirmatory lines for ICP-AES analysis.



(g) Digestion vessel decontamination for open-vessel

microwave digestion (MD2 ruggedness test).—The glass

volumetric flasks can be directly used after being washed in a

laboratory washing machine or soaked overnight in a water

bath with HNO3 30% (v/v) and rinsed with H2O to avoid or

minimize risk of iron contamination.

(h) Spiked food grade salt preparation (SLV).—Weigh

0.2 ± 0.01 g food-grade salt into a 100 mL volumetric flask.

Add deionized water and 10 mL HNO3. Add 0.8 mL stock

standard solution corresponding to standard Std4 [prepared

from CPI commercial solution (Ca, 60 mg/L; Cu, 0.4 mg/L;

Fe, 2 mg/L; K, 80 mg/L; Mg, 20 mg/L; Mn, 0.01 mg/L; Na,40 mg/L; P, 40 mg/L; Zn, 0.8 mg/L)]. Dissolve salt and dilute

to volume with deionized water.

ICP Analysis

(a) ICP-AES spectrometer (SLV-MD1 and MD2

ruggedness tests).—A Varian Vista-Pro axial ICP-AES

instrument with autosampler SP5 was used during SLV, using

the operating conditions shown in Table 4. The recommended

and alternative analytical lines (nm) used for elements to be

determined and internal standards (IS) are shown in Table 5.

A 3-channel peristaltic pump (Gilson, Middleton, WI) and a T

connector (id 1.5 mm/Socochim, Lausanne, Switzerland)

linked between the peristaltic pump and nebulizer were used

to avoid having to manually add ionization buffer and IS to

each sample solution. A thermostatted (15C) cyclonic spray

chamber (Jacketed ‘Althea’ Cyclonic Glass Spray Chamber,

water-cooled, EPOND SA, Vevey, Switzerland) equipped

with a micro-concentric nebulizer [C-Type ‘Caliber’ standard

Concentric Glass Nebulizer with ‘NebLink’ sample capillary

tube and argon connector (EPOND)] were used to obtain the

best method performance. Sample and IS pump tubes (e.g.,

orange/white, 0.64 mm id Socochim), and peristaltic pumprotation speed (15 rpm), were selected to keep sample and IS

pump tubes of similar size to maximize mixing accuracy,

while maintaining the required detection levels. Ionization

buffer (cesium chloride) was combined with IS solution to

compensate for easy ionizable element (EIE) effects (e.g., K,

Na, and Ca) in the plasma, because certain food materials can

contain substantial concentrations of these elements that

provide a significant source of electrons in the plasma. The

presence of ionization buffer in all samples and standards will

minimize the effects of varying concentrations of EIEs in the


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

ElementBaking chocolate(NIST SRM 2384)

Whole egg powder (NIST RM 8415)

Baby food composite(NIST SRM 2383)

Calcium Certified

a

840 ± 74 2480 ± 190 853 ± 28Found

b(recovery, %)

c 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

Foundb

(recovery, %)c

7750 ± 100 (95) 3125 ± 27 (98) 3793 ± 33 (105)

Magnesium Certifieda 2570 ± 150 305 ± 27 248 ± 5

Foundb (recovery, %)c 2567 ± 34 (100) 292 ± 1 (96) 245 ± 3 (99)

Manganese Certifieda 20.3 ± 1.3 1.78 ± 0.38 1.39 ± 0.11

Foundb

(recovery, %)c

19.9 ± 0.1 (98) 1.62 ± 0.01 (91) 1.42 ± 0.002 (102)Sodium Certifieda 40 ± 4 3770 ± 340 390 ± 28

Foundb

(recovery, %)c

NDd

3550 ± 222 (94) 378 ± 7 (97)

Phosphorus Certifieda 3330 ± 210 10010 ± 320 948 ± 33

Foundb (recovery, %)c 3301 ± 48 (99) 11643 ± 295 (116) 962 ± 10 (101)

Zinc Certifieda 36.6 ± 1.7 67.5 ± 7.6 10.5 ± 0.3

Foundb (recovery, %)c 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 singledetermination.

b SD from average of triplicates analysis.c Found value/certified value ratio expressed in %.d ND = Not determined (<LOQ).



sample. Power settings and nebulizer gas flow were optimized

to ensure that the Mg 280.271:Mg 285.213 ratio demonstrates

robust operating conditions (18) in accordance with the ratio

established by the instrument manufacturer. Five replicate

readings of the same sample were performed, with relatively

long integration times to minimize noise. The solution

presented to the nebulizer contains at the most 5000 mg/L

cesium ( Note: 500 mg/L Cs for ruggedness tests MD1, MD2);

20 mg/L indium; and 5 mg/L strontium, yttrium, and

chromium; less than half of each element concentration of the

higher working standard Std6 and <0.5 g/L total dissolved

minerals. All responses for both recommended and alternative

lines for each element are corrected using one IS line

(Table 5).

(b) ICP-AES spectrometers (MD3 and MD4 ruggedness

tests).—Operating conditions of both instruments (Table 1) as

well as sample and IS pump tubes and peristaltic pump

rotation speed used were selected according to manufacturer’s

recommendations and were adapted to optimize aerosol and

maximize precision, and to demonstrate robust operating

conditions. They were capable of determining at least the

recommended multiple lines for each element of interest

(Table 5). Combined ionization buffer/IS solution was

prepared such that after mixing digest sample and

IS/ionization buffer solutions using the instrument’s peristaltic pump, the combined solution presented to the

nebulizer contained at least 500 mg/L Cs; 20 mg/L indium;

5 mg/L strontium, yttrium, and chromium; and less than half

of each element concentration of the higher working standard

and <.5 g/L total dissolved minerals. All sample instrument

responses of recommended and alternate lines for each

element are corrected using one IS line (Table 5).

(c) Quantification.—The concentration (C) of each

element, in mg/kg, was calculated as follows:

C a V

m

F

where C = concentration in the test portion sample (mg/kg); a

= concentration (mg/L) of the element in the digest solution; V

= volume (mL) of the test solution after being made up [i.e.,

50 mL for closed vessel microwave digestion (MDC) for SLV

and 100 mL for open-vessel microwave digestion (MDO)

used for test MD2]; F = dilution factor of the test solution; m =

weight of the test portion (g).

Statisic Calculations

All data used for selectivity, accuracy and precision

performance tests were treated using robust statistics (29)

based in the concept of Rousseeuw and Croux (28). The

presence of some suspect values (outliers) can strongly distort

classical estimations. However, results must not be eliminated

without a valid justification. For that reason, robust statistics,

which provide good estimations even without the elimination

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.

The median has been used as a robust estimation of the central

value. The robust standard deviation SD(Sn) drawn from the

algorithm Sn of Rousseeuwand Croux (28) has been used as a

robust estimation of spread. This algorithm uses median

absolute distances between all the couples we can set up withthe data:

Sn = medi {medj *xi – xj *}

SD(Sn) = 1.1926 Sn

Statistical comparative t -test is the robust adaptation of the

classical t -test by replacing the mean with the median, and the

SD with the robust SD(Sn). Robust t -test was used for

selectivity (median comparison) and accuracy was evaluated

to check if the recovery is significantly different from 100%

(at 95 and 99% confidence intervals).


Table 7. Regression coefficients obtained from

weighed linear and nonlinear regression for

recommended and alternate lines

Calibration curvea Concn range, mg/L

Element line R² nonlinear b R² linear c Low High

Ca IId

0.99997 0.99980 0 240

Cu Id 0.99999 1.00000 0 2.1

Fe IId

0.99997 0.99999 0 8.5

K Id

0.99924 0.99326 0 320

Mg Id

0.99999 0.99891 0 75

Mn IId

1.00000 0.99998 0 0.04

Na Id

0.99996 0.99666 0 160

P Id

0.99998 0.99997 0 160

Zn Id

0.99999 0.99998 0 3.2

Ca IIe

0.99994 0.99993 0 240

Cu Ie

1.00000 0.99995 0 2.1

Cu Ie

0.99999 0.99999 0 2.1

Cu Ie 0.99999 0.99998 0 2.1

Fe IIe

0.99996 0.99997 0 8.5

Fe IIe

0.99999 0.99998 0 8.5

Mg Ie

0.99997 0.99929 0 80

Mg IIe

1.00000 0.99999 0 80

Mn IIe

1.00000 0.99997 0 0.04

Mn IIe

1.00000 0.99998 0 0.04

P Ie

0.99999 0.99967 0 160

P Ie

1.00000 0.99993 0 160

Zn Ie

0.99999 0.99991 0 3.2

Zn Ie

1.00000 1.00000 0 3.2

a Calibration with six standard solutions and a blank.b Weighed second degree quadratic regression.c Weighed linear regression without forcing zero.d Recommended lines for ICP-AES analysis.e Alternate confirmatory lines for ICP-AES analysis.



Recovery is judged satisfactory if it is not significantly

different from 100% at 95% confidence interval ( P -value

>0.05) between the median concentration and the certified or

reference values (Table 2). Recovery is judged questionable if

it is not significantly different from 100% at 99% confidence

interval (0.01 < P -value < 0.05) between the median

concentration and the certified or reference values (Table 2).

Recovery is judged unsatisfactory if it is significantly

different from 100% at 99% confidence interval ( P -value

<0.01) between the median concentration and the certified or

reference values (Table 2).

SLV

(a) Study material .—Ten materials included six reference

materials (RM) of LGC Promochem LGC RM 7104

(sterilized cream), of existing NIST Standard Reference

Material (SRM) NIST SRM 2384 (baking chocolate), NIST

RM 8415 (whole egg powder), NIST SRM 2383 (baby food

composite), NIST RM 8433 (corn bran), NIST SRM 1549

(nonfat milk powder), and four Nestlé well-characterized

reference samples of infant cereals with milk powder,

chocolate milk powder, dietetic milk powder (DMP1), and pet

food (PET). The reference values of element concentrations in

the four in-house reference materials and their associated

reproducibility SD were obtained from Nestlé proficiency

tests performed by a significant number of internal and

external laboratories (between 12 and 45 laboratories,

depending on element and materials analyzed) using robust

statistics (28).

(b) Microwave digestion optimization.—Main reagents

for microwave-assisted digestion are HNO3 and HCl. H2O2 is

generally used to remove any remaining organic residues of

rich-fat or rich-carbohydrate samples. MDC systems supplied

by manufacturers typically apply to a single food matrix or

minimize the analytical portion mass to account for the most

reactive food matrix. This approach is notefficient when a full

range of food matrixes is analyzed on a routine basis, and an

additional predigestion step is often used to allow various

matrixes to be digested concurrently. Thus, approach of a

single optimized MDC program for duty vessels with only

HNO3 operating up to 200C, which has been adapted on a

CEM Mars Xpress system, was favored: an optimal analytical

test portion mass of 0.5 g [based on an empirical maximum

energy release by the food of 3 kcal (8)] was used, and

concurrent digestions of triplicates of three NIST-certifiedRMs (baking chocolate, NIST SRM 2384; whole egg powder,

NIST RM 8415; and Baby Food Composite, NIST SRM

2383) were performed. Similar programs to that of CEM Mars

Xpress system were adapted on other MDC systems used for

MD3 and MD4 ruggedness tests. An optimized program for

MDO system CEM MDS 2000 (Table 3) was also developed

in parallel, and used for all matrixes during ruggedness test

MD2 and by one volunteer laboratory during the RT.

(c) Linearity.—A calibration curve was obtained from

seven standards, including a blank (Std0) and six

concentrations of the standard solution (Std1–Std6).

Weighted linear and quadratic regression analyses were

performed and correlation coefficient (R 2) values were

calculated.

(d) LOD and LOQ.—The instrumental detection (DL)

and quantification (QL) limits expressed in mg/L were

estimated using the SD approach by analyzing 10 replicates of

the low standard solution (Std1) according to the formula:

DL = 3 SD of the mean of Std1 determinations ( n = 10)

QL = 10 SD of the mean of Std1 determinations ( n = 10)

The DL and QL values of elements in real samples were

hence estimated assuming a sample weight of 0.5 g and a

dilution to 50 mL (i.e., a dilution factor of 100). Verification

of QL estimation was performed by checking satisfactory

performance characteristics (accuracy and precision) for

elements present in real matrixes at these estimated

concentrations, e.g., Ca, Cu, Fe, K, Na, P in corn bran (NIST

RM 8433), Mg, Zn in sterilized cream (LGC RM 7104), and

Mn in nonfat milk powder (NIST SRM 1549).

(e) Selectivity.—Element median recovery was calculated

using all the linesdisplayed in Table 5 from duplicates of eight

different solutions of spiked food grade salts (Table 2) with a

spiking concentration corresponding to Std4 prepared from

the CPI commercial solution. Robust t -test for comparison of

median recovery and SD (i.e., median of averages of

duplicates found per element using all analytical lines) of eachspiked element in all food-grade salts with and without

addition of ionization buffer Cs 1% (w/v) was performed.

(f ) Accuracy.—Three replicate test portions (r = 3) were

collected from each of seven (n = 7) different food samples

populating four of the nine food triangle sectors, and then

were digested and analyzed on 8 days (d = 8) corresponding to

nine series (i.e., 9 elements) of 168 data (n r d = 168).

Recovery median values (ratio of found and certified values)

and associated RSD were determined. Accuracy was

statistically evaluated.


Table 8. Estimated LOD and LOQ for the nine elements

in food matrixes obtained from recommended and

alternate element lines

Element LODa, mg/L LOQb, mg/L LOQc , mg/kg

Calcium 0.5 1.50 150

Copper 0.006 0.02 2

Iron 0.03 0.1 10Potassium 0.5 2 200

Magnesium 0.2 0.5 50

Manganese 0.0002 0.0005 0.05

Sodium 0.4 1 100

Phosphorus 0.4 1 100

Zinc 0.02 0.05 5

a Instrumental LOD calculated in solution.b Instrumental LOQ calculated in solution.c LOQ estimated in food matrixes (dilution factor 100).



(g) Precision.—Both repeatability standard deviation

(SDr ; within day) and intermediate reproducibility standard

deviation (SDiR , between day) were determined.

(h) Uncertainty.—Uncertainty (U) was estimated using

the simplified approach based on existing validation data

proposed by Barwick and Ellison (30, 31), mainly from

precision and trueness studies, which, if properly planned to

cover as many of the uncertainty sources previously identifiedas possible, provide the necessary data required to calculate

measurement uncertainty. SDs of intermediate precision

(SDiR ) and recovery (SDrec) contributions are combined

together as followed to obtain the overall uncertainty (U):

U SD SDiR

2

rec 2

(i) HorRat values.—Horwitz ratio (HorRat value) was

calculated for SLV according to the equation:

HorRat value = RSD , %

2C , %

iR

0.1505

where RSDiR is the relative intermediate reproducibility

standard deviation of each element in each of the 10 tested,

certified or in-house reference matrixes; C is the relative

concentration (expressed as a dimensionless mass fraction) of

the analyte in question (valid for concentration ratioabove 10 –9).

The HorRat value is a simple performance parameter that

reflects the acceptability of a chemical method of analysis

with respect to precision (32).

HorRat value of 1.0 is judged satisfactory with limits of

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

deviations on the high side (values >1.5) may indicate

inhomogeneity of the test samples, need for further method


Table 9. Recovery values (%) and standard deviations (%) found using recommended lines for nine elements spiked

in eight food-grade salts

Spikedelementline

Calciumcarbonate

Calcium citratetribasic

Calciumphosphate

tribasicPotassium

chlorideMagnesiumcarbonate

Magnesiumchloride

Manganesesulfate 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

Ca IIb

ND ND ND 104.5 ± 2.1 100.6 ± 0.2 103.4 ± 0.6 98.2 ± 6.8 105.4 ± 6.7

Cu Ia 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

Cu Ib

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

Cu Ib

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

Cu Ib

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

Fe IIa

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

Fe IIb

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

Fe IIb

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

K Ia

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

Mg Ia

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

Mg Ib

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

Mg Ib

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

Mn IIa

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

Mn IIb

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

Mn IIb

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

Na Ia

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

P Ia

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

P Ib

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

P Ib

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

Zn Ia

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

Zn Ib

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

Zn Ib

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 Alternate confirmatory lines for ICP-AES analysis.c ND = Not determined.



optimization or training, operating below the limit of

determination, or an unsatisfactory method. It is now one of

the acceptability criteria for many of the recently adopted

chemical methods analysis of AOAC INTERNATIONAL,

the European Union, and other European organizations

dealing with food analysis (33).

( j) Ruggedness.—Three replicate test portions (r = 3)were collected from each of the six (n = 6) different food

samples populating three of the nine food triangle sectors, and

then were digested using different closed-vessel (MD1, MD3,

and MD4) and open-vessel (MD2) microwave digestion

systems (Table 1). Triplicates were analyzed for 8 days (d = 8)

corresponding to nine series (i.e., nine elements) of 144 data

(n r d = 144), and using different ICP equipment with the

recommended lines (except the use of alternate 2MnII and2Zn I alternate lines for the MD4 test) according to adapted

ICP operating conditions used for SLV with addition of ion

buffer Cs 0.1% (w/v). Recovery median values, SDr (within

day), SDiR (between day), and HorRat values were

determined. Accuracy was statistically evaluated.

Ring Trial

(a) Design of the study.—Five materials that cover four of

the nine sectors of AOAC food triangle (Table 2) were

proposed for testing the proposed ICP-AES method for

determining the nine elements of interest in food matrixes

after microwave digestion through an internal RT.

(b) Study material .—Five materials were samples of

existing NIST SRM 2387 (peanut butter) and NIST RM 8418

(wheat gluten), and three in-house reference samples of infant

cereals with milk powder, chocolate milk powder, and dietetic

milk powder 2 (DMP2). The three in-house reference

materials were validated through Nestlé proficiency tests

performed by a significant number of internal and external

laboratories (between 12 and 45 depending on element and

materials analyzed). Their reference values and associated

SDR values were calculated using robust statistics (28).

(c) Protocol .—Three lots of each NIST material were

simply remixed in an amber container and sample portions of minimum 10 g were then extracted from random locations in

the container using a small weighing spatula and transferred

into numbered amber PVC 100 mL boxes (Greiner Bio-one,

Frickenhausen, Germany) for distribution to participants.

Sample portions of minimum 10 g were extracted from

random locations in each original P -test container of the

three in-house references using a small weighing spatula and

transferred into numbered amber PVC 100 mL boxes (Greiner

Bio-one) for distribution to participants. One set of five

numbered samples, including five amber 100 mL flasks

containing 10 g materials, was mailed to nine collaborators

who volunteered to participate in this study.

(d) Stock standard solution.—Prepare working standards

either from a multistandard commercial stock standard

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.

(e) Intermediate stock standard solution.—(Composition

of the intermediate standard solution, in mg/L: Ca = 1500; Cu

=10;Fe=50;K=2000;Mg=500;Mn=0.25;Na=1000;P=

1000; Zn = 20.) Add into a 500 mL volumetric flask, 75 mL

calcium 10 000 mg/L, 5 mL copper 1000 mg/L, 25 mL iron

1000 mg/L, 100 mL potassium, 25 mL magnesium

10 000 mg/L, 0.125 mL manganese 1000 mg/L, 50 mLsodium 10 000 mg/L, 50 mL phosphorus 10 000 mg/L, and

10 mL zinc 1000 mg/L. Add 10 mL of analytical grade HNO3

and dilute to volume with H2O.

(f ) Working standard solutions.—Standards prepared

from intermediate stock standard solution are designed to

have the same acid concentration as digested test solutions.

(1) Std6 .—Pipet 15.0 mL intermediate standard solution into a


grade HNO3, dilute to volume with H2O, mix, and transfer to

acid-washed polypropylene bottle. (2) Std5.—Pipet 10.0 mL

intermediate standard solution into a 100 mL acid-washed

volumetric flask. Add 10 mL analytical grade HNO3, dilute to

volume with H2O, mix, and transfer to acid-washed

polypropylene bottle. (3) Std4.—Pipet 5.0 mL intermediate

standard solution into a 100 mL acid-washed volumetric flask.

Add 10 mL analytical grade HNO3, dilute to volume with

H2O, mix, and transfer to acid-washed polypropylene bottle.

(4) Std3.—Pipet 2.0 mL intermediate standard solution into a


grade HNO3, dilute to volume with H2O, mix, and transfer to

acid-washed polypropylene bottle. (5) Std2.—Pipet 1.0 mL

intermediate standard solution into a 100 mL acid-washed

volumetric flask. Add 10 mL analytical grade HNO3, dilute to


Table 10. Median recovery (%) and standard deviations

(%) by element and for all elements in eight food-grade

salts using recommended lines

Recovery, %b

Spiked element Concn, mg/La Without Csc With Cs 1% w/vd

Calcium 60 93 ± 4 101 ± 4

Copper 0.4 92 ± 3 105 ± 4

Iron 2 92 ± 3 101 ± 4

Potassium 80 117 ± 18 105 ± 5

Magnesium 20 95 ± 5 102 ± 6

Manganese 0.01 95 ± 7 105 ± 6

Sodium 40 110 ± 14 103 ± 5

Phosphorus 40 90 ± 7 102 ± 4

Zinc 0.8 91 ± 7 103 ± 6

All elementse

0.01–60 94 ± 9 103 ± 5

a Concentration of spiked element in salt solution.b Average of spiked element recoveries in all food-grade salts

solutions.c Recovery value without Cs ion buffer addition.d Recovery value with Cs ion buffer 1% (w/v) addition.e Median of all spiked elements recoveries in all food-grade salts.



volume with H2O, mix and transfer to acid-washed

polypropylene bottle. (6 ) Std1.—Pipet 0.5 mL intermediate

standard solution into a 100 mL acid-washed volumetric flask.

Add 10 mL analytical grade HNO3, dilute to volume with

H2O, mix, and transfer to acid-washed polypropylene bottle.

All calibration solutions should be stable for 1 week.

(g) Test portion preparation.—Three single test portions

were first extracted using a small weighing spatula from each

of the five amber PVC 100 mL boxes (Greiner Bio-one)containing 10 g of test sample (Table 2), then weighed, and

finally prepared as described for SLV before MCD or MDO

microwave digestions.

(h) Sample digestion.—All 15 test portions (i.e.,

triplicates extracted from each of the five test samples) were

digested according to MCD or MDO programs adapted or

similar to those described for SLV (Mars Xpress and MDS

2000 systems, respectively). Information was provided to

collaborators concerning the maximum concentration for each

element in the five delivered food matrixes in order to

pre-adapt the final dilution of digest test portions before

ICP-AES analysis.

(i) Detection.—Digested test solutions, or appropriate

dilutions were presented to the ICP-AES instrument

calibrated as described for SLV with acid-matched standard

calibrant solutions.

( j) Calculations.—Element concentrations in the five

food matrixes were calculated using the equation described

for SLV in the ICP Analysis Section, part (c).

(k ) Statistical calculations.—Medians, SDr , and SDiR ,

repeatability (r) and reproducibility (R) limits, z -scores, and

HorRat values for nine elements in five blind samples

prepared in triplicate were determined by a statistical

treatment of 45 data sets.

(l) Repeatability and reproducibility limits.— Repeatability limit r, which is the maximum tolerated relative

variation between two measurements taken by a single person

or instrument on the same matrix and under the same

conditions, is expressed as follows:

2.772 SDr

where SDr is the repeatability standard deviation.

Reproducibility limit R is the maximum variation between

two measurements taken by several persons in different

laboratories, on different days, under different conditions, and

is expressed as follows:

2.772 SDR

where SDR is the reproducibility standard deviation.

(m) Z-score.—Evaluation of the whole laboratory

performance in term of accuracy was obtained based on the

calculation of the z -score. The z-score “ z ” is given by the

following equation:

z x X

RSDR

where x is the found mean value of analyte concentration in

the test material calculated from the nine means reported by

the nine laboratories; X is the certified (or reference) value of

the certified (or in-house) reference material; RSDR is the

relative reproducibility standard deviation.

If z 2, the result is satisfactory; if 2 < z < 3, the result is

questionable; if z > 3, the result is unsatisfactory.

(n) HorRat values.—HorRat values for each element in

each of the five matrixes were calculated as follows:

HorRat value = RSD , %

2C , %

R

0.1505

where RSDR is the relative reproducibility standard deviation

of each element in each of the five tested matrixes; C is the

relative concentration (expressed as dimensionless mass

fraction) of the analyte in question (valid for concentration

ratio above 10 –9).

The original data developed from interlaboratory studies

were assigned a HorRat value of 1.0 with limits of

acceptability of 0.5–2.0. Consistent deviations from the ratio

on the low side (values <0.5) may indicate unreportedaveraging or excellent training and experience; consistent

deviations on the high side (values >2) may indicate

inhomogeneity of the test samples, need for further method

optimization or training, operating below the LOD, or an

unsatisfactory method.

(o) Horwitz equation.—Horwitz equation expressed using

log scale (log SDR = 0.8495 log C 1.6991) was compared with

a similar equation found using the 45 SDR values and medians

of concentration (i.e., SDR and median values for nine

elements in five matrixes) obtained from the data treatment of

the RT.

Results and Discussion

SLV

(a) Closed-vessel microwave digestion optimization.—

Average values of each element concentration in whole egg

powder, baby food composite, and baking chocolate fell

within confidence intervals of reference value for the three

matrixes and are in agreement with AOAC recovery criteria,

except for iron and copper in baking chocolate, with recovery

values of 123 and 125%, respectively, and for phosphorus in

whole egg powder, with a recovery value of 116% (Table 6).

Significant variation of results for iron and copper in baking

chocolate have previously been observed (34). The higher

value of recovery for phosphorus in whole egg powder is due

to the use of the optimized microwave program in CEM Mars

Xpress, whereas generally lower values are obtained with a

program leading to incomplete digestion (6, 8). This single

optimized microwave digestion program on a CEM Mars

Xpress system is adapted to be applicable to various matrixes

covering the AOAC food triangle.

(b) Linearity.—The calibration curves constructed by

plotting element concentration versus peak ratio response

(element/IS) showed good linearity either in linear or in

weighted nonlinear regression (Table 7). Weighted nonlinear





T a b l e

1 1 .

S L V s t u d y : a c c u r a c

y , p r e c i s i o n , a n d H o r R a t v a l u e s f o u n

d f o r c a l c i u m

i n s e v e n c e r t i f i e d a n d i n - h o u s e r e f e r e n c e m a t e r i a l s u s i n g r e c o m m e n d e d a n d

a l t e r n a t e l i n e s

M a t e r i a l d e s c r i p t i o n

L i n e

R e f e r e n c e

v a l u e , m g / k g a , b

M e d i a n ± U ,

m g / k g c

R e c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g

S t e r i l i z e d c r e a m

C a

h

8 4 5 ± 1 8

7 5 6 ± 1 3 2

9 0 ± 5

0 . 0 7

7 2

1 2 5

9 . 5

1 6 . 5

2 . 8

C a

i

7 2 6 ± 1 3 2

8 6 ± 5

0 . 0 2

6 7

1 1 2

9 . 3

1 5 . 4

2 . 6

C h o c o l a t e m i l k p o w d e r

C a

h

9 8 7 0 ± 1 2 4

1 0 0 9 2 ± 3 4 3

1 0 2 ± 2

0 . 2 1

1 3 5

3 0 3

1 . 3

3 . 0

0 . 7

C a

i

9 8 8 2 ± 1 7 9

1 0 0 ± 1

0 . 9 3

1 1 7

1 3 3

1 . 2

1 . 3

0 . 3

I n f a n t c e r e a l s

C a

h

6 0 0 0 ± 4 4

6 1 8 9 ± 2 4 9

1 0 3 ± 1

0 . 0 5

1 5 1

2 1 9

2 . 4

3 . 5

0 . 8

C a

i

5 9 8 3 ± 2 1 5

1 0 0 ± 1

0 . 8 2

1 6 0

2 0 4

2 . 7

3 . 4

0 . 8

C o r n b r a n

C a

h

4 2 0 ± 1 9

4 3 5 ± 2 9

1 0 4 ± 5

0 . 6 0

6

2 0 . 0

1 . 4

4 . 5

0 . 7

C a

i

4 2 4 ± 2 4

1 0 1 ± 5

0 . 9 7

6

1 4

1 . 4

3 . 3

0 . 5

D i e t e t i c m i l k p o w d e r 1

C a

h

3 0 2 0 ± 1 5

3 0 4 7 ± 5 6

1 0 1 ± 1

0 . 3 5

3 3

4 9

1 . 1

1 . 6

0 . 3

C a

i

2 9 8 7 ± 5 4

9 9 ± 1

0 . 1 1

2 2

4 9

0 . 7

1 . 7

0 . 4

P e t f o o d

C a

h

6 1 9 0 ± 1 4 0

6 5 8 0 ± 3 1 7

1 0 6 ± 3

0 . 0 5

9 4

1 9 2

1 . 4

2 . 9

0 . 7

C a

i

6 4 1 0 ± 2 1 2

1 0 4 ± 3

0 . 1 8

9 2

1 5 0

1 . 4

2 . 3

0 . 5

N o n f a t m i l k p o w d e r

C a

h

1 3 0 0 0 ± 2 5 0

1 3 0 7 0 ± 9 8 8

1 0 1 ± 3

0 . 8 6

2 0 6

9 0 8

1 . 6

6 . 9

1 . 8

C a

i

1 2 9 1 0 ± 8 1 3

9 9 ± 3

0 . 8 0

2 2 2

7 3 5

1 . 7

5 . 7

1 . 5

a

S D e x p r e s s e d a s h a l f o f s t a t e d u n c

e r t a i n t y ( N I S T a n d L G C c e r t i f i e d m a t e r i a l s ) g

i v e n a s 9 5 % c o n f i d e n c e i n t e r v a l .

b

S D e x p r e s s e d a s a r e p r o d u c i b i l i t y S

D ( i n - h o u s e r e f e r e n c e m a t e r i a l s ) c a l c u l a t e d

a c c o r d i n g t o r o b u s t s t a t i s t i c s ( 2 9 ) .

c

U n c e r t a i n t y ( U ) e s t i m a t e d t h r o u g h s i m p l i f i e d a p p r o a c h ( 3 0 , 3 1 ) .

d

S t a t i s t i c e v a l u a t i o n o f r o b u s t t - t e s t :

S a t i s f a c t o r y r e s u l t ( P

> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P

< 0 . 0 5 ) ; u n s a t i s f a c t o r y r e s u l t ( P

< 0 . 0 1 ) .

e

S D r = S D o f r e p e a t a b i l i t y .

f

S D i R = S D o f i n t e r m e d i a t e r e p r o d u c

i b i l i t y .

g

H o r R a t = R a t i o u s i n g R S D i R a n d H o r w i t z d e n o m i n a t o r a c c o r d i n g t o r e f . 3 2 .

h

R e c o m m e n d e d l i n e f o r I C P - A E S a n a l y s i s .

i

A l t e r n a t e c o n f i r m a t o r y l i n e s f o r I C P

- A E S a n a l y s i s .




T a b l e

1 2 .



d f o r c o p p e r i n t h r e e c e r t i f i e d a n d i n

- h o u s e r e f e r e n c e m a t e r i a l s u s i n g r e c

o m m e n d e d a n d



L i n e

R e f e r e n c e


M e d i a n ± U ,

m g / k g c

R

e c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g

C o r n b r a n

C u

h

2 . 4 7 ± 0 . 2 0

2 . 6 9 ± 0 . 2 4

1 0 9 ± 9

0 . 3 6

0 . 0 5

0 . 0 9

1 . 8

3 . 1

0 . 2

C u i

2 . 6 1 ± 0 . 2 4

1 0 6 ± 9

0 . 5 3

0 . 0 5

0 . 1

1 . 9

3 . 9

0 . 3

C u i

2 . 5 6 ± 0 . 2 6

1 0 3 ± 9

0 . 7

0 . 0 6

0 . 1 5

2 . 4

5 . 6

0 . 4

C u i

2 . 5 2 ± 0 . 2 5

1 0 2 ± 8

0 . 8 2

0 . 0 6

0 . 1 4

2 . 5

5 . 4

0 . 4


C u

h

7 . 5 5 ± 0 . 1 3

7 . 7 8 ± 0 . 2 1

1 0 3 ± 2

0 . 1 5

0 . 0 9

0 . 1 5

1 . 1

2

0 . 2

C u i

7 . 6 3 ± 0 . 2 3

1 0 1 ± 2

0 . 6

0 . 0 8

0 . 1 8

1

2 . 4

0 . 2

C u i

7 . 7 2 ± 0 . 2 5

1 0 2 ± 2

0 . 2 8

0 . 0 8

0 . 2

1

2 . 6

0 . 2

C u i

7 . 5 8 ± 0 . 2 1

1 0 0 ± 2

0 . 8 4

0 . 0 8

0 . 1 5

1

2

0 . 2


C u

h

0 . 7 0 ± 0 . 0 5

0 . 7 5 ± 0 . 0 8

1 0 7 ± 8

0 . 4 4

0 . 0 6

0 . 0 6

8 . 3

8 . 5

0 . 5

C u i

0 . 7 8 ± 0 . 1 1

1 1 2 ± 9

0 . 2 2

0 . 0 6

0 . 0 9

8 . 3

1 1 . 4

0 . 7

C u i

0 . 7 8 ± 0 . 0 9

1 1 2 ± 8

0 . 2 1

0 . 0 5

0 . 0 7

7

8 . 5

0 . 5

C u i

0 . 7 9 ± 0 . 1 0

1 1 3 ± 9

0 . 1 7

0 . 0 5

0 . 0 8

6 . 8

1 0 . 2

0 . 6

a




b




c


d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P

< 0 . 0 5 ) ; u n s a t i s f a c t o r y r e s u l t

( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g


h


i






T a b l e

1 3 .



d f o r i r o n i n f o u r c e r t i f i e d a n d i n - h o u

s e r e f e r e n c e m a t e r i a l s u s i n g r e c o m m e n d e d a n d a l t e r n a t e

l i n e s


L i n

e

R e f e r e n c e


M e d i a n ± U ,

m g / k g c

R e c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g


F e

h

1 6 9 . 9 ± 2 . 4

1 6 6 . 9 ± 1 2 . 0

9 8 ± 2

0 . 3 3

1 3 . 2

1 1 . 7

7 . 9

7 . 0

1 . 0

F e

i

1 6 3 . 1 ± 1 2 . 4

9 6 ± 2

0 . 1 0

1 3 . 1

1 2 . 2

7 . 9

7 . 3

0 . 8

F e

i

1 6 6 . 6 ± 1 3 . 1

9 8 ± 2

0 . 3 5

1 3 . 3

1 2 . 7

8 . 0

7 . 6

1 . 2


F e

h

8 0 . 4 ± 0 . 6

7 9 . 6 ± 1 . 3

9 9 ± 1

0 . 2 8

1 . 0

1 . 1

1 . 2

1 . 4

1 . 0

F e

i

7 9 . 0 ± 1 . 2

9 8 ± 1

0 . 0 9

1 . 0

1 . 1

1 . 3

1 . 3

0 . 7

F e

i

7 9 . 5 ± 1 . 3

9 9 ± 1

0 . 2 5

1 . 1

1 . 2

1 . 3

1 . 4

0 . 3

C o r n b r a n

F e

h

1 4 . 8 ± 0 . 9

1 4 . 7 ± 1 . 3

9 9 ± 6

0 . 8 8

0 . 2

1 . 0

1 . 3

6 . 7

0 . 2

F e

i

1 4 . 7 ± 1 . 2

9 9 ± 6

0 . 9 2

0 . 2

0 . 8

1 . 3

5 . 4

0 . 3

F e

i

1 4 . 7 ± 1 . 1

9 9 ± 6

0 . 8 8

0 . 2

0 . 7

1 . 4

4 . 8

0 . 3


F e

h

5 8 . 5 ± 0 . 4

5 9 . 3 ± 0 . 9

1 0 1 ± 1

0 . 1 1

0 . 8

0 . 8

1 . 3

1 . 3

0 . 2

F e

i

5 9 . 1 ± 0 . 9

1 0 1 ± 1

0 . 2 4

0 . 7

0 . 8

1 . 1

1 . 4

0 . 3

F e

i

5 9 . 1 ± 1 . 0

1 0 1 ± 1

0 . 2 6

0 . 7

0 . 9

1 . 1

1 . 5

0 . 4

a




b




c


d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g


h


i





regression was used during SLV, MD1, and MD2 tests, as the

best regression coefficients are obtained with R 2 > 0.99995 for

all lines except for 1K766.491 (R 2 = 0.99924) and for 2Ca317.933 (R 2 = 0.99994) lines.

(c) LOD and LOQ.—The detection and quantification

limits of elements in real matrixes were estimated in Table 8

by affecting a dilution factor of 100 (i.e., corresponding to

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,

Fe, K, Na, P in corn bran; Mg, Zn in sterilized cream, and Mn

in nonfat milk powder).

(d) Selectivity.—Element mean recoveries and associated

SD values calculated from spiked element of interest in eight

different solutions of food-grade salts are displayed in

Table 9. Good recoveries ranging between 88 and 115% were

found in all food-grade salt solutions with SDs ranging

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 element spiked duplicates in all food-grade salt solutions with

and withoutadded 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

(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

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

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

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

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

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

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 fulfillAOAC 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

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

(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.

Calculated derived relative U values range from 1 to 26% for

all elements in the seven tested matrixes.


T a b l e

1 4 .



d f o r p o t a s s i u m

i n s i x c e r t i f i e d a n d i n - h o u s e r e f e r e n c e m a t e r i a l s u s i n g r e

c o m m e n d e d a n d



L i n e

R e f e r e n c e


M e d i a n ± U ,

m g / k g c

R e

c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g


K h

1 1 6 0 ± 5 0

1 3 9 5 ± 2 0 3

1 2 0 ± 7

0 . 0 2

5 4

1 4 6

3 . 9

1 0 . 5

2 . 0


K h

9 0 9 0 ± 1 3 3

8 7 2 6 ± 4 6 8

9 6 ± 2

0 . 0 3

1 2 5

3 9 9

1 . 4

4 . 6

1 . 1


K h

6 5 0 0 ± 5 0

6 4 2 3 ± 4 8 5

9 9 ± 3

0 . 6 6

1 0 8

4 5 4

1 . 7

7 . 1

1 . 7

C o r n b r a n

K h

5 6 6 ± 3 8

5 3 1 ± 6 0

9 4 ± 7

0 . 7 0

1 0

4 6

1 . 8

8 . 6

1 . 4


K h

5 5 7 0 ± 2 5

5 6 0 0 ± 1 7 5

1 0 1 ± 1

0 . 8 8

5 4

1 5 4

1 . 0

2 . 8

0 . 6


K h

1 6 9 0 0 ± 1 5 0

1 7 0 0 8 ± 1 3 4 2

1 0 1 ± 2

0 . 7 7

3 2 2

9 5 8

1 . 9

5 . 6

1 . 5

a




b




c


d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


< 0 . 0 1 ) .

e


f


i b i l i t y .

g


h





T a b l e

1 5 .



d f o r m a g n e s i u m

i n s e v e n c e r t i f i e d a

n d i n - h o u s e r e f e r e n c e m a t e r i a l s u s i n g r e c o m m e n d e d a n d



L i n e

R e f e r e n c e

v a l u e , m g / k g a ,

b

M e d i a n ± U ,

m g / k g c

R e

c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g


M g

h

8 4 . 4 ± 2 . 0

8 1 . 7 ± 4 . 5

9 7 ± 3

0 . 2 8

2 . 0

3 . 9

2 . 4

4 . 7

0 . 6

M g

i

7 9 . 9 ± 4 . 2

9 5 ± 3

0 . 0 8

1 . 7

3 . 6

2 . 1

4 . 5

0 . 5

M g

i

8 2 . 7 ± 4 . 4

9 8 ± 3

0 . 4 8

2 . 3

3 . 8

2 . 7

4 . 6

0 . 6


M g

h

1 7 7 9 ± 2 3

1 7 7 1 ± 2 2

1 0 0 ± 1

0 . 8 2

2 3

3 2

1 . 3

1 . 8

0 . 3

M g

i

1 7 5 7 ± 6 7

9 9 ± 2

0 . 4 8

2 2

5 9

1 . 3

3 . 4

0 . 7

M g

i

1 7 4 4 ± 5 4

9 8 ± 2

0 . 2 2

2 1

4 7

1 . 2

2 . 7

0 . 5


M g

h

8 8 1 ± 8

9 1 1 ± 4 6

1 0 3 ± 2

0 . 1 0

2 5

4 3

2 . 7

4 . 7

0 . 8

M g

i

9 0 2 ± 4 3

1 0 2 ± 2

0 . 2 2

2 1

4 0

2 . 3

4 . 5

0 . 8

M g

i

8 9 2 ± 4 8

1 0 1 ± 2

0 . 5 3

2 4

4 6

2 . 7

5 . 1

0 . 9

C o r n b r a n

M g

h

8 1 8 ± 3 0

7 9 1 ± 4 5

9 7 ± 4

0 . 5 3

9

3 5

1 . 1

4 . 4

0 . 8

M g

i

7 7 5 ± 4 5

9 5 ± 4

0 . 2 5

8

3 3

1 . 0

4 . 2

0 . 7

M g

i

7 8 5 ± 4 0

9 6 ± 4

0 . 4 1

9

2 6

1 . 1

3 . 3

0 . 6


M g

h

1 2 0 0 ± 8

1 2 0 6 ± 2 5

1 0 1 ± 1

0 . 8 8

8 . 0

2 3

0 . 7

1 . 9

0 . 3

M g

i

1 1 8 0 ± 3 0

9 8 ± 1

0 . 0 6

7 . 0

2 8

0 . 6

2 . 3

0 . 4

M g

i

1 1 8 1 ± 3 4

9 8 ± 1

0 . 0 3

9

2 9

0 . 8

2 . 5

0 . 5

P e t f o o d

M g

h

4 8 1 ± 2

4 8 3 ± 8

1 0 1 ± 1

0 . 3 1

7

8

1 . 4

1 . 6

0 . 3

M g

i

4 7 1 ± 1 2

9 8 ± 1

0 . 0 8

6

1 1

1 . 2

2 . 4

0 . 4

M g

i

4 7 7 ± 1 2

9 9 ± 1

0 . 5 2

8

1 1

1 . 6

2 . 3

0 . 4


M g

h

1 2 0 0 ± 1 5

1 2 0 6 ± 8 9

1 0 1 ± 3

0 . 8 7

1 8

8 3

1 . 5

6 . 9

1 . 3

M g

i

1 1 8 6 ± 7 0

9 9 ± 2

0 . 6 0

1 9

6 5

1 . 6

5 . 5

1 . 0

M g

i

1 1 5 6 ± 8 7

9 6 ± 3

0 . 2 1

1 6

8 1

1 . 4

7 . 0

1 . 3

a




b




c


d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g


h


i






T a b l e

1 6 .



d f o r m a n g a n e s e i n t h r e e c e r t i f i e d a n d i n - h o u s e r e f e r e n c e m a t e r i a l s u s i n

g r e c o m m e n d e d a n d



L i n e

R e f e r e n c e


M e d i a n ± U ,

m g / k g c

R e c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R S D i R , %

H o r R a t g

C o r n b r a n

M n

h

2 . 5 5 ± 0 . 1 5

2 . 3 3 ± 0 . 1 8

9 1 ± 6

0 . 1 5

0 . 0 3

0 . 1 2

1 . 1

4 . 9

0 . 4

M n

i

2 . 5 5 ± 0 . 2 5

1

0 0 ± 6

0 . 9 6

0 . 0 3

0 . 2 0

1 . 1

7 . 3

0 . 5

M n

i

2 . 3 9 ± 0 . 1 7

9 4 ± 6

0 . 2 5

0 . 0 3

0 . 0 9

1 . 1

3 . 6

0 . 3


M n

h

1 2 . 7 ± 0 . 8 5

1 2 . 8 ± 1 . 1

1

0 2 ± 7

0 . 8 3

0 . 2 7

0 . 6 8

2 . 1

5 . 3

0 . 5

M n

i

1 3 . 4 ± 1 . 3

1

0 5 ± 7

0 . 4 8

0 . 2 6

0 . 8 8

2 . 0

6 . 6

0 . 6

M n

i

1 2 . 8 ± 1 . 1

1

0 1 ± 7

0 . 8 8

0 . 2 4

0 . 6 7

1 . 8

5 . 2

0 . 5


M n

h

0 . 2 6 ± 0 . 0 3

0 . 2 2 ± 0 . 0 3

8 5 ± 1 0

0 . 1 6

0 . 0 1

0 . 0 2

3 . 1

6 . 8

0 . 3

M n

i

0 . 2 0 ± 0 . 0 3

7 9 ± 9

0 . 0 6

0 . 0 1

0 . 0 2

3 . 8

1 0 . 8

0 . 5

M n

i

0 . 2 2 ± 0 . 0 3

8 5 ± 1 0

0 . 0 8

0 . 0 1

0 . 0 1

3 . 6

5 . 9

0 . 3

a




b




c


d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g


h


i





(h) HorRat values.—HorRat values never exceed 1.5

except for several analytical lines of Ca (2.8 and 2.6), K (2.0),

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

values higher than 3.0. If several values are between 0.2 and

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

six matrixes tested. Global statistic recovery results were

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

MD4, K for MD1, Mg for MD1 and MD3, and Na for MD4.

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

milk powder for MD1. Unsatisfactory statistical recovery

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

applicability of the method is limited by less analytical

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).

However, AOAC requirements for recovery performance

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

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

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

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 thismethod 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.

Ring Trial

(a) Calcium.—Table 29 presents estimated performance

characteristics of the ICP-AES method for determination of

Ca, Cu, Fe, K, Mg, Mn, Na, P, and Zn. The RSDr of the


T a b l e

1 7 .



d f o r s o d i u m

i n s i x c e r t i f i e d a n d i n - h

o u s e r e f e r e n c e m a t e r i a l s u s i n g r e c o

m m e n d e d a n d a l t e r n a t e

l i n e s


L i n e

R e f e r e n c e


M e d i a n ± U ,

m g / k g c

R e c o v e r y , %

P - v a l u e

d

S D r , m g / k g

e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g


N A

h

5 0 5 ± 1 9

5 5 4 ± 6 7

1 1 0 ± 6

0 . 1 3

2

6

3 . 4

1 0 . 9

1 . 8


N A

h

1 9 9 0 ± 4 2

1 9 5 0 ± 7 4

9 8 ± 2

0 . 3 7

3 2

5 9

1 . 6

3 . 0

0 . 6


N A

h

1 0 5 0 ± 1 4

1 0 5 4 ± 6 4

1 0 0 ± 2

0 . 8 9

3 4

6 0

3 . 2

5 . 7

1 . 0

C o r n b r a n

N A

h

4 3 0 ± 1 6

4 2 4 ± 2 6

9 9 ± 4

0 . 4 0

6

1 9

1 . 5

4 . 4

0 . 7


N A

h

4 9 2 0 ± 3 0

4 8 9 0 ± 8 7

9 9 ± 1

0 . 2 0

4 0

7 9

0 . 8

1 . 6

0 . 4


N A

h

4 9 7 0 ± 5 0

4 9 1 0 ± 3 2 1

9 9 ± 1

0 . 6 1

6 5

2 9 7

1 . 3

6 . 1

1 . 4

a




b




c


d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g


h





T a b l e

1 8 .



d f o r p h o s p h o r u s i n s e v e n c e r t i f i e d a n d i n - h o u s e r e f e r e n c e m a t e r i a l s u s i n g r e c o m m e n d e d a n d



L i n e

R e f e r e n c e


M e d i a n ± U ,

m g / k g c

R e

c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g


P h

8 2 3 ± 2 2

8 0 7 ± 6 0

9 8 ± 3

0 . 5 6

3 2

5 3

4 . 0

6 . 6

1 . 1

P i

8 2 1 ± 5 6

9 9 ± 3

0 . 9 5

3 6

4 9

4 . 4

6 . 0

1 . 0

P i

7 8 3 ± 8 2

9 5 ± 4

0 . 2 6

3 2

7 6

4 . 1

9 . 6

1 . 6


P h

4 0 8 0 ± 3 0

4 1 1 0 ± 1 5 0

1 0 1 ± 1

0 . 6 0

6 7

1 3 8

1 . 6

3 . 4

0 . 7

P i

4 1 5 0 ± 8 8

1 0 2 ± 1

0 . 6 1

6 0

7 0

1 . 5

1 . 7

0 . 4

P i

4 0 5 0 ± 8 5

9 9 ± 1

0 . 5 9

6 0

7 0

1 . 6

1 . 8

0 . 4


P h

4 3 2 0 ± 2 1

4 4 7 0 ± 1 6 5

1 0 4 ± 1

0 . 0 2

5 3

1 3 6

1 . 2

3 . 1

0 . 7

P i

4 5 1 0 ± 2 9 7

1 0 4 ± 2

0 . 1 0

7 7

2 7 9

1 . 7

6 . 2

1 . 4

P i

4 3 4 0 ± 1 1 6

1 0 0 ± 1

0 . 6 9

6 3

1 1 0

1 . 5

2 . 5

0 . 6

C o r n b r a n

P h

1 7 1 ± 6

1 6 3 ± 1 8

9 5 ± 4

0 . 8 6

2

1 7

1 . 4

1 0 . 4

1 . 4

P i

1 6 6 ± 9

9 7 ± 4

0 . 4 4

2

7

1 . 3

4 . 4

0 . 6

P i

1 6 2 ± 8

9 5 ± 3

0 . 1 7

3

5

1 . 6

3 . 2

0 . 4


P h

3 0 8 0 ± 1 1

3 1 0 0 ± 6 5

1 0 1 ± 1

0 . 2 7

2 8

5 8

0 . 9

1 . 9

0 . 4

P i

3 1 2 0 ± 6 8

1 0 1 ± 1

0 . 1 2

3 7

6 3

1 . 2

2 . 0

0 . 4

P i

3 0 8 0 ± 4 3

1 0 0 ± 1

0 . 6 5

2 1

3 9

0 . 7

1 . 3

0 . 3

P e t f o o d

P h

4 5 1 0 ± 4 0

4 6 3 0 ± 1 2 2

1 0 3 ± 1

0 . 0 5

4 8

9 2

1 . 0

2 . 0

0 . 4

P i

4 6 2 0 ± 1 7 6

1 0 2 ± 2

0 . 1 6

6 3

7 2

1 . 5

1 . 7

0 . 4

P i

4 4 9 0 ± 8 8

1 0 0 ± 1

0 . 6 7

6 1 . 0

7 8

1 . 3

1 . 7

0 . 4


P h

1 0 6 0 0 ± 1 0 0

1 0 8 3 0 ± 8 4 5

1 0 2 ± 3

0 . 4 6

1 6 9

7 8 8

1 . 6

7 . 3

1 . 9

P i

1 0 8 4 0 ± 9 8 1

1 0 2 ± 3

0 . 5 0

2 0 0

9 2 2

1 . 8

8 . 5

2 . 2

P i

1 0 5 0 0 ± 7 0 7

9 9 ± 2

0 . 7 1

1 4 4

6 5 7

1 . 4

6 . 3

1 . 6

a




b




c


d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


< 0 . 0 1 ) .

e


f


i b i l i t y .

g


h


i






T a b l e

1 9 .



d f o r z i n c i n f o u r c e r t i f i e d a n d i n - h o u

s e r e f e r e n c e m a t e r i a l s u s i n g r e c o m m e n d e d a n d a l t e r n a t e

l i n e s


L i n e

R e f e r e n c e


M e d i a n ± U ,

m g / k g c

R e c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R S D i R , %

H o r R a t g


Z n

h

3 . 1 ± 0 . 2

2 . 6 ± 0 . 6

8 3 ± 7

0 . 0 5

0 . 4

0 . 6 0

1 6 . 9

2 2 . 5

1 . 6

Z n

i

2 . 7 ± 0 . 6

8 8 ± 7

0 . 1 4

0 . 5

0 . 5 0

1 6 . 7

1 9 . 3

1 . 4

Z n

i

2 . 6 ± 0 . 7

8 2 ± 8

0 . 0 5

0 . 4

0 . 6 0

1 6 . 8

2 3 . 3

1 . 7

C o r n b r a n

Z n

h

1 8 . 6 ± 1 . 1

1 7 . 6 ± 1 . 3

9 5 ± 6

0 . 3 8

0 . 8

2 . 1

1 . 8

4 . 5

0 . 4

Z n

i

1 7 . 8 ± 1 . 4

9 6 ± 6

0 . 5 0

0 . 2

0 . 9

1 . 2

5 . 0

0 . 4

Z n

i

1 7 . 5 ± 1 . 3

9 4 ± 6

0 . 3 5

0 . 2

0 . 7

1 . 4

4 . 3

0 . 4


Z n

h

7 4 . 8 ± 0 . 6

7 2 . 8 ± 1 . 4

9 7 ± 1

0 . 0 2

0 . 5

0 . 6

0 . 6

0 . 9

0 . 1

Z n

i

7 3 . 9 ± 1 . 7

9 9 ± 1

0 . 2 4

0 . 8

1 . 6

1 . 1

2 . 1

0 . 3

Z n

i

7 2 . 7 ± 1 . 3

9 7 ± 1

0 . 0 1

0 . 5

0 . 5

0 . 7

0 . 7

0 . 1


Z n

h

4 6 . 1 ± 1 . 1

4 5 . 6 ± 2 . 4

9 9 ± 3

0 . 6 8

0 . 8

2 . 1

1 . 8

4 . 5

0 . 5

Z n

i

4 4 . 1 ± 2 . 4

9 6 ± 3

0 . 1 4

0 . 8

2 . 1

1 . 8

4 . 7

0 . 5

Z n

i

4 5 . 2 ± 2 . 1

9 8 ± 3

0 . 5 1

0 . 8

1 . 8

1 . 7

3 . 9

0 . 4

a




b




c


d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g


h


i






T a b l e

2 0 .

M D 1 – 4 r u g g e d n e s s

t e s t s : a c c u r a c y , p r e c i s i o n , a n d H o r R

a t v a l u e s f o u n d f o r c a l c i u m

i n s i x c e

r t i f i e d a n d i n - h o u s e r e f e r e n c e m a t e r

i a l s u s i n g

r e c o m m e n d e d l i n e s


- t e s t c o d e a

R e f e r e n c e

v a l u e , m g / k g

b , c

M e d i a n ,

m g / k g

R e c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g


M D 1

9 8 7 0 ± 1 2 4

9 9 1 5

1 0 0 ± 1

0 . 9 2

9 1

1 7 1

0 . 9

1 . 7

0 . 3

M D 2

9 9 1 3

1 0 0 ± 2

0 . 9 3

9 7

2 5 0

1 . 0

2 . 5

0 . 4

M D 3

9 6 9 7

9 8 ± 1

0 . 2 0

1 5 5

2 5 0

1 . 6

2 . 6

0 . 5

M D 4

1 0 0 4 2

1 0 1 ± 2

0 . 3 6

6 8

2 1 1

0 . 7

2 . 1

0 . 4


M D 1

6 0 0 0 ± 4 4

6 7 5 2

1 1 3 ± 3

0 . 0 0 3

3 3 7

5 2 6

5 . 0

7 . 7

0 . 8

M D 2

6 0 2 7

1 0 0 ± 1

0 . 8 0

1 1 1

2 7 0

1 . 8

4 . 4

0 . 5

M D 3

5 7 6 4

9 6 ± 1

0 . 0 1

8 9

1 4 8

1 . 5

2 . 6

0 . 3

M D 4

5 7 3 1

9 6 ± 2

0 . 0 3

1 5 8

2 7 7

2 . 8

4 . 8

0 . 3

C o r n b r a n

M D 1

4 2 0 ± 1 9

3 7 4

8 9 ± 6

0 . 0 9

1 3

4 6

3 . 3

1 2 . 2

1 . 3

M D 2

4 0 5

9 6 ± 6

0 . 5 5

7

4 4

1 . 6

1 1 . 0

1 . 2

M D 3

4 1 1

9 8 ± 5

0 . 7 1

4

3 7

1 . 1

8 . 9

1 . 0

M D 4

3 1 3

7 5 ± 5

0 . 0 0 2

9

5 1

2 . 8

1 6 . 1

1 . 7


M D 1

3 0 2 0 ± 1 5

3 0 6 2

1 0 1 ± 1

0 . 0 7

3 3

4 3

1 . 1

1 . 4

0 . 2

M D 2

3 0 3 1

1 0 0 ± 1

0 . 7 7

2 6

9 5

0 . 9

3 . 1

0 . 5

M D 3

2 9 5 8

9 8 ± 2

0 . 2 3

3 0

1 3 0

1 . 0

4 . 4

0 . 7

M D 4

2 9 8 6

9 9 ± 1

0 . 2 1

4 3

6 6

1 . 4

2 . 2

0 . 3

P e t f o o d

M D 1

6 1 9 0 ± 1 4 0

6 3 6 5

1 0 3 ± 2

0 . 2 9

8 3

1 4 4

1 . 3

2 . 3

0 . 4

M D 2

6 4 5 7

1 0 4 ± 3

0 . 1 5

8 0

2 2 6

1 . 2

3 . 5

0 . 6

M D 3

6 1 9 8

1 0 0 ± 3

0 . 9 6

1 5 1

2 5 1

2 . 4

4 . 1

0 . 7

M D 4

6 3 8 4

1 0 3 ± 3

0 . 2 5

1 7 6

2 1 0

2 . 7

3 . 0

0 . 5


M D 1

1 3 0 0 0 ± 2 5 0

1 3 0 1 2

1 0 0 ± 2

0 . 9 7

9 3

1 9 2

0 . 7

1 . 5

0 . 3

M D 2

1 2 9 3 1

1 0 0 ± 2

0 . 8 1

1 7 0

3 7 7

1 . 3

2 . 9

0 . 5

M D 3

1 2 6 1 2

9 7 ± 2

0 . 1 7

1 1 3

2 2 0

0 . 9

1 . 7

0 . 3

M D 4

1 2 8 4 5

9 9 ± 2

0 . 5 7

1 5 7

2 5 7

1 . 2

2 . 0

0 . 4

a

R u g g e d n e s s t e s t u s i n g d i f f e r e n t m i c r o w a v e d i g e s t i o n s y s t e m s a n d I C P - A E S e q

u i p m e n t ( T a b l e 1 ) .

b




c




d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g





T a b l e

2 1 .



a t v a l u e s f o u n d f o r c o p p e r i n t h r e e c e r t i f i e d a n d i n - h o u s e r e f e r e n c e m a t e r i a l s u s i n g



- t e s t c o d

e a

R e f e r e n c e


b , c

M e d i a n ,

m g / k g

R e c o v e r y , %

P - v a l u e

d

S D r , m g / k g

e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g

C o r n b r a n

M D 1

2 . 4 7 ± 0 . 2 0

2 . 3 9

9 7 ± 9

0 . 7 4

0 . 0 6

0 . 3 2

2 . 7

1 3 . 1

0 . 7

M D 2

2 . 3 9

9 7 ± 1 1

0 . 7 8

0 . 0 6

0 . 5 5

2 . 5

2 2 . 9

1 . 2

M D 3

2 . 2 7

9 2 ± 8

0 . 3 2

0 . 0 7

0 . 1 7

3 . 0

7 . 5

0 . 4

M D 4

2 . 4 7

1 0 0 ± 1 0

0 . 9 9

0 . 1 1

0 . 1 2

4 . 3

4 . 7

0 . 2


M D 1

7 . 5 5 ± 0 . 1 3

7 . 7 3

1 0 2 ± 2

0 . 2 9

0 . 3 3

0 . 4 0

4 . 3

5 . 3

0 . 3

M D 2

7 . 5 9

1 0 1 ± 2

0 . 8 3

0 . 0 6

0 . 3 5

0 . 8

4 . 6

0 . 3

M D 3

7 . 6 1

1 0 1 ± 2

0 . 7 6

0 . 1 0

0 . 3 5

1 . 3

4 . 6

0 . 3

M D 4

7 . 7 9

1 0 3 ± 2

0 . 2 0

0 . 3 0

0 . 3 8

3 . 9

5 . 0

0 . 3


M D 1

0 . 7 0 ± 0 . 0 5

0 . 5 2

7 4 ± 2 0

0 . 2 3

0 . 0 8

0 . 4 1

1 3 . 1

6 7 . 6

2 . 7

M D 2

0 . 5 9

8 4 ± 2 1

0 . 4 6

0 . 0 4

0 . 3 9

6 . 0

6 7 . 0

2 . 8

M D 3

0 . 4 7

6 7 ± 8

0 . 0 0 4

0 . 0 6

0 . 1 3

1 2 . 8

2 9 . 6

1 . 2

M D 4

0 . 7 4

1 0 5 ± 2 7

0 . 8 5

0 . 1 3

0 . 5 2

1 8 . 9

7 3 . 6

3 . 1

a


u i p m e n t ( T a b l e 1 ) .

b




c




d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g





T a b l e

2 2 .



a t v a l u e s f o u n d f o r i r o n i n f o u r c e r t i f i e d a n d i n - h o u s e r e f e r e n c e m a t e r i a l s u s i n g r e c o m m e n d e d

l i n e s


- t e s t c o d e a

R e f e r e n c e


b , c

M e d i a n ,

m g / k g

R

e c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g


M D 1

1 6 9 . 9 ± 2 . 4

1 6 7 . 7

9 9 ± 2

0 . 4 8

1 . 7

5 . 2

1 . 0

3 . 1

0 . 3

M D 2

1 6 4 . 8

9 7 ± 2

0 . 1 0

1 . 4

3 . 9

0 . 8

2 . 4

0 . 2

M D 3

1 7 3 . 6

1 0 2 ± 2

0 . 2 2

2 . 1

3 . 8

1 . 2

2 . 2

0 . 2

M D 4

1 6 9 . 9

1 0 0 ± 4

0 . 9 9

9 . 3

2 0 . 9

5 . 4

1 2 . 2

1 . 2


M D 1

8 0 . 4 ± 0 . 6

7 9 . 9

9 9 ± 2

0 . 7 2

3 . 0

3 . 8

3 . 7

4 . 7

0 . 4

M D 2

7 7 . 8

9 7 ± 1

0 . 0 2

1 . 8

2 . 0

2 . 3

2 . 6

0 . 2

M D 3

7 7 . 7

9 7 ± 2

0 . 0 5

1 . 3

2 . 7

1 . 7

3 . 5

0 . 3

M D 4

7 2 . 8

9 1 ± 2

0 . 0 0 5

3 . 2

5 . 5

4 . 3

7 . 5

0 . 6

C o r n b r a n

M D 1

1 4 . 8 ± 0 . 9

1 2 . 5

8 5 ± 7

0 . 0 5

0 . 3

1 . 8

2 . 6

1 3 . 8

0 . 9

M D 2

1 3 . 9

9 4 ± 6

0 . 3 1

0 . 9

0 . 9

6 . 2

6 . 1

0 . 4

M D 3

1 2 . 4

8 4 ± 6

0 . 0 3

0 . 4

1 . 3

2 . 8

1 0 . 7

0 . 7

M D 4

1 0 . 7

7 2 ± 6

0 . 0 0 2

0 . 5

1 . 7

4 . 9

1 5 . 9

1 . 0


M D 1

5 8 . 5 ± 0 . 4

5 8 . 0

9 9 ± 1

0 . 5 1

0 . 9

1 . 8

1 . 5

3 . 2

0 . 3

M D 2

5 7 . 6

9 8 ± 1

0 . 2 4

0 . 7

1 . 8

1 . 2

3 . 1

0 . 3

M D 3

5 7 . 8

9 9 ± 1

0 . 3 1

1 . 0

1 . 8

1 . 8

3 . 2

0 . 3

M D 4

5 6 . 3

9 6 ± 1

0 . 1 0

0 . 9

1 . 6

1 . 6

2 . 9

0 . 2

a


u i p m e n t ( T a b l e 1 ) .

b




c




d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


< 0 . 0 1 ) .

e


f


i b i l i t y .

g





T a b l e

2 3 .



a t v a l u e s f o u n d f o r p o t a s s i u m

i n f i v e c e r t i f i e d a n d i n - h o u s e r e f e r e n c e m a t e r i a l s u s i n g



- t e s t c o d e a

R e f e r e n c e v a l u e ,

m g / k g

b , c

M e d i a n , m g / k g

R e c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g


M D 1

9 0 9 0 ± 1 3 3

9 1 4 5

1 0 1 ± 2

0 . 7 3

1 1 5

2 3 7

1 . 3

2 . 6

0 . 5

M D 2

8 9 1 5

9 8 ± 2

0 . 3 4

7 1

3 1 7

0 . 8

3 . 6

0 . 6

M D 3

9 0 2 5

9 9 ± 2

0 . 6 5

6 5

1 3 0

0 . 7

1 . 4

0 . 2

M D 4

9 0 3 1

9 9 ± 2

0 . 6 9

3 9

1 5 3

0 . 4

1 . 7

0 . 3


M D 1

6 5 0 0 ± 5 0

6 8 3 0

1 0 5 ± 2

0 . 0 0 6

2 0 0

2 5 0

2 . 9

3 . 7

0 . 6

M D 2

6 4 5 0

9 9 ± 1

0 . 5 6

8 0

1 8 0

1 . 2

2 . 8

0 . 5

M D 3

6 3 6 0

9 8 ± 1

0 . 0 6

5 0

1 2 0

0 . 8

1 . 9

0 . 3

M D 4

6 2 6 0

9 6 ± 1

0 . 0 3

1 0 0

2 1 0

1 . 6

3 . 3

0 . 5

C o r n b r a n

M D 1

5 6 6 ± 3 8

4 6 0

8 1 ± 7

0 . 0 2

1 5

5 9

3 . 3

1 2 . 6

1 . 4

M D 2

4 6 3

8 2 ± 6

0 . 0 2

1 0

4 1

2 . 1

8 . 8

1 . 0

M D 3

5 3 8

9 5 ± 7

0 . 4 8

7

3 5

1 . 4

6 . 3

0 . 7

M D 4

5 6 1

9 9 ± 8

0 . 9 1

1 3

7 3

2 . 4

1 3 . 1

1 . 5


M D 1

5 5 7 0 ± 2 5

5 6 7 8

1 0 2 ± 1

0 . 0 0 7

4 3

5 2

0 . 8

0 . 9

0 . 1

M D 2

5 6 3 3

1 0 1 ± 1

0 . 3 2

6 5

1 5 8

1 . 1

2 . 8

0 . 5

M D 3

5 5 6 1

1 0 0 ± 1

0 . 8 3

3 0

9 9

0 . 5

1 . 8

0 . 3

M D 4

5 6 4 0

1 0 1 ± 1

0 . 0 8

3 2

7 5

0 . 6

1 . 3

0 . 2


M D 1

1 6 9 0 0 ± 1 5 0

1 6 6 1 0

9 8 ± 1

0 . 1 0

1 8 0

2 0 0

1 . 1

1 . 2

0 . 5

M D 2

1 6 2 3 0

9 6 ± 1

0 . 0 1

2 2 0

4 5 0

1 . 4

2 . 8

0 . 3

M D 3

1 6 5 8 0

9 8 ± 1

0 . 1 0

8 0

2 5 0

0 . 5

1 . 5

0 . 3

M D 4

1 6 7 3 0

9 9 ± 1

0 . 3 0

2 2 0

2 3 0

1 . 3

1 . 4

0 . 9

a


u i p m e n t ( T a b l e 1 ) .

b




c




d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g





T a b l e

2 4 .



a t v a l u e s f o u n d f o r m a g n e s i u m

i n s i x c e r t i f i e d a n d i n - h o u s e r e f e r e n c e m

a t e r i a l s u s i n g



- t e s t c o d

e a

R e f e r e n c e v a l u e ,

m g / k g

b , c


R

e c o v e r y , %

P - v a l u e

d

S D r , m g / k g

e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g


M D 1

1 7 7 9 ± 2 3

1 7 8 2

1 0 0 ± 2

0 . 9 3

1 9

4 2

1 . 0

2 . 4

0 . 3

M D 2

1 7 8 0

1 0 0 ± 2

0 . 9 7

1 8

7 4

1 . 0

4 . 1

0 . 6

M D 3

1 7 8 9

1 0 1 ± 1

0 . 6 9

2 4

2 5

1 . 3

1 . 4

0 . 2

M D 4

1 7 7 0

1 0 0 ± 1

0 . 7 2

1 1

3 3

0 . 6

1 . 8

0 . 2


M D 1

8 8 1 ± 8

8 2 7

9 4 ± 1

0 . 0 0 2

2 8

3 2

3 . 4

3 . 9

0 . 5

M D 2

8 9 8

1 0 2 ± 2

0 . 2 9

1 7

3 7

1 . 9

4 . 1

0 . 5

M D 3

9 2 2

1 0 5 ± 1

0 . 0 0 4

2 1

2 2

2 . 2

2 . 4

0 . 3

M D 4

8 9 3

1 0 1 ± 1

0 . 3 3

1 9

2 6

2 . 1

2 . 9

0 . 4

C o r n b r a n

M D 1

8 1 8 ± 3 0

7 7 8

9 5 ± 3

0 . 2 0

9

1 1

1 . 1

1 . 4

0 . 2

M D 2

7 4 9

9 2 ± 3

0 . 0 4

1 4

1 6

1 . 8

2 . 1

0 . 3

M D 3

7 7 8

9 5 ± 4

0 . 2 1

7

1 5

1 . 0

1 . 9

0 . 2

M D 4

7 6 1

9 3 ± 3

0 . 0 8

5

9

0 . 7

1 . 2

0 . 1


M D 1

1 2 0 0 ± 8

1 1 9 1

9 9 ± 1

0 . 2 1

1 3

1 7

1 . 1

1 . 5

0 . 2

M D 2

1 1 9 8

1 0 0 ± 1

0 . 7 9

8

4 4

0 . 7

3 . 7

0 . 5

M D 3

1 2 1 6

1 0 1 ± 1

0 . 2 1

5

1 7

0 . 4

1 . 4

0 . 2

M D 4

1 1 9 3

9 9 ± 1

0 . 4 0

1 2

2 4

1 . 0

2 . 0

0 . 3

P e t f o o d

M D 1

4 8 ± 2

4 6 2

9 6 ± 3

0 . 1 7

7

8

1 . 5

1 . 8

0 . 2

M D 2

4 7 4

9 9 ± 3

0 . 6 7

4

1 4

0 . 9

3 . 0

0 . 3

M D 3

4 7 2

9 8 ± 3

0 . 5 5

6

1 2

1 . 3

2 . 5

0 . 3

M D 4

4 5 6

9 5 ± 3

0 . 1 2

7

2 1

1 . 5

4 . 6

0 . 5


M D 1

1 2 0 0 ± 1 5

1 1 8 8

9 9 ± 1

0 . 5 1

1 3

2 5

1 . 1

2 . 1

0 . 3

M D 2

1 1 9 4

1 0 0 ± 2

0 . 8 0

1 4

5 3

1 . 2

4 . 4

0 . 6

M D 3

1 1 8 5

9 9 ± 1

0 . 3 7

9

1 6

0 . 8

1 . 3

0 . 2

M D 4

1 1 7 2

9 8 ± 1

0 . 1 2

1 1

2 1

0 . 9

1 . 8

0 . 2

a


u i p m e n t ( T a b l e 1 ) .

b




c




d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


< 0 . 0 1 ) .

e


f


i b i l i t y .

g





T a b l e

2 5 .



a t v a l u e s f o u n d f o r m a n g a n e s e i n t h

r e e c e r t i f i e d a n d i n - h o u s e r e f e r e n c e

m a t e r i a l s u s i n g

r e c o m m e n d e d l i n e s ( M D 1 , M D 2

, M D 3 ) a n d a l t e r n a t e 2 M n I I l i n e ( M D 4 )


- t e s t c o d e a

R e f e r e n c e


b , c


R

e c o v e r y , %

P - v a l u e

d

S D r , m g / k g

e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g

C o r n b r a n

M D 1

2 . 5 5 ± 0 . 1 5

2 . 3 6

9 3 ± 6

0 . 2 2

0 . 0 3

0 . 0 5

1 . 1

2 . 1

0 . 1

M D 2

2 . 2 7

8 9 ± 5

0 . 0 8

0 . 0 4

0 . 0 8

1 . 9

3 . 5

0 . 2

M D 3

2 . 1 7

8 5 ± 5

0 . 0 2

0 . 0 4

0 . 0 8

2 . 0

3 . 7

0 . 2

M D 4

2 . 3 7

9 3 ± 6

0 . 2 3

0 . 0 3

0 . 0 4

1 . 4

1 . 7

0 . 1


M D 1

1 2 . 7 0 ± 0 . 8 5

1 2 . 4 5

9 8 ± 7

0 . 7 7

0 . 1 6

0 . 1 6

1 . 2

1 . 3

0 . 1

M D 2

1 2 . 3 1

9 7 ± 7

0 . 6 5

0 . 1 2

0 . 2 2

0 . 9

1 . 7

0 . 1

M D 3

1 1 . 7 1

9 2 ± 6

0 . 2 5

0 . 1 2

0 . 2 2

1 . 0

1 . 8

0 . 1

M D 4

1 2 . 7 7

1 0 1 ± 7

0 . 9 3

0 . 1 6

0 . 1 4

1 . 2

1 . 1

0 . 1


M D 1

0 . 2 6 ± 0 . 0 3

0 . 2 2

8 5 ± 1 0

0 . 1 8

0 . 0 1

0 . 0 2

4 . 5

8 . 3

0 . 3

M D 2

0 . 2 3

9 0 ± 1 1

0 . 3 7

0 . 0 1

0 . 0 2

4 . 9

6 . 9

0 . 2

M D 3

0 . 3 1

1 1 8 ± 1 4

0 . 2 5

0 . 0 2

0 . 0 3

6 . 7

9 . 7

0 . 4

M D 4

0 . 2 1

8 2 ± 1 0

0 . 1 0

0 . 0 1

0 . 0 1

4 . 8

4 . 3

0 . 2

a


u i p m e n t ( T a b l e 1 ) .

b




c




d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g





T a b l e

2 6 .



a t v a l u e s f o u n d f o r s o d i u m

i n f i v e c e r t i f i e d a n d i n - h o u s e r e f e r e n c e m a t e r i a l s u s i n g



- t e

s t c o d e a

R e f e r e n c e


b , c


R e c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R S D i R , %

H o r R a t g


M D 1

1 9 9 0 ± 4 2

2 0 2 9

1 0 2 ± 2

0 . 4 3

3 1

5 9

1 . 5

2 . 9

0 . 4

M D 2

2 0 0 0

1 0 1 ± 3

0 . 8 6

4 2

9 9

2 . 1

4 . 9

0 . 7

M D 3

1 9 8 1

1 0 0 ± 2

0 . 8 5

2 9

6 8

1 . 5

3 . 4

0 . 5

M D 4

1 9 4 9

9 8 ± 2

0 . 3 6

1 4

3 4

0 . 7

1 . 7

0 . 2


M D 1

1 0 5 0 ± 1 4

1 1 0 5

1 0 5 ± 3

0 . 1 2

5 3

8 9

4 . 8

8 . 1

1 . 0

M D 2

1 0 0 2

9 5 ± 2

0 . 0 4

2 1

4 2

2 . 1

4 . 2

0 . 5

M D 3

9 7 0

9 2 ± 3

0 . 0 1

1 2

5 9

1 . 3

6 . 1

0 . 8

M D 4

9 3 5

8 9 ± 3

0 . 0 0 2

2 3

6 3

2 . 5

6 . 8

0 . 8

C o r n b r a n

M D 1

4 3 0 ± 1 6

3 9 5

9 2 ± 3

0 . 0 5

4

1 0

1 . 1

2 . 4

0 . 3

M D 2

3 9 5

9 2 ± 4

0 . 0 8

4

2 7

1 . 1

6 . 8

0 . 7

M D 3

3 9 1

9 1 ± 5

0 . 0 9

1 0

4 0

2 . 5

1 . 0

0 . 1

M D 4

3 8 8

9 0 ± 4

0 . 0 3

4

1 9

1 . 0

5 . 0

0 . 5


M D 1

4 9 2 0 ± 3 0

4 8 5 0

9 9 ± 1

0 . 0 9

4 3

6 9

0 . 9

1 . 4

0 . 2

M D 2

4 8 7 0

9 9 ± 1

0 . 3 1

4 0

1 1 0

0 . 8

2 . 4

0 . 4

M D 3

4 9 1 0

1 0 0 ± 1

0 . 8 1

3 2

1 2 2

0 . 6

2 . 5

0 . 4

M D 4

5 0 0 0

1 0 2 ± 1

0 . 0 9

3 9

7 7

0 . 8

1 . 5

0 . 2


M D 1

4 9 7 0 ± 5 0

4 8 9 0

9 8 ± 1

0 . 1 5

6 6

7 1

1 . 3

1 . 4

0 . 2

M D 2

4 9 4 0

9 9 ± 1

0 . 6 0

6 2

1 0 6

1 . 3

2 . 2

0 . 4

M D 3

4 8 1 0

9 7 ± 1

0 . 0 2

6 6

9 8

1 . 4

2 . 0

0 . 3

M D 4

4 9 3 0

9 9 ± 1

0 . 5 1

7 5

1 1 0

1 . 5

2 . 2

0 . 4

a


u i p m e n t ( T a b l e 1 ) .

b




c




d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g





T a b l e

2 7 .



a t v a l u e s f o u n d f o r p h o s p h o r u s i n s

i x c e r t i f i e d a n d i n - h o u s e r e f e r e n c e m

a t e r i a l s u s i n g



- t e s t c

o d e a

R e f e r e n c e


b , c

M e d i a n ,

m g / k g

R e c o v e r y , %

P - v a l u e

d

S D r , m g / k

g e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g


M D

1

4 0 8 0 ± 3 0

4 1 1 0

1 0 1 ± 1

0 . 6 1

5 1

1 3 2

1 . 3

3 . 2

0 . 5

M D

2

4 1 5 0

1 0 2 ± 1

0 . 2 8

6 2

1 5 0

1 . 5

3 . 6

0 . 6

M D

3

4 1 4 0

1 0 1 ± 1

0 . 1 4

3 0

4 9

0 . 7

1 . 2

0 . 2

M D

4

4 1 7 0

1 0 2 ± 1

0 . 0 7

3 6

8 6

0 . 9

2 . 0

0 . 3


M D

1

4 3 0 ± 2 1

4 4 1 8

1 0 2 ± 1

0 . 1 0

1 0 1

1 5 5

2 . 3

3 . 5

0 . 5

M D

2

4 5 0 8

1 0 4 ± 1

0 . 0 1

7 5

1 6 1

1 . 7

3 . 6

0 . 6

M D

3

4 3 8 4

1 0 2 ± 1

0 . 1 0

5 7

8 7

1 . 3

2 . 0

0 . 3

M D

4

4 3 2 1

1 0 0 ± 1

0 . 9 7

7 7

9 7

1 . 8

2 . 3

0 . 4

C o r n b r a n

M D

1

1 7 1 ± 6

1 5 1

8 8 ± 6

0 . 0 6

4

2 3

2 . 7

1 5 . 1

1 . 4

M D

2

1 6 3

9 5 ± 6

0 . 4 5

4

2 4

2 . 4

1 5

1 . 4

M D

3

1 6 9

9 9 ± 4

0 . 3 0

2

9

1 . 0

5 . 3

0 . 5

M D

4

1 1 6

6 8 ± 6

0 . 0 0 1

4

2 7

3 . 3

2 3 . 4

2 . 1


M D

1

3 0 8 0 ± 1 1

3 0 9 5

1 0 1 ± 1

0 . 4 1

2 9

4 4

0 . 9

1 . 4

0 . 2

M D

2

3 1 7 0

1 0 3 ± 1

0 . 0 8

2 7

1 2 3

0 . 8

3 . 9

0 . 6

M D

3

3 1 3 3

1 0 2 ± 1

0 . 0 6

1 7

6 1

0 . 5

1 . 9

0 . 3

M D

4

3 1 1 1

1 0 1 ± 1

0 . 2 1

4 5

6 7

1 . 4

2 . 2

0 . 3

P e t f o o d

M D

1

4 5 1 0 ± 4 0

4 4 9

1 0 0 ± 1

0 . 6 0

2 8

4 9

0 . 6

1 . 1

0 . 2

M D

2

4 6 4

1 0 3 ± 1

0 . 1 1

3 0

1 7 3

0 . 6

3 . 7

0 . 6

M D

3

4 5 4

1 0 1 ± 1

0 . 5 6

3 6

7 8

0 . 8

1 . 7

0 . 3

M D

4

4 5 3

1 0 1 ± 1

0 . 6 0

6 6

8 4

1 . 4

1 . 9

0 . 3


M D

1

1 0 6 0 0 ± 1 0 0

1 0 5 9

1 0 0 ± 1

0 . 9 0

1 0 9

1 3 6

1 . 0

1 . 3

0 . 2

M D

2

1 0 8 8

1 0 3 ± 2

0 . 1 2

1 5 6

3 6 7

1 . 4

3 . 4

0 . 6

M D

3

1 0 4 4

9 9 ± 1

0 . 2 0

8 1

1 5 3

0 . 8

1 . 5

0 . 3

M D

4

1 0 4 0

9 8 ± 1

0 . 1 2

6 6

1 7 6

0 . 6

1 . 7

0 . 3

a


u i p m e n t ( T a b l e 1 ) .

b




c




d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g





T a b l e

2 8 .



a t v a l u e s f o u n d f o r z i n c i n t h r e e c e r t i f i e d a n d i n - h o u s e r e f e r e n c e m a t e r i a

l s u s i n g r e c o m m e n d e d

l i n e s ( M D 1 , M D 2 , M D 3 ) a n d a l t e

r n a t e 2 Z n I l i n e ( M D 4 )


- t e s t c o

d e a

R e f e r e n c e


b , c

M e d i a n ,

m g / k g

R

e c o v e r y , %

P - v a l u e

d

S D r , m g / k g e

S D i R , m g / k g

f

R S D r ,

%

R

S D i R , %

H o r R a t g

C o r n b r a n

M D 1

1 8 . 6 ± 1 . 1

1 7 . 6

9 5 ± 6

0 . 3 8

0 . 2

0 . 5

1 . 1

2 . 8

0 . 2

M D 2

1 7 . 0

9 2 ± 5

0 . 1 7

0 . 2

0 . 3

1 . 4

1 . 7

0 . 1

M D 3

1 7 . 5

9 4 ± 6

0 . 3 8

0 . 5

1 . 4

3 . 1

8 . 3

0 . 6

M D 4

1 8 . 3

9 8 ± 7

0 . 8 3

0 . 8

2 . 4

4 . 5

1 3 . 6

0 . 9


M D 1

7 4 . 8 ± 0 . 6

7 2 . 5

9 7 ± 1

0 . 0 0 6

1 . 1

0 . 9

1 . 5

1 . 3

0 . 1

M D 2

7 2 . 6

9 7 ± 1

0 . 0 3

0 . 6

1 . 8

0 . 9

2 . 4

0 . 2

M D 3

7 5 . 3

1 0 1 ± 3

0 . 7 8

1 . 0

5 . 0

1 . 3

6 . 7

0 . 6

M D 4

7 6 . 3

1 0 2 ± 1

0 . 1 2

1 . 2

1 . 9

1 . 6

2 . 5

0 . 2


M D 1

4 6 . 1 ± 1 . 1

4 5 . 6

9 9 ± 2

0 . 6 9

0 . 7

0 . 9

1 . 5

2 . 0

0 . 2

M D 2

4 4 . 8

9 7 ± 2

0 . 2 8

0 . 7

0 . 9

1 . 6

2 . 0

0 . 2

M D 3

4 5 . 1

9 8 ± 2

0 . 3 8

0 . 7

0 . 9

1 . 6

2 . 0

0 . 2

M D 4

4 6 . 4

1 0 1 ± 3

0 . 8 3

0 . 9

2 . 1

1 . 9

4 . 7

0 . 4

a


u i p m e n t ( T a b l e 1 ) .

b




c




d



> 0 . 0 5 ) ; q u e s t i o n a b l e r

e s u l t ( 0 . 0 1 < P


( P

< 0 . 0 1 ) .

e


f


i b i l i t y .

g




method for the determination of Ca ranged between 1.1%

(dietetic milk powder) and 3.9% (infant cereals). The RSDR

ranged between 2.9% (chocolate milk powder) and 5.0%

(peanut butter). The HorRat value should not exceed 1.5.

Table 29 shows that the present method has an acceptable

precision for the determination of Ca in all foodstuffs

analyzed with Ca concentration above the estimated QL. The

trueness as z -score (Table 30) is calculated to range from –0.6

(chocolate milk powder) to 0.2 (peanut butter). The resultsindicate that this method can determine, with sufficient

precision, from 9870 to 411 mg/kg.

(b) Copper .—Table 29 presents estimated performance

characteristics of the ICP-AES method for Cu. The RSDr of

the method for the determination of Cu ranged between 1.8%

(wheat gluten) and 5.0% (infant cereals). The RSDR ranged

between 4.4% (wheat gluten) and 12.0% (infant cereals). The

HorRat value should not exceed 1.0. Table 29 shows that the

present method has an acceptable precision for the

determination of Cu in all foodstuffs analyzed with Cu

concentration above or close to the estimated QL. The

trueness, expressed as z -score (Table 30), is calculated to

range from –0.7 (wheat gluten) to 0.03 (peanut butter). The

results indicate that this method can determine, with sufficient

precision, from 6.09 to 1.94 mg/kg.

(c) Iron.—Table 29 presents estimated performance

characteristics of the ICP-AES method for Fe. The RSDr of

the method for the determination of Fe ranged between 1.5%

(infant cereals) and 5.0% (peanut butter). The RSDR ranged

between 4.1% (wheat gluten) and 14.9% (peanut butter). The



determination of Fe in all foodstuffs analyzed with Fe

concentration above the estimated QL. The trueness as z -score

(Table 30) is calculated to range from –2.2 (wheat gluten) to0.2 (dietetic milk powder 2). The results indicate that this

method can determine, with sufficient precision, from 169.9

to 16.4 mg/kg.

(d) Potassium.—Table 29 presents estimated

performance characteristics of the ICP-AES method for K.

The RSDr of the method for the determination of K ranged

between 0.9% (dietetic milk powder) and 1.6% (peanut

butter). The RSDR ranged between 1.2% (dietetic milk

powder) and 7.8% (wheat gluten). The HorRat value should

not exceed 1.5. Table 29 shows that the present method has an

acceptable precision for the determination of K in all

foodstuffs analyzed with K concentration above the estimated

QL. The trueness as z -score (Table 30) is calculated to rangefrom –1.8 (wheat gluten) to 0.7 (peanut butter). The results

indicate that this method can determine, with sufficient

precision, from 9090 to 472 mg/kg

(e) Magnesium.—Table 29 presents estimated

performance characteristics of the ICP-AES method for Mg.

The RSDr of the method for the determination of Mg ranged

between 1.2% (dietetic milk powder) and 3.4% (infant

cereals). The RSDR ranged between 2.9% (dietetic milk

powder) and 5.4% (infant cereals). The HorRat value should

not exceed 1.5. Table 29 shows that the present method has an

acceptable precision for the determination of Mg in all

foodstuffs analyzed with Mg concentration above the

estimated QL. The trueness as z -score (Table 30) is calculated

to range from –1.8 (wheat gluten) to 0.6 (peanut butter). The

results indicate that this method can determine, with sufficient

precision, from 1779 to 444 mg/kg.

(f ) Manganese.—Table 29 presents estimated

performance characteristics of the ICP-AES method for Mn.

The RSDr of the method for the determination of Mn ranged

between 1.4% (wheat gluten) and 7.2% (infant cereals). The

RSDR ranged between 6.5% (chocolate milk powder) and

9.2% (infant cereals). The HorRat value should not exceed

1.0. Table 29 shows that the present method has an acceptable

precision for the determination of Mn in all foodstuffs

analyzed with Mn concentration above the estimated QL. The

trueness as z -score (Table 30) is calculated to range from –1.1

(wheat gluten) to 0.1 (dietetic milk powder). The results

indicate that this method can determine, with sufficient

precision, from 16.0 to 0.405 mg/kg.

(g) Sodium.—Table 29 presents estimated performance

characteristics of the ICP-AES method for Na. The RSDr of the method for the determination of Na ranged between 1.2%

(wheat gluten and dietetic milk powder) and 3.9% (infant

cereals). The RSDR ranged between 2.2% (chocolate milk

powder) and 5.5% (infant cereals). The HorRat value should

not exceed 1.0. Table 29 shows that the present method has

an acceptable precision for the determination of Na in all

foodstuffs analyzed with Na concentration above the

estimated QL. The trueness as z -score (Table 30) is calculated

to range from –1.6 (dietetic milk powder and wheat gluten)

to –0.03 (peanut butter). The results indicate that this

method can determine, with sufficient precision, from 4890

to 1420 mg/kg.

(h) Phosphorus.—Table 29 presents estimated

performance characteristics of the ICP-AES method for P.

The RSDr of the method for the determination of P ranged

between 1.2% (wheat gluten) and 2.0% (peanut butter). The

RSDR ranged between 2.1% (infant cereals) and 3.3% (wheat

gluten). The HorRat value should not exceed 1.0. Table 29

shows that the present method has an acceptable precision for

the determination of P in all foodstuffs analyzed with

P concentration above the estimated QL. The trueness as

z -score (Table 30) is calculated to range from –2.0 (wheat

gluten) to 0.8 (peanut butter). The results indicate that this

method can determine, with sufficient precision, from 4320

to 2190 mg/kg.(i) Zinc.—Table 29 presents estimated performance

characteristics of the ICP-AES method for Zn. The RSDr of the

method for the determination of Zn ranged between 1.4%

(wheat gluten) and 3.0% (infant cereals). The RSDR ranged

between 3.8% (infant cereals) and 6.0% (peanut butter). The



determination of Zn in all foodstuffs analyzed with Zn

concentration above the estimated QL. The trueness as z -score

(Table 30) is calculated to range from –1.7 (wheat gluten) to 1.1





T a b l e

2 9 .

E s t i m a t e d p e r f o r m a n c e c h a r a c t e r i s t i c s o f t h e I C P - A E S m

e t h o d f o r d e t e r m i n a t i o n o f n i n e e l e m

e n t s ( C a , C u , F e , K , M g , M n , N a , P , a n d Z n )

S a m p l e d e s c r i p t i o n

R e f e

r e n c e

v a l u e , m g / k g a


n b

S D

r , m g / k g c

S D R , m g / k g

d

R S D r ,

%

R S D R , %

r , m g / k g e

R , m g / k g

f

H o r R a t g

z - s c o r e

C a l c i u m

P e a n u t b u t t e r

4 1 1

± 1 8

4 1 6

9

1 4

2 1

3 . 3

5 . 0

3 9

5 7

0 . 8

0 . 2


9 8 7 0

± 2 4 8

9 7 1 2

9

1 6 2

2 8 0

1 . 7

2 . 9

4 4 8

7 7 5

0 . 7

– 0 . 6


6 0 0 0 ± 8 8

5 9 7 2

9

2 3 4

2 9 0

3 . 9

4 . 9

6 4 9

8 0 3

1 . 1

– 0 . 1


5 7 8 0 ± 5 6

5 6 9 6

9

6 1

2 1 8

1 . 1

3 . 8

1 7 0

6 0 6

0 . 9

– 0 . 4

W h e a t g l u t e n

3 6 9

± 3 5

3 6 5

9

5

1 5

1 . 4

4 . 0

1 5

4 1

0 . 6

– 0 . 3

C o p p e r


4 . 9 3

± 0 . 1 5

4 . 9 4

9

0 . 2 0

0 . 3 1

4 . 1

6 . 2

0 . 5 6

0 . 8 5

0 . 5

0 . 0 3


N

A h

5 . 9 2

9

0 . 1 1

0 . 4 1

1 . 9

6 . 9

0 . 3 1

1 . 1 3

0 . 6


N A

1 . 9 4

9

0 . 1 0

0 . 2 3

5 . 0

1 2

0 . 2 7

0 . 6 5

0 . 8


6 . 1 0

± 0 . 2 0

6 . 0 9

9

0 . 1 2

0 . 4 0

2 . 0

6 . 5

0 . 3 3

1 . 1 0

0 . 5

– 0 . 0 2


5 . 9 4

± 0 . 7 2

5 . 7 7

9

0 . 1 0

0 . 2 6

1 . 8

4 . 4

0 . 2 9

0 . 7 1

0 . 4

– 0 . 7

I r o n


1 6 . 4

± 0 . 8

1 5 . 9

9

0 . 8

2 . 4

5 . 0

1 4 . 9

2 . 2

6 . 5

1 . 4

– 0 . 2


1 6 9 . 9 ± 4 . 8

1 6 6 . 2

9

3 . 8

7 . 4

2 . 3

4 . 4

1 0 . 5

2 0 . 4

0 . 6

– 0 . 5


8 0 . 4

± 1 . 2

7 4 . 8

9

1 . 2

3 . 9

1 . 5

5 . 2

3 . 2

1 0 . 8

0 . 6

– 1 . 4


8 1 . 1

± 1 . 0

8 1 . 9

9

2 . 2

3 . 6

2 . 7

4 . 3

6 . 0

9 . 9

0 . 5

0 . 2


5 4 . 3

± 6 . 8

4 9 . 8

9

1 . 2

2 . 1

2 . 4

4 . 1

3 . 4

5 . 7

0 . 5

– 2 . 2

P o t a s s i u m


6 0 7 0

± 2 0 0

6 1 5 7

9

1 0 1

1 1 8

1 . 6

1 . 9

2 7 9

3 2 6

0 . 4

0 . 7


9 0 9 0

± 2 6 6

9 0 0 6

9

1 0 0

1 6 6

1 . 1

1 . 8

2 7 7

4 6 0

0 . 4

– 0 . 5


6 5 0 0

± 1 0 0

6 4 6 6

9

1 0 0

1 2 4

1 . 5

1 . 9

2 7 7

3 4 3

0 . 4

– 0 . 3


6 4 2 0 ± 5 2

6 4 3 1

9

6 1

7 6

0 . 9

1 . 2

1 6 9

2 1 2

0 . 3

0 . 1


4 7 2

± 6 1

4 1 3

9

6

3 2

1 . 5

7 . 8

1 7 . 1

8 9 . 3

1 . 2

– 1 . 8

M a g n e s i u m


1 6 8 0 ± 7 0

1 7 5 3

9

4 2

1 1 6

2 . 4

6 . 6

1 1 7

3 2 1

1 . 3

0 . 6


1 7 7 9 ± 4 6

1 7 5 1

9

3 3

5 4

1 . 9

3 . 1

9 1 . 7

1 5 1

0 . 6

– 0 . 5


8 8 1

± 1 6

8 6 0

9

2 9

4 7

3 . 4

5 . 4

8 1

1 2 9

0 . 9

– 0 . 5


4 4 4 ± 5

4 4 1

9

5 . 4

1 2 . 7

1 . 2

2 . 9

1 5 . 0

3 5 . 3

0 . 5

– 0 . 2


5 1 0

± 4 7

4 8 3

9

1 0

1 5

2 . 0

3 . 1

2 6

4 1

0 . 5

– 1 . 8




T a b l e

2 9 .

( c o n t i n u e d )

S a m p l e d e s c r i p t i o n

R e f e

r e n c e

v a l u e , m g / k g a

M e d i a n ,

m g / k g

n b

S D

r , m g / k g c

S D R , m g / k g

d

R S D r ,

%

R S D R , %

r , m g / k g e

R , m g / k g

f

H o r R a t g

z - s c o r e

M a n g a n e s e


1 6 . 0

± 0 . 6

1 6 . 0

9

0 . 4

1 . 3

2 . 4

7 . 8

1 . 1

3 . 5

0 . 7

0 . 0


N A

7 . 5 3

9

0 . 1 5

0 . 4 9

2 . 1

6 . 5

0 . 4 3

1 . 3 6

0 . 6


N A

1 6 . 5

9

1 . 2

1 . 5

7 . 2

9 . 2

3 . 3

4 . 2

0 . 9


0 . 4 0 5

± 0 . 0 3 4

0 . 4 0 7

9

0 . 0 0 7

0 . 0 2 9

1 . 7

7 . 1

0 . 0 2 0

0 . 0 8 0

0 . 4

0 . 1


1 4 . 3

± 0 . 8

1 3 . 0

9

0 . 2

1 . 2

1 . 4

9 . 1

0 . 5

3 . 3

0 . 8

– 1 . 1

S o d i u m


4 8 9 0

± 1 4 0

4 8 8 6

9

1 2 9

1 2 2

2 . 6

2 . 5

3 5 8

3 3 7

0 . 6

0 . 0 3


1 9 9 0 ± 8 4

1 9 3 7

9

2 8

4 2

1 . 4

2 . 2

7 7

1 1 7

0 . 4

– 1 . 3


1 0 5 0 ± 2 8

9 9 7

9

3 9

5 5

3 . 9

5 . 5

1 0 7

1 5 2

1 . 0

– 1 . 0


1 8 4 0 ± 2 0

1 7 6 7

9

2 2

4 6

1 . 2

2 . 6

6 0

1 2 8

0 . 5

– 1 . 6


1 4 2 0

± 1 1 0

1 3 5 3

9

1 7

4 1

1 . 2

3 . 0

4 7

1 1 2

0 . 6

– 1 . 6

P h o s p h o r u s


3 3 7 8 ± 9 2

3 4 4 6

9

7 0

8 8

2 . 0

2 . 5

1 9 3

2 4 3

0 . 5

0 . 8


4 0 8 0 ± 6 0

4 1 2 7

9

7 0

1 2 3

1 . 7

3 . 0

1 9 4

3 4 1

0 . 7

0 . 4


4 3 2 0 ± 4 2

4 3 0 2

9

5 8

9 1

1 . 3

2 . 1

1 6 0

2 5 3

0 . 5

– 0 . 2


3 7 2 0 ± 2 4

3 7 6 0

9

5 2

1 2 2

1 . 4

3 . 2

1 4 4

3 3 8

0 . 7

0 . 3


2 1 9 0

± 1 5 0

2 0 5 3

9

2 5

6 8

1 . 2

3 . 3

6 9

1 8 9

0 . 7

– 2 . 0

Z i n c


2 6 . 3

± 1 . 1

2 6 . 8

9

0 . 6

1 . 6

2 . 4

6 . 0

1 . 8

4 . 5

0 . 6

0 . 3


N A

1 8 . 5

9

0 . 4

0 . 9

2 . 1

4 . 8

1 . 1

2 . 5

0 . 5


N A

3 6 . 7

9

1 . 1

1 . 4

3 . 0

3 . 8

3 . 1

3 . 8

0 . 4


5 1 . 0

± 0 . 8

5 3 . 6

9

1 . 3

2 . 4

2 . 4

4 . 5

3 . 6

6 . 6

0 . 5

1 . 1


5 3 . 8

± 3 . 7

4 9 . 9

9

0 . 7

2 . 3

1 . 4

4 . 5

2 . 0

6 . 3

0 . 5

– 1 . 7

a

E x p a n d e d u n c e r t a i n t y e x p r e s s e d a s u n c e r t a i n t y w i t h a c o v e r a g e f a c t o r o f 2 .

b

n = N u m b e r o f l a b o r a t o r i e s .

c

S D r = R e p e a t a b i l i t y S D .

d

S D R = R e p r o d u c i b i l i t y S D .

e

r = R e p e a t a b i l i t y l i m i t .

f

R = R e p r o d u c i b i l i t y l i m i t .

g

H o r R a t v a l u e a s r a t i o u s i n g R S D R a n d H o r w i t z d e n o m i n a t o r a c c o r d i n g t o r e f . 3 2

.

h

N A = N o t a v a i l a b l e .




T a b l e

3 0 .

S t a t i s t i c a l a n a l y s i s

o f n i n e e l e m e n t s d e t e r m i n e d i n f o o d

p r o d u c t s t e s t e d b y I C P - A E S

S t a t i s t i c

C a

C u

F e

K

M g

M n

N a

P

Z n


F o u n d m e a n l e v e l , m g / k g

4 1 6

4 . 9 4

1 5 . 9

6 1 5 7

1 7 5 3

1 6 . 0

4 8 8 6

3 4 4 6

2 6 . 8

C e r t i f i e d v a l u e , m g / k g

4 1 1

4 . 9 3

1 6 . 4

6 0 7 0

1 6 8 0

1 6 . 0

4 8 9 0

3 3 7 8

2 6 . 3

S D R , m g / k g

2 1

0 . 3 1

2 . 4

1 1 8

1 1 6

1 . 3

1 2 2

8 8

1 . 6

z - s c o r e

0 . 2

0 . 0 3

– 0 . 2

0 . 7

0 . 6

0

– 0 . 0 3

0 . 8

0 . 3



9 7 1 2

5 . 9 2

1 6 6 . 2

9 0 0 6

1 7 5 1

7 . 5 3

1 9 3 7

4 1 2 7

1 8 . 5


9 8 7 0

N A a

1 6 9 . 9

9 0 9 0

1 7 7 9

N A

1 9 9 0

4 0 8 0

N A

S D R , m g / k g

2 8 0

0 . 4 1

7 . 4

1 6 6

5 4

0 . 4 9

4 2

1 2 3

0 . 9

z - s c o r e

– 0 . 6

– 0 . 5

– 0 . 5

– 0 . 5

– 1 . 3

0 . 4



5 9 7 2

1 . 9 4

7 4 . 8

6 4 6 6

8 6 0

1 6 . 5

9 9 7

4 3 0 2

3 6 . 7


6 0 0 0

N A

8 0 . 4

6 5 0 0

8 8 1

N A

1 0 5 0

4

3 2 0

N A

S D R , m g / k g

2 9 0

0 . 2 3

3 . 9

1 2 4

4 7

1 . 5

5 5

9 1

1 . 4

z - s c o r e

– 0 . 1

– 1 . 4

– 0 . 3

– 0 . 5

– 1

– 0 . 2



5 6 9 6

6 . 0 9

8 1 . 9

6 4 3 1

4 4 1

0 . 4 0 7

1 7 6 7

3 7 6 0

5 3 . 6


5 7 8 0

6 . 1

8 1 . 1

6 4 2 0

4 4 4

0 . 4 0 5

1 8 4 0

3 7 2 0

5 1

S D R , m g / k g

2 1 8

0 . 4

3 . 6

7 6

1 3

0 . 0 2 9

4 6

1 2 2

2 . 4

z - s c o r e

– 0 . 4

– 0 . 0 2

0 . 2

0 . 1

– 0 . 2

0 . 1

– 1 . 6

0 . 3

1 . 1



3 6 5

5 . 7 7

4 9 . 8

4 1 3

4 8 3

1 3

1 3 5 3

2

0 5 3

4 9 . 9


3 6 9

5 . 9 4

5 4 . 3

4 7 2

5 1 0

1 4 . 3

1 4 2 0

2 1 9 0

5 3 . 8

S D R , m g / k g

1 5

0 . 2 6

2 . 1

3 2

1 5

1 . 2

4 1

6 8

2 . 3

z - s c o r e

– 0 . 3

– 0 . 7

– 2 . 2

– 1 . 8

– 1 . 8

– 1 . 1

– 1 . 6

– 2

– 1 . 7

a

N A = N o t a v a i l a b l e .



(dietetic milk powder).The results indicate that this method candetermine, with sufficient precision, from 53.6 to 18.5 mg/kg.

( j) Horwitz equation.—Comparison of the Horwitz

function with that obtained from 45 results (i.e., nine elements

in five matrixes) of the collaborative study highlights

excellent precision as shown in Figure 1. This graph shows

that the trend of data from collaborative study follows the

theoretical Horwitz function, as the slope is similar. The

intercept is significantly different, and hence, this consistent

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

that were also informed of the maximum concentration of

each element in the five matrixes tested.

Conclusions

Method Performance

The MDO and MDC procedures used for SLV and RT

were shown to be effective with acceptable agreement toward

certified and in-house reference values in terms of recovery

for most samples covering the nine sectors of the food

triangle.

This ICP-AES analytical method was proven to be simple,

selective, accurate, and reliable for the determination of Ca,

Cu, Fe, K, Mg, Mn, P, Na, and Zn in most food matrixes.

Recommended and alternate analytical lines corrected byappropriate IS and Cs ion buffer concentration ranging

between 0.1 and 1% (w/v) were validated according the

statistic treatment of selectivity, sensitivity, linearity,

accuracy, and precision through SLV using ICP-AES

equipment with axially viewing plasma after MDC, and

through ruggedness tests using ICP-AES equipment with

radial and dual viewings of the plasma after MDC and MDO

procedures.

Performance characteristics reported for 13 certified and

in-house reference materials covering the triangle foodsectors

fulfilled AOAC criteria and recommendations in terms of accuracy (trueness, recovery, and z -scores) and precision

(repeatability and reproducibility RSDs, HorRat values)

regarding SLV and collaborative study.

The determination of nine nutritional minerals in fortified

foodproducts by ICP-AES is fit-for-purpose according to ISO

17025 norm and AOAC acceptability criteria.

Improvement of AOAC Official Method 984.27

Main improvements compared to the AOAC Method

984.27 are: (1) The use of microwavedigestion systems with a

single acid (nitric acid) for an optimized sample preparation inorder to improve element recoveries from difficult matrixes

and to increase sample throughput, favoring safety

precautions and time-savings for operators in laboratories. It

is a significant improvement regarding long, time-consuming

acid digestion with unsafe acid handling (HNO3/HClO4) used

for AOAC Method 984.27. (2) The use of appropriate

analytical wavelengths for each element of interest and the

automatic addition of a solution of appropriate IS and

ionization buffer to correct for physical and chemical

interferences (i.e., to compensate for matrix effects induced

by the complexity of the food samples). The aim is to improve

short-term accuracy (repeatability) and long-term stability(reproducibility and calibration curve validity in a long

analytical batch). Neither internal standardization nor ion

buffer are used for AOAC Method 984.27. (3) The application

for all food matrixes covering all of the nine AOAC food

triangle sectors, including infant formula, which were the

unique types of matrixes validated for AOAC Method 984.27.

This fully validated multielemental method is

cost-efficient, time-saving, accurate, and fit-for-purpose. It is

proposed as an improved version of AOAC Official Method

984.27 for fortified food products, including infant formula.


Figure 1. Ring trial: Comparison of Horwitz equation and experimental equation found for the ring trial.



Acknowledgments

We would like to thank Florence Monard and Thierry

Delatour from the Nestlé Research Center for useful

discussions and support given during the work. We are also

indebted to the following collaborators for their skilfull

participation in ruggedness tests MD3, MD4, and the

collaborative study:

Susanna Berger and Rafael Berrocal, PTC, Konolfingen,Switzerland

Ria Bos, Ton Noorlos, and Genevieve Daix, NQAC,

Nunspeet, The Netherlands

Anne Baillon, Christine Senechal, Laure Brullebaut, and

Caroline Gaudin, Creully Factory, Creully, France

Agnes Fortineau, Christian Dekussche, and Christine

Caseiro, Boué Factory, Boué, France

Rick Reba and Genevieve Cole, NQAC, Dublin, OH

Andy Abrahamson, Eau Claire Factory, Eau Claire, WI

Roberto Leal and J. Barrios, SQAL, Macul, Chile

Rey Oliver Mabiog and Ma. Josephine Gonzales, SQAL,

Cabuyao, The Philippines

Gursharan-Singh Dhillon, CQAL, Moga, India

Choo Lee Foon, NQAC, Shah Alam, Malaysia

References

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