Production and Certification of Standard Reference Materials for Food at NIM

Zhang Qinghe

National Institute of Metrology� P. R. China

Nov. 24 2012

food matrix 71: Milk, fish, corn, seafood, egg powder

65

42

68

Matrix CRMs

NIM FOOD MATRIX CRMs: A overview

Food Matrix CRMs – inorganic targets

CRM No. Matrix Methods Elements and Speices

GBW08509a Skimmed Milk Powder

IC, Kjeldahl, ICP-MS, ICP-OES, AFS, AAS

Se, Zn, Fe, As, K, Na, Ca, Cu, Mn, Pb, Mg, N, P, Cl

GBW08521 Laver Powder IDMS, INAA, ICP-MS, ICP-OES, AFS, AAS As, Pb, Cd

GBW10029 Fish Tissue Powder IDMS, ICP-MS, CVAFS, CVAAS, ID-GC-ICPMS, ID-LC-ICPMS, HPLC-CVAFS Hg, MeHg

GBW10035 Wheat Powder IDMS, ICP-MS, ICP-OES Pb, Cd, Cr

GBW10036 Corn Powder IDMS, ICP-MS, ICP-OES Pb, Cd, Cr

GBW10054 defatted soybean powder IDMS, ICP-MS, AAS, ICP-OES K�Mg�Ca�Fe�Zn�Mn�Cu�Na

GBW10055 whole fat soybean powder IDMS, ICP-MS, AAS, ICP-OES K�Mg�Ca�Fe�Zn�Mn�Cu�Na

GBW(E)100194 Rice Powder AAS, ICP-OES, ICP-MS K, Mg, Ca, Fe, Mn, Zn, Cu, Na

GBW(E)100195 Wheat Powder AAS, ICP-OES, ICP-MS K, Mg, Ca, Fe, Mn, Zn, Cu, Na

GBW(E)100196 Corn Powder AAS, ICP-OES, ICP-MS K, Mg, Ca, Fe, Mn, Zn, Cu, Na

GBW(E)100197 Bovine Muscle Powder AAS, ICP-OES, ICP-MS K, Mg, Ca, Fe, Mn, Zn, Cu, Na

GBW(E)100198 Egg Yolk Powder AAS, ICP-OES, ICP-MS K, Mg, Ca, Fe, Mn, Zn, Cu, Na

GBW(E)100199 Cushaw Powder AAS, ICP-OES, ICP-MS K, Mg, Ca, Fe, Mn, Zn, Cu, Na

GBW(E)100227 Infant Formula Milk Powder ICP-MS, ICP-OES, AAS, XRF, SP Se, Zn, Fe, K, Na, Ca, Cu, Mn, Mg, P

Food Matrix CRMs – organic targets

Most of them used IDMS methods for value assignment

NIM Modes of Value Assignment for Food RMs

• Certification at NIM using two independent, critically

evaluated methods (inorganic targets)

• Value-assignment based on NIM measurements using a single

method or measurements by an outside collaborating

laboratory using a single method (some organic targets)

• Value-assignment based on measurements by more laboratories

using different methods in collaboration with NIM

• Value-assignment based on selected data from interlaboratory

studies

Food CRMs Development using Isotope Dilution Mass Spectrometry (IDMS)

Development of IDMS as a definitive

method allowed the certification of

trace analytes in food-based CRMs

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Food Matrix CRMs at NIM:

Milk and milk powder

Chloramphenicol

Melamine

Nutrients

Elements

Some Current Examples:

Definition of the Measurand: CAP

Chloramphenicol (CAP):

Molecular Formula: Cl2CHCONHCH(CH2OH)CH(OH)C6H4NO2Molar Mass : 323.13 g mol–1

CAS Number : 56-75-7

Medium Polar, Stable and non Volatile

Medium

Molecular Weight

mass fraction range of 0.1 to 50 ng/g

USA

PRA

JAPAN

EU

Prohibited addition CAP in original animal food

BackgroundBackground

BackgroundBackground

Analysis Technology

4

ELISA….Disadvantages: •False positives•Low accuracy•Low qualitative

GC, LC….Disadvantages: •Low sensitivity•Low selectivity

Higher selectivity; Higher sensitivity; Higher throughout...

GC�HPLC-MS/MS

1.0 1.5 2.0 2.5 3.00.0

0.4

0.8

1.2

B/B

0

LgC

Y=-0.503X+1.697�R2=0.991

0�750ng/kg linear

Screening of Candidate materials by ELISA

• Using ELISA screening of 21 kinds of different brands of milk powder• Definition of Candidate materials and Keep portions in 5g brown bottle

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

0.0

100.0

200.0

300.0

400.0

500.0

600.0

700.0

Con

cent

ratio

n(ng

/kg)

The different brands of milk powder (n=4)

ELISA

629.8ng/kg

525.3ng/kg

blank

Screening of Candidate materials: Milk Powder

Composition of the milk

�� �� ��� ��� �� �� ��� ���

��� g 16 16.1 � mg 680 407

�� g 18 28.7 � mg 90 68

����� g 59.1 51 � mg 220 250.9

���A IU 1350 1541 � mg 790 587

���B1 ug 700 518 � mg 360 313

���B2 ug 800 781 � mg 520 543

���B6 ug 700 340 � mg 9 8.3

���B12 ug 1.2 1.8 � ug 500 357

���C mg 60 50 � mg 4.6 4.1

���D IU 280 258 � ug 25�80 82

���E IU 9 7 � ug 30�150 30-150

���K1 ug 32 27 � ug 6�15

�� ug 2000 2780 ���� 1.3

�� ug 8600 4445 ��������DHA� mg 70 50

�� ug 120 39.6 �������AA� mg 10

β-���� ug 100 ������� mg 340

��� ug 12 12.7 ��� mg 33 37

�� mg 50 72 ��� mg 22

��� mg 2000 3328 ��� mg 40

A Reference Measurement Procedure for CAP:HPLC-ID-MS/MS

Weight some samples

Add ID�water and sodium sulfate

Heat in bathEthyl acetate

Blending

supernatantClean-up

LLE

MCX

HLB

MIP

AB API5000

Agilent6490

Waters XEVOTQS

Centrifugal vortex

The sensitivity of GC-MS was not fitness this sample

Complex Matrix

Matrix Effect

Sensitivity�reproducibilityaccuracy�linearity…..

Spiked stable-isotope-labeled internal standards

Validated method

Difference between target drugs andstable-isotope-labeled internal standards

Problems ?

1�extract

2�cleaning

3�determination

LLE

Cation inion exchange MCX

Molecular imprint polymer MIP

AB API 5000

Agilent 6490

Waters XEVO TQS

Process

Waters HLB

A Reference Measurement Procedure for CAP:extracted conditions

Items The designed experimental programs The optimized results

Weight of water used dissolving samples 2.5, 3.5, 4.5g 2.5g

Extraction solvent ethyl acetate and acetonitrile The higher extraction efficiency of ethyl acetate�7.45%�

Protein degradation anhydrous sodium sulfate and trichloroacetic acid 3.0g of anhydrous sodium sulfate

Extraction times 1, 2, 3, 4 2 times

Temperature of bath 50, 55, 60, 65, 70� 55�

Time of ultrasonic 0, 10, 20, 30, 40, 50min 10min

Time of oscillation 0, 10, 20, 30, 40, 50min 10min

Stability of calibration solution

The calibration solution was detected in everyday �16days� 16 days

Effect of filters

nylon 6�0.45um and 0.22um of organic filter�0.45um of water filter�polyether sulphone�0.22um of water

filter

polyether sulphone�0.22um of water filter

Time of equilibration /h 0, 1, 2, 24h 0h

Combined analyte with addition HCL and without addition HCL without addition HCL

0 10 20 30 40 500.82

0.84

0.86

0.88

0.90

0.92

ÌáÈ

¡Ð§Â

Ê(ng

/g)

Õñµ ʱ¼ä t/min

0 10 20 30 40 500.80

0.82

0.84

0.86

0.88

0.90

0.92

ÌáÈ

¡Ð§Â

Ê(ng

/g)

³ ¬É ùÊ ±¼ä t/min

50 55 60 65 700.82

0.84

0.86

0.88

0.90

0.92

ÌáÈ

¡Ð§Â

Ê (ng

/g)

Ë ®Ô¡Î ¶ È T/¡ æ

Time of oscillation 10min

Time of ultrasonic10min

Tem bath 55�

Result of Extraction

1 2 3 4

0.86

0.88

0.90

0.92

Con

cent

ratio

n (

ng/g

)Extracted Time /n

Result of Extraction

0 1 4 240.75

0.80

0.85

0.90

0.95

1.00

Con

cent

ratio

n (n

g/g)

Equilibrium Time /h

Times of extracted by ethyl acetate

Time of isotopic CAP equilibration /h

Difference of filters before mass analysis

Recovery

Level ng/g Spiked /ng/g Detected /ng/g Recovery %

0.30.2926 0.2832 96.800.2939 0.2968 100.960.2849 0.2932 102.94

0.90.9303 0.9756 104.870.9393 0.9232 98.290.8901 0.8876 99.72

2.02.0198 1.9346 95.782.0250 1.9452 96.061.9968 1.9644 98.38

LOD and LOQ

LOD 0.004ng/g

LOQ 0.01ng/g

1 2 3 40

1 5

3 0

4 5

6 0

7 5

9 0

Inte

nsity

, cps

T im e , m in

MRM chromatogram of milk powder sample containing 0.004ng/g chloramphenicol

Matrix effect of IDMS7

Concentration�ng/g�

K�cap�/K�cap-D5�

AArea

CAPCAP-D5

Figure1 . Spiked five points�n=5� of CAP and CAP-D5 in standard solution�ACN/H2O=30/70�V/V�and blank matrix, comparison of the ratio target compound to stable-isotope-labeled internal standard.

Concentration�ng/g�

Area K�cap�/K�cap-D5�

B

CONTENTCONTENT

BB

EE

CC

DD

AAThe different apparatus and clean-up methods

The concentration of matrix

The different milk powder samples

The concentration of CAP and CAP-D5

The mobile phase and dilution solvent

Matrix effect of IDMS

40%35%

30%25%

0.80

0.85

0.90

0.95

1.00

1.05

30%

35%

40%

The

fact

or o

f mat

rix

effe

ct (

¦È)

Aceton

itrile in

disso

lution

solve

nt

Acetonitrile in mobile phase

Effect of mobile phase and dilution solvent

LLE

MIP

MCX

HLB0.9

1.0

1.1

1.2

The

fact

or o

f mat

rix

effe

ct

¦È

Methods of extraction

mobile phase dilution solventapparatus clean-up methods

Determination of factors of matrix effect in four pretreatment different conditions LLE�MIP �MCX �HLB�by API5000�Waters TQS and Agilent 6490.

Matrix effect of IDMS

Matrix effect of IDMS

The concentration of matrix

0.25 0.50 0.75 1.00

0.84

0.88

0.92

0.96

1.00

1.04Fa

ctor

of m

atri

x ef

fect

(¦È)

The ratio of diluted matrix

4 3 2 1

Effect of the concentration of matrix. The sample was diluted by 30%ACN solvent in water and detected in the four different mobile phase�1�40%ACN in water�2� 35%ACN in water�3� 30%ACN in water�4� 25%ACN in water.

0 .0 1 .0 2 .0 3 .0 4 .0 5 .00 .9 0

0 .9 5

1 .0 0

1 .0 5

1 .1 0

The

fact

or m

atri

x ef

fect

(¦È)

T h e co n cen tra tio n o f C A P a n d C A P -D 5 (n g /g )

The samples extracted by four different clean-up methods were spiked with the different concentration of CAP and D5-CAP. The datas were detected by API 5000�n=5�. The four clean-up methods were: ● MIP, ▼ HLB, ▲ MCX, ■ LLE.

Matrix effect of IDMS

Test of Homogeneity

2

ISO GUIDE 35: Reference materials-general and statistical principles for producers

Test of Homogeneity

If insufficient repeatability of the measurement method

If sufficient repeatability of the measurement method

ISO GUIDE 35: Reference materials-general and statistical principles for producers

Test of Homogeneity

Bottle No Result No.1 Result No.2 Result No.3 Average Variance count1 0.894 0.89 0.904 0.896 0.007 32 0.889 0.938 0.928 0.918 0.026 33 0.888 0.952 0.944 0.928 0.035 34 0.916 0.923 0.905 0.915 0.009 35 0.917 0.932 0.901 0.917 0.016 36 0.896 0.891 0.904 0.897 0.007 37 0.868 0.894 0.871 0.878 0.014 38 0.906 0.923 0.916 0.915 0.009 39 0.911 0.894 0.873 0.892 0.019 3

10 0.9 0.867 0.866 0.878 0.019 311 0.9 0.888 0.87 0.886 0.015 312 0.938 0.897 0.891 0.909 0.026 313 0.881 0.901 0.881 0.888 0.012 314 0.884 0.872 0.905 0.887 0.017 315 0.934 0.888 0.895 0.906 0.025 3

Source of variation SS Degrees of

freedom MS

Between bottles 0.011 14 7.86E-04Within bottles 0.021 30 7.00E-04

Table – Homogeneity data for CAP in milk powder

The between –bottle variance is estimated using

= (0.000786-0.0007)/3 =0.0000286

The between –bottle standard deviation is the square root of this variance

2.86E-04 = 0.0054 μg/kg

The repeatability standard deviation can be computed from MSwithin

= 0.0007

Test of Homogeneity

= 0.026 μg/kg

ISO GUIDE 35: Reference materials-general and statistical principles for producers

Test of Stability

There are two types of (in)stability to be considered in the certification of reference materials:

the long-term stability of the material (e.g. shelf life),and

the short-term stability(e.g. stability of the material under “transport conditions”

6/20/20117/19/20117/30/20118/31/201110/9/2011

4/7/20125/23/2012

6/1/20128/13/20128/29/2012

0 .8 0

0 .8 5

0 .9 0

0 .9 5

1 .0 0

Con

cent

ratio

n(ng

/g)

T h e d e t e c t e d t im e ( M o n t h / D a y /Y e a r )

Test of Stability

0.00341

0.90 6.67

0.898

Time months CAP content (mg/kg)

0 0.9026

1 0.8975

3 0.9008

6 0.9046

12 0.8921

18 0.905

Table –Stability data for CAP in milk powder

ISO GUIDE 35: Reference materials-general and statistical principles for producers

Test of Stability

3.84E-04

3.66E-05

2.78

ISO GUIDE 35: Reference materials-general and statistical principles for producers

Uncertainty evaluation

uncertainty valueus 0.0011%ubb 0.0024%Ws 0.0044%

ucal-cap 0.0092%ucal-capd5 0.0092%uw-capd5 0.11%upure-cap 0.916%

usample preparation 3.13%uprecision 1.73%u combine 3.700%

U(k=2) 7.400%

A

B

Uncertainty evaluation

Certified value �μg/kg�U�μg/kg�

�k=2�

<0.004 --

0.915 0.067

Chloramphenicol in Milk Powder (CRMS)

0 1 2 3 4 5 6

0

1x103

2x103

3x103

4x103

5x103

6x103

7x103

Inte

nsity

,cp

s

Time, min

Instrumentation view

Thermo MAT253 IRMS

Waters Synapt G2 HDMS IMMS

ABI-5500Qtrap linear ion trap

Thermo HPLC-ICP-MS