production and certification of standard reference
TRANSCRIPT
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