CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
Final Report on Key Comparison
CCQM-K81
“Chloramphenicol in Pig Muscle”
Joachim Polzer1, Andre Henrion2, and Petra Gowik1
1
Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (BVL)
D-12277 Berlin, Mauerstrasse 39-42, Germany
2
Physikalisch-Technische Bundesanstalt (PTB)
D-38116 Braunschweig, Bundesallee 100, Germany
With contributions from:
Norma Gonzalez / Esther Castro
Centro Nacional de Metrologia (CENAM)
Queretaro, Mexico
Twinnie Tso, W.O. Lee,
Government Laboratory (HKSAR)
HongKong, China
Kanjana Wiangnon
National Institute of Metrology (NIMT)
Patumthani, Thailand
Meg Croft
National Measurement Institute, Australia (NMIA)
Sydney, Australia
Ahmet Ceyhan Gören
Ulusal Metroloji Enstitüsü (TUBITAK-UME)
Gebze-Kocaeli/TR, Turkey
A key comparison and parallel pilot study agreed upon by the Organic Analysis Working
Group (OAWG) of the CCQM and coordinated by BVL and PTB.
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Index of Contents
1
Introduction................................................................................................................. 3
2
Study Measurand ........................................................................................................ 3
3
Study Material............................................................................................................. 3
3.1
3.2
Preparation of Study Samples by BVL and IRMM ............................................ 3
Homogeneity and Stability Testing of samples .................................................. 4
3.3
Instructions for Study Participants...................................................................... 4
4
Reporting instructions................................................................................................. 6
4.1
Study Schedule.................................................................................................... 6
5
Reference materials used by the participating Laboratories ....................................... 7
6
Overview on methods ................................................................................................. 8
7
Approaches to uncertainty ........................................................................................ 11
8
Participants results .................................................................................................... 12
8.1
Additional studies on bound CAP residues in the test material by the CL [1] ... 12
9
KCRV calculation..................................................................................................... 13
10
Degree of equivalence (DoE) calculation ............................................................. 14
11
Scope of the key comparison and core competencies........................................... 16
11.1 Comments on “How far does the light shine?”................................................. 16
12
Summary and Conclusions ................................................................................... 16
13
Annexes................................................................................................................. 18
13.1
Annex I : Details of reported measurement uncertainties (participants reports)
18
13.2
Annex II: Calculation of DoE ........................................................................... 24
13.3
Annex III (only for comparison): Calculations taking into account all reported
values (corrected values, which were submitted subsequently to the closing of the key
comparison and excluded values) ................................................................................. 25
13.4
Annex IV: Core competencies .......................................................................... 26
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Introduction
Even though chloramphenicol (CAP) is an effective broad-spectrum antibiotic, its use for
the treatment of food producing animals was forbidden e.g. in the European Union (in
1994) and in the U.S. due to severe side effects, as e.g., aplastic anemia. Since the toxic
effects of CAP are not dose dependent, a no effect level could not be established and
consequently a zero tolerance level was set for CAP in food. In 2003 with EC
Commission Decision 2003/181/EC, a minimum required performance limit (MRPL) of
0.3 µg/kg for residues of CAP in different matrices, as e.g. milk and muscle, was fixed.
Products intended as imports to the EU must not exceed this mass fraction level.
At the CCQM/OAWG meeting in April 2009 the conduct of a key comparison and
parallel pilot study “Chloramphenicol in pig muscle” as a follow-up to the pilot study
“CCQM-P90: chloramphenicol in milk” was agreed to by the WG. This study is intended
to provide a basis for documenting the capabilities of NMIs in this type of measurement
also with respect to the fulfillment of legal requirements to control this residue in food.
The study was classified as a “track C” study (studies in emerging areas of global
interest).
For the study lyophilised pig muscle material containing CAP (at a mass-fraction level
around the maximum allowable level for import for a number of countries) has been
produced as a candidate reference material by BVL and IRMM. The WG agreed to the
use of this material in this study.
2
Study Measurand
The study measurand is chloramphenicol (D(−)-threo-2,2-Dichloro-N-[β-hydroxy-α(hydroxymethyl)-β-(4-nitrophenyl)ethyl]acetamide, also known as “Chloromycetin”).
Molecular Formula
Cl2CHCONHCH(CH2OH)CH(OH)C6H4NO2
Molar Mass 323.13 g mol–1
CAS Number 56-75-7
Participants are to report the mass fraction of CAP in the reconstituted study material.
3
Study Material
3.1
Preparation of Study Samples by BVL and IRMM
The study samples were prepared from an incurred material produced at the BVL. For the
production of incurred muscle material a pig was treated for a few days with
“Chloromycetin palmitate”. Muscle samples were collected directly after slaughtering of
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the animal and pre-tested for its CAP content. The incurred material was then delivered
to the IRMM for further processing and testing of homogeneity and stability. Processing
to the final material comprised of freeze-drying, mixing of the obtained powder with
lyophilised blank pork meat powder, and homogenisation of the pooled materials by
milling and mixing techniques. The final product was filled in 7.5 gram portions into
amber glass bottles under inert gas atmosphere and stored at -20 ºC. A subset of these
bottles of material is being used as the study material for this study, for K81 and P122.
The material is meanwhile available to purchase as certified reference material “ERMBB130”.
3.2
Homogeneity and Stability Testing of samples
For the homogeneity study, 10 samples of ERM-BB130 were chosen using a random
stratified sample picking scheme and analysed in quadruplicate for their CAP content.
Measurements were performed using a validated reverse-phase liquid chromatography negative electrospray ionisation - tandem mass spectrometry method. 1.25 g of powder
(equivalent to 5.00 g of reconstituted material) was used as sample intake per analysis.
Samples were measured in a random order under repeatability conditions. Data were
technically scrutinised and statistically evaluated according to ISO Guide 35. The
material showed to be sufficiently homogeneous (calculated uncertainty contribution due
to possible heterogeneity 1.24% out of the overall RSD of 3.4%).
A four weeks isochronous study was performed to evaluate stability of ERM-BB130
during transport. Twenty samples were selected from the produced batch using a random
stratified sample picking scheme. Samples were stored at +4 °C, +18 °C, +60 °C and at a
reference temperature of -70 °C. Two bottles were stored at each temperature for 0, 1, 2,
and 4 weeks. After the indicated storage periods, the samples were transferred to storage
at -70 °C until analysis. Samples were analysed in quadruplicate under intermediate
precision conditions and in a random order. The same method and sample intake as
described for the homogeneity measurements was used. Data were technically scrutinised
and statistically evaluated according to ISO Guide 35. Regression lines were calculated to
detect possible degradation. Whereas the slope was found to be indistinguishable from
zero for storage temperatures of 4 ºC and 18 ºC, a significant slope was found when the
samples were stored at 60 ºC. The uncertainty of the short-term stability (usts) can be
assumed to be negligible if sample shipment is carried out with cooling elements or on
dry ice.
3.3
Instructions for Study Participants
Participants received four bottles of the study material, each of them containing 7.5 g of
lyophilised pork meat. Each vial was equivalent to approximately 30 g of fresh meat.
A result for each of three individual bottles and an overall, combined result for the study
material should be reported. On demand of a participant, whether free or bound
(conjugated) and free chloramphenicol should be reported it was clarified, that all
participants should indicate the "free CAP" as target analyte (i.e. a hydrolysis of the
sample should not be performed).
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The samples were to be stored at the laboratory's premises at a temperature of -20 ± 2ºC.
Laboratories should use their own methodology for the analysis of the chloramphenicol
in muscle (as used to deliver their services). The participating laboratories should use
their own CAP reference standard for any preparation of standard solutions and
calibration.
Appropriate portions of the samples had to be reconstituted according to the instructions
provided below so that the sample will be representative of a fresh meat sample. This
should be done gravimetrically so that the results could be reported as required as CAP in
the reconstituted material.
A protocol for reconstitution of the sample material with water was supplied to the
participants and is outlined below:
•
Each vial contains about 7.5 g of lyophilized pork meat. For the conversion
lyophilised meat/fresh meat, 0.25 g of the dry material corresponds to originally
1.000 g of meat.
•
Make sure the test bottles have warmed up to room temperature before reconstitution,
as the samples are hygroscopic. Avoid frequent warm up and freeze-store cycles with
the vials.
•
Before opening of the bottle, tap the bottom of the concerned test tube several times
against the table in order to loosen sample material that might stick to the cap, then
carefully open the test tube and stir it e.g. with a spatula .
•
To reconstitute a portion of the dry material, remove a minimum amount of 1.25 g of
the dry material (as this was the sample size used in the homogeneity evaluation) into
an appropriate tared vessel Weigh. Add the amount of water (HPLC grade) needed:
0.75 g of water per 0.25 g of the dry material used. Weigh again. This will enable
participants to report the analyte content in reconstituted meat as required for the
study.
•
If your method calls for addition of an internal standard(s) to the meat prior to
subsequent sample preparation, it may be added during or directly after
reconstitution.
•
Vortex the sample.
•
Proceed with this material according to your normal standard procedure for
determining CAP in fresh meat.
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Reporting instructions
A data reporting sheet was provided for the submission of the results. The following data
was to be included in the report:
 For each of three samples, the mass fraction of free CAP in the pig muscle as
ng/g expressed as analyte content in reconstituted meat and an overall,
combined result for these bottles should be reported with the standard
uncertainty, uc, and the expanded uncertainty .
 Outline of methodology of analytical method and uncertainty estimation
(including a measurement equation and sources considered for uncertainty)
 A full uncertainty budget
 For calibrant materials used: Source, purity, information as to who valueassigned the purity, and methods used to assess
 The source and details of any labelled materials used.
4.1
Study Schedule
Deadline for signup to study:
September 2009
Distribution of sample materials:
October 2009
Deadline for submission of results:
January 2010
Draft report to participants:
March 2010
Overview of study results /
participant presentations
:
April 2010 OAWG Meeting
Draft A report:
April 2011 OAWG Meeting
Draft B report:
April 2012 OAWG Meeting
The IRMM provided the samples to the BVL at the end of September 2009. The samples
were shipped on dry ice. The BVL distributed the samples to all participants between 9
October and the 19 October 2009; samples were shipped on dry ice too. Due to problems
with the customs in some countries, samples could not be sent at the same time.
Nevertheless all samples received the laboratories in good condition between the 12
October and the 21 October 2009.
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Table 1 : Study schedule
Laboratory
Short
name
National Measurement
Institute Australia
NMIA
Government Laboratory
(Hong Kong, China)
HKSAR
Centro Nacional de
Metrologia (Mexico)
CENAM
Ulusal Metroloji Enstitüsü,
TUBITAK (Turkey)
UME
National Institute of
Metrology (Thailand)
NIMT
Dispatch of
Samples
(2009)
Receipt of
Sample
(2009)
Submission
of result
16 October
21 October
January
2010
9 October
12 October
January
2010
19 October
21 October
January
2010
9 October
15 October
December
2009
15 October
19 October
December
2009
--
30 September
December
2009
Federal Office of Consumer
Protection and Food Safety
(Germany)
5
BVL
Reference materials used by the participating Laboratories
Table 2 summarises the calibration standards and the internal standards used by the
participants. All participants used D5-CAP as internal standard. As calibrant CAP from
different commercial sources was used and in-house tested for purity with different
methods. Considering the results of the purity test, it can be concluded that CAP is
commercially available in high purity.
Table 2 : Summary of Reference Standards, purity assessments and internal Standards
Materials used by the Participants
Laboratory
Source of calibrant purity
Methods used for Internal
purity assessment
standard
CENAM
(Mexico)
Sigma-Aldrich
100 – x approach
99.8 %
DSC
HKSAR
(China)
Sigma
98 %
Page 7 of 44
98 % (purity
statement of
supplier; verified by
comparison with
other sources)
D5-CAP
(Cambridge
Isotope
Laboratories)
D5-CAP
(Witega)
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
Laboratory
Source of calibrant purity
Methods used for Internal
purity assessment
standard
HKSAR
(China)#
Sigma-Aldrich
99.85 %
100 – x approach
HPLC; ICP-MS;
Headspace-GC;
Moisture;
NIMT
(Thailand)
Sigma-Aldrich
99.6 %
100 – x approach
HPLC;
DSC
Moisture;
D5-CAP
(Cambridge
Isotope
Laboratories)
NMIA
(Australia)
UME
(Turkey)
BVL
(Germany)
Fluka Vetranal;
99.8 %
Sigma-Aldrich
99.9 %
Sigma-Aldrich
98.0 %
qNMR
D5-CAP
(98,1 %)
(additional: 100 – x
approach:
LC/UV; HS-GC;
Moisture; TGA)
100 – x approach
LC/TOF;
LC/MSMS
Moisture;
(CHN analysis /
plausibility)
(Dr.
Ehrenstorfer)
Sigma-Aldrich
99.5 %
qNMR
D5-CAP
from
Cambridge
Isotope
Laboratories
D5-CAP
(Cambridge
Isotope
Laboratories)
#) comprehensive reference standard characterisation was done after the closure of the
key comparison
6
Overview on methods
The participating laboratories were encouraged to use a method of their own choice. The
details of sample preparation and measurement are summarised in table 3.
All laboratories used IDMS (isotope dilution mass spectrometry) technique with
deuterated chloramphenicol as internal standard. Very different sample preparation
techniques were applied. Different waiting times after reconstitution of the samples were
applied as well as different extraction procedures, solvents (aqueous/organic extraction)
and subsequent clean-up procedures (different types of SPE-cartridges, even simple
distribution between solvents).
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For measurement five of the six laboratories used the LC/MSMS technique, all of them
applying negative electrospray as ionisation technique. Different instruments of two
different suppliers were used. One laboratory used the GC/MS technique, also in the
negative ion mode (negative chemical ionisation).
Table 3 : Summary of Methods used by the Participants
Laboratory
Sample preparation
measurment
CENAM
(Mexico)
1.25 g sample;
reconstitution with water, addition of
internal standard D5-CAP;
extraction with ethyl acetate;
reduction by evaporation and addition
of NaCl solution; defattening with nhexane; clean-up on SPE C18;
elution with acetonitrile, evaporation
to dryness; addition of acetonitrile and
injection of an aliquot
LC/MSMS (API 5000)
Column: Symmetry shield
RP18
Gradient:
NH4 acetate – acetonitrile
Negative ESI mode
ions 321 -> 152, 257
323 -> 152
326 -> 157, 262
HKSAR
(China)
0.75 g sample;
reconstitution with 11 ml water,
addition of internal standard D5-CAP;
equilibrate 3 h at +4°C;
extraction with ethyl acetate;
change of solvent to NaCl solution;
defattening with n-hexane;
SPE clean-up on strata XC, elution
with methanol; addition of water,
evaporation of methanol,
inject
aliquot
LC/MSMS (API 4000)
Column: Alltima C18
Gradient: Water / Methanol
Negative ESI mode
ions 321 -> 152, 194, 257
326 -> 157, 198, 262
NIMT
(Thailand)
1.28 g sample;
reconstitution with Water, addition of
internal standard D5-CAP; equilibrate
for 1.5 h
extraction with phosphate buffer,
filtration;
clean-up on SPE C18, elution with
water/methanol; extraction with ethyl
acetate; evaporation to dryness,
addition of methanol and injection of
an aliquot
LC/MSMS (API 4000 Q Trap)
Column: Luna C18
Gradient: Water / Methanol
Negative ESI mode
ions 321 -> 152, 257
326 -> 157, 262
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Laboratory
28 August 2012
Sample preparation
measurment
NMIA
(Australia)
1.25 g sample;
reconstitution with Water, addition of
internal standard D5-CAP; equilibrate
for 0.4 h prior to addition of
acetate buffer pH5 and standing
overnight;
extraction with basified ethylacetate;
evaporation to dryness, redisolve in
dilute acetic acid and defat with
hexane
clean-up on SPE Strata XC: wash with
water/methanol;
elute
with
methanol/5% ammonia; evaporation to
dryness, addition of water, filtration
and injection of an aliquot
LC/MSMS (API 4000 Q Trap)
Column: Vision HT C18
Gradient: Water / Methanol /
0.01% formic acid
Negative ESI mode
ions 321 -> 152, 257
323 -> 152
326 -> 157, 262
UME
(Turkey)
1.25 g sample;
reconstitution with Water, addition of
internal standard D5-CAP;
extraction with ethyl acetate;
evaporation to dryness, add n-hexane
and MeOH/water;
filter aqueous phase and inject aliquot
LC/MSMS (Tandem Gold)
Column: Synergy max. RP
80A
gradient MeOH/water – MeOH
Negative ESI mode
ions 321 -> 151
326 -> 156
BVL
(Germany)
1.25 g sample;
reconstitution with Water, addition of
internal standard D5-CAP;
extraction with ethyl acetate;
evaporation to
dryness, add nwater/acetonitril 95/5, defat with nhexane;
clean-up on SPE (MIP), elution with
methanol;
evaporation to dryness, addition of
derivatisation
reagent
(BSTFA/TMCS/n-heptane), 45 min at
60°C;
inject aliquot
GC/MS (6890 / 5975)
Column: DB5
temperature program; splitless
injection of 2 µl at 280 °C;
Negative CI mode
ions 466, 468, 378, 376
471, 473
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Approaches to uncertainty
Table 4 summarises the standard uncertainties u, k-factors and expanded uncertainties U
as reported by the participants. Considered factors and main identified contributions are
listed in the table; the complete uncertainty estimation as reported by the participants is
attached in annex I.
Table 4: Summary of measurement uncertainty evaluations
Laboratory standard
Factor Expanded
considered contributions to
uncertainty k
uncertainty measurement uncertainty
(in bold: main contributions)
u [%]
U [%]
CENAM
internal standard mass fraction
(Mexico)
16.0
1.92
30.1
repeatability
0.047 ng/g
0.09 ng/g
calibration curve
mass of sample
HKSAR
stock standard solution
(China)
22.3
2
44.4
sample blend
0.0542 ng/g
0.108 ng/g
calibration blend
method precision
method bias
NIMT
calibration standard mass fraction
(Thailand)
3.4
2.04
6.8
calibration curve
0.01 ng/g
0.02 ng/g
internal standard mass fraction
mass fraction of internal standard
spike
mass of sample
method precision
interference from different ion
pairs
extraction effects factor
NMIA
(double
IDMS
measurement
(Australia)
3.8
2.78
11.0
equation)
0.011 ng/g
0.032 ng/g
calibration solution mass fraction
blend preparation masses
isotope amount ratios in blends
potential method biases
batch effect
UME
standard solution
(Turkey)
4.0
2
7.9
sample weight
0.00945 ng/g
0.01889 ng/g
sample volume
calibration curve
repeatability
recovery
BVL
CAP standard solution
(Germany) 4.0
2
7.9
weight of sample and IS spike
0.0097 ng/g
0.0194 ng/g
method precision
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Participants results
Table 5 summarises the results of the participants. 5 Laboratories reported values for
three of the four bottles; one laboratory reported results for all four bottles. Two
laboratories provided corrected results after the final deadline for submitting of the results
and the preliminary report.
Table 5: Overview on results as reported by the participants (if not indicated otherwise)
Laboratory
CENAM
(Mexico)
HKSAR
(China)
NIMT
(Thailand)
NMIA
(Australia)
UME
(Turkey)
BVL
(Germany)
Values for the
single bottles
[ng/g]
0.319; 0.335;
0.289
0.242; 0.244;
0.243
0.301; 0.280;
0.305
0.292; 0.294;
0.292; 0.284
0.237; 0.239
0.242
0.250; 0.239;
0.246
Mean value
[ng/g]
Uncertainty u
[ng/g] [%]
k value
0.313
0.047
1.921
0.243
0.0542 9.3
2
0.295
0.01
3.4
2.04
0.290
0.011
3.8
2.78
0.239
0.0095 4.0
2
0.245
0.0097 4.0
2
15.0
Corrected Values
(submitted after deadline / disclosure of results and preliminary evaluation)
CENAM
0.297; 0.293;
0.293
0.030 10.2
(Mexico)
0.289
NIMT
0.281; 0.277;
0.285
0.01
2.04
3.5
(Thailand)
0.296
Italic letters: values calculated by BVL from the given values
8.1
Additional studies on bound CAP residues in the test material by the CL [1]
Due to the question on bound residues and the distribution of the results additional
studies on the study material were performed by the CL. Taking into account data from
literature [e.g. 2, 3] the presence of glucuronide or sulfate residues was not expected.
Anyhow analysis of the study material with and without glucuronidase/arylsufatase (helix
pomatia) treatment showed that there is a significant percentage (> 20%) of conjugated
CAP present in the material. Additional studies showed that conjugated CAP is set free to
some extent already after reconstitution of the sample and storage at room temperature.
Hence it can not be excluded, that extended waiting times after reconstitution and
aqueous extraction procedures may lead to a partial release of bound CAP and
accordingly to a bias of methods including this sample preparation step.
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KCRV calculation
The OAWG has established criteria for including results in the calculation of the KCRV.
Among others required are the use of a method that has been verified as appropriate for
the measurand and of higher metrological order, and the use of a primary standard with a
metrologically traceable assigned purity – that is, either a Certified Reference Material
(from a NMI/DI with a demonstrated capability via the MRA) or a material the purity of
which has been suitably assessed by the reporting participant.
In total six results for the KCRV calculation were principally available. Based on the
discussion in the OAWG data from two laboratories were excluded from the KCRV
calculation. Data from HKSAR were not considered since the laboratory did not provide
metrologically traceable results (the reference compound characterisation was missing
when the study was closed; characterisation data were provided belatedly). Data from
CENAM were excluded since the laboratory informed the OAWG, that the provided data
were questionable due to instrument problems, which were discovered after the
submission of the results.
In order to select the appropriate KCRV calculation method [4] a consistency check for
the remaining data was done. This check showed inconsistent data, outliers could not be
identified. The most likely reasons for this inconsistency are unconsidered uncertainty
contributions due to the use of different methods and/or an underestimation of
uncertainties reported by the participants. For taking this into account different ways of
KCRV calculation were applied, using the simple arithmetic mean and different iterative
processes applying a maximum likelihood approach (uncertainty-weighted means). The
results of these calculations did not show a significant difference, neither in the KCRV
value nor in the corresponding uncertainty; hence according to “Data Evaluation
Principles for CCQM Key Comparisons” [5] it was decided by the OAWG working
group to use the simplest approach, i.e. the use of the arithmetic mean as the KCRV and
the standard deviation of the mean as the uncertainty.
The KCRV calculated as the arithmetic mean of the four remaining values was 0.267
ng/g. The standard deviation was calculated as 0.0293 ng/g (10.9 %), the standard
deviation of the mean as 0.0147 ng/g (5.5 %). The k-factor for the estimation of the
expanded uncertainty was chosen as k = 3.18 ( t (0.05;3) ), since the very limited number
of employable results (results of four laboratories, i.e. degrees of freedom=3) and their
distribution should be considered adequately.
The participants data (see table 5), the KCRV and its uncertainty (standard deviation of
the mean) are summarized in figure 1 (blue lines denoting the KCRV and KCRV ± u
values).
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CCQM K81 - CAP in Muscle: Results of Participants
0,43
__________
KCRV :
(arithmetic mean)
0.267 ng/g
0,39
0,35
_______
u = 0.0147 (5.5 %)
(standard deviation
of the mean)
ng/g
0,31
0,27
●:
0,23
Value considered for
KCRV calculation
0,19
◆:
0,15
Value excluded from
KCRV calculation
0,11
UME
HKSAR
BVL
NMIA
NIMT
CENAM
Figure 1: Results of participants in CCQM K 81 key comparison “chloramphenicol in
muscle” (results and expanded uncertainties as reported by the participants)
10
Degree of equivalence (DoE) calculation
The DoE for NMIi has a value component and an uncertainty component. The DoE and
its uncertainty between an NMI result and the KCRV has been calculated within CCQM
according to the following equations:
1) the value component is di = xi - xref
where di is the degree of equivalence between the NMI result xi and the KCRV xref. The
best possible di is zero, when the result is identical to xref .
2) the uncertainty component is Ui (di) = k * u(di)
where the expanded uncertainty Ui is calculated by combining the expanded uncertainties
kiui of xi and krefuref of xref as Ui =[(ki2ui2 + kref2 uref2)]1/2, using kref, uref as described under point 9
and ki, ui as reported by the participating laboratories.
Figures 2a and 2 b gives the absolute di and the relative di including with uncertainty
ranges (using the corresponding uncertainty Ui calculated as described above).
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DOE for CAP in Muscle CCQM K 81
0,200
0,150
Di [ng/g]
0,100
0,050
0,000
-0,050
-0,100
-0,150
CENAM
NIMT
NMIA
BVL
HKSAR
UME
-0,200
Labora tory
Figure 2a: Degree of equivalence (ng/g) of the originally reported results for CCQM–K81
for chloramphenicol using the arithmetic mean as KCRV (0.267 ng/g ; U95=0.047 ng/g)
DOE for CAP in Muscle CCQM K 81
60,0
40,0
Di [%]
20,0
0,0
-20,0
-40,0
CENAM
NIMT
NMIA
BVL
HKSAR
UME
-60,0
Laboratory
Figure 2b: Degree of equivalence (%) of the originally reported results for CCQM–K81
for chloramphenicol using the arithmetic mean as KCRV (0.267 ng/g ; U95=0.047 ng/g)
Page 15 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
11
28 August 2012
Scope of the key comparison and core competencies
This study was intended to demonstrate the capability of NMIs / DIs to analyse traces of
chloramphenicol in food at concentration levels resulting from legal requirements for
food control. Additionally the quality of this kind of analysis with respect to compliance
with legal requirements for food control methods and the international comparability of
measurements should be evaluated.
At a broader level this key comparison should demonstrate the capabilities of the
laboratories
 to measure selectively analytes at a sub –ng/g level
 to demonstrate the effective utilisation of IDMS for quantification
 to apply effectively extraction and clean-up procedures as part of a complex
sample preparation as it is required e.g. for biological matrices
 to provide reference values of samples as a service to customers
11.1 Comments on “How far does the light shine?”
The successful participation in this key comparison can demonstrate the ability to apply
IDMS in a mass fraction range of 0.1 to 50 ng/g (applying the respective analytical
technique, i.e. LC/MSMS or GC/MS).
An extension to other analytes should be done with care since there is a close linkage of
the analyte to other relevant parameters as e.g. sample preparation procedures and
detection techniques. Nevertheless an extension to other medium polar, stable and non
volatile drug residues of medium molecular weight (e.g. beta-agonists, benzimidazoles,
nitroimidazoles) can be justified, presuming that there is an isotopically labelled standard
available.
An extension to other matrices as unprocessed food of animal origin (primary animal
products#) in general and non-food matrices as blood, plasma and urine (important for
residue control) can also be justified, since the ability to apply effective sample
preparation steps for complex matrices and selective detection techniques was
demonstrated.
Anyhow in cases of extension of the CMC claims to other analytes or matrices additional
evidence of the capabilities of the laboratory (e.g. method validation data) has to be
provided.
12
Summary and Conclusions
CCQM-K81 demonstrated successfully the capability of the participating laboratories to
assign chloramphenicol values in tissue down to residue levels of around 0.3 ng/g*.
Although very different sample preparation techniques (different pre-treatments,
defattening steps, cartridges, solvents) as well as different analytical techniques (LC#
Primary animal products as defined in Council Directive 96/23/EC, Article 3 and Annex II (Council
Directive 96/23EC, Off. J. Eur. Comm. L0023, 1996).
*
The study was conducted with lyophilised muscle material. Nevertheless all reported values refer to the
analyte content in reconstituted muscle material.
Page 16 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
MSMS, GC-MS, different instruments, columns, eluents) were applied to detect
chloramphenicol at a very low concentration, a KCRV of 0.267 ng/g with a satisfying
expanded uncertainty of 17.4 % could be calculated. This result is in agreement with the
results of the preceding pilot study CCQM P90 chloramphenicol in milk whereupon an
additional contribution to the uncertainty due to the change of the matrix has to be
considered.
With respect to legal requirements for quantitative methods for residue control in food
(e.g. [6], [7]) the key comparison results prove the capabilities of the laboratories to fulfil
these settings with highest precision and to provide calibration and measurement services
to residue control laboratories.
References:
[1]
J. Polzer, K. Kindt, P.Gowik: Bound residues of Chloramphenicol in incurred muscle
samples; EuroResidue VII: Conference on Residues of Veterinary Drugs in Food,
Egmond aan Zee, The Netherlands (2012), accepted for publication.
[2]
Pascal Mottier,Véronique Parisod, Eric Gremaud, Philippe A. Guy and Richard H.
Stadler , Determination of the antibiotic chloramphenicol in meat and seafood products
by liquid chromatography–electrospray ionization tandem mass spectrometry
Journal of Chromatography A, Volume 994, Issues 1-2, 25 April 2003, Pages 75-84.
[3]
A. D. Cooper; J. A. Tarbin; W. H. H. Farrington; G. Shearer, Aspects of extraction,
spiking and distribution in the determination of incurred residues of chloramphenicol in
animal tissues, Food Additives and Contaminants, Volume 15, Issue 6, August 1998,
Pages 637-644.
[4]
OAWG 10/10 : CCQM Guidance note: Estimation of a consensus KCRV and associated
Degrees of Equivalence, Version 6 , 2010-03-01
[5]
CCQM KCRV WG: Data Evaluation Principles for CCQM Key Comparisons, 19 March
2008.
[6]
Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive
96/23/EC concerning the performance of analytical methods and the interpretation of
results (2002). Off. J. Eur. Comm. L221:8.
[7]
Draft CCRVDF Guidelines for the development of performance characteristics for
multiresidue analysis of veterinary drug residues, March 2011; draft of electronic
working group, 19th Session of CCVRDF, Burlington Vermont, USA, August 2010.
Page 17 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
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28 August 2012
Annexes
13.1 Annex I : Details of reported measurement uncertainties (participants reports)
13.1.1 UME
The uncertainty of the result of chloramphenicol was mainly affected by the following
sources:
- Purity of standard
- Standard preparation
- Sample weight
- Final volume of the sample extract
- Calibration curve
- Repeatability
- Recovery
Table:1. Parameters and their values taken into account in the calculation of uncertainty
of the results
Parameter
Value(X)
u(x)
u(x)/X
25
0.40
0.0160000
4998
0.0119
0.0000024
Standard solution (µg/kg)
Sample weight (mg)
Sample volume (dilution) (mL)
1.5
0.005
0.0033
Calibration curve (ng/kg)
239.49
6.762
0.0282
Repeatability
100.00
2.0700
0.0207
1.00
0.0079
0.0079
Recovery
Relative Combined Uncertainty
0.039
Result (ng/kg)
239.49
Standart Combined Uncertainty
9.45
Expanded Uncertainty ( k=2)
18.89
Relative Uncertainty (%)
7.89
Reported Value Expanded Uncertainty (k=2)
239.49
Page 18 of 44
±
18.89
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
13.1.2 NIMT
Expanded measurement equation used to estimate measurement uncertainty:
w 
Fw  x   w  m  0.25
Zc
y(x)
o
x
m
y(x)
 F .F .F
P
I
E
x
Where;
wx
= Mass fraction of CAP in sample (ng/g)
FwZc
= standard uncertainty of the mass fraction of the calibration standard factor
estimated from the purity and weighings
standard uncertainty of the mass ratio obtained from the calibration curve
estimated from standard error of the calibration curve
= standard uncertainty of the mass fraction of internal standard (D5-CAP)
= standard uncertainty of the mass of D5-CAP spiked into sample
= standard uncertainty of the mass of sample
= Parameter for converting from mass of lyophilized
pig muscle to mass of reconstituted sample
xo=
w y(x)
m y(x)
mx
0.25
FP
FI
FE
= standard uncertainty of the method precision factor
= standard uncertainty of the interference factor
= standard uncertainty of the extraction factor
Uncertainty budget:
Source of uncertainty
FwZc (1)
xo (1)
Wy(x) (ng/g)
My(x) (g)
mx (g)
FI (1)
FE (1)
FP (1)
Typical Value
1.0
0.89157
13.55055
0.12650
1.28653
1.0
1.0
1.0
Standard
uncertainty
0.0112
0.00420
0.00333
0.0000530
0.0000530
0.00803
0.0200
0.0228
Page 19 of 44
Degree of
freedom (Veff)
22
200
200
200
200
8
7
15
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
13.1.3 BVL
contributions to measurement uncertainty:
u
0,04
ng/g
u calibration solution:
target
12,66
ng/g
u(x)/X [%]
0,316
u sample weight:
0,000017
g
1,25
g
0,001
u sample spike:
0,000012
g
0,02
g
0,060
ng/g
0,2
ng/g
3,94
reproducibility method:
0,0079
“reproducibility method” : inhouse method reproducibility from validation experiment
(orthogonal experimental design), including uncertainty contributions from matrix, time,
run and calibration.
13.1.4 CENAM
We use the GUM general guide using the following general equation and we evaluated
the individual components of the sources of uncertainty summarized in the reference
shown below.
2
u CAPng / g

 2

  CAP
  CAP  u CC
 CC 
 ma
Description
2
 2   CAP
 u ma  
 M m

2
 2
 u M m  u r2

Standard uncertainty u(x)
value
Cis Internal
Isotopic
purity
and
balance B
standard
uncertainty. As the same isotopic
mass
analogue solution is added to both
fraction
added
the sample and the calibration
to solution its exact chemical purity,
each sample isotopic purity and concentration
(and
hence
their
associated
uncertainties) are unimportant.
Page 20 of 44
Type
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
F
28 August 2012
standard
As a relative standard deviation A
uncertainty
of
from the measurements.
repeatability
CC Calibration
curve
M
Mass
Low contribution
of Balance
sample
analysed
uncertainty.
A
All
the A
weights required are obtained by
weighing by difference, therefore
the errors associated with each
weighing will cancel and the
uncertainty contribution fall to
zero.
13.1.5 HKSAR
Quantifying the uncertainty components to obtain
the combined standard uncertainty, Ucx
Uc x 
u(S)
:
u (S ) 2  u (SB ) 2  u ( CB ) 2  u ( P ) 2  u ( R m ) 2
Stock standard solution
0.001274
u(SB) :
Sample blend
0.00001927
u(CB) :
Calibration blend
0.000004817
u(P)
Method precision
0.01897
Method bias
0.05077
:
u(Rm) :
Combined standard uncertainty, u(C) =
0.0542
Expanded uncertainty = Ucx x k where k = coverage factor of 2 = 0.108
Page 21 of 44
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28 August 2012
13.1.6 NMIA
The mass fraction of CAP in the sample was caclutated using the exact matching double
IDMS equation (1). Factors that contribute to the overall uncertainty are defined below.
Exact Matching Double IDMS Measurement equation:
w X  wZ .
where;
wX
wZ
m y  m zc
m x  m yc

R'b
 FME  FI  FBCS
R 'bc
(1
= mass fraction of analyte in sample
= mass fraction of analyte in the calibration standard solution used to
prepare calibration blend
my
myc
= mass of internal standard solution added to sample blend
= mass of internal standard solution added to calibration blend
mx
= mass of sample added to sample blend
mzc
= mass of calibration standard solution added to calibration blend
R b
Rbc
= observed isotope amount ratio in sample/internal standard blend
= observed isotope amount ratio in standard/internal standard calibration
blend
FI
= interference effects factor (nominal value of 1)
FBCS
FME
= batch blank correction and sampling effect factor (nominal value of 1)
= matrix effect factor (nominal value of 1)
A standard uncertainty with an estimation of degrees of freedom was calculated for each
component in the measurement equation (1), and these were combined using the derived
sensitivity coefficients to give a combined standard uncertainty. The determined total
effective degrees of freedom was used to calculate the appropriate k factor to expand the
combined standard uncertainty to a 95% confidence interval for reporting.
Calibration Solution Mass Fraction: w Z
The uncertainty in the mass fraction of CAP in the calibration blends was calculated by
combining estimated standard uncertainties for the purity of the reference standard of
CAP used to make the calibration stock solution with uncertainty components for the
dilution masses (including bias and precision data from calibration certificates). The
uncertainty in the purity of the reference material was estimated from in-house analysis
Page 22 of 44
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28 August 2012
by QNMR.
Blend Preparation Masses: mx, my, mzc, myc
Only bias effects on the weighed masses of standard solutions were considered as the
precision is captured in the method precision. These were derived from balance
calibration certificates.
Observed isotope amount ratio in blends: R´b and R´bc
It was assumed that the exact matching procedure minimises biases on the observed
ratios and therefore only precision was considered. The ratios are corrected for
instrumental drift by bracketing sample blends with calibration blends. The standard
deviation of the mean of the replicate analyses was used as an estimate for the method
precision. This factor includes the uncertainty in these two components and in the
sampling precision as well as all other precision components resulting from multiple
measurements carried out over multiple days.
Potential Method Biases: FME, FI, and FBCS,
The effect of the matrix on the calibration was examined by preparing calibration blends
in a blank freeze dried pork matrix and in aqueous solution that was either taken through
the entire extraction procedure or only the SPE step. Differences in results obtained using
the two matrices for preparation of calibration blends was determined to be insignificant
at the 95%confidence level by ANOVA. The maximum possible effect from this cause
was calculated (as described in ISO Guide 35 for ubb) and incorporated in the uncertainty
estimate.
Differences between results calculated from the three MRM transitions monitored by
LC/MS/MS were also determined to be statistically insignificant at the 95% confidence
level by ANOVA. The maximum possible effect from this cause was calculated as it was
for the matrix effect and incorporated in the uncertainty budget
Analyses were performed over four analytical batches.
ANOVA demonstrated a
significant effect of analytical batch on the results. Probable reasons for this finding
consistent with the trend observed in the data include the necessity of performing blank
corrections in the two earlier batches, and the likely uptake of water by the hygroscopic
sample material affecting later batches. This factor was the major contributor to the
overall measurement uncertainty. Due to the low degrees of freedom (3) for this major
uncertainty contributor, the k factor used to expand the combined standard uncertainty to
a 95% confidence interval for reporting was relatively large; 2.78.
Page 23 of 44
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28 August 2012
13.2 Annex II: Calculation of DoE
Basic data:
KCRV (arithmetic mean) of 0.267 ng/g with the standard deviation of the mean as u = 0.0147 ng/g (5.5 %), k = 3.18 ( t (0.05;3) ).
Participant
Mean as
reported
u as
reported
U as
reported
:
[ng/g]
[ng/g]
[ng/g]
k=
Value
component
Di (xi - xr)
Ui ²
component
for DoE *
Ui
component
for DoE
Ui
Di
[ng/g]
[ng/g]²
[ng/g]
[%]
[%]
Values used for KCRV calculation:
UME
0,239
0,00945
0,0189
2
-0,028
0,00253
0,05032
18,8
-10,6
BVL
0,245
0,0097
0,0194
2
-0,022
0,00255
0,05051
18,9
-8,3
NMIA
0,290
0,011
0,032
2,78
0,023
0,00311
0,05577
20,9
8,5
NIMT
0,295
0,01
0,02
2,04
0,028
0,00259
0,05090
19,0
10,4
Values excluded from KCRV calculation :
CENAM
0,313
0,047
0,09
1,92
0,046
0,01027
0,10136
37,9
17,1
HKSAR
0,243
0,0542
0,108
2
-0,024
0,01393
0,11801
44,2
-9,1
Corrected values after closing of the key comparison and disclosure of results:
CENAM
(corrected)
0,293
0,03
0,06
2
0,026
0,00577
0,07599
28,4
9,6
NIMT
(corrected)
0,285
0,01
0,0204
2,04
0,018
0,00259
0,05090
19,0
6,6
Page 24 of 44
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28 August 2012
13.3 Annex III (only for comparison): Calculations taking into account all reported
values (corrected values, which were submitted subsequently to the closing of the key
comparison and excluded values)
Participant Value (ng/g)
1
2
3
mean u
(ng/g) (ng/g)
U
(ng/g)
k
UME
0,237
0,239
0,242
0,239
0,00945
0,0189
2
HKSAR
0,242
0,244
0,243
0,243
0,0542
0,1084
2
BVL
0,250
0,239
0,246
0,245
0,0097
0,0194
2
NMIA
0,292
0,294
0,292
0,290
0,011
0,032
2,78
NIMT (corr.)
0,281
0,277
0,296
0,285
0,01
0,02
2,04
CENAM
(corr.)
0,297
0,293
0,289
0,293
0,030
0,060
2
Arithmetic mean:
Standard deviation:
Standard deviation
of the mean:
4
0,284
0.266 ng/g
0.0259 ng/g (9.76 %)
0.0196 ng/g (3.96 %)
Page 25 of 44
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28 August 2012
13.4 Annex IV: Core competencies
Annex IV provides the list of competencies (participants own appraisal)gained with this
key comparison based on a template of core competencies as proposed by the OAWG
(Sidney 2011).
13.4.1 UME
Purity of medium molecular weight, polar
CCQM
NMI
organic compounds
General description of competency coverage including mass fraction (similar to previous HFTLS
statements)
Tick or
Capability
no tick
Additional information
• Value assignment of Primary References: Mass fraction and associated uncertainty
Purity assessment of purchased compound done by
External SI traceable "pure substance"

TUBITAK UME using mass balance approach and
qNMR.
Identity verification

qNMR, HPLC, TGA, Karl Fischer and LC-MS
Molecular weight range

High (300-500 amu)
Polarity

Polar
Assignment method(s)

Mass balance

qNMR

TGA

Verification method(s) (if used)
Karl Fischer
Mass balance
qNMR

HPLC

LC-MS
Applicable to mass fraction range

950 mg/g - 1000 mg/g
Standard uncertainty estimation range

1.5 mg/g - 5 mg/g
Page 26 of 44
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28 August 2012
• Value-assignment (including verification) of single and multi-component formulated solutions: Mass
fraction and associated uncertainty
External SI traceable "calibration solution"

Identify components

Assess solubility

LC-MS/MS (IDMS)
Control volatility (measurand/solvent)
Assess solution stability
Selectivity of analytes of interest
Assess multicompenent effects
Serial dilution

Assignment method(s)

Gravimetry
Calibration against external standard

Verification method(s) – if used
Internal standard (IDMS)
Gravimetry
Other (specify)
Applicable to mass fraction range

25 ng/kg -750 ng/g
Standard uncertainty estimation range

2 %
• Extraction of analytes of interest from matrix

Liquid/liquid
Soxhlet
ASE

Incorporation of isotopic analogue?
Other
• Cleanup - separation of analytes of interest from other undesirable/interfering matrix/extract components
SPE
If neccesary
GPC
Chromatographic

LC-MS, GC-MS
Immunoaffinity
Other
• Transformation
Hydrolysis
Derivatization
Other
Page 27 of 44
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28 August 2012
• Analytical separation/specificity
LC

LC
GC

GC

LC-MS/MS (IDMS), GC-MS/MS (IDMS)
MS
MS/MS
HRMS
FAIMS
Other
• Value-assignment of analytes in matrix: Mass fraction and uncertainty

LC-MS/MS (IDMS), GC-MS/MS
Applicable to mass fraction range

0.05 ng/g - 500 ng/g
Standard uncertainty estimation range

3.9 % - 7.5 %
Assignment method(s)
Verification method(s) – if used
CCQM-K81
NMI
Chloramphenicol in Pig Muscle
General description of competency coverage including mass fraction (similar to previous HFTLS
statements)
Tick or
Capability
no tick
Additional information
• Value assignment of Primary References: Mass fraction and associated uncertainty
Sigma Alrich Product No:22792 Chloramphenicol
used. Purity assessment of
External SI traceable "pure substance"

purchased compound
done by TUBITAK UME using mass balance
approach and qNMR (In-house)
(98.0 ± 0.3) %
Identity verification

NMR, HPLC, TGA, Karl Fischer and LC-MS
Molecular weight range

High (300-500 amu)
Polarity

Polar
Assignment method(s)

Mass balance

qNMR

TGA

Karl Fischer
Verification method(s) (if used)
Mass balance
qNMR
Page 28 of 44
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28 August 2012

LC-MS

HPLC
Applicable to mass fraction range

950 mg/g - 990 mg/g
Standard uncertainty estimation range

1.5 mg/g - 3 mg/g
• Value-assignment (including verification) of single and multi-component formulated solutions: Mass
fraction and associated uncertainty
External SI traceable "calibration solution"

Chloramphenicol (98.0 ± 0.3) %
Identify components

LC-MS/MS (IDMS)
Assess solubility

Methanol in water
Control volatility (measurand/solvent)
Assess solution stability
Selectivity of analytes of interest
Assess multicompenent effects
Serial dilution

Assignment method(s)

Gravimetric preparation (50 ng/kg, 100 ng/kg, 250
ng/kg, 500 ng/kg, 750 ng/kg, 1000 ng/kg)
Gravimetry
Calibration against external standard

Verification method(s) – if used
Internal standard D5-chloramphenicol (IDMS)
Gravimetry
Other (specify)
Applicable to mass fraction range
50 ng/kg -1000 ng/kg
Standard uncertainty estimation range
1.6 %
• Extraction of analytes of interest from matrix
Liquid/liquid

Solvent: Ethylacetate

D5-chloramphenicol
Soxhlet
ASE
Incorporation of isotopic analogue?
Other
Page 29 of 44
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28 August 2012
13.4.2 HKSAR
CCQM-K81
GLHK Chloramphenicol in Pig Muscle
This study provides a means for assessing measurement capabilities for determination of mid-polarity
measurands by a procedure that requires extraction, clean-up, and analytical separation, and selective detection
in a food matrix. Generally, it provides demonstration of the capabilities of GLHK in determining the mass
fraction in range from 0.1 to 1 µg/kg of analytes with the molecular mass range 100-350 and having
intermediate polarity (-log Kow in range 0 to 2) in tissue matrices.
Tick or
Capability
no tick
Additional information
• Value assignment of Primary References: Mass fraction and associated uncertainty
External SI traceable "pure substance"

The neat standard used as calibrant was purity
assessed by GLHK after submission of data.
Identity verification

LC-MS/MS
Molecular weight range

Medium: <300-500 amu>
Polarity

pKOW value: ~1
Mass balance
Assignment method(s)
By
an
indirect
approach
through
consecutive
determination of possible impurities:
(i)

Moisture: Karl Fischer coulometry with oven
processor
(ii)
Organic related substance: LC-UV
(iii)
Inorganic
non-volatiles:
ICPMS
with
microwave digestion
(iv)
Volatile organic compounds: Headspace
GCMS
qNMR
Verification method(s) (if used)
Mass balance
qNMR
Other (specify)
Applicable to mass fraction range
Purity assessed to be 99.85 %w/w
Standard uncertainty estimation range
0.034 % w/w
Page 30 of 44
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28 August 2012
• Value-assignment (including verification) of single and multi-component formulated solutions: Mass
fraction and associated uncertainty
External
SI
traceable
"calibration
<Identity of supplier & CRM>
solution"
Identify components
<Methods used to confirm structure>
Assess solubility
Control volatility (measurand/solvent)
Assess solution stability
Selectivity of analytes of interest
Assess multicompenent effects
Serial dilution
Assignment method(s)
Gravimetry
Calibration against external standard
Other (specify)
Verification method(s) – if used
Gravimetry
Calibration against external standard
Other (specify)
Applicable to mass fraction range
## - ## mg/kg
Standard uncertainty estimation range
## - ## mg/kg
• Extraction of analytes of interest from matrix
Liquid/liquid

Soxhlet
ASE
Incorporation of isotopic analogue?

Other
• Cleanup - separation of analytes of interest from other undesirable/interfering matrix/extract components
SPE

GPC
Chromatographic
Immunoaffinity
Other
Page 31 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
• Transformation
Hydrolysis
Derivatization
Other
• Analytical separation/specificity
LC

GC
MS
MS/MS

HRMS
FAIMS
Other
• Value-assignment of analytes in matrix: Mass fraction and uncertainty
Assignment method(s)

IDMS
Applicable to mass fraction range

0.1 to 1 µg/kg
Standard uncertainty estimation range

0.0054 to 0.054 µg/kg
Verification method(s) – if used
Page 32 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
13.4.3 BVL
CCQM-K81
BVL
Chloramphenicol in Pig Muscle
Quantification of medium polare, stable and non volatile drug residues of medium molecular weight in
complex biological matrices (tissue) using chromatography/mass spectrometry coupled techniques
Tick or
Capability
no tick
Additional information
• Value assignment of Primary References: Mass fraction and associated uncertainty
External SI traceable "pure substance"
√
Sigma-Aldrich; In-house purity assessed
Identity verification
√
LC-TOF; LC-MSMS; GC-HRMS
Molecular weight range
√
Medium: <300-500 amu>
Polarity
√
pKOW ~1 (moderate polare)
Assignment method(s)
√
Mass balance
qNMR
Other: moisture; organic impurities: GC-NCI-MS;
√
LC-TOF; LC-MSMS; CHN-analysis (consistency
check)
Verification method(s) (if used)
Mass balance
qNMR
Other (specify)
Applicable to mass fraction range
√
990 - 1000 mg/g
Standard uncertainty estimation range
√
5 mg/g to 10 mg/g
• Value-assignment (including verification) of single and multi-component formulated solutions: Mass
fraction and associated uncertainty
External SI traceable "calibration solution"
<Identity of supplier & CRM>
Identify components
√
Assess solubility
√
Mass spectrometry (GC/MS, LC/MS); retention time
Control volatility (measurand/solvent)
Assess solution stability
√
Tests on stability in solution (isochronous study at
different temperatures)
during validation (experimental design based in-house
Selectivity of analytes of interest
validation approach)
Assess multicompenent effects
during validation (experimental design based in-house
Page 33 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
validation approach)
√
Serial dilution
Assignment method(s)
Gravitmetric preparation of dilutions of a stock
solution (1 mg/g)
Gravimetry
Calibration against external standard
√
Verification method(s) – if used
√
Other: Calibration against external standard using
isotopically labeled internal standard
Gravimetry
Calibration against external standard
√
Other: GC/NCI/MS, GC/NCI/HRMS
Applicable to mass fraction range
13 ng/g – 1.3 mg/g
Standard uncertainty estimation range
0.4 % (rel.)
• Extraction of analytes of interest from matrix
Liquid/liquid
√
Soxhlet
ASE
Incorporation of isotopic analogue?
√
Deuterated chloramphenicol (D5-CAP)
Other
• Cleanup - separation of analytes of interest from other undesirable/interfering matrix/extract components
SPE
√
Molecular imprinted polymer cartridges
√
Silylation
GPC
Chromatographic
Immunoaffinity
Other
• Transformation
Hydrolysis
Derivatization
Other
Page 34 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
• Analytical separation/specificity
LC
GC
MS
√
GC/MS and GC/HRMS with negative chemical
ionisation
√
MS/MS
HRMS
√
FAIMS
Other
• Value-assignment of analytes in matrix: Mass fraction and uncertainty
Assignment method(s)
Verification method(s) – if used
√
√
GC/MS and GC/HRMS with negative chemical
ionisation – IDMS
GC/MS and GC/HRMS with negative chemical
ionisation – IDMS
Applicable to mass fraction range
0.1 - 10 ng/g
Standard uncertainty estimation range
0.008 – 0.4 ng/g
Page 35 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
13.4.4 NMIA
CCQM-K81
NMI
Chloramphenicol in Pig Muscle
Quantification of sub ng/g mass fraction of mid-polarity, non-volatile, low MW drug in a heterogeneous
tissue matrix
Tick or
Capability
no tick
Additional information
• Value assignment of Primary References: Mass fraction and associated uncertainty
Material sourced not traceable. Traceability provided
External SI traceable "pure substance"
in-house though qNMR
Identity verification

NMR, LCMSMS, 2 sources of material
Molecular weight range

Low – Medium 100-500 amu
Polarity

Moderate polarity
Assignment method(s)
Mass balance

qNMR (25 analyses, 2 sources of material)
Other (specify)
Verification method(s) (if used)
Mass balance
qNMR
Other (specify)
Applicable to mass fraction range

990 – 1000 mg/g
Standard uncertainty estimation range

5 - 10 mg/g
• Value-assignment (including verification) of single and multi-component formulated solutions: Mass
fraction and associated uncertainty
Calibration
External SI traceable "calibration solution"
Identify components
solutions
prepared
in-house
using
materials with purity assigned as per above

Chromatographic retention,
MS/MS - 3 SRM transitions,
Assess solubility

Assessed previously
Control volatility (measurand/solvent)

Non-volatile
Assess solution stability

Assessed previously
Analytical separation/specificity achieved using LC

Selectivity of analytes of interest
separation, negative ion ESI MSMS, 3 SRM
transitions, solvent and matrix calibration blends
Page 36 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report

Assess multicomponent effects
28 August 2012
Use of a deuterated internal standard to overcome
matrix enhancement/suppression effects
Gravimetric preparation and dilution of standard

Serial dilution
solutions (stocks, working solutions, calibration
blends)

Assignment method(s)
Gravimetry
Calibration against external standard
Other (specify)

Verification method(s) – if used
Gravimetry - multiple calibration solutions prepared
and compared using two sources of pure material.
Calibration against external standard
Other (specify)
Applicable to mass fraction range

0.003 – 600 mg/kg
Standard uncertainty estimation range

0.00003 - 5 mg/kg
• Extraction of analytes of interest from matrix

Liquid/liquid
Soxhlet
ASE

Incorporation of isotopic analogue?
deuterated isotope, 3 SRM ratios
Equilibration of isotopic analogue
Other
• Cleanup - separation of analytes of interest from other undesirable/interfering matrix/extract components
Effectiveness of clean up confirmed as equivalent

SPE
results were obtained using each of 3 SRM ratios with
either solvent or matrix calibration blends
GPC
Chromatographic
Immunoaffinity
Other
• Transformation
Hydrolysis
Derivatization
Other
Page 37 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
• Analytical separation/specificity
LC

LC MSMS

LC MSMS
GC
MS
MS/MS
HRMS
FAIMS
Other
• Value-assignment of analytes in matrix: Mass fraction and uncertainty

Bracketed exact matching double IDMS
Applicable to mass fraction range

0.00025 – 0.003 mg/kg
Standard uncertainty estimation range

0.00001 – 0.00003 mg/kg
Assignment method(s)
Verification method(s) – if used
Page 38 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
13.4.5 NIMT
CCQM-K81
NIMT Chloramphenicol in Pig Muscle
Quantification of sub ng/g mass fraction of mid-polarity, non-volatile, mid MW drug in a heterogeneous
tissue matrix
General description of competency coverage including mass fraction (similar to previous HFTLS
statements)
Tick
Capability
no tick
or
Additional information
• Value assignment of Primary References: Mass fraction and associated uncertainty
External SI traceable "pure substance"
<Identity of supplier & CRM>

Identity verification

Molecular weight range

Polarity

Assignment method(s)
HPLC-UV, LC-MS/MS
Medium: <300-500 amu>
Molar Mass =323.13 g mol–1
List pKOW value
Moderate polarity
Mass balance
qNMR

Verification method(s) (if used)
Other (DSC, KFT, HPLC-UV)
Mass balance
qNMR
Other (specify)
Applicable to mass fraction range

0.98 - 1 g/g
Standard uncertainty estimation range

0.002 g/g to 0.005 g/g
• Value-assignment (including verification) of single and multi-component formulated solutions: Mass
fraction and associated uncertainty
External
SI
traceable
"calibration
<Identity of supplier & CRM>
solution"
Identify components
<Methods used to confirm structure>
Assess solubility

Control volatility (measurand/solvent)

Assess solution stability
Page 39 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
Selectivity of analytes of interest
Assess multicompenent effects

Serial dilution

Assignment method(s)
gravimetric preparation and dilution of standard
solutions
Gravimetry
Calibration against external standard
Other (specify)
Verification method(s) – if used
Gravimetry
Calibration against external standard
Other (specify)
Applicable to mass fraction range
## - ## mg/kg
Standard uncertainty estimation range
## - ## mg/kg
• Extraction of analytes of interest from matrix
Liquid/liquid

Extraction efficiency/internal standard equlibration

Deuterated isotope, 2 SRM ratios
Soxhlet
ASE
Incorporation of isotopic analogue?
Other
• Cleanup - separation of analytes of interest from other undesirable/interfering matrix/extract components
Effective clean-up provides statistically insignificant

SPE
different results obtained using either with solvent or
matrix-matched calibration solutions
GPC
Chromatographic
Immunoaffinity
Other
• Transformation
Hydrolysis
Derivatization
Other
Page 40 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
• Analytical separation/specificity
LC

LC-MS/MS

LC-MS/MS
GC
MS
MS/MS
HRMS
FAIMS
Other
• Value-assignment of analytes in matrix: Mass fraction and uncertainty
Assignment method(s)

IDMS (using multipoint calibration curve)
Verification method(s) – if used

Exact-matching double IDMS
Applicable to mass fraction range

0.05 - 3 g/kg
Standard uncertainty estimation range

0.0025 g/kg( low mass fraction) – 0.105 g/kg (high
mass fraction)
Page 41 of 44
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
13.4.6 CENAM
CCQM-K81
CENAM Chloramphenicol in Pig Muscle
Quantification of mass fraction of medium polarity, non-volatile, low molecular weight drug in a tissue
matrix
Tick or no
Capability
tick
Additional information
• Value assignment of Primary References: Mass fraction and associated uncertainty
External SI traceable "pure substance"
√
√
Identity verification
Molecular weight range
√
USP grade from Sigma-Aldrich
LC-MS/MS chromatographic retention time,
mass profile
Medium 300 u – 500 u
Polarity
Assignment method(s)
Mass balance
qNMR
√
Verification method(s) (if used)
Other (specify) DSC
Mass balance
Other (specify)
Applicable to mass fraction range
√
0.998 – 1.000 g/g
Standard uncertainty estimation range
√
0.0005 g/g to 0.0007 g/g
• Value-assignment (including verification) of single and multi-component formulated solutions: Mass
fraction and associated uncertainty
External
SI
traceable
"calibration
<Identity of supplier & CRM>
solution"
Identify components
Assess solubility
√
√
LC-MS/MS chromatographic retention time, mass
profile
Solubility test using the solvent to be used in
extraction and measurement process
Control volatility (measurand/solvent)
Assess solution stability
√
Selectivity of analytes of interest
Page 42 of 44
Comparison analyte concentration in stored and
freshly made standard solutions
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
Assess multicompenent effects
√
Serial dilution
Assignment method(s)
√
Gravimetric dilution from a concentrated solution
of chloramphenicol
Gravimetry
Calibration against external standard
Other (specify)
Verification method(s) – if used
Gravimetry
Calibration against external standard
Other (specify)
Applicable to mass fraction range
0.8 µg/kg – 20 µg/kg
Standard uncertainty estimation range
0.3 µg/kg – 0.05 µg/kg
• Extraction of analytes of interest from matrix
Liquid/liquid
√
Extraction with ethyl acetate and hexane
√
Addition of d5-CAP
Soxhlet
ASE
Incorporation of isotopic analogue?
Other
• Cleanup - separation of analytes of interest from other undesirable/interfering matrix/extract components
SPE
√
GPC
Chromatographic
Immunoaffinity
Other
• Transformation
Hydrolysis
Derivatization
Other
Page 43 of 44
C18 cartridge was used to cleanup the sample,
CAP was eluted with acetonitrile
CCQM-K81 “Chloramphenicol in Pig Muscle” Final Report
28 August 2012
• Analytical separation/specificity
Separation of CAP using a RP-18 column, mobile
√
LC
phase ammonium acetate 5mM / acetonitrile,
gradient, chromatographic retention time
GC
MS
Quantification
of
CAP
was
carried
out
considering the presence of two ions originating
√
from CAP (m/z 152, used as quantifier, m/z 257),
the presence of ion originating from labelled CAP
MS/MS
(m/z 157)
HRMS
FAIMS
Other
• Value-assignment of analytes in matrix: Mass fraction and uncertainty
Assignment method(s)
√
IDMS
Applicable to mass fraction range
√
0.1 µg/kg – 1 µg/kg
Standard uncertainty estimation range
√
0.047 µg/kg – 0.15 µg/kg
Verification method(s) – if used
Page 44 of 44