SIST EN 17049:2018

SLOVENSKI STANDARD
01-april-2018
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Animal feeding stuffs: Methods of sampling and analysis - Identification of tylosin,
spiramycin, virginiamycin, carbadox and olaquindox at sub-additive levels in compound
feed - Confirmatory analysis by LC-MS
Futtermittel: Probenahme- und Untersuchungsverfahren - Identifizierung von Tylosin,
Spiramycin, Virginiamycin, Carbadox und Olaquindox in Konzentrationen unterhalb von
Zusatzstoffen in Mischfuttermitteln - Bestätigungsanalyse mittels LC-MS
Aliments des animaux: Méthodes d'échantillonnage et d'analyse - Identification de la
tylosine, spiramycine, virginiamycine, du carbadox et de l’olaquindix dans les aliments
composés pour animaux à des concentrations inférieures à celles des additifs - Analyse
de confirmation par CL-SM
Ta slovenski standard je istoveten z: EN 17049:2018
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17049
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2018
EUROPÄISCHE NORM
ICS 65.120
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Identification of tylosin, spiramycin, virginiamycin,
carbadox and olaquindox at sub-additive levels in
compound feed - Confirmatory analysis by LC-MS
Aliments des animaux: Méthodes d'échantillonnage et Futtermittel: Probenahme- und
d'analyse - Identification de la tylosine, spiramycine, Untersuchungsverfahren - Identifizierung von Tylosin,
virginiamycine, du carbadox et de l'olaquindix dans les Spiramycin, Virginiamycin, Carbadox und Olaquindox
aliments composés pour animaux à des concentrations in Konzentrationen unterhalb von Zusatzstoffen in
inférieures à celles des additifs - Analyse de Mischfuttermitteln - Bestätigungsanalyse mittels LC-
confirmation par CL-SM MS
This European Standard was approved by CEN on 8 January 2018.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17049:2018 E
worldwide for CEN national Members.

Contents Page
European foreword . 5
1 Scope . 6
2 Normative references . 6
3 Principle . 6
4 Reagents and materials . 6
4.1 General . 6
4.2 Reagents and materials . 7
4.2.1 Acetonitrile (LC-MS grade) . 7
4.2.2 Methanol (LC-MS grade) . 7
4.2.3 Formic acid (LC-MS grade) . 7
4.2.4 Tylosin . 7
4.2.5 Spiramycin . 7
4.2.6 Virginiamycin . 7
4.2.7 Carbadox . 7
4.2.8 Olaquindox . 7
4.3 Solutions . 7
4.3.1 HPLC Mobile phase A: Formic acid 5mM . 7
4.3.2 HPLC Mobile phase B: Formic acid 50 mM/ acetonitrile (10/90, v/v) . 7
4.4 Standard solutions . 7
4.4.1 Stock solution tylosin (500 μg/ml) . 7
4.4.2 Stock solution spiramycin (500 μg/ml) . 7
4.4.3 Stock solution virginiamycin (500 μg/ml) . 7
4.4.4 Stock solution carbadox (500 μg/ml) . 8
4.4.5 Stock solution olaquindox (500 μg/ml) . 8
4.4.6 Mixed stock solution 1 . 8
4.4.7 Mixed stock solution 2 . 8
4.4.8 Calibration standard . 8
5 Apparatus . 8
6 Sampling . 9
7 Sample preparation . 9
7.1 Sample pre-treatment . 9
7.2 Quality control samples . 9
7.3 Sample extraction . 10
7.4 Sample purification . 10
7.5 Sample preparation for LC-MS/MS . 10
7.6 Confirmation control . 10
8 LC-MS/MS analysis . 10
8.1 General . 10
8.2 LC-MS/MS experimental conditions . 10
8.3 Initial test . 11
8.4 Analysis of samples . 11
9 Data processing and interpretation of results . 11
9.1 Data processing . 11
9.2 Recording and calculation of identification parameters . 11
10 Criteria for acceptance of the analytical results . 12
10.1 General . 12
10.2 Run acceptance . 12
10.3 Identification of the analyte . 12
10.3.1 General . 12
10.3.2 Retention time criterion . 12
10.3.3 Ion ratio criterion . 12
11 Test report . 13
Annex A (informative) Results of the interlaboratory study . 14
A.1 Procedure . 14
A.2 Materials. 14
A.3 Statistical analysis of results . 15
A.4 Results and interpretation - Precision . 16
Annex B (informative) Run and sample acceptance form . 24
Annex C (informative) Quantitative analysis . 25
C.1 General . 25
C.2 Procedure quantitative analysis. 25
C.2.1 Sample pre-treatment quantitative analysis . 25
C.2.2 Quality control samples . 25
C.2.3 Sample extraction . 26
C.2.4 Sample purification . 26
C.2.5 Sample preparation for LC-MS/MS . 26
C.2.6 Recovery control . 26
C.2.7 Confirmation control . 26
C.2.8 LC-MS/MS analysis . 27
C.2.8.1 LC-MS/MS experimental conditions . 27
C.2.8.2 Initial test . 28
C.2.8.3 Analysis of samples . 28
C.3 Data processing and interpretation of results . 29
C.3.1 Data processing . 29
C.3.2 Recording and calculation of identification parameters . 29
C.3.3 Calculation of the amount of analyte in the sample . 29
C.3.4 Calculation of recovery percentage . 29
C.4 Criteria for the acceptance of the analytical result . 29
C.4.1 General . 29
C.4.2 Run acceptance . 30
C.4.3 Identification of the analyte . 30
C.4.3.1 General . 30
C.4.3.2 Retention time criterion . 30
C.4.3.3 Ion ratio criterion . 30
C.5 Notes on the procedure . 31
C.5.1 Influence of ionization suppression . 31
C.5.2 Comments on the quantitive accuracy . 31
C.5.3 Comments on the relevance of recovery percentage . 31
Annex D (informative) Run and sample acceptance form . 32
Bibliography . 33

European foreword
This document (EN 17049:2018) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by August 2018, and conflicting national standards shall
be withdrawn at the latest by August 2018.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
WARNING — The method described in this standard implies the use of reagents that pose a hazard to
health. The standard does not claim to address all associated safety problems. It is the responsibility of
the user of this standard to take appropriate measures for the health and safety protection of the
personnel prior to use of the standard and to ensure that regulatory and legal requirements are
complied with.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
1 Scope
This European Standard specifies a high performance liquid chromatography – mass spectrometry (LC-
MS/MS) method for the identification of tylosin, spiramycin, virginiamycin, carbadox and olaquindox in
animal feeds.
The method is suitable for the identification of low concentrations of tylosin, spiramycin, virginiamycin,
carbadox and olaquindox in compound animal feeds. A limit of identification of 1 mg/kg for tylosin,
spiramycin and virginiamycin, 4 mg/kg for carbadox and 3 mg/kg for olaquindox should be obtained by
using the described method. The method was fully validated during a collaborative study (see Annex A).
Since tylosin, spiramycin and virginiamycin are fermentation products consisting of a mixture of several
closely related compounds, the analysis is based on detection and identification of the most abundant
constituents. For tylosin the marker is tylosin A, for spiramycin the marker is spiramycin I and II and for
virginiamycin the marker is virginiamycin M1 and S1. The other isomers and forms can be readily
detected with the same method but adjustment of the MS parameters according to the molecular mass
of precursor and product ions need to be made. Carbadox and olaquindox are analysed as such.
2 Normative references
There are no normative references in this document.
3 Principle
The compounds are extracted from the feed with a mixture of water and methanol. An aliquot of the
liquid phase is diluted and applied to a pre-conditioned SPE column. After washing of the SPE column,
compounds of interest are eluted with methanol. The obtained extract is evaporated and re-dissolved in
dilute formic acid. The resulting extract is analysed by LC-MS/MS. Separation is carried out on a silica-
based C18 bonded phase column and detection is performed by mass spectrometry in multiple reaction
monitoring mode.
The validation of this method was performed at concentration levels that were calculated on a weight
(w/w) basis. Expression of working ranges in terms of w/w concentration is common practice in
residue analysis of veterinary drugs, in fact Maximum Residue Limits (MRL) are exclusively expressed
on a w/w basis. For feed additives however, tolerances have been expressed traditionally as
microbiological activity. To translate the validation experiments concerning the level at which they
were performed, to units expressed as microbiological activity, the w/w concentrations should be
corrected for the microbiological potency of the preparation used for spiking experiments.
4 Reagents and materials
WARNING — Use all solvents and solutions in a fume hood. Wear safety glasses, protective clothing and
avoid skin contact.
4.1 General
All reagents are of 'Analytical reagent' grade or better unless otherwise stated. Throughout this method,
−1
“water” means demineralized water with a conductivity of at least 10 MΩ.cm . Guaranteed purity is
required for each lot of reference standard.
4.2 Reagents and materials
4.2.1 Acetonitrile (LC-MS grade)
4.2.2 Methanol (LC-MS grade)
4.2.3 Formic acid (LC-MS grade)
4.2.4 Tylosin
4.2.5 Spiramycin
4.2.6 Virginiamycin
4.2.7 Carbadox
4.2.8 Olaquindox
4.3 Solutions
4.3.1 HPLC Mobile phase A: Formic acid 5mM
Measure 200 μl formic acid (4.2.3) and transfer to a volumetric flask of 1 000 ml, make up to the mark
with water. Filter and degas before use.
4.3.2 HPLC Mobile phase B: Formic acid 50 mM/ acetonitrile (10/90, v/v)
Measure 200 μl formic acid (4.2.3) and transfer to a volumetric flask of 1 000 ml, add 100 ml water and
make up to the mark with acetonitrile (4.2.1). Filter and degas before use.
4.4 Standard solutions
4.4.1 Stock solution tylosin (500 μg/ml)
Weigh between 10 and 50 mg of tylosin standard substance (4.2.4) and transfer to a brown glass bottle.
Calculate the required amount of methanol (4.2.2) and add that amount (on a weight basis) to obtain a
standard solution of 500 μg/ml. Store this stock solution in the dark at 4-8 °C. Under these conditions it
is stable for at least one month.
4.4.2 Stock solution spiramycin (500 μg/ml)
Weigh between 10 and 50 mg of spiramycin standard substance (4.2.5) and transfer to a brown glass
bottle. Calculate the required amount of methanol (4.2.2) and add that amount (on a weight basis) to
obtain a standard solution of 500 μg/ml. Store this stock solution in the dark at 4-8 °C. Under these
conditions it is stable for at least one month.
4.4.3 Stock solution virginiamycin (500 μg/ml)
Weigh between 10 and 50 mg of virginiamycin standard substance (4.2.6) and transfer to a brown glass
bottle. Calculate the required amount of methanol (4.2.2) and add that amount (on a weight basis) to
obtain a standard solution of 500 μg/ml. Store this stock solution in the dark at 4-8 °C. Under these
conditions it is stable for at least one month.
4.4.4 Stock solution carbadox (500 μg/ml)
Weigh between 10 and 50 mg of carbadox standard substance (4.2.7) and transfer to a brown glass
bottle. Calculate the required amount of methanol (4.2.2) and add that amount (on a weight basis) to
obtain a standard solution of 500 μg/ml. Store this stock solution in the dark at 4-8 °C. Under these
conditions it is stable for at least one month.
4.4.5 Stock solution olaquindox (500 μg/ml)
Weigh between 10 and 50 mg of olaquindox standard substance (4.2.8) and transfer to a brown glass
bottle. Calculate the required amount of methanol (4.2.2) and add that amount (on a weight basis) to
obtain a standard solution of 500 μg/ml. Store this stock solution in the dark at 4-8 °C. Under these
conditions it is stable for at least one month.
4.4.6 Mixed stock solution 1
Measure 1,0 ml of stock solutions 4.4.1, 4.4.2 and 4.4.3 and transfer into a 25 ml volumetric flask.
Accurately measure 4,0 ml of stock solution 4.4.4 and transfer to the same volumetric flask. Accurately
measure 3,0 ml of stock solution 4.4.5 and transfer to the same volumetric flask. Make up to the mark
with water and mix. The concentration of tylosin, spiramycin, virginiamycin, carbadox and olaquindox
in this stock solution is 20, 20, 20, 80 and 60 mg/l respectively. Store the stock solution in the dark at 4-
8 °C. Under these conditions it is stable for at least one week.
4.4.7 Mixed stock solution 2
Mix equal volumes of mixed stock solution 1 (4.4.6) and water. Store the stock solution in the dark at 4-
8 °C. The concentration of tylosin, spiramycin, virginiamycin, carbadox and olaquindox in this stock
solution is 10, 10, 10, 40 and 30mg/l respectively. Prepare this stock solution fresh daily.
4.4.8 Calibration standard
Measure 50 μl of mixed stock solution 1 (4.4.6) and transfer to a volumetric flask of 10 ml. Make up to
the mark with water. The concentration of tylosin, spiramycin, virginiamycin, carbadox and olaquindox
in this calibration standard is 100, 100, 100, 400 and 300 μg/l respectively. Prepare this calibration
standard fresh daily.
5 Apparatus
Usual laboratory equipment and, in particular, the following:
5.1 Analytical balance suitable to accurately weigh between 0 and 10 g with an accuracy of 0,1 mg
5.2 Balance suitable to accurately weigh between 0 and 1 500 g with an accuracy of 0,1 g
5.3 Centrifuge
5.4 Ultrasonic bath
5.5 Evaporation unit
5.6 Centrifuge tubes of different volumes, adapted to the centrifuge
5.7 SPE Vacuum manifold
® 1
5.8 Oasis HLB cartridges polymer phase, 60 mg, 3 ml (Waters WAT094226 or equivalent)
5.9 Sample vials suitable for the auto-sampler system that is used (5.11.1)
5.10 Head-over-head shaker
5.11 LC-MS/MS equipment
5.11.1 LC-MS/MS equipment comprised of gradient HPLC system ®
5.11.2 LC-MS/MS equipment comprised of analytical column Symmetry 300 C18 150 × 3 mm,
5 μm particle size (Waters WAT106154 or comparable)
NOTE During the method validation, this recommended LC column has proved to be fit for purpose.
5.11.3 LC-MS/MS equipment comprised of mass spectrometer suitable for tandem MS
measurement (triple quadrupole or ion trap) and equipped with an electrospray interface.
6 Sampling
The laboratory should receive a sample that is truly representative and has not been damaged or
changed during transport or storage.
NOTE 1 Sampling is not part of the method specified in this European Standard. Sampling is described in
Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis
for the official control of feed. [7]
NOTE 2 For quantification of the content multi-level standard addition is applied to account for the
considerable variability of feed composition. This procedure is described in Annex C.
7 Sample preparation
7.1 Sample pre-treatment
Weigh (5,0±0,1) g each test sample and transfer in a 50 ml centrifuge tube (5.6) and proceed with the
procedure at 7.3.
7.2 Quality control samples
A known negative sample, preferably of approximately the same composition, is included in each series
(code S0). Weigh (5,0±0,1) g of the known negative sample as indicated in 7.1 and proceed with the
procedure at 7.3.
Also in each series a confirmation control sample is prepared by spiking an aliquot of the extract of the
negative control sample S0 obtained after sample extraction (see 7.6).

1 ®
Oasis HLB cartridges polymer phase, 60 mg, 3 ml is an example of a suitable product available commercially.
This information is given for the convenience of users of this European standard and does not constitute an
endorsement by CEN of this product.
2 ®
Symmetry 300 C18 150 × 3 mm, 5 μm particle size is an example of a suitable product available
commercially. This information is given for the convenience of users of this European standard and does not
constitute an endorsement by CEN of this product.
7.3 Sample extraction
To each sample add 10 ml water, close the tubes and shake vigorously by hand. Add 10 ml of methanol
(4.2.2), close the tubes and mix manually. Place the tubes in a head-over-head shaker (5.10) during 20
min. Centrifuge (5.3) the tubes at 3500 rpm for 10 min.
Accurately measure 0,5 ml of the supernatant and transfer to a clean centrifuge tube (5.6). Add 3,5 ml
water and mix.
NOTE For the preparation of the confirmation control sample proceed as described in 7.6.
7.4 Sample purification ®
Condition an Oasis HLB cartridge (5.8) with subsequently 3 ml methanol (4.2.2) and 5 ml water.
Apply the extract obtained in 7.3 (7.6 in case of the recovery and confirmation control samples) to the
cartridge and apply mild vacuum to transfer at a flow rate of approximately 1 ml/min. Wash the
cartridge with 3 ml of water. Place a clean centrifuge tube (5.6) under the cartridge and subsequently
elute the analytes of interest with 3 ml of methanol (4.2.2).
7.5 Sample preparation for LC-MS/MS
Evaporate the extract obtained in 7.4 TO dryness at 40 °C under a mild stream of nitrogen in an
evaporation unit (5.5). Redissolve the residue in 1,0 ml of 5 mM formic acid (4.3.1).
Transfer the extract to a sample vial (5.9). Proceed as described in 8.
7.6 Confirmation control
Take an aliquot of 0,5 ml of the extract of sample S0 obtained in 7.3 and transfer to a clean centrifuge
tube (5.6). Add 3,5 ml water and 12,5 μl of the mixed stock solution 2 (4.4.7) and mix. The concentration
in the extract is comparable to the addition of 1 mg/kg of virginiamycin, spiramycin and tylosin,
4 mg/kg carbadox and 3 mg/kg olaquindox before sample preparation. Proceed as described in 7.4.
8 LC-MS/MS analysis
8.1 General
Quantification of the content is performed by multi-level standard addition to account for the
considerable variability of feed composition. This procedure is described hereunder.
The procedure is fully validated in-house and is described in Annex C 'Quantitative analysis' for
information. Other conditions may be used provided that they give equivalent results.
8.2 LC-MS/MS experimental conditions
— Mount the analytical column in the HPLC system, flush the solvent lines and adjust the gradient
settings. See Table C.2 for guidance;
— Mount the electrospray interface and connect the analytical column exit to the flow splitter and
subsequently the flow splitter transfer line to the MS interface;
— Make sure the mass spectrometer is properly calibrated according to the manufacturer's
instructions;
— Optimize the mass spectrometer for the compounds of interest according to the manufacturer's
instructions.
The feed additives are measured in MRM (Multiple Reaction Monitoring) mode, monitoring two product
ions originating from one precursor ion. For information, details concerning the MRM acquisition
parameters, suitable for a Micromass Quattro Ultima mass spectrometer, are given in Table C.3 for
guidance.
8.3 Initial test
Inject the calibration standard (4.4.8) the negative control S0 and the confirmation control (7.6). Verify
that the sensitivity is sufficient (S/N > 10 for the least abundant MRM ion (9.1)) and that carry-over in
the LC-MS system is absent.
8.4 Analysis of samples
— Calibration standard (4.4.8)
— blank solvent
— S0 (C.2.2)
— Confirmation control sample (C.2.7)
— Test portions S1, S2, S3, S4 and S5 of test sample A
— Recovery control sample of sample A (C.2.6)
— Calibration standard (4.4.8)
— Blank solvent
— Test portions S1, S2, S3, S4 and S5 of test sample B
— Recovery control sample of sample B (C.2.6)
— Calibration standard (4.4.8)
— Confirmation control sample (C.2.7)
— Calibration standard (4.4.8)
9 Data processing and interpretation of results
9.1 Data processing
Record each of the MRM traces separately and integrate the peaks of analytes of interest. Record the
peak area of each individual MRM trace.
9.2 Recording and calculation of identification parameters
Record the retention time of each MRM peak of each of the analytes.
The ion ratio of each of the analytes in each of the samples, as well as for the control samples should be
calculated according to Formula (1).

peak area least abundant MRM ion
R × 100 % (1)

peak area most abundant MRM ion

=
Calculate for all samples the deviation D of the ion ratio from the average ion ratio recorded for the
confirmation control (R ) according to Formula (2).
cc

RR−
sample cc

D × 100 % (2)

R
cc

10 Criteria for acceptance of the analytical results
10.1 General
For registration use Annex B.
10.2 Run acceptance
To accept the experiments, the following criteria have to be met:
The signal that may be present in the negative control sample S0 should not exceed the equivalent of
0,1 mg/kg for the antibiotics, 0,4 mg/kg for carbadox and 0,3 mg/kg for olaquindox.
10.3 Identification of the analyte
10.3.1 General
For identification of the analyte two parameters are evaluated:
a) The retention time;
b) The ion ratio.
NOTE Regulation (EC) No 657/2002 [6] is applicable.
10.3.2 Retention time criterion
The maximum deviation of the retention time of the analyte should be identical with the retention time
recorded in the confirmation control sample within a margin of ± 2,5 %. The peaks for each of the two
MRM product ions should essentially coelute within the time resolution of the mass spectrometric
acquisition method.
10.3.3 Ion ratio criterion
The ion ratio deviation should not exceed pre-set limits (Table 1). Record the ion ratios (Formula (1)) of
all samples, as well as the deviation from the ion ratio in the confirmation control (Formula (2)).
Compare the deviation with the critical values in Table 1.
Table 1 — Critical values for the identification of the analytes by LC-MS/MS
Ion ratio (R) Maximum deviation (D)
R > 50 % ±20 %
20 % < R ≤ 50 % ±25 %
10 % < R ≤ 20 % ±30 %
R ≤ 10 % ±50 %
=
11 Test report
Results are reported as either positive (confirmed) or negative.
The test report shall specify:
a) information necessary for complete identification of the sample;
b) the sampling procedure if known;
c) the test method used, with reference to this European Standard;
d) the calculation result(s) obtained;
e) operating details not specified in this European Standard, or regarded as optional, together with
details of any incidentals which may have influenced the calculation result(s).
Annex A
(informative)
Results of the interlaboratory study
A.1 Procedure
An inter-laboratory comparison was carried out to assess the method performance characteristics of
the analytical method based on high performance liquid chromatography – mass spectrometry (LC-
MS/MS) for the identification of tylosin, spiramycin, virginiamycin, carbadox and olaquindox in feeding
stuffs.
The inter-laboratory (for validation) study was jointly conducted by the European’s Commission Joint
Research Centre (JRC) Institute for Reference Materials and Measurements (IRMM) and the RIKILT
Institute for Food Safety Wageningen (The Netherlands).
RIKILT was the overall coordination of the SIMBAG-Feed project Specific tasks of the JRC included the
coordination of the validation study, the material preparation, the statistical evaluation of the results.
RIKILT was the expert laboratory responsible for the LC-MS/MS method evaluation.
The study was conducted with a step-by-step approach, where the laboratories were first involved in a
training period consisting of specific training on the method through audio-visual material and known
test samples. In a second step nineteen laboratories were involved in the validation period where they
were asked to participate in the final inter-comparison trial. Ten laboratories delivered final results for
the validation period.
A.2 Materials
The SIMBAG-Feed validation studies targeted in parallel different methods for the detection of five
antibiotics (spiramycin, tylosin, virginiamycin, zinc bacitracin, avoparcin) and two growth promoters
(olaquindox, carbadox) in feed. For this LC-MS/MS method validation 10 materials (MAT) were sent in
blind duplicates to each laboratory participating in the validation phase of the study for the qualitative
determination of virginiamycin, tylosin phosphate, spiramycin, carbadox and olaquindox. The test
materials were compound feeds for cattle, pig, poultry and calf (milk replacer) containing typical
ingredients using a realistic recipe. The banned antibiotics and growth promoters were then added at
specific concentrations and in different combinations. The composition of the test material and the
selected compounds are shown in Table A.1.
The pure ingredients were first tested for the identity, purity and content of the active compounds
together with water determination and particle size analysis. After mixing with the various compound
feeds, aliquoting and bottling, the homogeneity and stability were evaluated. The homogeneity principle
was that if a test material was proven homogeneous for one banned compound, it would also be for the
other banned ones. This allows the analysis of only one of the compound present in each material in
order to give homogeneity results. Full reports describing the preparation of the test materials and the
outcome of the homogeneity study are available at RIKILT and IRMM as deliverable of the SIMBAG-Feed
project [3,4].
European project SIMBAG-Feed, Screening and Identification Methods for official control of Banned
Antibiotics and Growth promoters in Feeding stuffs, Competitive and sustainable growth programme (GROWTH),
project GRD1-200-00413.
Table A.1 — Design of the sample composition and the selected compounds
MAT1 MAT2 MAT3 MAT4 MAT5 MAT6 MAT7 MAT8 MAT9 MAT10
Feed matrix Pig Pig Poultry Poultry Calf Cattle Cattle Calf Pig Poultry
Spiramycin 0,7 3,6    1,4 2,9 2,2 5,0 7,2
Tylosin   1,1 4,4 3,3 2,2 5,5  11,0 7,7
Phosphate
Virginiamycin 4,0 2,0  1,0   5,0 3,0 10,0 7,0
Olaquindox 10,0 5,0  12,0 3,0  7,0  15,0
Carbadox  8,0 4,0  6,0 16,0  12,0 10,0 20,0
As shown in Table A.2, all materials taken in consideration in this study resulted to be homogeneous on
the basis of the ANOVA analysis. In fact the F-value in 8 out 10 cases is lower compared to the “F critic”.
In our case “F critic” is 3,02 (10 groups and 2 counts per group). Calculating the sampling error of MAT4
and MAT9 showed that these materials are also fit for the intended purpose since the sampling error is
below 50 % of the reference standard deviation of the study defined by the Horwitz Formula (9).
Table A.2 — Summary of the homogeneity study
Material Feed Compound Analysing F Theoretical Measured
analysed a concentration concentration
laboratory
(mg/kg) b
(mg/kg)
1 Pig Zinc IRMM 1,15 3,2 3,7 ± 0,1
Bacitracin
2 Pig Spiramycin IRMM 1,86 3,6 3,8 ± 0,2
3 Poultry Carbadox ISS 2,60 4,0 4,6 ± 0,1
4 Poultry Olaquindox ISS 8,90 12,0 12,0 ± 0,2
5 Calf Olaquindox ISS 1,10 3,0 3,0 ± 0,1
6 Cattle Spiramycin IRMM 0,33 1,4 1,3 ± 0,1
7 Cattle Spiramycin IRMM 1,72 2,9 2,5 ± 0,1
8 Calf Spiramycin IRMM 0,22 2,2 1,8 ± 0,1
9 Pig Carbadox ISS 8,80 10,0 9,3 ± 0,2
10 Poultry Carbadox ISS 3,13 20,0 20,0 ± 0,7
a
JRC-IRMM = Joint Research Centre- Institute for Reference Materials and Measurements (Geel, BE)
ISS = Istituto Superiore di Sanità (Rome, IT)
b
range given defined by ± standard error of the analytical method.
All concentrations are in mg/kg (gravimetric basis).
A.3 Statistical analysis of results
The major aim of the validation study in this context was the evaluation of the performance of the LC-
MS method in detecting the banned substances in feed samples. Since the results taken into account
were qualitative in nature, usual procedures for quantitative results could not be applied. In order to
obtain a measure of the method performance, the results were analysed from different aspects. First,
the straight results (positive, negative and no results) were calculated for each test material, thus
looking at the overall results by considering the compound, its concentration and the matrix. Then,
correct positive, correct negative, false positive and false negative were calculated considering the
compound, its concentration and the matrix in order to look at the performance of the method not only
for each banned substances but also at the chosen concentrations and in the different feed matrices. The
accuracy was then used to describe the capability of the method to correctly classify both positive and
negative samples. In order to obtain this parameter at first the following formula was applied:
PA+ NA
Accuracy AC= (A.1)
PA+ ND++PD NA
As some laboratories were not able to analyse all samples thereby reporting less results than other
laboratories the Adjusted Accuracy was finally used. This parameter is taking into consideration the
number of “no results” reported (data obtained for all materials are pooled):
PA+ NA
Adjusted Accuracy aAC= (A.2)
PA+ ND++PD NA+ NR
where
PA is the number of correct positive identifications (positive agreements),
NA is the number of correct negative identifications (negative agreements),
PD is the number of false positives (positive deviations),
ND is the number of false negatives (negative deviations), and
NR is the number of reported “no results”.
The statistics were expressed as percentages after multiplication of AC by 100. A score of 100 % means
that all positive samples were correctly reported as positive and all negative samples were correctly
reported as negative.
Since a score below 100 % means that part of the samples was incorrectly classified, it was of interest to
know whether the reason of this result was a number of false positive or false-negative results.
Therefore, the sensitivity and the specificity were also calculated, applying the following formulae:
PA
Sensitivity SE= (A.3)
PA+ ND
NA
Specificity SP= (A.4)
PD+
...