Analysis of a large number of anabolic compounds in animal tissues and urine by GC-MS/MS

12 Pages·2008·0.15 MB·English

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Analysis of a large number of anabolic compounds in animal
tissues and urine by GC-MS/MS

Document: SOP ARO/534 versie 1 Auteur: Hennie van Rossum
Documentgebied: ARO Methods Autorisator: Leen van Ginkel
(labhoofd)
Status: Definitief

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1 Introduction
Here, we present a method which is capable to detect and confirm the identity of a large number (26)
of different growth promoters in meat and urine at the suggested level of control of 0.5 µg/kg or 0.5
µg/L. The samples are purified using a simple clean-up, detection is performed using highly specific
GC-MS/MS. The method was fully validated with values for CCα for most compounds in the range 0.1
– 0.3 µg/kg or µg/L. The method is validated for samples of meat and urine.
1.1 Objective
This SOP describes a quantitative method of analysis of anabolic compounds in meat and urine. The
method consists of the following steps: destruction of meat matrix, TBME extraction and defattening.
For samples of urine hydrolysis is performed and TBME extraction. Further clean-up for all matrices is
performed by C18 purification, derivatization and detection by gas-chromatography - mass
spectrometry (GC-MS-MS), ionisation mode is electron impact (EI). Confirmation can be performed in
the same extracts (Table 3).
1.2. Scope
The method is used to perform quantitative analysis of samples of meat and urine. The identity of
detected residues can be confirmed. The CCß and the measurement of uncertainty for all compounds
are given in table 6.
1.3 Definitions
The definitions of CCα, CCß and measurement of uncertainty can be found in SOP ARO/501.
1.4 Background information
The use of anabolic compounds is permitted in a number of countries, e.g. the USA, Brazil and
Australia. However, the European Union has prohibited the use of hormones for the purpose of animal
husbandry. In order to monitor imported meat products, it is necessary to use highly sensitive
confirmatory methods for the detection of low levels of banned hormones. Currently, the suggested
level for control is 0.5 µg/kg. However, until recently it was not possible on a routine scale to enforce
this level since the available multi-residue methods were not able to confirm the identity according to
the current criteria [1]. For this reason, we developed and validated a method based on GC-MSMS.
This technique has the required sensitivity and diagnostic power necessary for confirmatory analyses.
The multi residue method was developed for 26 anabolic compounds. In this paper the analytical
procedure is described and validation data are presented.
Note 1: DES has two isomers trans-DES and cis-DES. These isomers are in dynamic balance, this
balance depends on pH, light, temperature etc. For a standard DES in ethanol this balance is
approximately trans-DES: cis-DES 95:5. In this method trans-DES-D6 is used as internal standard,
during clean-up isomerizes trans-DES-D6 to cis-DES-D6. This isomerization will also occur to trans-
DES. In this SOP the sum of trans+cis-DES peak area’s and trans+cis-DES-d6 peak area’s are used
for quantification.
Note 2: For Medroxyprogestroneacetate (MPA), Melengestrolacetate (MLA) and Megestrolacetate
(MGA) derivatization with MSTFA++ is not working.

2 Apparatus
For operating instructions and maintenance status files see ARO Cardbox data-bases.
Standard laboratory glassware and equipment is used, with in addition:
2.1 Polypropylene tubes 50 ml (Greiner).
2.2 Ultrasonic finger with microtip, 50% duty cycle (Branson sonifier 250).
2.3 Moulinette S (Moulinex).
2.4 Vortex (Vortex-genie, Wilton & Co).
2.5 Automatic pipettes (Gilson P100, P200, P1000 and P5000).

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2.6 Refrigerated centrifuge (RC-3, Sorvall).
2.7 Test tubes, 10 ml, glass (55 mm x 11.5 cm) (Renes, RB55).
2.8 Electric water bath with thermostat adjustable with nitrogen facility (Turbo Vap).
2.9 Heating module thermostat adjustable (Pierce 18790) with nitrogen facility.
2.10 Ultrasonic water bath (Bransonic).
2.11 Rotating apparatus (Heidolph REAX2).
2.12 Glass injection vials (Agilent 5182-0714) with glass 50 µl inserts (Alltech 98024) and
caps (Agilent 5182-0717).
2.13 Incubator thermostat adjustable 37°C for hydrolysis.
2.14 Incubator thermostat adjustable 60°C for derivatization.
2.15 Gas chromatograph (Varian, type CP-3800).
2.16 Automatic injector (Varian, type CP-8400).
2.17 Mass spectrometer (Varian, type 1200L).
2.18 GC-column VF-17MS, 30 m x 0.25 mm ID, film thickness 0.15µm (Varian CP8981).

3 Safety and environment
3.1 Safety
The RIVM have defined rules to work safe. These rules can be found on the intranet.
3.2 Waste disposal
Waste is disposed according to SOP 487. More information how to handle waste is found on the RIVM
intranet.

4 Chemicals and reagents
Materials
All chemicals and reagents are of high purity quality.
4.1 Standards
Relevant data of the analytes are listed in table 1. Stock solutions containing 1 mg/ml are prepared by
dissolving the appropriate amount of the analyte in ethanol. These solutions are stored in the dark at -
20 ºC for a maximum period of 5 year. Working solutions are prepared by 10-fold dilutions of the stock
solutions with ethanol. These solutions are stored in the dark at 4 ºC (range 1 – 10 ºC) for a maximum
period of 12 months.
Table 1. Information about analytes.
Analyte CAS # Formula Mol. Weight
(g/mol)
DIENESTROL DE 84-17-3 C H O 266.3
18 18 2
HEXESTROL HEX 84-16-2 C H O 270.4
18 22 2
BENZESTROL Benz 85-95-0 C H O 298.4
20 26 2
Trans-DIETHYLSTILBESTROL DES 56-53-1 C H O 268.4
18 20 2
α-NORTESTOSTERONE α-NT 4409-34-1 C H O 274.4
18 26 2

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α-BOLDENONE α-Bold 27833-18-7 C H O 286.4
19 26 2
α-TESTOSTERONE α-T 481-30-1 C H O 288.4
19 28 2
ß-NORTESTOSTERONE ß-NT 434-22-0 C H O 274.4
18 26 2
ß-BOLDENONE ß-Bold 846-48-0 C H O 286.4
19 26 2
ß-TESTOSTERONE ß-T 58-22-0 C H O 288.4
19 28 2
α-ESTRADIOL α-E2 57-91-0 C H O 272.4
18 24 2
ß-ESTRADIOL ß-E2 50-28-2 C H O 272.4
18 24 2
NORMETHANDROLONE Normeth 514-61-4 C H O 288.4
19 28 2
METHYLTESTOSTERONE MTT 58-18-4 C H O 302.4
20 30 2
METHYLBOLDENONE MBold 72-63-9 C H O 300.2
20 28 2
ETHYNYLESTRADIOL EE2 57-63-6 C H O 296.4
20 24 2
NORETHANDROLONE Norethan 52-78-8 C H O 302.4
20 30 2
MEGESTROL Meg n.a. C H O 342.5
22 30 3
MEDROXYPROGESTERONE MP 520-85-4 C H O 344.5
22 32 3
PROGESTERONE P 57-83-0 C H O 314.5
21 30 2
MELENGESTROL Meleng n.a. C H O 354.5
23 30 3
NORCLOSTEBOL NorCT n.a. C H ClO 308.8
18 25 2
CHLOROTESTOSTERONE CT 1093-58-9 C H ClO 322.9
19 27 2
CHLOROMADINONE CM n.a. C H ClO 362.5
21 27 3
NORCLOSTEBOL-ACETATE NorCTA n.a. C H ClO 350.8
20 27 3
CHLOROTESTOSTERONE-ACETATE CTA 855-19-6 C H ClO 364.9
21 29 3
n.a not available
4.1.1 Internal standards: Relevant internal standards are trans-DES-D6, HEX-D4, DE-D2, ß-NT-D3,
ß-Bold-D3, ß-T-D2, ß-E2-D3, MTT-D3, EE2-D4, Meg-D3, MP-d3, P-d5, Meleng-D3, CT-D3,
37-Cl-CM, and CTA-d3.
4.1.2 An internal standard-mixture is prepared by mixing 0.1 ml containing 10 ng/µl of each internal
standard to a final volume of 20 ml ethanol.
This mixture contains 2.5 ng / 50 µl.
4.1.3 A standard mixture is prepared as the internal standard-mixture: the final mixture contains 1
ng / 50 µl.
4.1.4 Standards used for the calibration curve are prepared according to table 2.
Table 2. Calibration curve for samples of meat.
Amount µl standard Corresponding µl internal standard
(ng)
(mixture 1 ng/50µl) Amount in µg/kg 2.5 ng/50 µl ( = 2.5 ng)
2.5 125 2.5 50
2.0 100 2.0 50
1.5 75 1.5 50
1.0 50 1.0 50
0.5 25 0.5 50

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0.25 12.5 0.25 50
0 0 0 50
After pipetting the required amount of standard solution into derivatization vials the standards are
further processed from section 5.4.
4.2 Chemicals
Chemicals
4.2.1 Methanol.
4.2.2 Tertiair-Butyl-Methyl-Ether (TBME).
4.2.3 NaOH, 1 mol/l, dissolve 40 g sodium hydroxide in water and add water to a final volume of
1000 ml.
4.2.4 HCl solution, 1 mol/l
4.2.5 Ethanol.
4.2.6 n-Heptane.
4.2.7 Isooctane.
4.2.8 Water; Milli-Q, Waters
4.2.9 C SPE columns (Varian 12102052)
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4.2.10 N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) (Macherey-Nagel).
4.2.11 Ammonium iodide (Fluka).
4.2.12 Dithioerythreitol (Sigma).
4.2.13 MSTFA++: consists of a mixture of N-methyl-N trimethylsilyltrifluoroacetamide (MSTFA):
ammonium iodide: dithioerythreitol (1000:2:4, v/w/w-%).
4.2.14 Acetic acid (Merck, art. 63)
4.2.15 Sodium acetate (Merck, art. 6268).
4.2.16 Acetate buffer 2 mol/L, pH=5.2. Dissolve 25,2 g acetic acid and 129.5 g sodium acetate in 800
ml of water. Adjust the pH to 5,2 ± 0,1 and add water to a final volume of 1000 ml.
4.2.17 Beta-glucuronidase/sulfatase (suc d’Helix Pomatia containing 100.000 units ß-
glucuronidase/sulfatase per ml. (Industr.Biol. France, 213473).
4.2.18 C washing solution methanol/water 40/60 (v/v).
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4.2.19 C elution solution methanol/water 80/20 (v/v).
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4.2.20 n-pentane.

5 Procedure
Samples of urine and meat are stored in the dark at approximately -20°C, but not higher than -10°C,
until analysis, or at approximately 4°C if analysis is foreseen to be within 1 day. As a rule quality
control samples are included. Details on these samples are always included within the study plan.
If a laboratory sample is considered suitable for analysis (adequate sample size, proper storage
history and representative for the analysis) the first step in the analytical procedure is the preparation
of a primary extract.
5.1 Preparation of a primary meat-extract
5.1.1 Weigh 1 g of well homogenized meat in a 50-ml tube; add the internal standard-mixture and
vortex for a few seconds.
5.1.2 Add 10 ml of water and vortex meat/water mixture for 30 seconds.
5.1.3 Ultrasonificate the meat/water mixture for 30 seconds.
5.1.4 Place the tube in an ultrasonic water bath for 15 minutes.
5.1.5 Add 10 ml TBME and rotate for 15 minutes.
5.1.6 Centrifugate 10 minutes at 4000 rpm, place the tubes for 20 minutes at -80°C and decantate
the TBME layer into a 10 ml tube and evaporate at 55°C under a gentle stream of nitrogen.

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5.1.7 Repeat 5.1.5 and 5.1.6 and evaporate the combined TBME.
5.1.8 Dissolve the extract in 4 ml methanol/water (80/20).
5.1.9 Add 4 ml n-heptane and vortex for 30 seconds.
5.1.10 Centrifugate 10 minutes at 3000 rpm and discard the n-heptane layer.
5.1.11 Repeat 5.1.8 and 5.1.9 and evaporate till a volume < 0.5 ml and add water till about 4 ml.
5.1.12 Continue at 5.3.
5.2 Preparation of a primary urine-extract
5.2.1 Pipette 5 ml of urine in a 50-ml tube, add the internal standard-mixture.
5.2.2 Add 1 ml of 2.0 mol/L acetate buffer, check pH (5.2 ± 0.2) and add NaOH, 1 mol/l or HCl
solution 1 mol/l, if necessary.
5.2.3 Add 0.05 ml ß-glucuronidase/sulfatase and incubate during the night at 37°C or 2 hours at
50°C. Cool down to room temperature.
5.2.4 Add 10 ml of TBME, vortex for 30 seconds.
5.2.5 Centrifugate 10 minutes at 4000 rpm, place the tubes for 20 minutes at -80°C and decantate
the TBME layer into a 10 ml tube and evaporate at 55°C under a gentle stream of nitrogen.
5.2.6 Repeat 5.2.4 and 5.2.5. and evaporate the combined TBME.
5.2.7 Dissolve the extract in 0.2 ml methanol followed by 4 ml of water.
5.2.8 Continue at 5.3.
5.3 C18 purification
5.3.1 Pre wash a C cartridge with 5 ml methanol followed by 5 ml of water.
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5.3.2 Pass the extract over the column.
5.3.3 Wash the column with 5 ml of water.
5.3.4 Wash the column with 5 ml of methanol/water 40/60 (v/v).
5.3.5 Elute the column with 5 ml of methanol/water 80/20 (v/v).
5.3.6 Evaporate at 55°C under a gentle stream of nitrogen.
5.3.7 Dissolve the extract in 3 ml of water and extract with 5 ml of n-pentane.
5.3.8 Centrifugate 10 minutes at 3000 rpm and transfer the n-pentane layer into a clean tube.
5.3.9 Repeat the n-pentane extraction and combine the n-pentane layers, evaporate.
5.3.10 Dissolve the dried extract in 0.4 ml ethanol.
5.4 Derivatization
5.4.1 Transfer the residue into a derivatization vial.
5.4.2 Pipette standards used for the calibration curve according to table 2 into derivatization vials.
5.4.3 The ethanol of sample(s) and from the calibration curve are evaporated and 25 µl MSTFA++
is added.
5.4.4 The vials are vortexed and the reaction mixture is incubated during 1 hour at 60°C.
5.4.5 After incubation the reaction mixture is evaporated to dryness under a stream of nitrogen at
55°C.
5.4.6 Dissolve the residue in 50 µl of isooctane and transfer into a glass injection insert and transfer
into the automatic injector of the GC-MS-MS.
5.5 GC-MS-MS detection
• injection 2 µl pulsed splitless 260°C
• initial oven temperature 110°C (1 minute)
• temperature is increased by 20°C/min to 340°C, remains for 5 minutes at 340°C
• temperature transfer line 330°C
• Constant flow mode, 1.1 ml/min

The suitability of the GC-MS-MS system is checked by autotune conform the manual of the apparatus.
Injection of a derivatized standard of 20 pg (on-column) estradiol-D3 should have a S/N at MRM 419.3

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> 285.3 (-28V) higher then 100. In that case the system is considered as suitable for analysis of all the
samples.

Measurements are for screening (quantification) performed on MRM I (Multiple Reaction Monitoring).
For confirmation measurements is performed on both MRM I and MRM II. Both methods can be
combined. If a screening result looks non-compliant, MRM II is used for confirmation. The ratio MRM
I/II should fulfil 2002/657/EC [1].

Table 3. GC-MS-MS Measured transitions with characteristic retention times.
Analyte Retentiontime MRM I Col. MRM II Col.
(quantification) Energy Energy
(min) (confirmation)
(Volt) (Volt)
cis-DIETYLSTILBESTROL-d6 8.62 418.3 > 220.3 -25
cis-DIETHYLSTILBESTROL 8.64 412.3 > 217.3 -20 412.3 > 383.3 -15
HEXESTROL-d4 8.98 209.0 > 180.0 -5
HEXESTROL 8.99 207.0 > 179.0 -10 207.0 > 191.0 -10
DIENESTROL-d2 9.21 412.3 > 397.3 -20
DIENESTROL 9.21 410.3 > 381.3 -5 410.3 > 395.3 -5
t-DIETHYLSTILBESTROL-d6 9.06 418.3 > 220.3 -25
t-DIETHYLSTILBESTROL 9.07 412.3 > 217.3 -20 412.3 > 383.3 -15
BENZESTROL 9.82 207.0 > 179.0 -10 207.0 > 191.0 -10
α-NORTESTOSTERONE 10.09 418.3 > 313.3 -12 418.3 > 328.3 -10
α-BOLDENONE 10.15 430.0> 206.0 -18 430.0 > 325.0 -12
α-TESTOSTERONE 10.18 432.3 > 209.3 -10 432.3 > 327.3 -5
ß-NORTESTOSTERONE-d3 10.25 421.3 > 316.3 -20
ß-NORTESTOSTERONE 10.26 418.3 > 313.3 -12 418.3 > 328.3 -10
ß-BOLDENONE-d3 10.34 433.0 > 206.0 -15
ß-TESTOSTERONE-d2 10.35 434.0 > 211.0 -11
ß-BOLDENONE 10.35 430.0 > 206.0 -18 430.0 > 325.0 -12
ß-TESTOSTERONE 10.35 432.3 > 209.3 -10 432.3 > 327.3 -5
α-ESTRADIOL 10.42 416.3 >285.3 -16 416.3 > 326.3 -18
ß-ESTRADIOL-d3 10.59 419.3 > 285.3 -28
ß-ESTRADIOL 10.60 416.3 >285.3 -16 416.3 > 326.3 -18
NORMETHANDROLONE 10.62 432.0 > 287.0 -25 432.0 > 342.0 -15
METHYLTESTOSTERONE-d3 10.69 449.3 > 301.3 -10
METHYLTESTOSTERONE 10.70 446.3 > 301.3 -30 446.3 > 356.3 -5
METHYLBOLDENONE 10.70 444.3 > 206.3 -15 444.3 >339.3 -10
ETHYNYLESTRADIOL-d4 11.09 429.0 > 233.0 -15
ETHYNYLESTRADIOL 11.10 425.0 > 231.0 -14 425.0 >205.0 -14
NORETHANDROLONE 11.11 446.0 > 356.0 -10 446.0 > 287.0 -20

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MEGESTROL 11.29 453.3 > 273.3 -17 Not present
MEGESTROL-d3 11.29 456.3 > 276.3 -15
MEDROXYPROGSTERONE-d3 11.31 563.3 > 331.3 -25
MEDROXYPROGSTERONE 11.32 560.3 > 328.3 -15 560.3 > 315.3 -15
PROGESTERONE-d5 11.31 463.3 > 448.3 -15
PROGESTERONE 11.33 458.3 > 443.3 -5 458.3 > 157.3 -20
MELENGESTROL 11.34 570.3 > 480.3 -10 570.3 > 465.3 -15
MELENGESTROL-d3 11.33 573.3 > 483.3 -10
NORCLOSTEBOL 11.39 452.3 > 417.3 -5 452.3 > 321.3 -5
CHLOROTESTOSTERONE-d3 11.43 469.3 > 338.3 -5
CHLOROTESTOSTERONE 11.43 466.3 > 335.3 -15 466.3 > 431.3 -10
37-CHLORO-MADINONE 11.89 580.3 > 231.3 -5 Not present
CHLORO-MADINONE 11.89 578.3 > 231.3 -15 Not present
NORCLOSTEBOL-ACETATE 12.28 422.3 > 216.3 -10 422.3 > 387.3 -5
CHLOROTESTOSTERONE-ACETATE-
d3 12.32 439.3 > 404.3 -5
CHLOROTESTOSTERONE-ACETATE 12.33 436.3 > 401.3 -15 436.3 > 230.3 -20

6 Calculation
6.1 Calculation
Areas of the selected ion of the standard and of the internal standard are calculated. The ratio is the
response variable. Quantitative results are obtained by constructing linear curve fitting using least
squares linear regression calculation of the response variable versus the concentration. Unknown
concentrations are calculated by interpolation.
Quantification is only valid if:
• The maximum of the signal originating from the analyte has an S/N ratio >3.
• In the blank control sample all the internal standards are present. (S/N ratio >3 for internal
standards).
• In the spiked control sample all components are present (S/N ratio >3 for internal standards and
for the non-deuterated compounds).
6.2 Process control
Each series contains at least a blanc and a spiked sample (0.5 µg/kg) and all samples are spiked with
deuterated internal standards, both should fulfill the criteria as stated in 6.1.
To judge if a sample is compliant or not-compliant Commission Decision 2002/657 is used [1].

7 Validation and Measurement uncertainty
7.1 Validation of method
The validation (2, 3) was performed conform SOP 501 for the compounds given in table 5. The
validation level was set at 0,5 µg/kg. In table 5 an overview is given of the validation results.

Table 5. Overview of the validation results.

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ANALYT CCa (µg/kg) CCß (µg/kg) Measurement uncertainty (%)
HEXESTROL 0.07 0.12 52
DIENESTROL 0.14 0.23 87
DIETHYLSTILBESTROL 0.06 0.10 48
BENZESTROL 0.07 0.12 50
a-NORTESTOSTERONE 0.07 0.11 73
a-ESTRADIOL 0.05 0.09 63
a-TESTOSTERONE 0.11 0.18 50
ß-NORTESTOSTERONE 0.13 0.22 55
ß-ESTRADIOL 0.05 0.09 45
NORMETHANDROLONE 0.07 0.12 116
ß-TESTOSTERONE 0.07 0.11 47
ETHYNYLESTRADIOL 0.12 0.21 52
METHYLTESTOSTERONE 0.05 0.08 35
NORETHANDROLONE 0.10 0.17 83
NORCLOSTEBOL 0.22 0.37 123
PROGESTERONE 0.12 0.21 81
CHLOROTESTOSTERONE 0.16 0.27 93
NORCLOSTEBOL-ACETATE 0.13 0.22 145
CHLORO-TESTOSTERONE-ACETATE 0.09 0.16 56
MEDROXYPROGESTERONE 0.22 0.38 110
CHLORO-MADINONE 0.36 0.61 181
a-BOLDENONE 0.16 0.27 181
ß-BOLDENONE 0.12 0.21 50
METHYLBOLDENONE 0.18 0.31 128
MELENGESTROL 0.57 0.97 470
MEGESTROL 1.43 2.43 1375

The CCα is lower then 0.3 µg/kg for most compounds. The uncertainty of measurement is acceptable
for all compounds except for melengestrol and megestrol. It is known that gestagens are difficult to
analyse with GC-MS. however the CCα is low enough to detect trace amounts in samples of meat.
7.2 Limited validation for urine
To determine if the validation results of meat samples are representative for urine samples, spiked
samples (0.5 ng/ml or 0.5 ng/g) of each matrix were analysed simultaneously and calculated (n=8). In
table 6 an overview of the results is given.

To compare the results of the different matrices, the following statistic tests were used:
• T-test [5] for the comparison of means (T-critical for n=8 samples for 95% confidence = 2.31)
• calculation conform : http://www.physics.csbsju.edu/stats/t-test_NROW_form.html
• F-test [5] for the comparison of standard deviations (F-critical for n=8 samples for 95% confidence
= 3.69)
• F calculated = (SD samples)2/ (SD samples)2 (where F > 1)

Table 6. T- and F-test results for meat and urine 0.5 ng/ml. Gray shaded fields are significant different.
meat urine

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mean SD mean SD T F
calculated calculated
DE 0.55 0.08 0.44 0.05 3.47 2.14
HEX 0.46 0.05 0.44 0.07 0.78 2.03
Benz 0.39 0.04 0.33 0.07 2.24 4.12
DES 0.46 0.09 0.43 0.05 1.03 4.00
α-NT 0.71 0.06 0.76 0.20 -0.68 9.77
α-Bold 0.87 0.52 1.10 0.60 0.71 1.38
α-T 0.66 0.09 8.90 3.72 -6.26 1635
ß-NT 0.47 0.14 0.40 0.09 1.19 2.25
ß-Bold 0.54 0.10 0.76 0.45 -1.26 21.2
ß-T 0.49 0.16 0.78 0.43 -1.75 6.97
α-E2 0.64 0.07 13.1 14.9 -2.37 40542
ß-E2 0.64 0.18 0.55 0.19 0.95 1.19
Normeth 0.71 0.34 0.63 0.15 0.67 5.09
MTT 0.49 0.06 0.47 0.07 0.63 1.23
MBold 0.70 0.17 0.27 0.15 4.88 1.24
EE2 0.46 0.07 0.43 0.13 0.72 2.99
Noreth 0.50 0.09 0.44 0.23 0.72 5.91
Meg
MP 0.75 0.37 0.50 0.16 1.77 5.23
P 1.35 1.78 1.01 1.21 0.42 2.16
Meleng
NorCT 0.61 0.56 0.66 0.19 -0.24 8.48
CT 0.59 0.20 0.41 0.13 2.08 2.64
CM
NorCTA 0.45 0.16 0.83 0.10 -8.23 2.31
0.45 0.12 0.54 0.12 -1.47
CTA 1.02
T-max F-max
significant difference =2.31 =3.69

Overall it can be concluded that the calculated average concentrations are comparable for both
matrices. The variance is in general better for samples of meat. It can therefore concluded that the
method is suitable for analysis of both matrices. It was found that the method is not suitable for the
analysis of melengestrol, chlorotestosterone and megestrol. See for the analysis of these compounds
SOP ARO/435.
7.3 Determination of confirmation capabilities of method
To determine if it is possible confirm the identity of compounds conform EU 2002/657 (1) samples
were spiked at different levels and confirmatory analysis was performed. In table 7 an overview of the
confirmation analysis is given.

Table 7. Overview of confirmation analysis on samples of meat which were spiked with different concentrations of compounds
(µg/kg). Gray shaded fields are confirmed according to 2002/657/EC
Compound Ratio limits Measured Ratio for spiked (µg/kg) samples of meat
(low-high) 0.25 0.5 0.75 1.0 2.5

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