REPORT Proficiency Test May-September 2014 · LNR Chimie, 96/23 & biotoxines marines NRL Chemie, 96/23 & mariene biotoxines DOC.QA/GEN29-8 v.01_131004 2/31 Summary A proficiency test

LNR Chimie, 96/23 & biotoxines marines NRL Chemie, 96/23 & mariene biotoxines

SOCIETE MOMENTANEE – TIJDELIJKE VENNOOTSCHAP

CER Groupe – Département Santé ILVO-T&V Rue du Point du Jour, 8 Brusselsesteenweg 370 BE-6900 Marloie BE-9090 Melle www.cergroupe.be www.ilvo.vlaanderen.be

DOC.QA/GEN29-8 v.01_131004

� : +32 (0)84 31 00 98 [email protected]

REPORT Proficiency Test

Quantitative analysis of prednisolone and cortisol and qualitative analysis of prednisolone metabolites in porcine liver

May-September 2014

CER Groupe, Département Santé

Nathalie Gillard Michel Dubois

Philippe Delahaut


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Summary A proficiency test on the quantitative determination of prednisolone and cortisol and on the qualitative determination of prednisolone metabolites (20α-hydroxyprednisolone and 20β-hydroxyprednisolone, prednisone) in porcine liver was organized by the CER Groupe. Eight laboratories have participated in the proficiency test. The purpose of this proficiency test was to evaluate available quantitative methods for prednisolone and cortisol residues, as done in 2013 in porcine urine but this time in porcine liver. This study also provided an evaluation of the same methods for prednisolone metabolites identification in porcine liver.


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Table of contents 1. Introduction .................................... ................................................................................... 4 2. Test material ................................... ................................................................................... 4

2.1. Animal experiment ...................................................................................................... 4 2.2. Sample identification................................................................................................... 5 2.3. Homogeneity study ..................................................................................................... 5 2.4. Stability study.............................................................................................................. 5

3. Participants, instructions and schedule ......... ................................................................ 6 3.1. Participants ................................................................................................................. 6 3.2. Sample distribution and instructions ........................................................................... 6

4. Results and methods ............................. ........................................................................... 6 5. Conclusions..................................... .................................................................................. 7 6. Recommendation .................................. ............................................................................ 8 Appendix 1. Invitation letter ...................... ........................................................................... 9 Appendix 2. Registration form ...................... ..................................................................... 10 Appendix 3. List of the participating laboratories . ........................................................... 11 Appendix 4. Codification of the samples ............ .............................................................. 12 Appendix 5. Instructions to participating laborator ies ................................................ .... 13 Appendix 6. Acknowledgment of receipt form ......... ........................................................ 15 Appendix 7. Method description form................ ............................................................... 17 Appendix 8. Homogeneity study...................... .................................................................. 19 Appendix 9. Stability test for prednisolone and cor tisol in the 4 liver samples ............ 21 Appendix 10. Reporting of results form............. ............................................................... 22 Appendix 11. Results of participating laboratories. ......................................................... 24 Appendix 12. Graphical representation of prednisolo ne quantification in the 4 PT samples ……………………………………………………………………………………………...26 Appendix 13. Graphical representation of cortisol q uantification in the 4 PT samples 28 Appendix 14. Method description of participating la boratories...................................... 30


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1. Introduction Prednisolone is a synthetic glucocorticoid with anti-inflammatory and immuno-modulatory properties. Its use as growth-promoting agent is forbidden in animal breeding in Europe. However, several cases of detection of residues of prednisolone in low concentrations in urine of pigs were observed over the last years in the framework of the control plan of the Belgian Federal Agency for the Safety of the Food Chain (FASFC). The Scientific Committee of FASFC recommends continuing the investigations on the presence of residues of prednisolone in pigs in order to collect more information on the issue. In 2013 and 2014, animal studies were performed in Belgium on pigs. They had shown that prednisolone couldn’t be detected in liver of non treated animals, pointing out that the presence of prednisolone in porcine liver could be a good marker of treatment. As already mentioned in the urine PT organized in 2013, sensitive methods have to be available by the Food Authorities to guarantee the quality of the control. The aim of this proficiency test is therefore to evaluate available quantitative confirmatory methods for prednisolone and cortisol in porcine liver, as well as the capacity of participating laboratories to analyze prednisolone metabolites with the same methods.

2. Test material

2.1. Animal experiment For this proficiency test, materials were obtained from an animal experiment performed in the animal facilities of the CER Groupe (Marloie, Belgium). Samples were collected from sows (Pietrain x Landrace) untreated or treated with prednisolone (single intramuscular injection of around 1 mg kg-1 body weight; Prednisolone 2.5 % sol, V.M.D., Arendonk S.A., Belgium) or tetracosactide hexaacetate (single 5-mg intramuscular injection of 1 mg/mL Synacthen® Depot, Defiante Farmaceutica S.A., Funchal, Portugal). Liver samples were collected during the animal study and analyzed for their content in the five targeted compounds (prednisolone, cortisol, 20α-hydroxyprednisolone and 20β-hydroxyprednisolone). Four liver samples were selected:

• a liver sample with levels of prednisolone < 0.18 ppb and about 1 ppb cortisol (sample A)

• a liver sample with levels of prednisolone < 0.18 ppb and about 2 ppb cortisol (sample B)

• a liver sample with about 0.7 ppb prednisolone and level of cortisol < 0.8 ppb (sample C)

• a liver sample with about 2.5 ppb prednisolone and level of cortisol < 0.8 ppb (sample D)

Each sample was sent in blind duplicate to the participant, to check the repeatability of the analysis.


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2.2. Sample identification After homogenization, each sample material batch was divided in sub-portions of 20 g weighted in 50 mL plastic tubes. Samples were randomly selected and coded “PT14-01-xxx” (“xxx” = vial number randomly generated). For each laboratory, a sample set was prepared, including two liver samples of each material batch. Eighteen samples of each sample batch (A, B, C and D) were also selected for homogeneity and stability testing.

2.3. Homogeneity study From each sample batch a set of 12 samples was randomly selected and analyzed. Six samples were mixed. From the mixture, 6 individual samples were prepared. Mixed samples, as well as the six remaining units were analyzed. The homogeneity was determined based on CV comparison by a F-test (Table 1, 2, 3, 4). The CV had to be ≤ 20 %. As shown in Appendix 8, the homogeneity wasn’t tested for concentrations below the CCα (prednisolone in samples A and B and cortisol in samples C and D). Comparison of the CV values of individual and mixed samples by F-test shows no significant differences for prednisolone in samples C and D and cortisol in samples A and B. Therefore homogeneity (repeatability) was considered as acceptable.

2.4. Stability study The stability of prednisolone and cortisol in liver was tested for each sample batch. The aim was to prove that both prednisolone and cortisol are stable during shipment and samples storage in the PT conditions. From each batch, a set of 6 samples was randomly selected before shipment of samples to participants (June 10th 2014) and analyzed after reception of participant results (July 29th 2014). Three samples were stored at 4°C from June 10th 2014 to June 16st 2014 and then stored at -20 °C until July 29th 2014. The remaining three units were stored at -20 °C between June 10th 2014 and July 29th 2014. The results are presented in Appendix 9. Again, the stability wasn’t tested for concentrations below the CCα (prednisolone in samples A and B and cortisol in samples C and D). The CV values of the six samples (stored at 2 different conditions) for prednisolone in samples C and D and cortisol in samples B is below 7 % and is acceptable. For cortisol in sample A, the CV is 17.6 %; this higher CV could be explained by the concentration close to the CCα (0.8 ppb).


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3. Participants, instructions and schedule

3.1. Participants The invitation (see Appendix 1) was sent to potentially interested laboratories on May 14th 2014. Both Belgian laboratories (official control laboratories and academic teams) and members of EURL network were invited to participate in this proficiency test. In the registration form (see Appendix 2), it was also proposed to participants to provide them standard solutions of targeted prednisolone metabolites (prednisone, 20α-hydroxyprednisolone and 20β-hydroxyprednisolone). Eight laboratories, including the organizing laboratory, subscribed for participation in the study. The list with the addresses of the participating laboratories is included in Appendix 3. Four participants to this 2014 PT have also taken part to the 2013 PT for porcine urine. Standards solutions (individual vials containing 1 mL of prednisone, 20α-hydroxyprednisolone or 20β-hydroxyprednisolone at 10 µg/mL) were provided on request of 6 of them.

3.2. Sample distribution and instructions The sample sets were sent to the participants on June 10th 2014 (frozen liver samples send on dry ice). The codes of the samples provided to each participant are presented in Appendix 4. An instruction form (Appendix 5), an acknowledgement of receipt form (Appendix 6), a method description form (Appendix 7) and a results reporting form (Appendix 10) were enclosed to the shipment of samples. The deadline for the reporting of the results was July 31th 2014. All laboratories confirmed the receipt of the samples in good condition.

4. Results and methods Seven laboratory submitted results and method description, as shown in Appendices 11 and 14. Indeed, one laboratory was unable to provide results for the PT samples; this laboratory is therefore not included in the result and method summaries. Considering the non submission of results from one lab and therefore a small number of participating laboratories (below 8), no statistical evaluation of results was performed. However, some differences are observed between participant results. Prednisolone:

- Method sensitivities go from a CCα of 0.12 ppb to 1.4 ppb for prednisolone - Prednisolone was detected by every participant in sample D (highest prednisolone

concentration) and sample C, even if the detected concentration of prednisolone was below the CCα for 2 participants

- In samples C and D, a factor of 3 could be observed among participants for prednisolone quantification

- For lower prednisolone concentrations (sample A and sample B), only few labs were able to report the presence of prednisolone


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Cortisol :

- Method sensitivities go from a CCα of 0.5 ppb to 1.29 ppb for cortisol. - One laboratory does not perform the analysis of cortisol. The other participants were

able to detect cortisol in almost every sample, with levels below their CCα especially in samples C and D.

- In samples A and B, a factor of 2 to 4 could be observed among participants for cortisol quantification, while cortisol quantification is more reproducible for sample C.

Prednisolone metabolites:

- One laboratory does not perform prednisolone metabolites analysis. CCα and CCβ were not provided by 4 participants that have reported results for prednisolone metabolites, probably because these compounds are not included in their routine method

- Prednisone was detected in sample D by one participant - 20α-hydroxyprednisolone was detected by one participant in sample D and by 2

participants in one duplicate of sample B - 20β-hydroxyprednisolone was detected by 4 participants in sample D (corresponding

to the highest prednisolone concentration) and by one participant in sample B and sample C

Differences between applied methods are also to be underlined:

- The sample amount vary from 1 g to 15 g of liver - Use of internal standards : four labs use labeled prednisolone and 2 labs use labeled

cortisol; some participants uses fludrocortisones, triamcinolone acetonide or fluoroprednisolone as internal standard

- Six participant perform an enzymatic hydrolysis - pH adjustment is performed in 3 labs targeting pH 4.8-5.2 - All participants perform purification on SPE columns and one participant performs an

additional immuno-affinity clean-up - All laboratories apply liquid chromatography coupled to mass spectrometry of which 3

use HPLC-MS/MS and 4 UPLC-MS/MS. Ionization is performed with electrospray in negative or positive mode and with APCI for one participant

5. Conclusions Based on the results of this proficiency test, it is concluded that : - A high variability is observed between participant results for both prednisolone (factor

3) and cortisol (factor 2-4) quantification. Compared to the 2013 PT for urine, it could however be noticed that less variation is observed between participants

- Prednisolone metabolites are detected by some labs, especially when higher prednisolone concentration is present in the sample

- The analysis of prednisolone, cortisol and prednisolone metabolites in porcine liver is performed with a variety of methods. In regard of PT results, there is no preferred method but one can notice that the sensitivity is strongly impacted by the sample amount.


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6. Recommendation For the quantitative determination of prednisolone in porcine liver, the availability of certified reference material would be of great value.


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Appendix 1. Invitation letter


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Appendix 2. Registration form


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Appendix 3. List of the participating laboratories

Country Laboratory name Address Belgium Federaal Laboratorium voor de

Veiligheid van de Voedselketen - Gentbrugge

Braemkasteelstraat 59 9050 GENTBRUGGE

Belgium CER Groupe Département Santé

Rue du Point du Jour,8 6900 Marloie

Denmark Danish Veterinary and Food Administration (FVST)

S0ndervang 4 DK-41 00 Ringsted

France LABERCA - ONIRIS Route de Gachet Site de la Chantrerie CS 40706 44307 NANTES Cedex 3

Romania Institute for hygiene and veterinary public health

5 Cimpul Mosilor, Sector 2, 021201 BUCHAREST

Spain Unidad Zoosanitarios (Lab. 51-03-020) Centro Nacional de Alimentación (AECOSAN)

Ctra. Pozuelo-Majadahonda km 5,1 MAJADAHONDA 28220 MADRID

The Netherlands RIKILT Akkermaalsbos 2 6708WB WAGENINGEN

United Kingdom. AFBI Chemical Surveillance Branch.

Stoney Road. Stormont. Belfast BT4 3SD.


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Appendix 4. Codification of the samples

Participant Sample A1

number Sample A2

number Sample B1

number Sample B2

number Sample C1

number Sample C2

number Sample D1

number Sample D2

number

Lab 1 14-01-239 14-01-115 14-01-26 14-01-282 14-01-367 14-01-118 14-01-252 14-01-164

Lab 2 14-01-225 14-01-213 14-01-6 14-01-231 14-01-4 14-01-180 14-01-328 14-01-290

Lab 3 14-01-323 14-01-176 14-01-32 14-01-61 14-01-371 14-01-111 14-01-130 14-01-74

Lab 4 14-01-31 14-01-34 14-01-58 14-01-194 14-01-348 14-01-33 14-01-84 14-01-156

Lab 5 14-01-335 14-01-285 14-01-217 14-01-121 14-01-83 14-01-43 14-01-23 14-01-344

Lab 6 14-01-198 14-01-357 14-01-189 14-01-353 14-01-216 14-01-105 14-01-274 14-01-66

Lab 7 14-01-346 14-01-272 14-01-63 14-01-354 14-01-329 14-01-295 14-01-54 14-01-310


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Appendix 5. Instructions to participating laborator ies


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Appendix 6. Acknowledgment of receipt form


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Appendix 7. Method description form


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Appendix 8. Homogeneity study Table 1: Homogeneity test for sample A

Table 2: Homogeneity test for sample B

Prednisolone concentration (ppb) Cortisol concentr ation (ppb)

Samples Mix Single Mix Single 1 < CCα < CCα 0.68 0.62 2 < CCα < CCα 0.64 0.68 3 < CCα < CCα 0.68 0.65 4 < CCα < CCα 0.66 0.62 5 < CCα < CCα 0.67 0.64 6 < CCα < CCα 0.67 0.68

Average - - 0.67 0.65

STD - - 0.02 0.03

CV(%) - - 2.26 4.19

F-test - 3.25 critical value of f(p=0.05)is 5.05(N-1)


Samples Mix Single Mix Single

1 < CCα < CCα 2.42 2.23

2 < CCα < CCα 2.47 2.26

3 < CCα < CCα 2.42 2.43

4 < CCα < CCα 2.40 2.28

5 < CCα < CCα 2.37 2.32

6 < CCα < CCα 2.55 2.29

Average - - 2.44 2.30

STD - - 0.06 0.07

CV(%) - - 2.61 3.03

F-test - 1.20

critical value of f(p=0.05)is 5.05(N-1)


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Table 3 Homogeneity test for sample C

Table 4: Homogeneity test for sample D



1 2.51 2.56 < CCα < CCα

2 2.57 2.60 < CCα < CCα

3 2.54 2.61 < CCα < CCα

4 2.53 2.63 < CCα < CCα

5 2.60 2.50 < CCα < CCα

6 2.44 2.52 < CCα < CCα

Average 2.53 2.57 - -

STD 0.05 0.05 - -

CV(%) 2.17 2.03 - -

F-test 0.90 -




1 0.75 0.79 < CCα < CCα

2 0.74 0.78 < CCα < CCα

3 0.76 0.80 < CCα < CCα

4 0.77 0.76 < CCα < CCα

5 0.74 0.77 < CCα < CCα

6 0.79 0.80 < CCα < CCα

Average 0.76 0.78 - -

STD 0.02 0.02 - -

CV(%) 2.56 2.08 - -

F-test 1.41 -



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Appendix 9. Stability test for prednisolone and cor tisol in the 4 liver samples

Sample type

Sample ID Storage Temperature (RT=Room Temperature)

Prednisolone (ppb)

Cortisol (ppb)

CV for prednisolone (%)

CV for cortisol (%)

14 - 01 -240 < CCα 0.71

14 - 01 -241 < CCα 0.88 A 14 - 01 -279

+4°C (from 10/06/2014 to 16/06/2014)

-20°C (from 16/06/2014 to 29/07/2014) < CCα 0.81

14 - 01 -87 < CCα 1.09

14 - 01 -204 < CCα 1.03 A 14 - 01 -303

- 20°C (from 10/06/2014 to 29/07/2014

< CCα 1.12

- 17.6

14 - 01 -135 < CCα 2.10

14 - 01 -337 < CCα 1.97 B 14 - 01 -365

+4°C (from 10/06/2014 to 16/06/2014)

-20°C (from 16/06/2014 to 29/07/2014) < CCα 1.91

14 -01 -50 < CCα 2.18

14 - 01 -201 < CCα 2.19 B 14 - 01 -190

- 20°C (from 10/06/2014 to 29/07/2014

< CCα 2.26

-

6.5

14 - 01 -210 0.70 < CCα

14 - 01 -258 0.73 < CCα C 14 - 01 -361

+4°C (from 10/06/2014 to 16/06/2014)

-20°C (from 16/06/2014 to 29/07/2014) 0.64 < CCα

14 - 01 -40 0.73 < CCα

14 - 01 -304 0.72 < CCα C 14 - 01 -345

- 20°C (from 10/06/2014 to 29/07/2014

0.66 < CCα

5.5 -

14 - 01 -10 2.61 < CCα

14 - 01 -233 2.57 < CCα D 14 - 01 -212

+4°C (from 10/06/2014 to 16/06/2014)

-20°C (from 16/06/2014 to 29/07/2014) 2.63 < CCα

36174 2.64 < CCα

14 - 01 -174 2.72 < CCα D 14 - 01 -263

- 20°C (from 10/06/2014 to 29/07/2014

2.61 < CCα

1.9 -


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Appendix 10. Reporting of results form


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Appendix 11. Results of participating laboratories


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Appendix 12. Graphical representation of prednisolo ne quantification in the 4 PT samples (when participant report the presence of a target compounds but below its CCα, the CCα concentration is reported on the graph as a white box with red line)

Sample A1

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lab 1 lab 2 lab 3 lab 4 lab 5 lab 6 lab 7

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Sample C1

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Appendix 13. Graphical representation of cortisol q uantification in the 4 PT samples (when participant report the presence of a target compounds but below its CCα, the CCα concentration is reported on the graph as a white box with red line)

Sample A1

0.0

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(pp

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Sample C1

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Appendix 14. Method description of participating la boratories


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Documents

REPORT Proficiency Test May-September 2014 · LNR Chimie, 96/23 & biotoxines marines NRL Chemie, 96/23 & mariene biotoxines DOC.QA/GEN29-8 v.01_131004 2/31 Summary A proficiency test