Click to edit Master title style - RIVM · Saskia S. Sterk Henk A. Herbold, October 2007....

Saskia S. SterkHenk A. Herbold,

October 2007.

EVALUATION AND INNOVATION OF OUR PROFICIENCY TESTING PROGRAM.

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CRL workshop 2005 2ARO: Food and ResiduesARO: Food and Residues

Content of the presentation.

1. Evaluation of the results, proficiency test ‘Estradiol in lyophilisedbovine urine and plasma’

2. Evaluation of the results, research study ‘Metabolites of Norandrostenedione in lyophilised bovine urine’

3. General proficiency test information: ‘evaluation last 3 years’

4. Proficiency testing program 2007 - 2008: ‘future plans’


Overview of the proficiency test “Estradiol in lyophilised bovine urine

and plasma”.


Overview of the proficiency tests “Estradiol in lyophilised bovine urine and plasma”.

Urine.• Batch A. High concentration (approximately 5 ppb) of 17ß-Estradiol.

• Batch B. Low concentration (approximately 1 ppb) of 17ß-Estradiol.

• Batch C. Blank samples of urine.

Plasma.• Batch D. High concentration (approximately 1 ppb) of 17ß-Estradiol.

• Batch E. Low concentration (approximately 0.1 ppb) of 17ß-Estradiol.

• Batch F. Blank samples of plasma.



9.75 9.80 9.85 9.90 9.95 10.00 10.05 10.10 10.15 10.20 10.25 10.30

10000200003000040000

Time-->

Abundance

Ion 664.00 (663.70 to 664.70): 06020232.D

9.91 10.21

9.75 9.80 9.85 9.90 9.95 10.00 10.05 10.10 10.15 10.20 10.25 10.30

50001000015000200002500030000

Time-->

Abundance

Ion 451.00 (450.70 to 451.70): 06020232.D 9.90 10.21

9.75 9.80 9.85 9.90 9.95 10.00 10.05 10.10 10.15 10.20 10.25 10.30

5000100001500020000250003000035000

Time-->

Abundance

Ion 409.00 (408.70 to 409.70): 06020232.D

9.90 10.21

9.75 9.80 9.85 9.90 9.95 10.00 10.05 10.10 10.15 10.20 10.25 10.30

50001000015000200002500030000

Time-->

Abundance

Ion 356.00 (355.70 to 356.70): 06020232.D 9.90

10.21

3003203403603804004204404604805005205405605806006206406606807000

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m/z-->

Abundance

Average of 10.208 to 10.221 min.: 06020232.D (-)664

389

409

451

356

485584

329

698558

614427 540297 512 637

GCMSD


Excretion of 17alpha and 17 beta estradiolin samples of urine.

17α-Estradiol samples of bovine urine.

0

200

400

600

-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13

Day

ppb'

s

Free SHP Gluc. 17ß-Estradiol samples of bovine urine.

02468

10

-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13

Day

ppb'

s

Free SHP Gluc.


Excretion of 17alpha and 17 beta estradiolin samples of plasma.

17α-Estradiol samples of plasma.

0

2

46

8

10

-1 0 1 2 3 4 5 6 7 8 9 10 11 12

Days

ppb'

s

Free SHP Gluc. 17ß-Estradiol samples of plasma

0

2

4

6

8

-1 0 1 2 3 4 5 6 7 8 9 10 11 12

Days

ppb'

s

Free SHP Gluc.



Conclusions.

• From the results obtained for urine it can be seen that the hydrolyses step is necessary to liberate estradiol in samples of urine.

• Also for plasma, there is a significant influence on the mass concentrations, probably due to liberation form binding proteins (Sex Hormone Binding Globulin - SHBG)

• The traditional way of enzymatic hydrolysis of sulphate and glucuronide-conjugates, with Suc d’helix pomatia (SHP), is an efficient method.


Number of reporting participants (Only 17ß-estradiol).

Number of participants that didn’t send us any results.2No results

Number of participants that submitted confirmatory results.10Confirmed

Number of participants that submitted any results.915Analysed

Plasma

Number of participants that didn’t send us any results.3No results

Number of participants that submitted confirmatory results.12Confirmed

Number of participants that submitted results.1115Analysed

RemarksNRLParticipants

Urine


Overview of analytical results of all laboratories (ppb) (17ß Estradiol).

780.150.140.200.600.062005M2176

630.780.961.244.240.222005M2175

Samples of plasma

240.471.901.953.741.142005M1511

211.014.704.826.991.962005M1510

Samples of urine

CV%s.d.MedianMeanMax.Min.Sample code


• Hydrolysis step (temperature and time)

• Standards (Purity)

• The use of an internal standard (Deuterated).

• Detection technique (GC-MS, LC-MS, Elisa)

• Validated method (reproducibility, repeatability, CCα, CCß)

Analytical steps in the procedures used by the participants.Possible reasons of the variation.


Analytical steps in the procedures used by the participants.Detection technique

17ß-Estradiol (ppb)Sample of plasma: 2005M2175LCMS-MS Elisa GCMS-MS0.92 – 0.93 2.21 – 1.95 0.60 – 0.80

Sample of plasma: 2005M2176LCMS-MS Elisa GCMS-MS0.10 – 0.11 2.36 – 0.87 0.10 – 0.12


Analytical steps in the procedures used by the participants.

Samples of urine.

• 14 laboratories included a hydrolysis step in the sample preparation prior to extraction and just as much labs extracted the analytes by solid phase extraction.

• 7 labs extracted the analytes by liquid/liquid extraction.

• 5 laboratories used a preparative HPLC- technique too.

• 2 laboratories used LCMS or LCMSMS detection.

• 13 laboratories used GC-HRMS or GCMS.


Analytical steps in the procedures used by the participants.

Samples of plasma.• 4 laboratories included a hydrolysis step in their sample preparation prior

to extraction.

• 7 laboratories extracted the analytes by solid phase extraction.

• 8 laboratories by liquid/liquid extraction.

• 2 laboratories used a preparative HPLC- technique.

• 1 laboratory used a LCMS detection technique.

• 9 laboratories used GCMS or GCMS-MS.

• 2 laboratories used the Elisa-kit and one Radio-Immuno-Assay.


Confirmatory methods ( samples of urine).

Confirmation in samples of urine.

0

10

20

30

40

50

60

70

80

90

2005M1510 2005M1511 2005M1512

Sample nr.

Num

ber

of L

abs.

perf

omin

g a

posi

tive

resu

lt, e

xpre

ssed

as a

per

cent

of a

ll th

epa

rtic

ipan

ts.

17α-Estradiol17ß-Estradiol


Confirmatory methods ( samples of plasma).

Confirmation in samples of plasma.

0

10

20

30

40

50

60

70

2005M2175 2005M2176 2005M2177

Sample nr.

Num

ber

of L

abs.

perf

omin

g a

posi

tivre

sult,

exp

ress

ed a

s a p

erce

nt o

f all

the

part

icip

ants

.

17α-Estradiol17ß-Estradiol


Overview of the Research study “Metabolites of Norandrostenedione in

lyophilised bovine urine”.


Overview of the Research study “Metabolites of Norandrostenedione in lyophilised bovine

urine”.

Norandrostendione, prohormone as feedadditive


Overview of the Research study “Metabolites of Norandrostenedione in lyophilised bovine urine”.

19-Nor-4-androstene -3,17-dione

19-Nor-4-androstene-17α-ol-3-one5α-estrane-3ß, 17α-diol

5α-estrane-3ß-ol-17-one5ß-estrane-3α-ol-17-one



8.00 9.00 10.00 11.00 12.00 13.00 14.00

200000

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800000

1000000

1200000

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4000000

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4800000

5000000

5200000

5400000

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5800000

6000000

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6600000

6800000

Time-->

Abundance

TIC: 06050401.D 8.24

200 250 300 350 400 450 500 550 600 650 7000

50000

100000

150000

200000

250000

300000

350000

400000

450000

m/ z-->

Abundance

Scan 202 (8.234 min): 06050401.D (-)239

666

453

306

355

213331279

410381 497 639 695530 561 596476

17α – Nortestosterone (HFBA derivative)



8.00 9.00 10.00 11.00 12.00 13.00 14.00

500000

1000000

1500000

2000000

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Time-->

Abundance

TIC: 06050403.D 8.63

200 250 300 350 400 450 500 550 600 650 7000

100000

200000

300000

400000

500000

600000

700000

800000

900000

m/ z-->

Abundance

Scan 271 (8.628 min): 06050403.D (-)472

202

428

241

401

373283310 339 504 599538 657450 695629571

5α-estran-3ß-ol-17-one (19-Norepiandrosterone) (HFBA derivative)



8.00 9.00 10.00 11.00 12.00 13.00 14.00

500000

1000000

1500000

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Time-->

Abundance

TIC: 06050307.D 8.54

200 250 300 350 400 450 500 550 600 650 7000

100000

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m/ z-->

Abundance

Scan 255 (8.537 min): 06050307.D (-)202

428 472

241

401

373 499302269 348 649527325 556579 695605449 671

19-Noretiocholanolone (HFBA derivative)



8.00 9.00 10.00 11.00 12.00 13.00 14.00

500000

1000000

1500000

2000000

2500000

3000000

3500000

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Time-->

Abundance

TIC: 06051803.D 8.08

200 250 300 350 400 450 500 550 600 650 7000

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m/ z-->

Abundance

Scan 177 (8.091 min): 06051803.D (-)456

670

243 415216

375339 588 633525306 483 697610272 566

19-Norandrostanediol (HFBA derivative)


Overview of the Research study “Excretion of the metabolites in samples of urine”.

-1--2004M228012

----2004M227911

-5--2004M227810

-14--2004M22779

-93--2004M22768

-282-82004M22757

-73514372004M22746

-3693862012004M22735

2188411514772004M22724

36385621644242004M22713

144894902515922004M22702

13156711434212004M22691

352132902507652004M22680

----2004M2267-1

17α-Nor-testosterone

19-Nor-andostanediol

19-Noretio-cholanolone

19-Norepi-androsterone

RIVMnrDays aftertreatment.



Metabolites of Norandrosteendion

0200400600800

1000120014001600

-1 0 1 2 3 4 5 6 7 8 9 10 11 12

Days after treatment

Con

cent

ratio

n - p

pb

Norepiandrosterone Noretiocholanolone 17α-Nortestosterone

Metabolite of Norandrosteendion

0

2000

4000

60008000

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-1 0 1 2 3 4 5 6 7 8 9 10 11 12

Days after treatment

conc

entr

atio

n - p

pb

Norandrostanediol



Conclusions.

• The metabolites 19-Norepiandrosterone, 19-Noretiocholanolone, 19-Norandrostanediol and 17α-Nortestosterone, were detected after oral administration of 19-Norandrostenedion.

• The main metabolite is 19-Norandrostanediol, the concentration is about 10 times higher then the other metabolites.


Overview of the Research studyPreparation of the samples.

• Batch A. Incurred residues of 19-Norandrostanediol (Main metabolite).

• Batch B and C. Incurred residues of 17α-Nortestosterone, 19-Norepiandrosterone and 19-Noretiocholanolone.

• Batch D. No residues concentration range < 0.1 μg/l.


Overview of analytical results of all laboratories

410.551.301.352.810.642006M1433

290.722.462.494.301.202006M1432

19-Norepiandrosterone

870.740.540.862.830.102006M1433

730.840.911.143.640.012006M1432

19-Noretiocholanolone

450.410.800.911.740.072006M1433

510.951.501.875.060.492006M1432

17α-Nortestosterone

410.741.881.803.400.592006M1431

19-Norandrostanediol

CV%s.d.MedianMeanMax.Min.Sample code


Analytical steps in the procedures used by the participants

- 14 laboratories included a hydrolysis step in their sample preparation prior to extraction and just as much labs extracted there analytes by solid phase extraction.

- 7 labs extracted there analytes by liquid/liquid extraction

- 5 laboratories used a preparative HPLC- technique too.

- 2 laboratories used a LCMS or LCMSMS detection technique.

- 13 laboratories used GC-HRMS or GCMS.


Results confirmatory analysis.

Confirmation analyses.

0102030405060708090

Num

ber o

f lab

s per

form

ing

resu

lts,

expr

esse

d as

a p

erce

nt o

f al

l the

par

ticip

ants

.

Batch A 71Batch B 71 79 50 57Batch C 64 57 43 50

Nor- androstanediol

17α-NT Nor- etiocholanolon

Nor- epiandrosteron


Evaluation performance individual labs.

2732515161224232115210257Total

10101111111011Noretiocholanolone

1010101111101117α-NT

10101111011011Norepiandrosterone

10101111011011Norandrostanediol

2006M1434 (Blank)


3232222233303017α-NT



2006M1433


3132123233302017α-NT



2006M1432


2006M1431

CRL4138*33*32*31*30*262522*21*19*15*12*Labcodes

Total scores of all participants

0

5

10

15

20

25

12* 15* 19* 21* 22* 25 26 30* 31* 32* 33* 38* 41 CRL

Lab.codes (* = NRL)

scor

es


Conclusion.

•Most of the participating Labs (include NRLs) had no problems in confirming norandrostanediol.

•The results of the proficiency tests accentuated some problems regarding the quantification of the metabolites.

•It is obvious that quantification of low concentrations gave more variations in the results.

•Information of the used in-house method in detail, is interesting. Because it is the only way to screen for factors which could have an effect on the results.


Overview of participants (NRL’s) Period 2005 - 2007


+++++++Nantes Cedex -France

-++--+-Mödling - Austria

+++++++Madrid - Espana

-+---+-Ljubjana - Slovenia

+++-+++Lisboa - Portugal

-++++++Dublin - Ireland

+-+-+--Brussel - België

++++---Budapest - Hungary

+++++++Brno - Czech Republic

+++++++the Netherlands (RIVM)

-+++-++Berlin - Germany

-++--++Belfast - United Kingdom

2007200720062006200520052005

bovine urine

Urineplasma

bovine urine

bovine urine

bovine urine

bovine urine

bovine urine

NorandrostenedioneEstradiol

Diethylstilbestrol

Ethynylestradiol

Metabolism Norclostebol

Trenbolo-ne

Methyl-boldenone

Overview participants (NRL's)


--+--+-Tallinn - Estonia

--+----Helsinki - Finland

--+--+-Nicosia Acropolis -Cyprus

-+-----Vilnius - Lituania

-++--+-Uppsala - Sverige

-++--++Soborg - Denmark

-+++-++Serres - Greece

+++----Roma - Italy

++++-++Ringsted - Denmark

-+-+---Riga - Latvia

+++++++Pulawy - Poland

2007200720062006200520052005

Bovineurine

Urineplasma

bovine urine

bovine urine

bovine urine

bovine urine

bovine urine

NorandrostenedioneEstradiol

Diethylstilbestrol

Ethynylestradiol

Metabolism Norclostebol

Trenbolone

Methyl-boldenone

Overview participants (NRL's)


Overview participantsScores 100 - 71%

020406080

100120

RIVM

Czech

Rep

ublic

Espan

a

Fran

ce

Polan

d

Irelan

d

Portu

gal

Denmark

German

y

Greece

Institutes

Num

ber

of la

bs e

xpre

ssed

as a

perc

ent o

f all

the

prof

icie

ncy

test

s

Overview Participantsscores 57 - 14%

0

10

20

30

40

50

60

Hunga

ry

Belfas

t

Sobo

rg - D

enmark

Brusse

l - B

elgië

Austri

a

Sveri

ge

Italy

Slov

enia

Estonia

Latvia

Cypru

sLitu

ania

Finla

nd


Future plans.


Proficiency testing program 2007 – 2008.

1. Gestagens in samples of porcine kidney fat (Ongoing – evaluation report this year)

2. Thyreostatica in samples of porcine urine (end of 2007)

3. Profile of the future plans.

Proficiency testing program for 2008

•Trenbolone in samples of porcine urine (spring 2008)

•1-Testosterone, Methyl-prednisone, Estradiol, Dromostanolone: in samples of bovine urine


http://www.rivm.nl/residues


Extension of the CRL web-application‘Start 2008’

• Digital form for the results of our proficiency tests

• Database ‘Naturel Hormones’

• Forum discussion

• Future plans


Thank you for your attention.

Thank you for your attention.

Click to edit Master title style - RIVM · Saskia S. Sterk Henk A. Herbold, October 2007....

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