Question 1. Whichdiffrentprocedureshavebeenused by the Laboratory?

Question 1. Whichdiffrentprocedureshavebeenused by the laboratory?

Answer 1. Proceduresused by laboratory:

1)Procedure one: obtain ratio T/E (meantestosterone, E meanepitestosterone) in urinesampleusing GC/MS – preliminary test

2)Proceduretwo: determinerange of value13C/12C for exogenousorendogenoustestosteroneusing IRMS

Question 2. For eachprocedure, describebriefly the importantsteps.

Answer 2. For bothproceduresampleisdividedinto A and B sample. We useonlysample A

Importantsteps for procedure one:

1)Injectsample to GC column and useepitestosterone as internal standard ( in the inital screening of A samples for exogenoustestosterone)

2)Obtain ratio T/E

3)Determine ratioof freeepitestosteroneto glucuroconjugates (firstcalculateaveragevalue for glucuroconjugates.)Eachvalue expressed as a percentage

4)Iftheseresultsareabove 5%, the sampleiseithercontaminatedordegraded and proces shouldstoppedatthis point.

5)If the T/E isabove a certainvalue (itused to be 6 but was lowered to 4), the sampleisconsidered to be presumptivelypositive for exogenoustestosterone. In thiscaseanalysisproceeds to the nextlevel.

Importantsteps for proceduretwo:

1)Checklinearity for measurning isotopic ratio

2)Injectsample A to GC column

3)Calculatediffrencebetweenrange of values for the ratio 13C/12C orgininating for the exogenous and endogenoustestosterone (carbonstableisotope ratio arelookedat for fourtestosteronebreakdown products)

4)Test ispositiveifany one of the ratios for fourmetabolitesareabnormal (valueabove 30.8 is consider to be beyond the range of normal values)

Question 3. For eachprocedure, define - as exactly as possible – whatisintended to use

Answer 3. The aim of procedure one:

1)checkingif T/E isabovecertainlimits

2)checkingif the sampleiscontaminatedordegraded

The aimproceduretwo:

1)obtaindifferencerange of values for the ratio of C-13/C-12 originating for endogenous and exogenoustestosterone.

2)checkwhether the sampleis a synthetictestosterone

Question 4. Idenfity and describedwhatare – in Bob Blackledge`sopinion – the non complicanceswhentheseprocedure wereappiled.

Answer 4. Non complicances:

1)The ratio of freetestosterone to glucuroconjugates was above 5 % ( sample was contaminatedordegraded), solaboratoryshouldhavestopped proces atthis point. Theydid not do this. (results of thissample was unrealiable)

2)Peak of epitestosterone was unsatisfactory (separation from otherspeakswasinadequate and peaksize was too small), soacurrary and precision was suffer.

3)LNDD techniciansclaimthattheyusedthreeions for SIM, but LNDDhave not provided data of it.

4)Experimentalresults was saved with twosignificantsfigures, all the wayup to five

5) LNDDknewidentify of athletewhoprovidesample

6)Linearity of instruments was checked less than one in month

7)Data fileshadbeendeleted and anotherfiles was overwritten (used the same file name to diffrentrepeat)

8)LNDDconsiderif any one of the ratio of fourmetabolitesisabnormal test ispositive ( otherscertificedlabsconsideratleasttwometabolitesisabnormal, then test ispositive)

9)Laboratoryaccepted the relativeretentiontimes (theyshouldn`taccept)

10)LNDD did not takeinto a countsourses of sample ( theydid not includedfractioneffects)

11)Total testosterone and epitestosteroneshould be elevated.

12)The errors in laboratoryidentificationnumber and athlete’sidentificationnumber. There was no certainty, that the report of the T/E ratio from LNDD isrelated to Landis’s A sample, orthere was a mix up and it was from someotherathlete.

Question 5. For each of theseprocedureyoudefined, imaginethatyouwouldneed to validatethesein yourlaboratory. Canyouthen list the most importantparameters to validate and brieflydescribedyoureasoning.

Answer 5. Procedure one:

1)LOD - in a urinesamplethere wasvery small concentration of epitestosterone, so LODshould be as small as possible

2)LOQ - itshould be as small as possible to obtain the lowestconcentration of epitestosterone in a sample withappropriateuncertainty

3)Precision – reducerandomeffects

Proceduretwo:

1)LOD - in a urinesamplethereisvery small concentration of epitestosterone, so LOD should be as small as possible

2)LOQ - itshould be as small as possible to obtain the lowestconcentration of epitestosterone in a sample withappropriate uncertainty

3)Precision – reducerandomeffects

4)Linearity - to getessentially the same isotopic ratio for a givencompound, independently we introduceinto the instrument largeor small amount of that component.

5)Rugedness – we have to takeintoaccountfactthatchanges in experimentalconditionscanmakedeviations.