Question 1. Whichdiffrentprocedureshavebeenused by the Laboratory?

Question 1. Which diffrent procedures have been used by the laboratory?

Answer 1. Procedures used by laboratory:

1) Procedure one: obtain ratio T/E (mean testosterone, E mean epitestosterone) in urine sample using GC/MS – preliminary test

2) Procedure two: determine range of value 13C/12C for exogenous or endogenous testosterone using IRMS

Question 2. For each procedure, describe briefly the important steps.

Answer 2. For both procedure sample is divided into A and B sample. We use only sample A

Important steps for procedure one:

1) Inject sample to GC column and use epitestosterone as internal standard ( in the inital screening of A samples for exogenous testosterone)

2) Obtain ratio T/E

3) Determine ratio of free epitestosterone to glucuroconjugates (first calculate average value for glucuroconjugates.) Each value expressed as a percentage

4) If these results are above 5%, the sample is either contaminated or degraded and proces should stopped at this point.

5) If the T/E is above a certain value (it used to be 6 but was lowered to 4), the sample is considered to be presumptively positive for exogenous testosterone. In this case analysis proceeds to the next level.

Important steps for procedure two:

1) Check linearity for measurning isotopic ratio

2) Inject sample A to GC column

3) Calculate diffrence between range of values for the ratio 13C/12C orgininating for the exogenous and endogenous testosterone (carbon stable isotope ratio are looked at for four testosterone breakdown products)

4) Test is positive if any one of the ratios for four metabolites are abnormal (value above 30.8 is consider to be beyond the range of normal values)

Question 3. For each procedure, define - as exactly as possible – what is intended to use Answer 3. The aim of procedure one:

1) checking if T/E is above certain limits

2) checking if the sample is contaminated or degraded

The aim procedure two:

1) obtain difference range of values for the ratio of C-13/C-12 originating for endogenous and exogenous testosterone.

2) check whether the sample is a synthetic testosterone

Question 4. Idenfity and described what are – in Bob Blackledge`s opinion – the non complicances when these procedure were appiled.

Answer 4. Non complicances:

1) The ratio of free testosterone to glucuroconjugates was above 5 % ( sample was contaminated or degraded), so laboratory should have stopped proces at this point. They did not do this. (results of this sample was unrealiable)

2) Peak of epitestosterone was unsatisfactory (separation from others peaks was inadequate and peak size was too small), so acurrary and precision was suffer.

3) LNDD technicians claim that they used three ions for SIM, but LNDD have not provided data of it.

4) Experimental results was saved with two significants figures, all the way up to five

5) LNDD knew identify of athlete who provide sample

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

7) Data files had been deleted and another files was overwritten (used the same file name to diffrent repeat)

8) LNDD consider if any one of the ratio of four metabolites is abnormal test is positive ( others certificed labs consider at least two metabolites is abnormal, then test is positive)

9) Laboratory accepted the relative retention times (they shouldn`t accept)

10) LNDD did not take into a count sourses of sample ( they did not included fraction effects)

11) Total testosterone and epitestosterone should be elevated. 12) The errors in laboratory identification number and athlete’s identification number. There was no certainty, that the report of the T/E ratio from LNDD is related to Landis’s A sample, or there was a mix up and it was from some other athlete.

Question 5. For each of these procedure you defined, imagine that you would need to validate these in your laboratory. Can you then list the most important parameters to validate and briefly described you reasoning.

Answer 5. Procedure one:

1) LOD - in a urine sample there was very small concentration of epitestosterone, so LOD should be as small as possible

2) LOQ - it should be as small as possible to obtain the lowest concentration of epitestosterone in a sample with appropriate uncertainty

3) Precision – reduce random effects

Procedure two:

1) LOD - in a urine sample there is very small concentration of epitestosterone, so LOD should be as small as possible

2) LOQ - it should be as small as possible to obtain the lowest concentration of epitestosterone in a sample with appropriate uncertainty

3) Precision – reduce random effects

4) Linearity - to get essentially the same isotopic ratio for a given compound, independently we introduce into the instrument large or small amount of that component.

5) Rugedness – we have to take into account fact that changes in experimental conditions can make deviations.