The First Procedure

1. Which different procedures have been used by the laboratory?

The first procedure:

Determining testosterone/epitestosterone (T/E) ratio in urine sample by GC-MS (using selected ion monitoring –SIM ). This is preliminary test.

The second procedure:

Determining the range of values for the ratio of carbon 13/carbon 12 for testosterone from endogenous (within the body) sources or for testosterone originating from exogenoussources by using IR-MS.

1. For each procedure, describe briefly the important steps.

Used steps for the first procedure:

1)Dividing the urine sample into A and B.

2)Choosing appropriate temperature program and column.

3)Using epitestosterone like an internal standard in initial screening of A sample for exogenous testosterone and injecting the A sample into GC capillary column

4)Obtaining the ratio T/E.

5)Dividing the amount of free epitestosterone by amount of glucuroconjugates (expressed in a percentage) and comparing with cutoff levels.

Used steps for the second procedure:

1)Checking instrument for linearity.

2)Injecting urine sample (A sample) to GC capillary column.

3)Obtaining range of the ratio of carbon 13/carbon 12

4)Calculating the differences between range of 13C/12C originating from exogenous and endogenous testosterone.

5)Checking if range for four metabolites of testosterone and checking.

For two procedures above, LNDD should save the records and maintain the protocols.

1. For each procedure, define - as exactly as possible - what is the intended use.

The first procedure:

The preliminary test was used to indicate a various drugs from A sample, check if the sample is contaminated or degraded and check if T/E is above certain limits.

The second procedure:

IR-MS was used to obtain the range of 13C/12C and finally check the presence of synthetic testosterone in urine sample.

1. Identify and describe what are - in Bob Blackledge's opinion - the non-compliances when these procedures were applied.

The non-compliances:

The ratio of free epitestosterone to glucuroconjugates exceed 5% and LNDD should have stopped the process here, but they proceeded on to IRMS against WADA guidelines.

The peak separation and peak size of epitestosterone is unsatisfactory. They should have used longer capillary column to get better separation and added internal standard might have provided better precision/reliability/accuracy.

WADA required at least three ions to be used for SIM, but LNDD have not provided that data.

LNDD reported their result with two significant figures, all the way up to five.

A rate of detecting exogenous testosterone was over 300% of certified WADA labs and six times more the UCLA lab.

Total T and E should be elevated.

Identity of athlete, who provided the urine sample was known by LNDD.

LNDD should have optimized the parameters of measurement for both procedures. They should have used proper temperature program for the IRMS, another for GCMS, also different type of capillary column.

IRMS data from Landis case shows that the relative retention times fell outside the limits specified in LND SOP.

Instrument for IRMS need to be checked frequently for linearity, but the data had been deleted.

LNDD did not include fractionation effect to examine the urine sample.

There were many mistakes in protocols, wrong numbering of samples, wrong laboratory identification number and the 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.

1. For each of the procedures you identified, imagine that you would need to validate these in your laboratory. Can you then list the most important parameters to validate and briefly describe your reasoning.

For the first procedure:

LOD – the lowestanalyteconcentration that is possible to detect (in a urine sample there is very small amounts of epitestosterone, so LOD should be as small as possible)

LOQ–as small as possible to quantity amount of epitestosterone

Recovery – to get information about contamination or degradation of the sample

Precision – to describe how scattered replicate test results are (Repeatability;Robustness) and to decrease random effects

For the second procedure:

LOD, LOQ, Precision – reasoning as well as above

Linearity – for a given compound we get essentially the same isotopic ratio whether a comparatively large or small amount of that compound is introduced into the instrument

Ruggedness – changes in experimental conditions can make deviations

Karolina Szymanek