Dominance of pre-analytical over analytical variation for measurement of methadone and its main metabolite in postmortem femoral blood

On the basis of simultaneously sampled postmortem blood specimens from the left and right femoral veins the pre-analytical variation of methadone measurements was evaluated and compared to the analytical variation. The material consisted of a series of 27 duplicate samples from routine autopsy cases comprising mainly drug addicts. A chiral LC-MS/MS method was used for measurement of the R- and S-enantiomers of methadone and its main metabolite 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium (EDDP). The analytical CV% was determined to be in the range 3-4% for methadone enantiomers and 4-6% for EDDP enantiomers. The total measurement uncertainty (CV(T)) was estimated from the pre-analytical variation (CV(PA)), analytical variation proper (CV(A)), and variation related to calibration (traceability) (CV(Cal)) according to the relationship CV(T) = [CV(2)(PA) + CV(2)(A) + CV(2)(cal)](0.5). Uncertainty related to calibration concerned a component related to the purity of drug reference compound and a contribution from the production of calibrator solutions (CV(Cal)<1%). Pre-analytical sampling variation was estimated from the duplicate measurements of blood samples after subtraction of the analytical component. The pre-analytical variation amounted to a CV% of 19-21% for R- and S-methadone and 30-38% for R- and S-EDDP, i.e. considerably larger than the other components. Due to the squared addition principle, the resulting total uncertainty (CV(T)) became largely identical to the CV(PA), i.e. 19-21% for R- and S-methadone and 31-38% for R- and S-EDDP enantiomers. Accordingly, CV(T) exceeded CV(A) by a factor 5 or more. Dominance of the pre-analytical component of variation may also be likely for other compounds measured in postmortem blood samples. Thus, the width of the 95%-uncertainty interval (+/-2CV(T)) for a postmortem measurement is largely determined by the pre-analytical component of variation. This should be kept in mind when judging on the uncertainty of postmortem measurement results.