Reference materials for analytical toxicology including doping control: freeze-dried urine samples

Abstract

The feasibility of freeze-dried urine samples containing doping agents to be used in intercomparison exercises and/or as reference materials has been evaluated. Freeze-dried urine samples containing caffeine, ephedrine derivatives (ephedrine, methylephedrine, norephedrine, pseudoephedrine and norpseudoephedrine), amphetamine derivatives (amphetamine, metamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine) and testosterone and epitestosterone glucuronides have been evaluated. For preparation of the samples, blank urines previously subjected to filtration for clarification were fortified with standard solutions of the corresponding compounds and filtered under sterile conditions. Some aliquots of the sterile liquid samples were used for homogeneity testing, others were stored at -80 degree C for reference purposes, and the rest were subjected to lyophilisation. Freeze-dried urine samples were stored at 4-8 degree C and their stability was assessed for a period up to 18 months. Results obtained showed minimal differences (lower than 5%) between lyophilised and non-lyophilised aliquots (stored at -80 degree C) at all time periods except for amphetamine (up to 18%) and norpseudoephedrine (up to 10%). Nevertheless, such differences remained constant over the entire period of study, indicating that the loss of analytes was due to the initial lyophilisation process. The loss of analytes in freeze-dried samples was due to their volatility. Furthermore, an increase in pH by 1 unit was observed following reconstitution of samples prepared from drug-free urine of commercial origin.

Stability studies of testosterone and epitestosterone glucuronides in urine

“…As well as the storage conditions studied in this work (liquid urine samples stored at 4 and −20°C), the protocols for long‐term stability testing usually include the evaluation of lyophilized aliquots of the samples stored at 4°C. Results obtained for the lyophilized samples containing TG and EG have recently been published 13…”

“…Sterilized aliquots were placed in appropriate containers for storage at the different temperatures intended for stability testing. Some of the aliquots were subjected to freeze‐drying 13…”

“…The misuse of drugs as ergogenic aids by athletes constitutes an offence in regulated sport practice and conceptually the control of these substances in biological fluids, known as antidoping control, is similar to drugs of abuse testing. However, few stability studies of doping agents have been reported 11–13…”

Stability studies of amphetamine and ephedrine derivatives in urine

“…Apart from the storage conditions studied in this work (liquid urine samples stored at 4 • C and −20 • C), a similar protocol for longterm stability testing of lyophilized aliquots stored at 4 • C has been applied and the results obtained for samples containing the ephedrine and amphetamine derivatives have recently been published [48]. The stability of some of this substances in non-sterile samples was also evaluated in usual storage conditions of samples analyzed in routine antidoping control (4 • C and −20 • C).…”

Screening method for stimulants in urine by UHPLC‐MS/MS: identification of isomeric compounds

“…Dilute‐and‐shoot methods are also especially interesting for compounds with high volatility (e.g. some compounds of the group of stimulants) that may be lost during the evaporation step of the organic solvent used in liquid‐liquid or solid‐phase extractions …”