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More than 25 years of developing doping control methods have led to comprehensive screening and confirmation procedures for stimulants, narcotics and beta-blockers. Much of this work has been initiated and/or improved by the late Prof. Dr. Manfred Donike. The methodological approach covered in this overview was applied to doping control procedures during the XXV Summer Olympics in Barcelona, Spain, in 1992 and the XVII Winter Olympics in Lillehammer, Norway, in 1994. Urine samples are screened through a combination of two analytical methods that are complementary: (a) gas chromatographic analysis of the parent compound and unconjugated metabolites, following single-step sample extraction and detection by a nitrogen-specific detector based on a retention index identification system and (b) gas chromatographic analysis including also conjugated drugs and metabolites after hydrolysis, solid-phase extraction, derivatisation and mass spectrometric detection. Confirmation and identification is always performed by gas chromatographic separation and full scan mass spectrometric detection. These methods facilitate the rapid screening and confirmation of more than 100 stimulants, narcotic analgesics and beta-blockers in urine for at least 24 h after the intake of a pharmaceutical dose. Application of the methods ensures high quality standards for the unequivocal identification of doping agents as well as a rapid turnaround time for sample analyses.

This paper details various rapid and sensitive methods for the extraction and derivatisation of propranolol, metoprolol, sotalol, atenolol, pindolol, timolol, oxprenolol, alprenolol and penbutolol in equine urine and in human post mortem whole blood and urine. Three solid-phase extraction methods are described involving the use of either XtrackT XRDAH515, Bond Elut Certify or Sep-Pak C18 cartridges. Two derivatisation methods are also described involving the formation of cyclised silyl or pentafluoropropionate derivatives with either chloromethyldimethylchlorosilane or pentafluoropropionic anhydride, respectively. Gas chromatographic-mass spectrometry analysis was carried out in select-ion monitoring mode. All these methods were evaluated using drug-free human post mortem blood, urine and equine urine fortified at various levels with the beta-blockers mentioned above. The application of some of these methods on a forensic case study is also presented. This work does not include samples from equine administration trials of beta-blockers.

The most convenient way to perform supercritical fluid extraction (SFE) of liquid sample matrices is to combine it with solid-phase extraction (SPE). beta-Blockers from urine were collected on an Empore disc, which was then placed into an extraction cell for derivatization and SFE. SPE recovery was best at pH 10. Effects of temperature, pressure and volume of pyridine on the acetylation and SFE processes were studied. Without acetylation the beta-blockers were not significantly soluble in CO2. SFE temperatures of 70 degree C and 150 degree C together with 200 microliters of acetic anhydride and 400 microliters pyridine gave the best results. With the SPE-SFE-GC-MS method developed here, beta-blockers like oxprenolol, metoprolol and propranolol could easily be detected in urine samples, and the limit of detection (LOD) for these compounds was found to be 20 ng/ml, 30 ng/ml and 40 ng/ml, respectively.

A column-switching HPLC method using an internal-surface phenylboronic acid precolumn for the selective extraction of beta-blockers from biological fluids has been developed. Filtered urine and plasma samples (50 microliters) were injected onto the precolumn equilibrated with methanol-0.05 M disodium hydrogenphosphate (5:95, v/v). After the precolumn had been washed briefly, the selectively retained beta-blockers were eluted with methanol-0.05 M phosphate buffer (pH 2.0) and transferred to a reversed-phase analytical column, on which they were then separated. Even after exposure to at least 160 injections of non-treated urine and plasma samples, the retention efficiency of the precolumn was maintained with no increase in back pressure. Quantitative recoveries and good reproducibility were demonstrated with pindolol.

A high-performance liquid chromatographic method with electrochemical detection has been developed for the determination of six beta-blockers: atenolol, nadolol, timolol, metoprolol, oxprenolol, and alprenolol. The chromatographic separation was performed using a mu Bondapack C18 column, a mobile phase of acetonitrile-water (40:60), containing 5 mM KH2PO4/K2HPO4 proved to be optimal at a 1.3 ml/min flow-rate, and a pH of 6.5. The temperature was optimized at 30 +/- 0.2 degrees C. The amperometric detector, equipped with a glassy carbon electrode, was operated at 1300 mV versus Ag/AgCl in the direct current mode. The method was applied to the determination of these compounds at two concentration levels: ppm and ppb (ng/ml), obtaining relative standard deviations lower than 5% at ppm levels and lower than 10% at ppb levels, and quantitation limits ranging from 15 ppb to 500 ppb. The method was applied to the screening of beta-blockers in spiked urine samples, with a total elution time lower than 12 min, obtaining the best recoveries for timolol and metoprolol (never greater than 93%). These recoveries together with the low limits of quantitation achieved, allows its application to doping analysis in human urine.

Simple, sensitive and reliable high-performance liquid chromatographic methods are reported for the determination of pindolol enantiomers in human serum and urine. The methods involved a solid-phase extraction of serum and a direct injection of urine samples. The separation of R(+)- and S(-)-pindolol was accomplished on a reversed-phase cellulose-based chiral column with a mobile phase of 40:60 (v/v) acetonitrile-0.3 M aqueous sodium perchlorate at a flow-rate of 0.5 ml/min. The detection was achieved by monitoring the fluorescence emission of pindolol enantiomers at 310 nm with excitation at 270 nm. The limits of detection were 1.2 ng/ml of R(+)- and 4.3 ng/ml of S(-)-pindolol in serum, and 21 ng/ml of R(+)- and 76 ng/ml of S(-)-pindolol in urine. The external standard method was used for quantitation. The methods have been applied to the analysis of human serum and urine samples in a pharmacokinetic study.

A fast method is described for the screening of eleven beta-blockers, two narcotic analgesics and two stimulants in urine by HPLC with column switching. The urine sample (100 microliters), buffered to pH 9-9.5, is injected onto a short extraction column packed with CN stationary phase. The extraction column is flushed with water for 2.5 min to elute polar matrix components to waste. The retained components are then backflushed by means of a six-port valve onto the ODS analytical column where they are separated. Phosphate buffer pH 3.0 and acetonitrile were used as mobile phase. Gradient elution was applied in the screening method to improve separation. Detection was performed with a diode-array detector at 220, 235 and 300 nm. Recoveries were near 100%, precision was excellent and sensitivity about 0.25 micrograms/l. To speed up the quantitative analysis, the same method but with isocratic elution was successfully applied to the determination of acebutolol and metoprolol in urine samples collected 4 h after administration of the compounds as single doses.

The objective of this study was to determine the structure of talinolol metabolites formed and the amounts excreted in urine. Talinolol metabolites in urine were identified by comparing their HPLC retention times and their GC-MS profile with those of previously characterized reference compounds. The metabolites were quantified by HPLC with a normal-phase silica column, a single chloroform extraction and UV detection. Less than 1% of an administered dose was found in urine as hydroxylated talinolol. Other metabolites could be excluded. A sensitive method to determine talinolol in serum and a simple method for analysis of talinolol in urine are described. These methods were found to be precise and accurate for the measurement of talinolol in samples obtained from patients during chronic talinolol treatment as well as from healthy volunteers after a single dose of talinolol.