Identification and characterization of urinary prenylamine metabolites by means of liquid chromatography-tandem mass spectrometry.

Prenylamine is a vasodilator of phenylalkylamine structure and was used for the treatment of angina pectoris, until reports of undesirable effects including ventricular tachycardia led to a decreasing use of the drug in the 1980s. Metabolic N-dealkylation of orally ingested prenylamine can liberate amphetamine in humans and cause positive findings for amphetamine in doping and forensic analysis. In 2010, the World Anti-Doping Agency (WADA) classified prenylamine as a non-specified stimulant according to the 2010 Prohibited List, thus banning its use in sports in-competition. Supporting the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based detection method, a post-administration urine sample following a single oral prenylamine ingestion (Segontin(®) 60 mg) was analyzed for urinary metabolites. The LC-separated analytes were ionized in positive electrospray ionization (ESI) mode and detected as protonated ions using an AB Sciex TripleTOF 5600 quadrupole-time-of-flight hybrid mass spectrometer. Over 40 phase I metabolites were detected, including previously unknown mono- bis-, tris- and tetra-hydroxylated prenylamine, several hydroxylated and methoxylated prenylamine metabolites and (hydroxylated) diphenylpropylamine. Investigation of the collision-induced dissociation behaviours of the metabolites by high resolution/high accuracy mass spectrometry allowed for the assignment of the nature and the site of observed metabolic transformations. The most abundant phase I metabolite was confirmed as p-hydroxy-prenlyamine by chemical synthesis and stable isotope labelling of reference material. An existing routine screening assay based on direct injection and LC-MS/MS analysis of urine was modified and validated according to common guidelines, in order to allow for the detection of p-hydroxy-prenylamine in sports drug testing. The assay demonstrated the ability to detect the target metabolite at 0.1 ng/ml at intra- and inter-day imprecisions below 10%.

Determination of propranolol enantiomers in plasma and urine by spectrofluorimetry and second-order standard addition method.

The determination of propranolol enantiomers in human plasma and urine by spectrofluorimetry and a second-order standard addition method is described. The methodology is based on chiral recognition of propranolol by formation of an inclusion complex with beta-cyclodextrin, a chiral auxiliary, in the presence of 1-butanol. The adopted strategy combines the use of PARAFAC, for extraction of the pure analyte signal, with the standard addition method, for determinations in the presence of an individual matrix effect caused by the quenching action of the proteins present in the plasma and urine. A specific PARAFAC model was built for each sample, in triplicate, and the scores were related to (R)-propranolol mole fraction using a linear regression in the standard addition method. Using a propranolol with concentration of 260 ng mL(-1), good results were obtained for determinations in the mole fraction range from 50 to 80% of (R)-propranolol, providing absolute errors between 0.4 and 3.6% for plasma and between 0.9 and 6.0% for urine.

Temperature sensitive dopamine-imprinted (N,N-methylene-bis-acrylamide cross-linked) polymer and its potential application to the selective extraction of adrenergic drugs from urine.

A temperature sensitive dopamine-imprinted polymer was prepared in 80% aqueous methanol solution by free-radical cross-linking co-polymerisation of methacrylic acid and acrylamide at 60 degrees C in the presence of N,N-methylene-bis-acrylamide as the cross-linker and dopamine hydrochloride as template molecule. The resulting molecularly imprinted polymer (MIP) formed temperature responsive materials, which could be used for the selective separation of appropriate dopamine and adrenergic compounds from a liquid matrix at ambient temperatures. The thermoresponsive MIP exhibited a swelling-deswelling transition in 80% aqueous methanol solution at about 35 degrees C. The capacity of the thermoresponsive MIP to recognise the template molecule when present in aqueous methanol solution changed with temperature, with the highest selectivity found at 35 degrees C. Additionally, binding parameters obtained from Scatchard analyses indicate that increasing temperature resulted in an increased affinity and binding capacity of specific binding sites, but had less effect on non-selective binding sites. Subsequently, the thermoresponsive MIP was tested for its application as a sorbent material, utilisable in the selective solid-phase extraction (SPE) of dopamine and other adrenergic compounds (epinephrine, isoproterenol, salbutamol and serotonin) from urine samples. It was shown that the compounds that were structurally related to dopamine could be removed by elution, while dopamine and serotonin, the analytes of interest, remained strongly adsorbed to the adsorbent during SPE applications. The thermoresponsive MIP displayed different efficiency in clean-up and enrichments using the SPE protocol at different temperatures.

Identification of the aromatase inhibitor aminoglutethimide in urine by gas chromatography/mass spectrometry.

Aminoglutethimide is used therapeutically as an aromatase inhibitor in the treatment of metastatic breast cancer in post-menopausal women. For doping purposes, aminoglutethimide may be used for treatment of adverse effects of an extensive abuse of anabolic androgenic steroids (gynaecomastia) and to increase the testosterone concentration and stimulation of testosterone biosynthesis. The use of aromatase inhibitors has been prohibited for male athletes since September 1, 2001. The purpose of this study was to develop methods for the identification of the parent compound or its main metabolite and the inclusion of this information into established screening procedures in doping analysis. An excretion study was conducted using oral application of one single therapeutic dose (500 mg) of Orimeten. The analysis was performed by gas chromatography/mass spectrometry (GC/MS). Aminoglutethimide is excreted almost totally as unconjugated parent compound and is detectable by different screening procedures for up to 165 h. Most suitable for the detection of aminoglutethimide is the screening procedure for heavy volatile nitrogen-containing drugs ('Screening 2'). However, since only competition samples are analysed in that screening procedure, the additional inclusion of aminoglutethimide in the screening procedure for anabolic androgenic agents ('Screening 4') is recommended. Full mass spectra and diagnostic ions for the analysis of aminoglutethimide are presented.

Evidence for the polymorphic oxidation of debrisoquine and proguanil in a Khmer (Cambodian) population.

The frequency distributions of the urinary metabolic ratios of debrisoquine and proguanil were measured in a population of unrelated Khmers. Out of 98 Khmer subjects studied, two were identified as poor metabolisers of debrisoquine when a metabolic ratio of 12.6 was used as the cut off point. This represents a prevalence of debrisoquine poor metabolisers of 2.1% (95% confidence interval 0.25-7.3%) which is similar to other Asian populations. Based on the distribution of the ratio of proguanil to cycloguanil excreted in urine, and using an antimode value of 10, the prevalence of poor metabolisers of proguanil in a Khmer population was estimated to be 18.4% (95% confidence interval 10.9-28.1%). The frequency of poor metabolisers of proguanil in Khmers was higher than that described for Caucasian populations, but similar to most reported results in Asian populations.

Comprehensive screening procedure for detection of stimulants, narcotics, adrenergic drugs, and their metabolites in human urine.

An analytical procedure for the detection of stimulants, narcotics, beta-blockers, beta-agonists, and many of their metabolites in urine using a solid-phase extraction procedure and gas chromatography-mass spectrometry (GC-MS) is described. These substances have been specifically banned by the Medical Commission of the International Olympic Committee (IOC) in order to prevent their abuse in sports. Urine samples are submitted to an enzymatic hydrolysis (beta-glucuronidase arylsulfatase) and extracted by means of Bond-Elut Certify columns. The residues are then selectively derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), which enables the formation of trimethylsilyl derivatives of hydroxyl, acidic, and phenolic groups, and N-methyl-bis-trifluoroacetamide (MBTFA), which enables the formation of trifluoroacetamide derivatives of primary and secondary amines. A GC-MS system working in scan mode is sensitive and specific enough to detect and identify approximately 100 compounds and metabolites in urine for at least 24 h after the administration of doses typically encountered in therapeutics. Detection in selected ion monitoring mode is needed for the determination of beta-agonist agents. The method was successfully used in doping control of urine samples during the 25th Olympic Games, July 1992, in Barcelona, Spain.