Stability studies of amphetamine and ephedrine derivatives in urine

Knowledge of the stability of drugs in biological specimens is a critical consideration for the interpretation of analytical results. Identification of proper storage conditions has been a matter of concern for most toxicology laboratories (both clinical and forensic), and the stability of drugs of abuse has been extensively studied. This concern should be extended to other areas of analytical chemistry like antidoping control. In this work, the stability of ephedrine derivatives (ephedrine, norephedrine, methylephedrine, pseudoephedrine, and norpseudoephedrine), and amphetamine derivatives (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA)) in urine has been studied. Spiked urine samples were prepared for stability testing. Urine samples were quantified by GC/NPD or GC/MS. The homogeneity of each batch of sample was verified before starting the stability study. The stability of analytes was evaluated in sterilized and non-sterilized urine samples at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 4 degrees C and -20 degrees C was determined at different time intervals for 24 months for sterile urine samples, and for 6 months for non-sterile samples. For short-term stability testing, analyte concentration was evaluated in liquid urine stored at 37 degrees C for 7 days. The effect of repeated freezing (at -20 degrees C) and thawing (at room temperature) was also studied in sterile urine for up to three cycles. No significant loss of the analytes under study was observed at any of the investigated conditions. These results show the feasibility of preparing reference materials containing ephedrine and amphetamine derivatives to be used for quality control purposes.     

Detection of diuretic agents in doping control

Since the inclusion of diuretics in the list of banned substances in sports in 1988, a large number of screening and confirmation procedures to detect the presence of these substances in urine samples have been developed. In this paper, a review of the analytcal methodology described to analyze diuretics is presented. The paper has been focused on the needs of doping control and mainly screening procedures including sample preparation and liquid or gas chromatographic separation have been considered. More relevant papers using capillary zone electrophoresis have been also considered. Mass spectrometry is mandatory in doping control for confirmation purposes, and finally, mass spectrometric techniques described for diuretics have been reviewed.     

Liquid chromatographic method for the simultaneous determination of caffeine and fourteen caffeine metabolites in urine

An HPLC method has been developed for the separation and the determination of caffeine and its metabolites in urine samples using a one extraction–analysis run and UV detection. The compounds were extracted by liquid–liquid extraction using chloroform–isopropylalcohol (85:15, v/v). Chromatographic separation was accomplished on an ODS analytical column with a mobile phase containing 0.05% acetic acid/methylalcohol (92.5:7.5, v/v). Compounds were monitored at 280 nm. The method was validated for the determination of AFMU, 1X, 1U, 17X and 17U caffeine metabolites required to assess the metabolic activity of the enzymes subject to in vivo caffeine testing. The validated assay was applied to urine samples from ten healthy volunteers. The method was proved to be suitable to assess simultaneously the enzymatic activity of cytochrome P450 CYP1A2 and CYP2A6, as well as N-acetyltransferase and xanthine oxidase.     

Micellar electrokinetic chromatography as a fast screening method for the determination of the doping agents furosemide and piretanide in urine

The possibility of using micellar electrokinetic chromatography for the screening of the loop diuretics piretanide and furosemide in urine was studied. A fast and simple method with good repeatability is described. The method was applied to urine samples collected from a healthy volunteer after oral administration of therapeutic doses of each compound. Positive identification in the urine matrix was possible through recording diode array spectra.     

Felinine stability in the presence of selected urine compounds

Summary. The stability of felinine, an amino acid present in feline urine, was investigated. Synthetic felinine was unstable in the urine of a selection of mammals. Felinine was found to stable in feline urine in which urea had been degraded. Synthetic felinine was found to react specifically with urea and did not react with urea analogues such as biuret or thiourea or other nucleophilic compounds such as ammonia which is more nucleophilic or acetamide and water which are less nucleophilic than urea. The reaction of urea and felinine was independent of pH over the range of 3–10. Urea did not react with N-acetyl-felinine suggesting a felinine N-terminal interaction with urea. Mass spectral analysis of the reaction products showed the presence of carbamylated felinine and fragmentation ions derived from carbamyl-felinine. The physiological relevance of felinine carbamylation is yet to be determined.     

Detection of human growth hormone doping in urine: out of competition tests are necessary

The misuse of human growth hormone (hGH) in sport is deemed to be unethical and dangerous because of various adverse effects. Thus, it has been added to the International Olympic Committee list of banned substances. Until now, the very low concentration of hGH in the urine made its measurement difficult using classical methodology. Indeed, for routine diagnosis, only plasma measurements were available. However, unlike blood samples, urine is generally provided in abundant quantities and is, at present, the only body fluid allowed to be analysed in sport doping controls. A recently developed enzyme-linked immunosorbent assay (Norditest) makes it now possible, without any extraction, to measure urinary hGH (u-hGH) in a dynamic range of 2–50 ng hGH/1. In our protocol, untreated and treated non-athlete volunteers were followed. Some of them received therapeutical doses of recombinant hGH (Norditropin) for one week either intramuscularly (three increasing doses) or subcutaneously (12 I.U. every day). The u-hGH excretion after treatment showed dramatic increases of 50–100 times the basal values and returned to almost the mean normal level after 24 h. u-hGH was also measured in samples provided by the anti-doping controls at major and minor competitions. Depending on the type of efforts made during the competition, the hGH concentration in urine was dramatically increased. Insulin-like growth factor binding proteins and β2-microglobulins in urine and/or in blood could be necessary for the correct investigation of any hGH doping test procedure.     

Potentiating effects of caffeine on teratogenicity of alkylating agents in mice

Teratogenic to subteratogenic doses of x-ray, mitomycin C, MNNG, thio-TEPA, cyclophosphamide, and chlorambucil were administered to pregnant ICR mice together with caffeine at doses of 12.5, 25, or 50 mg/kg on day 11 of gestation. Fetuses were examined for gross malformations on day 18 of gestation. The teratogenicity of mitomycin C was significantly potentiated by caffeine at a dose as low as 12.5 mg/kg. The teratogenicity of chlorambucil was also significantly potentiated by caffeine at 50 mg/kg, but similar potentiation was not observed for x-ray, MNNG, thio-TEPA, and cyclophosphamide.     

Inhibition of monoamine oxidase B by selected benzimidazole and caffeine analogues

We have recently reported that a series of (E)-8-styrylcaffeines and (E)-2-styrylbenzimidazoles are moderate to very potent competitive inhibitors of monoamine oxidase B (MAO-B). The most potent member of the series was found to be (E)-8-(3-chlorostyryl)caffeine (CSC) with an enzyme-inhibitor dissociation constant (K(i) value) of 128 nM. In the present study, we have prepared additional caffeine and benzimidazole analogues in an attempt to identify compounds with improved MAO-B inhibition potency while still acting reversibly. The most potent inhibitor among the caffeine analogues was (E)-8-(3,4-dichlorostyryl)caffeine with a K(i) value of 36 nM, approximately 3.5 times more potent than CSC. The most potent inhibitor among the benzimidazole analogues was (E)-2-(4-trifluoromethylstyryl)-1-methylbenzimidazole with a K(i) value of 430 nM. An SAR analysis indicated that the potency of MAO-B inhibition by (E)-2-styryl-1-methylbenzimidazole analogues depended upon the Taft steric parameter (E(s)) of the substituents attached to C-4 of the styryl phenyl ring. Substituents with a large degree of steric hindrance appear to enhance inhibition potency. The proposal that potent MAO-B inhibition by (E)-8-styrylcaffeines and (E)-2-styrylbenzimidazoles can be explained by a mode of binding that involves traversing both the entrance and substrate cavities was supported by the finding that 1-methylbenzimidazole only weakly inhibited MAO-B with a K(i) value of 2084 microM. Without the styryl side chain, 1-methylbenzimidazole is not expected to be able to bind simultaneously to both the entrance and substrate cavities.     

Stabilization of human urine doping control samples: II. Microbial degradation of steroids

The transportation of urine samples, collected for doping control analysis, does not always meet ideal conditions of storage and prompt delivery to the World Anti-Doping Agency (WADA) accredited laboratories. Because sample collection is not conducted under sterile conditions, microbial activity may cause changes to the endogenous steroid profiles of samples. In the current work, funded by WADA, a chemical mixture consisting of antibiotics, antimycotic substances and protease inhibitors was applied in urine aliquots fortified with conjugated and deuterated steroids and inoculated with nine representative microorganisms. Aliquots with and without the chemical mixture were incubated at 37 °C for 7 days to simulate the transportation period, whereas another series of aliquots was stored at −20 °C as reference. Microbial growth was assessed immediately after inoculation and at the end of the incubation period. Variations in pH and specific gravity values were recorded. Gas chromatography-mass spectrometry (GC-MS) analysis was performed for the detection of steroids in the free, glucuronide, and sulfate fractions. The addition of the chemical stabilization mixture to urine samples inhibited microorganism growth and prevented steroid degradation at 37 °C. On the other hand, four of the nine microorganisms induced alterations in the steroid profile of the unstabilized samples incubated at 37 °C.     

Rapid detection of erythropoiesis-stimulating agents in urine and serum

A rapid and easy-to-use test kit, EPO WGA MAIIA, which can be used for distinguishing various endogenous human erythropoietins (hEPOs) and several recombinant hEPO and EPO analogues, has been evaluated. The test is based on chromatographic separation of the glycosylated isoforms of EPO using wheat germ agglutinin (WGA) and a sensitive immunoassay using anti-EPO carbon black nanostrings and image scanning for quantification. All of the reactions take place along the porous layer of a lateral flow microcolumn containing WGA and anti-EPO zones. The presence of molecules resembling hEPOs, such as Mircera, was detected by the aberrant affinity interaction with the antibody zone on the strip. It was possible to distinguish nine recombinant hEPOs expressed in hamster and human cell lines, as well as Aranesp and Mircera, from endogenous urine hEPO. The required amount of EPO in the samples, a few picograms, is very low compared with other methods for EPO isoform identification. This EPO isoform determination method opens the possibility to monitor recombinant EPO therapy for clinical research and seems to be a valuable candidate to the arsenal of EPO doping control tests.     

Response of selected fetal organs to antineoplastic agents

This study was designed to quantitate the effects of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DIC) and 5-(3,3-bis(2-chlorethyl)-1-triazeno)-imidazole-4-carboxamide (BIC) on growth and selected components of rat fetal organs. Twelve-day pregnant rats were given single intraperitoneal injections of 600 mg/kg of DIC and 900 mg/kg of BIC and autopsied on day 21 of gestation. Fetal liver, brain, kidney, and placenta were removed, weighed, and assayed for total DNA, RNA, and protein. DIC significantly reduced weight, total DNA, RNA, and protein of all four fetal organs as compared to age-matched controls. The brain was most severely affected by this compound. BIC also significantly reduced weight, DNA, RNA, and protein of fetal brain, kidney, and placenta, but in fetal liver only weight and total protein were significantly depressed, while DNA and RNA remained essentially unchanged. The effect of BIC was maximal on the placenta.     

Interactions between purine derivatives: Electronic spectral studies. I. Electronic transitions in caffeine monomer and exciton coupling in caffeine dimer

The concentration dependence of the ultraviolet absorption spectrum of aqueous solutions of caffeine has been studied. Individual species spectra have been derived for the monomer, dimer, and tetramer of caffeine. The emission spectrum of caffeine in aqueous solution and the dichroic spectra in oriented poly(vinyl alcohol) and polyethylene films have been measured. The long-wavelength tail of the absorption spectrum of caffeine in non-polar environment has been found to incorporate at least one carbonyl(π*, n) transition. Dichroic spectral data and molecular orbital calculations have been used to assign transition moment directions to the (π*,π) transitions. The lowest energy (π*,π) transition, responsible for the near-ultraviolet absorption peak in aqueous solution of caffeine, has been used for the study of degenerate exciton interactions in the dimeric species of caffeine. Assuming that the caffeine molecules in the dimer are stacked in parallel planes, theoretical calculations of the ground-state interactions and of the degenerate exciton interactions have been combined with experimental data and a unique model for the dimer of caffeine has been derived. The transfer rate of energy between the molecules in the dimer is of the order of 10¹³_S^?1.     

Effects of chronic caffeine on brain adenosine receptors: Regional and ontogenetic studies

The effect of chronic caffeine treatment on three different binding sites in five brain areas of mice is characterized. The sites studied were the adenosine receptor, using [³H] diethylphenylxanthine, the benzodiazepine receptor, using [³H] diazepam and the adenosine uptake site, using [³H] nitrobenzylthioinosine. Significant increases were only observed in adenosine receptors with the greatest degree of change seen in the cerebellum and brain stem at both 16 and 23 days of caffeine treatment. The lack of significant effects of chronic caffeine on benzodiazepine receptors and adenosine uptake sites indicates that the caffeine effect is specific. The effect of chronic caffeine treatment on the ontogency of adenosine receptors was also studied with the result showing a significantly accelerated development of the receptor in the caffeine treated animals. The adult adenosine receptor levels were 20–30% higher than those observed in control animals. The observed alterations in adenosine receptor number which occur as a consequence of caffeine consumption may underlie some of the behavioral effects of this cortical stimulant as well as provide insights concerning the mechanisms of tolerance to and dependence on caffeine.     

Systematic quantification of peptides/proteins in urine using selected reaction monitoring

The evaluation of biomarkers in bodily fluids necessitates the development of robust methods to quantify proteins in a complex background, using large sets of samples. The ability to multiplex numerous analytes in a single assay expedites the process. Liquid chromatography-mass spectrometry (LC-MS) analyses performed in selected reaction monitoring (SRM) in conjunction with stable isotope dilution MS present an effective way to detect and quantify biomarker candidates in bodily fluids. The strategy presented involves an initial qualification of predefined sets of proteins in urine. The technique was applied to detect and quantify peptides in urine samples as surrogates for a few endogenous proteins. Multiplexed assays were developed to analyze proteins associated with bladder cancer; a few exogenous proteins were added as internal standards. The sample preparation and the analytical protocols were optimized to ensure reproducibility, analytical precision, and quantification limits in the low nanogram per milliliter range. Analyses were performed using known amounts of isotopically labeled peptides. Systematic replication of the measurements indicated intra-assay and inter-assay variability, with CVs in the range of 10%. The differences measured for two targeted proteins were correlated with their level of expression in the corresponding tumors using immunohistochemistry.     

N-acylglycines: gas chromatographic mass spectrometric identification and determination in urine by selected ion monitoring

Eleven biologically interesting N-acylglycines have been synthesized and the gas chromatographic and mass spectrometric properties of their trimethylsilyl derivatives studied, A sharp and reproducible gas chromatographic peak could be obtained for each N-acylglycine as the N, O-bis-(trimethylsilyl)-N-acylglycine. By the use of these derivatives a sensitive and specific selected ion monitoring method for the determination of N-acylglycines in human urine has been developed.