Stabilization of human urine doping control samples: III. Recombinant human erythropoietin.

BACKGROUND: The tampering of athlete's urine samples by the addition of proteolytic enzymes during the doping control sampling procedure was reported recently. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in urine samples to chemically inactivate proteolytic enzymes and improve the electrophoteric signal of erythropoietin (EPO) in human urine. METHODS: The stabilization mixture applied was a combination of antibiotics, antimycotic substances and protease inhibitors. A series of incubation experiments were conducted under controlled conditions in the presence and absence of the stabilization mixture in urine aliquots spiked with six proteases. Two different analytical techniques were applied for the qualitative and quantitative EPO measurement: isoelectric focusing (IEF) and chemiluminescent immunoassay respectively. RESULTS: The addition of the chemical stabilization mixture into urine aliquots substantially improved EPO detection in the presence of proteolytic enzymes following incubation at 37 degrees C or storage at -20 degrees C. CONCLUSIONS: The results of this study indicated that the stabilization of urine prior to the sample collection procedure with the proposed chemical mixture might prove to be a useful tool for the preservation of anti-doping samples.

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 degrees C for 7 days to simulate the transportation period, whereas another series of aliquots was stored at -20 degrees 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 degrees C. On the other hand, four of the nine microorganisms induced alterations in the steroid profile of the unstabilized samples incubated at 37 degrees C.

Two-step silylation procedure for the unified analysis of 190 doping control substances in human urine samples by GC-MS.

BACKGROUND: While a number of different derivatization procedures for screening GC-MS analysis of prohibited substances are followed by doping control laboratories, a unified derivatization procedure for the GC-MS analysis of 190 different doping agents was developed. RESULTS: Following preliminary experiments, a two-step derivatization procedure was selected. The evaluation of various silylation parameters, such as reagent composition, reaction time, reaction temperature, catalysts and microwave oven reaction time, for this procedure was carried out. CONCLUSION: The suitability of the developed procedure was demonstrated through application on urine samples at concentration levels of the minimum required performance limit for all tested substances. This new derivatization procedure, which significantly decreases time and cost, is suitable for a routine basis application.

Spectrophotometric determination of lisinopril in tablets using 1-fluoro-2,4-dinitrobenzene reagent.

A spectrophotometric method for the determination of lisinopril (LN) in single and multicomponent tablets also containing hydrochlorothiazide (HCT), based on the derivatization reaction with 1-fluoro-2,4-dinitrobenzene (FDNB, Sanger reagent) is described. Aqueous solutions of LN (4.5-27.2 x 10(-5) M) react with FDNB (in acetonitrile) at pH 8.2 (borate buffer) in dark at 60 degrees C for 45 min. After acidification with HCl to decolourize 2,4-dinitrophenolate (the alkaline hydrolysis product of FDNB), the LN-derivative is measured at 356.5 or 405.5 nm (only at 405.5 nm if HCT is present). The calibration curves are linear (r>0.996 at both wavelengths) with a between days precision of slopes of 1.8 and 2.3% at 405.5 and 356.5 nm, respectively. The quantification limit is 3.49 x 10(-5) M (0.014 mg) at 405.5 nm and 5.69 x 10(-5) M (0.023 mg) at 356.5 nm. The accuracy and precision of the method were evaluated with the analysis of synthetic mixtures (Er%: 0.30-0.60 and 0.27-1.00 at 405.5 and 356.5 nm, respectively; RSD%: 0.48-0.92 and 0.35-0.51 at 405.5 and 356.5 nm, correspondingly; recovery%: 99.2-100.4 at 405.5 nm and 97.9-104.3 at 356.5 nm). Results obtained from the analysis of commercial preparations with the proposed method are in good agreement with those obtained with the official HPLC method (% relative difference 0.2-2.5%). The developed method can be used for rapid routine analysis for content uniformity, dissolution profile studies and assay of pharmaceutical preparations.

In vitro study on fluoxetine adsorption onto charcoal using potentiometry.

This in vitro investigation was performed to study the adsorption rate constant as well as the adsorption characteristics of fluoxetine (F) to activated charcoal and its commercial formulation Carbomix powder in simulated gastric (pH 1.2) fluid environment. Ion-selective electrode (ISE) potentiometry, based on the selective, direct and continuous monitoring of F with an F-ISE constructed in our laboratory was used. The method used in the kinetic experiments consists of the rapid addition of a slurry containing the charcoal into the drug solution under stirring and continuous recording of the F-ISE potential until the establishment of equilibrium. The free ionized drug concentration at appropriate time intervals was calculated from the recorded adsorption curve and the apparent adsorption rate constant was estimated assuming pseudo first order kinetics. Within run R.S.D. of the estimates ranged from 0.24 to 11.5%, while between run R.S.D. (n=3-4) ranged from 0.90 to 13.8%. A linear relationship was found between the apparent adsorption rate constants and the amount of charcoal used with slopes (+/-S.D.) for activated charcoal and Carbomix equal to 1.14(+/-0.21) and 0.146(+/-0.009) s(-1)g(-1), respectively. Successive additions of microvolumes of F solution were made into a charcoal slurry with measurement of the F-ISE potential at equilibrium. The maximum adsorption capacity values (+/-S.D.) of activated charcoal and Carbomix were 254.8+/-1.8 and 405+/-41 mg/g, respectively while the affinity constant values (+/-S.D.) were 45.6+/-2.2 and 55.5+/-2.9 l/g, respectively. The adsorption of F to charcoals was rapid and for amounts of charcoal 10 times greater than the amount of the drug, 95% of F was adsorbed within the first 5 min. Relative to the toxic and lethal doses in cases of F intoxications, both types of charcoals tested adsorbed effectively F at gastric pH. Carbomix can be considered as appropriate charcoal formulation for medical treatment in cases of F poisoning.

An in vitro evaluation of fluoxetine adsorption by activated charcoal and desorption upon addition of polyethylene glycol-electrolyte lavage solution.

BACKGROUND: In drug overdoses, treatment with activated charcoal is frequently used because of its adsorptive properties. Recently, whole-bowel irrigation with polyethylene glycol-electrolyte lavage solution has been used as a gastrointestinal decontamination procedure for ingestions of toxins not well adsorbed to activated charcoal and for toxins with a delayed absorption phase, although well adsorbed to activated charcoal. While a combined approach using activated charcoal and whole-bowel irrigation could theoretically enhance the efficacy of both modalities, this improvement remains speculative, since data demonstrating its clinical advantage in overdose treatment are lacking. Fluoxetine, a selective serotonin uptake inhibitor, is one of the most frequently prescribed antidepressants. Fluoxetine is well adsorbed onto activated charcoal. This in vitro investigation was undertaken to study: a) the effect of polyethylene glycol, as well as polyethylene glycol-electrolyte lavage solution, on the adsorption of fluoxetine to laboratory grade-activated charcoal and a commercial activated charcoal formulation (Carbomix powder) in simulated gastric (pH= 1.2) and intestinal (pH=7.2) fluid environment; b) whether the order of polyethylene glycol-electrolyte lavage solution addition would have any effect on the adsorption of fluoxetine to activated charcoal. METHODS: Adsorption of fluoxetine to charcoal in the presence of polyethylene glycol was examined: a) by the simultaneous addition of polyethylene glycol and charcoal to fluoxetine solution and b) by the addition of charcoal to fluoxetine solution and subsequent addition of polyethylene glycol. In both cases, the slurries were incubated at 37 degrees C for 1 hour and filtered. Free fluoxetine concentration was determined in the diluted filtrate by a reversed-phase high-performance liquid chromatography method. RESULTS: The amount of fluoxetine adsorbed to activated charcoal (or Carbomix) was dramatically decreased at gastric and intestinal pH by the presence of polyethylene glycol or polyethylene glycol-electrolyte lavage solution added either concurrently or sequentially to activated charcoal. CONCLUSIONS: In cases of fluoxetine overdose, administration of activated charcoal is recommended, while a combined approach using activated charcoal and whole-bowel irrigation with polyethylene glycol-electrolyte lavage solution is not recommended since it causes a significant desorption of the drug from activated charcoal.

In vitro adsorption study of fluoxetine onto activated charcoal at gastric and intestinal pH using high performance liquid chromatography with fluorescence detector.

BACKGROUND: This in vitro investigation was performed to study the adsorption characteristics of fluoxetine to activated charcoal and its commercial formulation Carbomix powder in simulated gastric (pH = 1.2) and intestinal (pH = 7.2) fluid environments. METHODS: Solutions containing fluoxetine and charcoal were incubated at 37 degrees C for one hour. Reversed phase high performance liquid chromatography was used for the determination of free fluoxetine concentrations (range 0.2-8 micrograms/mL) in the diluted filtrate. RESULTS: The maximum adsorption capacities at pH 1.2 for activated charcoal and Carbomix were 223 and 333 mg/g, respectively; at pH 7.2 they were 301 and 453 mg/g, respectively. The affinity constant values at pH 1.2 of activated charcoal and Carbomix were 441 and 122 L/g, respectively, while at pH 7.2 they were 482 and 589 L/g, respectively, indicating a strong binding of fluoxetine onto charcoals. CONCLUSIONS: Relative to the toxic and lethal doses in cases of fluoxetine intoxications, both types of charcoals tested were found effective for adsorption at gastric and intestinal pH. Adsorbed fluoxetine was significantly increased at intestinal pH, consistent with predominant adsorption of the undissociated form of the drug. We conclude that activated charcoal and Carbomix have adsorptive properties appropriate to medical treatment in cases of fluoxetine overdose.

The effect of polyethylene glycol on the charcoal adsorption of chlorpromazine studied by ion selective electrode potentiometry.

BACKGROUND: This investigation was undertaken to study: a) the adsorption characteristics of chlorpromazine to activated charcoal and its formulations Carbomix powder and Ultracarbon tablets at gastric pH; b) the effect on chlorpromazine adsorption of polyethylene glycol and its combination with electrolyte lavage solution; c) the effect of the order of addition of polyethylene glycol-electrolyte lavage solution. METHOD: Ion selective electrode potentiometry, based on the selective, direct and continuous response of a chlorpromazine-ion selective electrode to the concentration of the free drug, was used. Successive additions of microvolumes of a chlorpromazine solution were made into a charcoal slurry in acidic medium of pH 1.2 with measurement of the chlorpromazine-ion selective electrode potential at equilibrium. RESULTS: The maximum adsorption capacity values of activated charcoal, Carbomix and Ultracarbon, were 297, 563, and 382 mg/g respectively, while the affinity constant values were 40.2, 70.4, and 40.5 L/g, respectively. The adsorption of chlorpromazine to each of the Ultracarbon and Carbomix components was compared to the total adsorption of the formulations. The addition of polyethylene glycol-electrolyte lavage solution causes a slight desorption of chlorpromazine from activated charcoal at gastric pH, more pronounced when polyethylene glycol-electrolyte lavage solution follows the addition of activated charcoal, suggesting the possibility of a nonspecific binding of chlorpromazine to polyethylene glycol. The amount of chlorpromazine absorbed to Carbomix and Ultracarbon was not significantly affected at gastric pH by the presence of polyethylene glycol or polyethylene glycol-electrolyte lavage solution added either concurrently or sequentially to these formulations.