Towards internationally acceptable standards for food additives and contaminants based on the use of risk analysis.

Internationally acceptable norms need to incorporate sound science and consistent risk management principles in an open and transparent manner, as set out in the Agreement on the Application of Sanitary and Phytosanitary Measures (the SPS Agreement). The process of risk analysis provides a procedure to reach these goals. The interaction between risk assessors and risk managers is considered vital to this procedure. This paper reports the outcome of a meeting of risk assessors and risk managers on specific aspects of risk analysis and its application to international standard setting for food additives and contaminants. Case studies on aflatoxins and aspartame were used to identify the key steps of the interaction process which ensure scientific justification for risk management decisions. A series of recommendations were proposed in order to enhance the scientific transparency in these critical phases of the standard setting procedure.

Disposition of human drug preparations in the horse. IV. Orally administered fenoprofen.

Plasma and urinary concentrations of the non-steroidal anti-inflammatory drug fenoprofen were determined by a high-performance liquid chromatographic procedure following oral administration of a dose of 3 g to fed and fasted horses. In plasma, fenoprofen was present in detectable concentrations for 6-12 h. Free access to hay significantly reduced the peak plasma concentration and bioavailability of fenoprofen, and large interindividual differences in absorption and elimination pattern occurred. In fasted horses, fenoprofen was rapidly absorbed with a mean half-life of 0.10 h. Maximum concentrations were found 0.63 +/- 0.21 h after dosing. The elimination half-life was 0.9 h. As early as 1 h after dosage, fenoprofen could be detected in hydrolysed and unhydrolysed urine, and remained detectable up to 48 h. The maximum excretion rate and peak concentration occurred 2 h after administration, irrespective of the feeding schedule. In fed horses, a second maximum occurred after 9 h. The percentage of the dose excreted as unchanged fenoprofen in 12 h was 13.0 +/- 6.8%. A recovery of 21.9 +/- 7.4% and 42.2 +/- 7.0% of the dose was obtained after enzymatic and alkaline hydrolysis, respectively. At least three hydroxylated metabolites were detected in hydrolysed urine.

The influence of a heavy infection with sensitive and resistant strains of Ostertagia circumcincta and with Trichostrongylus colubriformis on the pharmacokinetics of febantel in lambs.

Plasma concentrations of febantel and its major metabolites, fenbendazole, oxfendazole and fenbendazole sulphone, were determined after oral administration of 7.5 mg/kg febantel in lambs before and 28 days after infection with 100,000 L3 larvae of a benzimidazole (BZ)-sensitive or BZ-resistant strain of Ostertagia circumcincta or with 75,000 L3 larvae of a BZ-sensitive Trichostrongylus colubriformis strain. The febantel concentrations were always low, and in only a few samples were higher than the limit of detection. A mean decrease in the area under the curve (AUC) for the three metabolites of 10.2%, 16.4% and 4.9% in lambs infected, respectively, with BZ-sensitive O. circumcincta, BZ-resistant O. circumcincta and T. colubriformis was observed. The Cmax for all the metabolites was higher in the BZ-sensitive O. circumcincta group than in the naive sheep, while the Tmax occurred earlier. The Cmax and the Tmax values for all the metabolites were lower in the BZ-resistant O. circumcincta group than in their own naive controls. In the T. colubriformis group the Cmax values of the metabolites were lower and the Tmax occurred much later.

The abuse of doping agents in competing body builders in Flanders (1988-1993).

Analytical findings of unannounced doping control in body builders in Flanders during 1988-1993 are reported. In some federations between 38 and 58% of the controlled athletes were found positive during this period. The results show that there was no fall in drug abuse over this period and steroids were taken even by body builders associated with an organisation against the use of anabolic steroids. In 1988 and 1989 popular anabolic steroids were nandrolone and testosterone. From 1990 on, a wide range of mostly injectable steroids including nandrolone, metenolone, drostanolone and testosterone were administered. Polydrug abuse including several anabolics in combination with stimulants and diuretics was also widespread. The beta agonist clenbuterol was detected in several urine samples taken in 1991, one year before its appearance in other sports.

Disposition of human drug preparations in the horse. III. Orally administered alclofenac.

Concentrations of the non-steroidal anti-inflammatory drug (NSAID) alclofenac were determined by a sensitive high performance liquid chromatographic procedure in plasma and urine of horses following oral administration of a dose of 3 g. In plasma, alclofenac was present in detectable concentrations for 72 h. The plasma disposition in individual horses was best described by a bi-compartmental model with two successive rate constants ka1 = 0.05 +/- 0.06 h-1 and ka2 = 0.06 +/- 0.01 h-1. Alclofenac half-lives t1/2 alpha and t1/2 beta were 1.0 +/- 0.8 h and 6.9 +/- 1.5 h, respectively. Maximal concentrations (38.9 +/- 16.2 micrograms/ml) were obtained after 8.5 +/- 2.4 h. Alclofenac was detected in urine for at least 48 h after dosing. The percentage of the dose excreted as unchanged alclofenac in 12 h was very low (0.68 +/- 0.19%), total (free+conjugated) alclofenac accounted for 2.16 +/- 0.55% of the dose.

A liquid chromatographic method for the determination of fenoprofen in equine plasma and urine.

A high performance liquid chromatographic method to measure plasma and urine fenoprofen levels in equine biofluids is described. Liquid-liquid extraction with diethylether was used to isolate the drug from plasma and urine. The accuracy and reproducibility of the method were within acceptable limits over the concentration range 0-10 micrograms/mL and 0-20 micrograms/mL respectively from plasma and urine. Detection limits were 0.05 microgram/mL (2 mL plasma) and 0.2 microgram/mL (0.5 mL urine). This procedure was applied to ascertain the pharmacokinetics of a 3 g dose of fenoprofen calcium in a horse.

The intramuscular bioavailability of a phenylbutazone preparation in the horse.

The plasma concentrations of phenylbutazone (PBZ) and its major metabolites, oxyphenbutazone (OPBZ) and gamma-OH-phenylbutazone (OHPBZ) were determined for up to 72 h in six horses, following intravenous (i.v.) and intramuscular (i.m.) administration of 4 g phenylbutazone, 20 ml Phenylarthrite Ventoquinol (Vetoquinol Spécialités Pharmaceutiques Vétérinaires, Magny-Vernois, 70200 Lure, France). After i.v. dosing the plasma disposition was best described by a two-compartment open model. The hydroxylated metabolites OPBZ and OHPBZ were present in detectable concentrations for 72 h and 48 h, respectively. After 36 h the OPBZ concentrations exceeded plasma PBZ concentrations. The plasma disposition following i.m. injection could be described by a one-compartment open model. The hydroxylated metabolites OPBZ and OHPBZ were present in detectable concentrations for 72 h and 36 h, respectively. Only after 72 h was the concentration of OPBZ in plasma higher than the concentration of PBZ. The mean i.m. bioavailability of phenylbutazone was calculated to be 91.7 +/- 10.1%.

Determination of alclofenac in equine plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method to measure plasma and urinary alclofenac levels in equine biofluids is described. Isolation of the drug from plasma is achieved using liquid-liquid extraction with diethyl ether. Reversed-phase C18 solid phase extraction is used for the extraction of free and conjugated alclofenac from urine. The reproducibility and accuracy of the method were well within acceptable limits over the concentration ranges 0-10 and 0-20 micrograms/ml, respectively, for plasma and urine. Starting with 2 ml of plasma, a concentration of 0.1 microgram/ml could easily be measured; the limit of quantification in urine (0.5 ml) was 1 microgram/ml. Hydrolysis of urine with strong alkali resulted in the decomposition of alclofenac. A pharmacokinetic profile of alclofenac in the horse is shown.

The influence of Ostertagia circumcincta and Trichostrongylus colubriformis infections on the pharmacokinetics of febantel in lambs.

Plasma concentrations of febantel and its major metabolites fenbendazole, oxfendazole and oxfendazole sulphone were determined after oral administration of 7.5 mg/kg febantel in lambs before and 28 days after infection with 50,000 L3 larvae of Ostertagia circumcincta or Trichostrongylus colubriformis. The febantel concentrations were always very low and only in a few samples higher than the detection limit. The mean decrease in AUC for the three metabolites for the infected sheep in comparison to the parasite naïve sheep was 13.9% +/- 4.1% (mean +/- SEM) and 23.7% +/- 5.3% in the O. circumcincta infected and the T. colubriformis infected lambs respectively. This reduction was only significant for the T. colubriformis infected group. In order to determine a more complete pharmacokinetic profile, febantel was injected intravenously at a dose of 2.5 mg/kg in a further study.

The disposition of suxibuzone in the horse.

A high performance liquid chromatographic method is described to determine the anti-inflammatory drug suxibuzone (SXB) and its major metabolites phenylbutazone (PBZ) and oxyphenbutazone (OPBZ) in equine plasma and urine. When suxibuzone (6 mg/kg) was administered intravenously (i.v.) or orally (p.o.) no parent drug was detected in plasma or in urine. The disposition of the metabolite PBZ (i.v.) could be described by a 2 compartment model with a beta half-life varying from 7.40 to 8.35 h. Due to severe side effects the use of i.v. suxibuzone should not be encouraged in the horse. PBZ and OPBZ were detected in plasma and urine after p.o. SXB administration. Peak plasma PBZ concentrations (8.8 +/- 3.0 micrograms/ml) occurred 6 h after oral dosing and the terminal exponential constant was 0.11 +/- 0.01 h-1. Phenylbutazone and oxyphenbutazone were detectable in urine (> 1 microgram/ml) for at least 36 h, after p.o. administration. SXB was not hydrolyzed in vitro by horse plasma. Equine liver homogenates however appeared to have a very high capacity for hydrolysing SXB, indicating that first-pass effect could be responsible for the rapid disappearance of this NSAID in the horse.

ELISA screening with GC-MS confirmation of the tranquilizer chlorprothixene administered in subtherapeutic doses to horses.

A commercially available generic promazine ELISA kit is available which shows cross-reactivity for the tranquilizer chlorprothixene (CPT). The ELISA test readily detects the presence of CPT or its metabolites in equine urine for up to 24 h after the i.v. and i.m. administration of sub-therapeutic doses (4.5 mg) to three horses. Maximum concentrations (CPT equivalents) are obtained 2 h after i.v. dosing. No distinct concentration peak values are observed after i.m. administration. Following solid-phase extraction, confirmation of CPT and its metabolites by electron impact mass spectrometry after sub-therapeutic administration is not successful. The use of chemical ionization mass spectrometry however revealed the presence of at least four metabolites including; chlorprothixene sulphoxide, hydroxylated chlorprothixene and hydroxylated chlorprothixene sulphoxide.

Influence of hydrolysis procedures on the urinary concentrations of codeine and morphine in relation to doping analysis.

A method is described for the GC-NPD determination of urinary codeine and morphine after derivatization with trifluoroacetic anhydride. The lower limit for accurate quantitative determination was 0.05 microgram ml-1. After the oral administration of Bisolvon Griblettes corresponding to 30 mg codeine phosphate to seven subjects maximum codeine concentrations were obtained after 1-2 h and codeine remained detectable generally 24 h post dosing. The mean maximum level was 5.1 +/- 2.8 micrograms ml-1 found after enzymatic hydrolysis with Suc Helix pomatia juice (SHP). Based on these and previous results (mean 6.3 +/- 3.4 micrograms ml-1) a threshold level for codeine of 16 micrograms ml-1 is proposed. Significant differences were noticed between urinary codeine concentrations found after enzymatic hydrolysis with SHP, beta-glucuronidase from Patella vulgata and acid hydrolysis, respectively. Generally, highest values were obtained after SHP, while beta-glucuronidase and especially acid hydrolysis resulted in much lower levels. No morphine could be detected after acid hydrolysis. Concerning doping analysis, in particular the uniformity of methods and interpretation of the results, it is recommended that the hydrolysis method should be specified in the rules of those sporting federations allowing codeine and/or morphine.

A high performance liquid chromatographic method for the determination of febantel and its major metabolites in lamb plasma.

A high performance liquid chromatographic (HPLC) method for the determination of the anthelminthic pro-benzimidazole febantel and its major metabolites in lamb plasma has been developed. Samples were extracted after addition of albendazole as internal standard, NH4OH and distilled diethyl ether. The extracted phase was dried under a stream of nitrogen redissolved in methanol and chromatographed by HPLC. Detection was by UV absorbance at 292 nm. Recovery from the plasma was 97.2, 97.1, 54.5 and 88.0% for febantel, fenbendazole, oxfendazole and oxfendazole sulphone respectively, and within-day and between-day coefficients of variation were 4.03, 4.69, 3.57 and 5.06% and 4.25, 3.73, 5.12 and 4.12%, respectively, for febantel, fenbendazole, oxfendazole and oxfendazole sulphone. The specificity and sensitivity of this method (limit of detection in plasma 0.025 micrograms/mL and < or = 0.0125 micrograms/mL for febantel and its metabolites, respectively) were sufficiently high to enable us to characterize the time course of the drug in the plasma after oral administration of therapeutic doses to sheep.

Plasma ronidazole concentrations in sheep after intravenous, oral, intraruminal and intraabomasal administration.

Plasma ronidazole concentrations were examined after intravenous (i.v.) and oral administration of ronidazole in sheep (n = 6) at a dosage of 5 mg/kg body weight. In three sheep a ruminal and an abomasal fistula were inserted. The ronidazole determinations were performed by an HPLC method. Oral bioavailability in the fistulated sheep was only 5.5 +/- 1.8% (mean +/- SE). Somewhat lower values (4.6 +/- 1.4%) were obtained when the drug was administered through the ruminal fistula in the rumen. After administration of the same dose directly in the abomasum through the intraabomasal fistula, bioavailability was increased to 86.0 +/- 8.9%. In the non-fistulated sheep, oral biodisponibility was 2.6 +/- 0.5%. After water was restricted for 48 h before the oral ronidazole administration to these sheep, bioavailability was slightly increased (6.0 +/- 3.1%). When desmopressin acetate was injected i.v. before the oral ronidazole administration, bioavailability was 10.6 +/- 6.5%. When glypressin, another vasopressin analogue, was used, oral bioavailability was not influenced: 2.4 +/- 1.3%. Ronidazole was also incubated with ruminal contents and the ronidazole concentration decreased with a first order rate constant of 0.122 +/- 0.050 min-1 (mean +/- SE). These results suggest that oral administration of ronidazole to sheep is of little therapeutic use, because most is metabolised by the ruminal micro-organisms before it can reach the circulation. A second conclusion we can make is that it is very difficult, if not impossible, at least with the methods used, to influence gastro-intestinal motility in sheep to get a reproducible closure of the oesophageal groove.

Disposition of human drug preparations in the horse. II. Orally administered fencamfamine.

A gas chromatographic method to measure urinary levels of the central nervous system stimulant fencamfamine and some of its metabolites is described. When 100 mg fencamfamine was given orally to four horses the parent drug could not be detected in the urine. After enzymatic hydrolysis of the urine the major human metabolite, N-desethylated fencamfamine, only accounted for 1% of the dose in 12 h. The major equine metabolites were conjugated parahydroxylated compounds representing 18% of the dose. With regard to horse doping control and analysis, the injudicious use of human doping routine methods for the detection of fencamfamine in equine urine could lead to false negative results.

Disposition of human drug preparations in the horse. I. Rectally administered indomethacin.

A high-performance liquid chromatographic method to measure urinary indomethacin levels is described. In 0.5 ml urine, 1 micrograms/ml of indomethacin could be detected. Alkaline hydrolysis of urine resulted in the decomposition of indomethacin. When two suppositories of Indocid corresponding to 200 mg indomethacin were administered rectally to four horses the drug was rapidly absorbed and remained detectable in urine from 1 to 12 h. The excretion rate peaked after 2-3 h while the maximal concentration ranged from 18.5 to 80.6 micrograms/ml. Only 8 to 16% of the indomethacin dose was eliminated in urine after 12 h. A fraction of the dose was excreted as the glucuronide conjugate.

The influence of diuretics on the excretion and metabolism of doping agents--V. Dimefline.

A sensitive method for the quantitative determination of the respiratory stimulant dimefline in 5 ml urine using capillary gas chromatography with nitrogen specific detection is presented. After the oral administration of a therapeutical amount of 16 mg dimefline to five subjects only 0.26 +/- 0.16% of the dose is excreted as the conjugated drug in 24 h. The maximum excretion rate occurred 3 h after dosing, the peak concentration being 154 +/- 60 ng ml-1. The influence of diuretics taken 2 h after the administration of dimefline was studied in three subjects. From these results it appeared that the use of acetazolamide and hydrochlorthiazide in order to circumvent a positive dimefline doping case is questionable. Due to the potent diuretic effect of furosemide, the intake of this diuretic could result in a suppression of the dimefline concentration below the detection limit of 10 ng ml-1.

Urinary excretion of theobromine in horses given contaminated pelleted food.

A high pressure liquid chromatographic (HPLC) method for measuring the theobromine content in cocoa husks, pelleted food and horse urine is described. Starting with 2 ml of urine, concentrations of 500 ng/ml could easily be detected. When feed containing 38.4 mg of theobromine was given twice daily to horses for 2 1/2 days, two days were needed after the last intake before the theobromine concentrations fell below the threshold value of 2 micrograms/ml. The time at which the peak excretion rate occurred varied from 2 to 12 h after the last administration, while the excretion rate seemed to be dependent on the urinary flow. Theobromine could not be detected in plasma after administration in this way.

The influence of diuretics on the excretion and metabolism of doping agents: Part VI. Pseudoephedrine.

A capillary gas chromatographic method with nitrogen specific detection is presented for measuring pseudoephedrine and its major metabolite norpseudoephedrine in urine after derivatization with trifluoroacetic anhydride. After the oral intake of 49.2 mg pseudoephedrine (ACTIFED) the active substance is nearly quantitatively excreted in urine over a 48 hr period. From 1 to 7 percent is metabolized to norpseudoephedrine. The intake of acetazolamide results in a suppression of the pseudoephedrine concentration for at least 12 h. The diuretic effect after the intake of 1.51 mineral water could be compared with the effect obtained with 1 mg bumetanide and results in a decrease in pseudoephedrine concentration by a factor 4 for several hours.

Urinary concentrations of codeine and morphine after the administration of different codeine preparations in relation to doping analysis.

A capillary GC method with nitrogen-specific detection is described for the analysis of codeine and morphine in urine. Both drugs were determined after enzymatic hydrolysis of the urine. Morphine was derivatized with trifluoroacetic anhydride. For 5-ml samples of urine, the lower detection limits for accurate quantitation were 50 ng ml-1 and 100 ng ml-1 for morphine and codeine, respectively. Both codeine and morphine were already detectable in urine 1 h after the intake of the analgesic preparation Perdolan. Codeine excretion and concentration peaked 2 h after administration of a dose. The percentage of the dose excreted as codeine was 3.0-6.2%. Administration of the antitussive preparation Bisolvon Griblettes resulted in detectable codeine and morphine levels for at least one day; 5.6-9% was excreted as total codeine over 24 h, the conjugated metabolite morphine accounting for 1.7-7.4% of the dose. Nearly the same amounts of codeine and morphine were excreted after administration of the antitussive syrup Bronchodine. The maximum excretion rate of codeine occurred after 1 h. Generally codeine and morphine remained detectable for 12 h. The results of these administration studies are discussed in relation to the codeine and morphine threshold levels recently introduced by the International Cyclist Union.