Michel Audran

Validation of liquid chromatographic and gas chromatographic methods. Applications to pharmacokinetics.

Validations of analytical methods are important for the generation of data for bioavailability, bioequivalence and pharmacokinetic studies. It is essential to use well defined and fully validated analytical methods to obtain reliable results that can be satisfactorily interpreted. This manuscript is intended to provide guiding principles for the evaluation of a methods overall performance. For this purpose, all of the variables of the method are considered, including sampling procedure, sample preparation, chromatographic separation, detection and data evaluation. The criteria considered are as follows: stability, selectivity, limits of quantification and of detection, accuracy, precision, linearity, recovery and ruggedness. Models used for analytical calibration curves are explained in term of validity and limitations, along with a presentation of the most common statistical considerations used to validate the model. Appropriate means of testing precision and accuracy, the most important factors in assessing method quality, are presented. Other issues, such as re-validation, cross-validation, partial sample volume, endogenous drugs and biological matrix of limited availability, are also discussed.

Effects of erythropoietin administration in training athletes and possible indirect detection in doping control

PURPOSE This study investigated the effects of repeated subcutaneous injection of rHuEpo (50 IU x kg(-1)) in athletes and proposes a method based on the measurement in blood samples of the sTfR/serum protein ratio to determine if the observed values of this marker are related to rHuEpo abuse. METHODS Serum erythropoietin concentrations, and hematological and biochemical parameters were evaluated, during treatment and for 25 d posttreatment in nine training athletes. Moreover, the effect of rHuEpo administrations on the maximum oxygen uptake (VO2max) and ventilatory threshold (VT) of these athletes was also studied. Threshold values for sTfr and the sTfr/serum protein ratio were determined from 233 subjects (185 athletes, 15 athletes training at moderately high altitude, and 33 subjects living at >3000 m). RESULTS Significant changes in reticulocytes, hemoglobin (Hb) concentration, hematocrit (Hct), sTfr, and sTfr/serum proteins were observed during and after rHuEpo treatment. The maximal heart rate of 177 beats x min(-1) at the beginning of the study was significantly higher than the value of 168 beats x min(-1) after 26 d of rHuEpo administration. Compared with the values measured at baseline, the VT measured after rHuEpo administration occurred at a statistically significant high level of oxygen uptake. CONCLUSIONS When oxygen uptake measured at the VT was expressed as a percentage of V02 max, the values obtained were also significantly higher. The increased values of Tfr and sTfr/serum proteins, respectively, above 10 microg x mL(-1) and 153, indicated the probable intake of rHuEpo.

Cyclodextrins and enantiomeric separations of drugs by liquid chromatography and capillary electrophoresis: basic principles and new developments

Investigation of individual drug enantiomers is required in pharmacokinetic and pharmacodynamic studies of drugs with a chiral centre. Cyclodextrins (CDs) are extensively used in high-performance liquid chromatography as stationary phases bonded to a solid support or as mobile phase additives in HPLC and capillary electrophoresis (CE) for the separation of chiral compounds. We describe here the basis for the liquid chromatographic and capillary electrophoretic resolution of drug enantiomers and the factors affecting their enantiomeric separation. This review covers the use of CDs and some of their derivatives in studies of compounds of pharmacological interest.

Drugs for Increasing Oxygen Transport and Their Potential Use in Doping

Blood oxygenation is a fundamental factor in optimising muscular activity. Enhancement of oxygen delivery to tissues is associated with a substantial improvement in athletic performance, particularly in endurance sports. Progress in medical research has led to the identification of new chemicals for the treatment of severe anaemia. Effective and promising molecules have been created and sometimes used for doping purposes. The aim of this review is to present methods, and drugs, known to be (or that might be) used by athletes to increase oxygen transport in an attempt to improve endurance capacity. These methods and drugs include: (i) blood transfusion; (ii) endogenous stimulation of red blood cell production at altitude, or using hypoxic rooms, erythropoietins (EPOs), EPO gene therapy or EPO mimetics; (iii) allosteric effectors of haemoglobin; and (iv) blood substitutes such as modified haemoglobin solutions and perfluorochemicals. Often, new chemicals are used before safety tests have been completed and athletes are taking great health risks. Such new chemicals have also created the need for new instrumental strategies in doping control laboratories, but not all of these chemicals are detectable. Further progress in analytical research is necessary.

EFFECTS OF MODERATE EXERCISE ON THE PHARMACOKINETICS OF CAFFEINE

SummaryThe effect of moderate exercise on the kinetics of caffeine in 12 healthy volunteers-6 heavy coffee drinkers (HD) and 6 light coffee drinkers (LD) has been studied. Kinetics at Rest was measured first (R): the subjects remained at rest for 8 h after a single 250 mg dose of caffeine. One week later, the Exercise Kinetics (E) was measured under the same conditions, but with the subjects performing moderate exercise (30% of VO2 max) during the first hour of the study.Exercise raised the maximal plasma caffeine concentrations (R: 7.28; E: 10.45) and reduced both the half-life (R 3.99 h; E 2.29 h) and the volume of distribution (R 371; E 20.91). Both during exercise and at rest, HD had a greater half-life elimination and volume of distribution than LD.The results suggest potentiation of the effects of caffeine during exercise and an increase in its distribution due to regular heavy coffee intake.

High-performance liquid chromatographic assay for minocycline in human plasma and parotid saliva.

A sensitive and specific high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of minocycline in human plasma and parotid saliva samples. Samples were extracted using an Oasis HLB cartridge and were injected into a C8 Nucleosil column. The HPLC eluent contained acetonitrile-methanol-distilled water-0.1% trifluoroacetic acid (25:2:72.9:0.1, v/v). Demeclocycline was used as internal standard. The assay showed linearity in the tested range of 0.1-25 microg/ml. The limit of quantitation was 100 ng/ml. Recovery from plasma or parotid saliva averaged 95%. Precision expressed as %CV was in the range 0.2-17% (limit of quantitation). Accuracy ranged from 93 to 111%. In the two matrices studied at 20 and 4 degrees C, rapid degradation of the drug occurred. Frozen at -30 degrees C, this drug was stable for at least 2 months, the percent recovery averaged 90%. The methods ability to quantify minocycline with precision, accuracy and sensitivity makes it useful in pharmacokinetic studies.

Population Pharmacodynamics for Monitoring Epoetin in Athletes

SummaryEpoetin (recombinant human erythropoietin) is used by some endurance athletes to increase oxygen transport and aerobic power in an attempt to improve endurance capacity and recovery during training and competition. Although currently on the list of banned substances issued by the medical commission of the International Olympic Committee, the use of epoetin as an ergogenic agent remains uncontrollable using classical analytical techniques. In the present paper, after subcutaneous administration of repeated high doses (200 units/kg), the pharmacokinetics of epoetin were evaluated in 18 athletes. The mean elimination half-life was 42.0 hours. The total clearance/bioavailability (F) and the volume of distribution/F averaged 0.05 L/h/kg and 2.95 L/kg, respectively. Significant changes in ferritin (fr), soluble transferrin receptor (sTfR) and ratio (sTfR/fr) were observed after administration. A population sigmoidal asymptotic maximum expected effect concentration (emax) model has been developed to assess and quantify the relationship between the changes in sTfR, fr and the ratio sTfR/fr secondary to the repeated administration of epoetin. The mean population parameters were as follows: emax: 12.6 mg/L, 44.9 µtg/Land 2313.5; the concentration producing 50% of the emax(C50): 18.3,18.1 and 37.7 IU/L; the sigmoidicity factor (gamma): 2.96, 4.17 and 5.15; and ke0 (rate constant of transfer between central and effect compartment): 1.29 × 10−3, 7.14 × 10−3 and 3.47 × 10−3/h for the 3 markers, respectively. Moreover, in this paper, we propose an appropriate statistical methodology based on the measurement of sTfR, fr and sTfR/fr from blood samples to decide if the observed values of these markers could be related, at a given probability risk, to the administration of epoetin. Values greater than 10 mg/L for sTfR and 403 for sTfR/fr indicate a probable intake of epoetin. Because of the large interindividual variability of fr, it has not been possible to define a threshold value for this parameter. The sTfR seems to be the most effective marker in doping control. An increased haematocrit with concomitant changes in sTfR and sTfR/fr values seems to supply a valuable diagnostic tool that avoids false-positive doping and allows the identification of epoetin abusers. Blood samples could be collected using capillary blood from the fingertip or earflap. These controls could be carried out during training because they are easy to perform and do not add substantially to total screening costs. At present, this is the only tool available for detecting epoetin abusers during competition.

Pharmacokinetics/Pharmacodynamics of Recombinant Human Erythropoietins in Doping Control

The purpose of this paper is: (i) to compare recombinant human erythropoietin (rHuEPO) pharmacokinetics in athletes and healthy individuals; and (ii) to report pharmacokinetic/pharmacodynamic (PK/PD) studies performed in athletes. Effect parameters in PK/PD studies included: (i) red blood cell variables (haematocrit, reticulocyte count); and (ii) markers of iron metabolism (serum soluble transferrin receptors [sTfR], ferritin [fr] and sTfR: fr ratio). To understand the choice of these markers, we first performed a brief review of the pharmacological effects of rHuEPO. Few studies have been conducted in healthy individuals and there are minimal references concerning pharmacokinetics in athletes. A ‘flip-flop’ phenomenon was noted after subcutaneous administration. The pharmacokinetics appeared linear from 50–1000 U/kg, but this linearity was not observed at the lowest dose of 10 U/kg. A negative-feedback loop of endogenous erythropoietin production occurred at the end of treatment. The half-life of the terminal part of the curves seemed to be slightly higher in athletes (36–42 vs 32 hours) than in untrained individuals and total clearance tended to be greater (17.5 vs 6.5 mL/h/ kg).In conclusion, more investigations are needed to better understand the relationship between rHuEPO administration and changes in haematological and iron-metabolism parameters in athletes, particularly after chronic low-dose administration of rHuEPO.

High-performance liquid chromatographic determination of tazobactam and piperacillin in human plasma and urine.

A high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) absorbance was developed for the analysis of piperacillin-tazobactam (tazocillin), in plasma and urine. The detection was performed at 218 nm for tazobactam and 222 nm for piperacillin. The procedure for assay of these two compounds in plasma and of piperacillin in urine involves the addition of an internal standard (ceftazidime for tazobactam and benzylpenicillin for piperacillin) followed by a treatment of the samples with acetonitrile and chloroform. To quantify tazobactam in urine, diluted samples were analysed using a column-switching technique without internal standard. The HPLC column, LiChrosorb RP-select B, was equilibrated with an eluent mixture composed of acetonitrile-ammonium acetate (pH 5). The proposed technique is reproducible, selective, and reliable. The method has been validated, and stability tests under various conditions have been performed. Linear detector responses were observed for the calibration curve standards in the ranges 5-60 micrograms/ml for tazobactam, and 1-100 micrograms/ml for piperacillin and spans what is currently though to be the clinically relevant range for tazocillin concentrations in body fluids. The limit of quantification was 3 micrograms/ml for tazobactam and 0.5 microgram/ml for piperacillin in plasma and urine. Extraction recoveries from plasma proved to be more than 85%. Precision, expressed as C.V., was in the range 0.4-18%.

Pharmacokinetics and haematological parameters of recombinant human erythropoietin after subcutaneous administrations in horses

The pharmacokinetics of recombinant human Epo (rHuEpo) were investigated after subcutaneous administration to horses. Four horses received a single 30IU kg-1 dose of rHuEpo. One horse received three repeated doses of 120 IU kg-1 at 48 h intervals. Plasma erythropoietin (Epo) was measured by radioimmunoassay. In both cases pharmacokinetic parameters were evaluated using a one-compartment open model and first-order input and output rates. The mean values (+/-SD) for elimination half-life, CL/F, and Vd/F after a single dose were 12.9 +/- 3.34 h, 11.8 +/- 4.96 L h-1, and 233 +/- 126 L, respectively. After repeated doses, elimination half-life, CL/F, and Vdss/F were 11.3 h, 8.94 L h-1, and 145.6 L, respectively. No significant differences were observed between the haematological parameters after a single 30 IU kg-1 administration compared to baseline values. Multiple and high doses of rHuEpo modified red blood cells, haemoglobin, and hematocrit. According to our results, plasma Epo assay can help, during an antidoping control procedure, to support a positive result only up to 72 h after the last rHuEpo.