Study of antifungal agent caspofungin adsorption to laboratory materials.

Treatment of invasive fungal infections with Caspofungin is used as the first-line antifungal agents. The minimum inhibitory concentration value is a test which indicates the degree of sensitivity of a strain regarding a drug. However, no value of minimum inhibitory concentration for caspofungin is available because very variable value is obtained. In this work, we study the link with the adsorption phenomenon of CSF previously described in literature and the lack of minimum inhibitory concentration value. A systematic study of the impact of different parameters on CSF adsorption is reported. The effect of the nature of container material, the aqueous solution pH and the organic solvent proportion was studied. In addition, the possibility of using a coating agent to minimize the adsorption was assayed and evaluated. Results obtained showed the importance of the material used during the manipulation of CSF. The use of acidic pH aqueous solution or the addition of acetonitrile or methanol proportions (50 % and 70 %, respectively) were found efficient to avoid adsorption of CSF on glassware material, which is the relevant strategy for analytical samples of caspofungin. The treatment of HPLC glass vials and 96-well plates with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane reduced the adsorption. The significant adsorption observed in this work especially with plastic materials, questions the results obtained before in different assays and explained the absence of MIC value.

Determination of antifungal caspofungin in RPMI-1640 cell culture medium by column-switching HPLC-FLD.

The actual scenario in the fight against fungal infections forces researchers to carry through with resistance studies to improve the therapies. These studies, which are performed in cell culture media, need accurate and sensitive analytical methodologies. That is why, in this work, an analytical method for caspofungin (CSF) concentration determination in RPMI-1640 cell culture medium with on-line sample treatment was developed and validated. CSF concentration was determined by HPLC-FLD using a column-switching procedure. The chromatographic analysis was carried out in less than 10 min using a C8 column (4 × 4 mm, 5 µm) as extraction stationary phase and a HSS T3 column (4.6 × 100 mm, 5 µm) as the analytical column. The used mobile phases were mixtures of phase A: pH 2 (adjusted with TFA) aqueous phase and phase B: ACN. For the extraction, the composition was (95:5, A:B v/v) and for the analysis (60:40, A:B v/v), both done in isocratic elution mode. These chromatographic conditions allowed reaching a limit of quantification of 10 µg/L, using 100 µL of sample with an injected volume of 40 µL. The proposed method was successfully validated in terms of selectivity, carryover, linear concentration range, accuracy and precision according to the criteria established by the Food and Drug Administration. Available amount of CSF in RPMI-1640 solution was found critical. CSF concentrations remained stable up to 2 h at room temperature. The developed method was applied for the direct analysis of CSF concentrations from in vitro experiments in presence of C. glabrata (CAGL18). The results highlight the decrease of cell proliferation even if the CSF amount decreases too, which asks question about the real value of the efficient concentration for CSF antifungal activity.

Determination of moxifloxacin in growth media by high-performance liquid chromatography.

A direct injection high-performance liquid chromatographic method with column switching has been developed to determine moxifloxacin in Mueller-Hinton broth. A LiChrocart 4-4 pre-column filled with a LiChrospher 100 RP 18, 5 microm and a 150 x 4.6 mm I.D. column packed with a Supelcozil ABZ+ Plus were used and led to a retention time of 5.70 min. Fluorescence detection allowed one to reach a quantification limit of 0.05 microg/ml with a 100-microl sample size. The standard curves were linear from 0.05 to 3.2 microg/ml. Intra- and inter-day imprecisions within the linearity range were < or =4.76 and < or =5.75%, respectively. The mean relative errors for the same day and the day-to-day inaccuracies ranged from -2.93 to +4.50% and from -1.10 to +6.00%, respectively. The method was demonstrated to be useful for pharmacokinetic-pharmacodynamic studies of moxifloxacin in an in vitro model.

New approach for accurate simulation of human pharmacokinetics in an in vitro pharmacodynamic model: application to ciprofloxacin.

An in vitro pharmacodynamic model using a disposable dialyser unit and computer-controlled devices was developed. Feedback control of peristaltic pump flow rates is used to maintain constant flow rates, thus avoiding the problem of the modification of the physical properties of the tubing that generally occurs. Fast equilibrium is obtained with capillaries, which allows simulation of the same kinetic profile in the central and the peripheral compartments. Thus, more accurate simulation of plasma, extracapillary fluid or whole tissue kinetics can be performed. Our model was validated by simulation of a 30 min infusion of a 200 mg dose, and of an oral administration of a 500 mg dose of ciprofloxacin.

Separation methods for antiviral phosphorus-containing drugs.

Among antiviral drugs, phosphorus-containing compounds, foscarnet and cidofovir, present adverse effects including renal toxicity. Since their main therapeutic target is the treatment of CMV retinitis, which needs lifelong maintenance therapy, accurate analytical methods are required for drug monitoring. According to the high hydrophilic property of the two compounds, ion pair reversed-phase HPLC methods were proposed for their separation in drug formulations and biological samples. Their lack of UV absorption at wavelengths above 205 nm does not allow the use of this detection technique for biological fluids. Electrochemical detection methods (coulometry and amperometry) led to a quantification limit of 15 microM for foscarnet. Fluorescent derivatives obtained by modification of cidofovir cytosine nucleus with alpha-haloketones offered advantage over UV detection and allowed to reach a detection limit of 5 ng/ml, making possible investigations on the drug time-course in biological fluids.

Determination of phosphonoformate (foscarnet) in calf and human serum by automated solid-phase extraction and high-performance liquid chromatography with amperometric detection.

An isocratic high-performance liquid chromatographic method with automated solid-phase extraction has been developed to determine foscarnet in calf and human serums. Extraction was performed with an anion exchanger, SAX, from which the analyte was eluted with a 50 mM potassium pyrophosphate buffer, pH 8.4. The mobile phase consisted of methanol-40 mM disodium hydrogenphosphate, pH 7.6 containing 0.25 mM tetrahexylammonium hydrogensulphate (25:75, v/v). The analyte was separated on a polyether ether ketone (PEEK) column 150x4.6 mm I.D. packed with Kromasil 100 C18, 5 microm. Amperometric detection allowed a quantification limit of 15 microM. The assay was linear from 15 to 240 microM. The recovery of foscarnet from calf serum ranged from 60.65+/-1.89% for 15 microM to 67.45+/-1.24% for 200 microM. The coefficient of variation was < or = 3.73% for intra-assay precision and < or =7.24% for inter-assay precision for calf serum concentrations ranged from 15 to 800 microM. For the same samples, the deviation from the nominal value ranged from -8.97% to +5.40% for same day accuracy and from -4.50% to +2.77% for day-to-day accuracy. Selectivity was satisfactory towards potential co-medications. Replacement of human serum by calf serum for calibration standards and quality control samples was validated. Automation brought more protection against biohazards and increase in productivity for routine monitoring and pharmacokinetic studies.

Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and Mueller-Hinton broth for pharmacokinetic/pharmacodynamic studies.

An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller-Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 microm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)-methanol (97:3, v/v) and the transfer of the analyte by a back-flush mode to a 150 x 4.6 mm I.D. column packed with a Kromasil C8 5 microm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)-acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 microg/ml with a 40-microl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.

Pharmacokinetic modeling of ethyl loflazepate (Victan) and its main active metabolites.

A simultaneous consideration of plasma and urine data of unchanged drug and active metabolites, taking into account the metabolic process of the precursor, is described. The maximum likelihood principle was used to estimate parameters. This methodology is highly efficient in determining the contribution of the two main and active metabolites in the pharmacological response of ethyl loflazepate. It also may serve in the search for optimum dosage regimens in clinical practice.

Effect of renal failure on the pharmacokinetics of ethyl loflazepate (Victan) in man.

The kinetics of ethyl loflazepate were studied in patients with various degrees of renal failure. A strong correlation was noted between urinary excretion of metabolite loflazepate and creatinine clearance. In contrast, elimination half-life and total plasma clearance of the sum of loflazepate + descarboxyloflazepate seemed to be independent of the degree of renal impairment. These results indicate the absence of a risk of accumulation of the 2 main and active metabolites of ethyl loflazepate in patients with renal failure.

New approach in bioavailability study of two formulations of ethyl loflazepate.

The disposition of ethyl loflazepate (Victan) was studied on 12 healthy male volunteers after administration of the two oral formulations of the drug: one 4-mg tablet and two 2-mg tablets according to an open crossover single dose comparison. A model-independent analysis and a model-dependent approach were used to treat experimental data. Appropriate statistical tests (ANOVA, Wilcoxon test, Westlake test for model-independent parameters; principal components analysis, Hotelling T2 test for model-dependent parameters) demonstrated the bioequivalence of the two formulations.