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Benzodiazepines are some of the most often co-abused substances in methadone treatment. Their identification in biological samples is of great importance in toxicological screening and in methadone maintenance program. The aim of this report is to describe, the analytical data making possible the identification of benzodiazepines and/or metabolites using a reversed phase liquid chromatography coupled with diode array detection.

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HPLC using fluorescence detection has already become the most accepted method for the determination of aflatoxins due to its several advantages over other analytical methods. Both normal- and reversed-phase HPLC can be used. However the reversed-phase HPLC methods are more popular. Liquid chromatographic determination of aflatoxins using fluorescence detection and its application in food analysis is reviewed in this article.

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Cerium is used for its antiseptic and immunomodulatory properties in burn injury. We have developed a cerium-doped clay to replace existing ointments. Adsorption and release of cerium (Ce3+) by diosmectite were studied at 22+/-2 degrees C, in the presence of various other cationic species. Simple spectrofluorimetric determination of cerium was used (lambdaexc=240 nm/lambdaem=360 nm). Cerium binding reached a plateau within 2 min and was a function of the electrolyte content of the solution in contact with the clay. Langmuir isotherm treatment led to a maximal binding capacity of 66 mg of Ce3+ per gram of clay. Partial release occurred within 2 min (19% in the presence of isotonic NaCl solution). The ionic strength of the solution, and the ionic radius and charge of the electrolytes present in the bathing solution significantly influenced cerium release, in contrast to pH and temperature changes. These results strongly point to a cationic exchange mechanism between diosmectite and cerium solution.

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The fluorescence properties of the aflatoxins M1, Q1, P1 in solution and the effect of various cyclodextrins (alpha-, beta-, gamma-, hydroxypropyl-beta- and alpha-beta-heptakis-di-O-methyl-beta-) on their fluorescence emission were studied. Among the aflatoxins, a substantial enhancement of the fluorescence emission of aflatoxin Q1 in the presence of aqueous solutions of alpha-, beta-, hydroxypropyl-beta, and alpha-beta-heptakis-di-O-methyl-beta-cyclodextrin, was observed. On the contrary, gamma-cyclodextrin proved to be inefficient to enhance the fluorescence properties of this compound. No important fluorescence enhancement was found for aflatoxins P1 or M1 for any of the cyclodextrin derivatives tested. The complex formation constant (Kf) of these compounds with beta-cyclodextrin was chromatographically determined, and from the results obtained, we can conclude that Kf cannot be used alone to explain the fluorescence increase. Thermodynamic studies showed that delta-H and delta-S parameters, associated with the partition of aflatoxins in RP-HPLC, increased when beta-cyclodextrin was added to the eluent.

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Some epoxyethane-/ethynesulfonamides had shown strong filaricidal activity with inconstant reproducibility as a result of a lack of stability in aqueous solution. The degradation in hydroxylic and aprotic solutions of two epoxyethanesulfonamides and one ethynesulfonamide was investigated using TLC, HPLC, GC and mass spectrometry. For both epoxydes, the degradation rate followed first-order kinetics and was more rapid in hydroxylic than in aprotic solutions. The degradation increased with the temperature whereas it was not modified with and without light exposure. Four kinds of degradation products were found: the first one involved the oxidation of the epoxyde bond, the second the breaking of the N-S bond, the third a desulfonation product and the fourth was not identified. In contrast, the stability of ethynesulfonamide was better than those of epoxyethanesulfonamide. These results suggest that epoxyethanesulfonamides should be kept at +4 degrees C before being injected to animals during the study of biological activity. Since epoxyde compounds are known to have inhibitory effects on parasite energy metabolism enzymes, the compunds were evaluated on two major filarial enzymes: lactate dehydrogenase (LDH) and cytoplasmic malate dehydrogenase (MDH). Both epoxyethanesulfonamides showed only a slight inhibitory effect on filarial LDH and MDH confirming the evidence that the main mode of action of these compounds remains to discover. Moreover, ethynesulfonamide and the degradation products of both epoxyethane-sulfonamides had no effect on LDH and MDH.

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The photochemically induced fluorescence (PIF) properties of tianeptine and some of its metabolites were investigated in acidic (pH 2.3) water-alcohol mixtures at room temperature. Two PIF methods were developed, including bulk solution and flow injection analysis (FIA). Linear calibration plots were established over a concentration range of more than one order of magnitude. Limits of detection ranged from 15 ng ml-1 for FIA-PIF to 25 ng ml-1 in bulk solution. The RSDs were between 3 and 5%. The PIF methods were applied to the determination of tianeptine in a pharmaceutical preparation with recoveries varying from 96 to 106% in bulk solutions and from 98 to 106% for FIA-PIF.

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Lanthanide sensitized luminescence is a very attractive alternative to UV detection and other luminescence techniques, i.e., fluorescence and phosphorescence, in separation science for the detection of drugs and xenobiotics because of the large Stokes shift, narrow emission bands and long lifetime. Some published applications of HPLC determination with lanthanide (Ln3+) sensitized luminescence detection are reviewed. Advantages and limitations of this technique are discussed. Normal-phase (NP) HPLC is not influenced by the quenching effect of water whereas reversed-phase (RP) HPLC is applicable to more compounds than NP-HPLC. However, pH adjustment and the quenching effect of water on Ln3+ luminescence are the main drawbacks of RP-HPLC. Elution properties and the need for pH adjustment are two arguments for selecting the mode of addition of Ln3+, i.e., pre- or post-column in the HPLC system. Sensitized Ln3+ luminescence detection is a much more specific method of detection than UV or fluorescence detection after HPLC separation but nevertheless, in some cases, does not always exhibit a significant increase in analytical performance when the donor itself is a strong fluorophore. The development of more powerful excitation sources could improve the limit of detection of the Ln3+ sensitized detection technique. This review suggests that it would be useful to obtain predicting factors about the drug to establish whether the latter is suitable to be measured using an HPLC-Ln3+ approach.

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1. The tissue distribution and metabolism of a new filaricidal agent P903 (N-[(2-phenylethynyl)sulfonyl]morpholine) were studied in rat. 2. After s.c. administration of 14C and 13C P903, the Tmax in the blood was observed on day 2. Elimination was slow and > 95% was bound to protein. Radioactivity was distributed in the whole organism but particularly in erythrocytes and the lymphatic channel. Four days later, > 60% of the radioactivity was excreted in urine and faeces at equal amounts and 15% remained at the injection point. 3. In all biological fluids tested no P903 was found but only its metabolites. 4. One principal metabolite, the N-[(2-phenyloxo-2-ethane) sulphonyl] morpholine or oxosulphonamide was identified in blood, urine and faeces as compared with the reference compound by GC/MS and NMR. This latter molecule was detected following hydrolysis by hydrochloric acid but not with beta glucuronidase/sulphatase. 5. Unconjugated and conjugated oxosulphonamide represented > 85% of the radioactivity at all times tested in blood but only 38 and 35% respectively of urinary and faecal radioactivity on day 1 after the administration of the labelled drug. 6. Thus, P903 is rapidly converted to a reactive metabolite, probably an oxirene, which is then conjugated with endogenous components to form conjugated oxosulphonamide and an unknown metabolite. The role of this reactive metabolite in antifilarial activity seems to be very important in understanding the mechanism of action of P903.

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The fluorescence properties of five substituted benzamides, including alizapride, metoclopramide, sulpiride, sultopride and tiapride, were investigated at several pH values and in various solvents (dimethyl sulfoxide, ethanol, ethylene glycol, methanol, propan-2-ol, tetrahydrofuran and water). Except for alizapride, the fluorescence intensities were found to be higher at acidic (1-6) than at alkaline (8-12) pH values. Using the optimum solvent (aqueous solutions) and appropriate pH conditions, linear spectrofluorimetric calibration curves were established over a concentration range of about two orders of magnitude, with correlation coefficients larger than 0.996. Limits of detection were between 1 and 13 ng ml-1, depending on the compound. The method was applied to the determination of benzamides in pharmaceutical preparations and in human urine, with recoveries ranging from 94 to 108% and from 93 to 104%, respectively.

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A new and highly sensitive method for the detection of the important mycotoxin, citrinin, has been developed. Spectroscopic studies demonstrate that the fluorescence of this metabolite is influenced by the pH of the environment. This fact was exploited in the chromatographic determination of citrinin with fluorescence detection. The proposed method, based on the addition of 1 M hydrochloric acid as an acidic post-column reagent, has a limit of detection of 0.9 center dot 10(-7) M. Analytical validation shows that linearity can be assumed from 2 center dot 10(-7) to 10(-4) M citrinin. The repeatability and reproducibility are satisfactory, with R.S.D. = 5.1% (n = 9, c = 10(-5) M) and R.S.D. = 7.2% (n = 9, c = 10(-5) M). The method was also applied to the determination of this mycotoxin produced by mould cultures isolated from soft cheese and also from soft cheese and also from cheese extracts spiked with citrinin. The specificity of the method is demonstrated and the necessity for post-column acidification is illustrated on real samples.

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Measurement of fluorescence increase was used for the comparative quantification of the effect that several cyclodextrins (alpha-, beta-, heptakis-2,6-beta-omicron-dimethyl- and gamma-) produce on the fluorescent response of aflatoxins B1 and G1. This constitutes a new chromatographic method with stability of the mobile phase, and shows general improvements in the chromatographic conditions with respect to other methods (especially those using an iodine reservoir as a postcolumn reactor). A C18-type column was used, with methanol-water (60:40, v/v) as the mobile phase. The excitation phase of the natural fluorescence of aflatoxins, a 10(-2) M solution of each cyclodextrin, was introduced postcolumn. The determination of the elution order aflatoxin G2 > G1 > B2 > B1 was performed for each phase in less than 15 min. As expected using an aqueous-alcoholic medium, an increase in the fluorescence response of aflatoxins with an unsaturated furanic ring was found to occur with all the cyclodextrins studied, except gamma-cyclodextrin. The observed increase was larger for heptakis-2,6-beta-omicron-dimethyl- than for beta-cyclodextrin (to our knowledge, the only cyclodextrin previously described in the literature to serve for the determination of aflatoxins). The difference is of the order of 70.1-fold in the case of aflatoxin G1 and 45.2-fold in the case of aflatoxin B1. The detection limit in the mobile phase used was determined (for aflatoxin B1) for beta-cyclodextrin and 2,6-beta-omicron-dimethylcyclodextrin (signal-to-noise ratio 1:3) to be 4 and 9 mg 1(-1), respectively.

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Therapeutic monitoring of patients participating in methadone maintenance programmes requires the qualitative or quantitative determination of methadone in urine before and during treatment. Generally immunoassay techniques are used for this determination, but more and more often measurement of plasma or serum levels are required by the physician. The possibility of adapting a commercially available FPIA kit (TDX ABBOTT) for urinary assays to the serum determinations was investigated. The specific modifications of the method and the dilution conditions for high concentrations are described. Results were compared with those obtained by liquid chromatography.

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Diacetyl, in deaerated solutions, is a major room temperature phosphorescent compound. Its phosphorescence quenching is proposed in order to quantify results on phenothiazine derivatives. It is established from the luminescent data on phenothiazines that diacetyl phosphorescence can be quenched quantitatively by these compounds. This detection mode is applied to liquid chromatography limitations as well as the mechanism of the quenching are discussed.

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Antibiotic susceptibility of 948 bacterial strains isolated from varied samples essentially proceeding from urinary infections in five Paris psychiatric Hospitals was determined by disk diffusion method. E. coli, P. mirabilis, Klebsiella spp., P. aeruginosa et S. aureus are the predominant bacteria. 40% of S. aureus are methicilline resistant. Enterobacteriaceae are progressively becoming resistant to aminopenicillines, but remain sensitive to third generation cephalosporines. They are still susceptible to first generation quinolones. At least, if no resistance of P. aeruginosa to imipeneme has been reported, 30% of strains are resistant to ciprofloxacine. Resistance phenotypes to antibiotics of the strains isolated in patients from psychiatric Hospitals are located between those observed in out patients and in patients from general Hospitals. However, we noticed a worrying evolution of resistance to those encontered in psychiatric Hospitals. Therefore, a multiresistant strains emergence monitoring must be carried out regulary.

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Cyclosporin A (CsA) is in vivo mainly metabolized by hepatic cytochrome P450 IIIA to more than 21 metabolites, the major ones known as: M1, M17 and M21. The aim of this work is to explore the in vitro metabolism of CsA after incubation, in the presence of NADPH, with renal or hepatic microsomes obtained from rabbits pretreated with rifampycin (enzyme inducer) or erythromycin (enzyme inhibitor). The presumed metabolites were separated by semi-preparative high-performance liquid chromatography (HPLC) and identified in each collected fraction by fluorescence polarization immunoassay (FPIA) (HPLC-FPIA) using a non-specific polyclonal antibody. They were also analyzed by HPLC-mass spectrometry (MS) using fast atom bombardment (HPLC-MS-FAB). Five collected fractions gave positive results with FPIA. The major metabolites found were M1, M17 and M21 after identification by HPLC-MS-FAB and comparison with three corresponding standard metabolites. The CsA biotransformation rates were calculated by the amount of unmetabolized CsA and were linear with time. These mean rates (Vm) for 12-min incubation by renal microsomes of rabbits treated with rifampicin or erythromycin or untreated (control) were 0.11, 0.02 and 0.04 nmol/min x mg microsomal protein, respectively. These rates were 15-, 37-, and 30-fold lower than those obtained with hepatic microsomes of rabbits treated identically. As CsA metabolites are less cytotoxic than the parent drug, this weak renal biotransformation of CsA after in vitro incubation should be one of the mechanisms of its in vivo nephrotoxicity.

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Chemiluminescence avoiding the fluctuations of the light of excitation, Rayleigh and Raman scattering, allows to get detection threshold 100 to 1,000 times lower than fluorimetry. Processes using the luminol, lucigenin, aryloxalic esters and ozone are employed in liquid chromatographic detection. Some pharmaceutical, biological and toxicological examples illustrate their applications.

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Capillary zone electrophoresis with indirect ultraviolet detection was used for the determination of fosfomycin in serum. Running buffer consisted of a mixture of 200 mM sodium borate with 10 mM phenylphosphonic acid used as ultraviolet absorbing background electrolyte. Relationships between the pH of the buffer and the efficiency of the separation (migration times and selectivities) or the sensitivity of detection were investigated. The method was then validated over a 10-100 micrograms ml-1 concentration range to be applied to further therapeutic drug monitoring. The choice of ethylphosphonic acid as internal standard is discussed. The specificity and the linearity of the technique are demonstrated. The inter-day precision was satisfactory with a relative standard deviation of less than 2%. Accuracy was calculated with a standard error near 0.5 and 18% for 100 and 10 micrograms ml-1, respectively.

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Under the words of sensitized luminescence, the processes who include the transfers of energy from a donor to an acceptor are described. They are to the origin of new possibilities of detection in liquid chromatography by emission or inhibition of phosphorescence and fluorescence. These news technologies are illustrated by examples in pharmaceutic, biologic or food applications.

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The emissions of light by biorganisms or these obtained by alchemists were known since long time ago but the are used in analytical chemistry only when STOKES discovered that the intensity of this light was proportional to the quantity of the matter. The very large sensibilities reached, associated with the great separation's ability of the liquid chromatography allows to develop new processes for quantification of very low concentrations of luminescent or no luminescent molecules. Many pharmaceutical, biological toxicological environmental or alimentary applications show that it is possible in liquid chromatography to obtain a detection limit about the pico or femtomole when simple chemical process are used: direct potentialization of luminescence by addition of modifiers of the chemical environment of the analytes: solvents, cyclodextrins, surfactants, metallic ions, indirect potentialization of the luminescence by transfer of energy from an excited molecule: sensitized fluorescence and phosphorescence, excitation of the molecule by a chemical reaction or chemiluminescence. These aspects are emphasized and illustrated by some examples in three articles.

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