Annie Cailleux

Determination of LSD and its metabolites in human biological fluids by high-performance liquid chromatography with electrospray tandem mass spectrometry

A liquid chromatographic procedure with electrospray ionization tandem mass spectrometric detection has been developed and validated for LSD and iso-LSD determination. A one-step liquid-liquid extraction on 1 ml blood or urine was used. The lower limit for quantitative determination was 0.02 microg/l for LSD and iso-LSD. The analytical procedure has been applied in two positive cases (case 1: LSD=0.31 microg/l, iso-LSD=0.27 microg/l in plasma and LSD=1.30 microg/l, iso-LSD=0.82 microg/l in urine; case 2: LSD=0.24 microg/l, iso-LSD=0.6 microg/l in urine). LSD metabolism was investigated using MS-MS neutral loss monitoring for the screening of potential metabolites. The main metabolite was 2-oxo-3-hydroxy-LSD (O-H-LSD) present in urine at the concentrations of 2.5 microg/l and 6.6 microg/l, respectively, for case 1 and 2, and was not present in plasma. Nor-LSD was also found in urine at 0.15 and 0.01 microg/l levels. Nor-iso-LSD, lysergic acid ethylamide (LAE), trioxylated-LSD, lysergic acid ethyl-2-hydroxyethylamide (LEO) and 13 and 14-hydroxy-LSD and their glucuronide conjugates were detected in urine using specific MS-MS transitions.

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of diazepam, atropine and pralidoxime in human plasma

A high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) procedure for the simultaneous determination of diazepam from avizafone, atropine and pralidoxime in human plasma is described. Sample pretreatment consisted of protein precipitation from 100microl of plasma using acetonitrile containing the internal standard (diazepam D5). Chromatographic separation was performed on a X-Terra MS C8 column (100mmx2.1mm, i.d. 3.5microm), with a quick stepwise gradient using a formate buffer (pH 3, 2mM) and acetonitrile at a flow rate of 0.2ml/min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges of 1-500ng/ml for diazepam, 0.25-50ng/ml for atropine and 5-1000ng/ml for pralidoxime. The coefficients of variation were always <15% for both intra-day and inter-day precision for each analyte. Mean accuracies were also within +/-15%. This method has been successfully applied to a pharmacokinetic study of the three compounds after intramuscular injection of an avizafone-atropine-pralidoxime combination, in healthy subjects.

Comparison of the reactivity of oxaliplatin, Pt(diaminocyclohexane)Cl2 and Pt(diaminocyclohexane1)(OH2)22+with guanosine and L-methionine

The initial rates of reactivity of oxaliplatin, its metabolites Pt(dach)Cl2 and Pt(dach)(OH2)2(2+) with guanosine and L-met in water, NaCl and phosphate were compared. Versus guanosine, the most reactive molecule was Pt(dach)(OH2)2(2+), about 40 fold that of oxaliplatin, the least reactive was Pt(dach)Cl2, Versus L-met, Pt(dach)(OH2)2(2+), was also the most reactive species but only about 2 fold more reactive than Pt(dach)Cl2 and oxaliplatin. Pt(dach)(OH2)2(2+) was approximately 3 fold less reactive versus methionine than guanosine whereas oxaliplatin and Pt(dach)Cl2 were about seven fold more reactive versus methionine than guanosine. Thus, the three platinum compounds oxaliplatin, Pt(dach)Cl2 and Pt(dach)(OH2)2(2+) react with L-met but only the Pt(dach)(OH2)2(2+) has a high reactivity with guanosine. Oxaliplatin, which is stable in water, has to be transformed in the presence of chloride in chloro-derivatives which are aquated to become active particularly versus guanosine. These data demonstrate that oxaliplatin has similarities with cisplatin in terms of chloride versus water coordination and in terms of dependence on chloride concentration for transformations.

Transport measurements across Caco-2 monolayers of different organic and inorganic selenium: influence of sulfur compounds.

The transport and uptake of the most common Se compounds, selenate (SeO42−), selenite (SeO32−), selenomethionine, and selenocystine, were investigated using confluent monolayers of Caco-2 cells, a human carcinoma cell line. Comparative measurements were performed in the absorptive (apical to basolateral side) and exsorptive (basolateral to apical side) directions. Apparent permeability coefficients (Papp), calculated from transport experiments in the absorptive direction, showed increasing values in the following rank order: about 1×10−6 cm/s ≤ mannitol ≤ SeO32− ≤ selenocystine < selenomethionine < SeO42− ≤ about 16×10−6 cm/s. The ratios of the Papp measured in the absorptive versus exsorptive directions indicated that only the organic forms presented a net polarized transport (Papp ratio ≫1), suggesting the presence of a transcellular pathway. No significant excretion was observed. The transport of selenomethionine was inhibited by its sulfur analog, methionine, suggesting a common transport mechanism. In contrast, an inhibition of the transport of selenocystine by cysteine was not observed. From the two substrates tested, sulfate and thiosulfate, only thiosulfate inhibited the transport of SeO42−. This effect was also observed for SeO32− (i.e., was unspecific), which questioned the assertion of a common transport for sulfate and SeO42− and may confirm the paracellular pathway of SeO42− suggested by the Papp ratio of about 1. The addition of glutathione (GSH) in large excess had no consequence on the passage of SeO32− but strongly increased the uptake (about fourfold). The liquid chromatography — mass spectrometry (LC-MS) data showed that, in the ionic condition of incubation medium, GSH promptly reduced SeO32− (≤2 min) in its elemental form Se0, which cannot ascribe to selenodiglutathione a direct role in the effect of GSH.

Plasma sialic acid as a marker of the effect of the treatment on metastatic colorectal cancer

The concentration of total sialic acid (TSA) is increased in the plasma of patients with many types of cancer. The purpose of this study was to assess the usefulness of the TSA marker in predicting the efficacy of the treatment, and to compare TSA with two common markers, carcinoembryonic antigen (CEA) and the carbohydrate antigen 19-9 (CA 19-9). The study was performed on 44 patients treated for advanced colorectal carcinoma by a weekly 8 h continuous infusion of 5-fluorouracil (1300 mg/m2) plus bolus injection of L-folinic acid (100 mg/m2). TSA, CEA and CA 19-9 levels were measured before and after 3 months of treatment and their variations analysed as a function of the response to the treatment. TSA levels of patients with metastatic colorectal carcinoma before treatment (959 +/- 265 mg/l) were significantly higher than those of 32 healthy people (584 +/- 99 mg/l). The percentage of patients with TSA concentration above the cut-off level (782 mg/l) was 73% before treatment and 23% after. All patients who experienced an objective response to the treatment (complete, partial or minor response) (n = 29) had a significant decrease of TSA levels (t = 5.96; P < 0.001). When the disease was considered as stabilised (n = 10), TSA changed slightly, but it increased with progressive disease (4 out of 5 patients). Changes in CEA and CA 19-9 did not correlate as well as TSA to the treatment efficacy. Initial levels of TSA did not permit prediction of the efficacy of the treatment since they were not significantly different between the five response groups. TSA seems to be more likely involved in tumour changes than in tumour volume. Its determination could provide useful information about the spreading and metastatic properties of the tumour. TSA normalisation is an indicator of probable tumour growth arrest and its elevation could be a marker of relapse.