Felipe Barreto

Application of a LC–MS/MS method for evaluating lung penetration of tobramycin in rats by microdialysis

HIGHLIGHTSA sensitive, precise and accurate LC–MS/MS method was validated to quantify to bramycin in plasma and microdialysate samples.A very low injection volume (4 &mgr;L) was used and a short retention time (˜6 min) was obtained.Microdialysis probe relative recovery for tobramycin is 30% in vitro and in vivo for a flow rate of 1.0 &mgr;L min−1.Tobramycin relative recovery is independent of drug concentration and technique used (dialysis or retrodialysis).Tobramycin has a high penetration in interstitial fluid with similar free lung and plasma concentrations in healthy rats. ABSTRACT A bioanalytical LC–MS/MS method was developed and validated to determine tobramycin in plasma and lung microdialysate samples. Tobramycin was separated using a C18 column and a mobile phase consisting of water and acetonitrile, both with 10 mM of heptafluorobutyric acid, eluted as gradient. Apramycin was used as internal standard (IS) for plasma samples. Drugs were monitored using electrospray ionization operating on positive mode (ESI+) monitoring the transitions 468.2 > 163.3 m/z for tobramycin and 540.3 > 217.2 m/z for IS. The method showed linearity in the concentration range of 0.1–50 &mgr;g mL−1 for microdialysate and 0.5–100 &mgr;g mL−1 for plasma with coefficients of determination ≥0.991. The inter‐ and intra‐day precision, the accuracy and the stability of the drug in different conditions were in accordance with the limits established by US Food and Drug Administration guideline. The concentrations of tobramycin in plasma and lung microdialysate, determined using CMA/20 probes and a Ringer solution at a flow rate of 1 &mgr;L min−1, were evaluated in healthy Wistar rats after a 10 mg kg−1 i.v. bolus dose. Samples were harvested up to 12 h post‐dose. Before animals experiments, probe recovery was determined by dialysis and retrodialysis in vitro and by retrodialysis in vivo. Probes recovery was independent of drug concentration and method of determination. In vivo recovery was 27.74 ± 7.70%. Pharmacokinetic parameters were estimated by non‐compartmental analysis using the software Phoenix®. The estimated area under the curve (AUC0‐∞) was 128 ± 19 &mgr;g h mL−1 and 105 ± 12 &mgr;g h mL−1 for plasma and lung, respectively. Tobramycin plasma clearance was 0.07 ± 0.01 L/h/kg and the volume of distribution was 0.49 ± 0.09 L/kg. Elimination half‐life in plasma was 4.4 ± 0.7 h and in lung, 4.2 ± 0.56 h. The free tissue/free plasma AUC0‐∞ ratio was 0.94. This is the first study showing a validated method to quantify tobramycin in microdialysate samples and to evaluate the lung interstitial concentration of the drug.

Neurochemical responses to chromatic and achromatic stimuli in the human visual cortex

In the present study, we aimed at determining the metabolic responses of the human visual cortex during the presentation of chromatic and achromatic stimuli, known to preferentially activate two separate clusters of neuronal populations (called “blobs” and “interblobs”) with distinct sensitivity to color or luminance features. Since blobs and interblobs have different cytochrome-oxidase (COX) content and micro-vascularization level (i.e., different capacities for glucose oxidation), different functional metabolic responses during chromatic vs. achromatic stimuli may be expected. The stimuli were optimized to evoke a similar load of neuronal activation as measured by the bold oxygenation level dependent (BOLD) contrast. Metabolic responses were assessed using functional 1H MRS at 7 T in 12 subjects. During both chromatic and achromatic stimuli, we observed the typical increases in glutamate and lactate concentration, and decreases in aspartate and glucose concentration, that are indicative of increased glucose oxidation. However, within the detection sensitivity limits, we did not observe any difference between metabolic responses elicited by chromatic and achromatic stimuli. We conclude that the higher energy demands of activated blobs and interblobs are supported by similar increases in oxidative metabolism despite the different capacities of these neuronal populations.

Sensitive analytical method to quantify clindamycin in plasma and microdialysate samples: Application in a preclinical pharmacokinetic study

HighlightsThe LC–MS/MS method validated to quantify clindamycin in plasma and microdialysate was selective, precise, accurate, and sensitive.The method advantages were the minimization of analysis time and sample volume, increasing sensitivity and the upper limit of detection.The method was successfully applied in an investigation of clindamycin’s subcutaneous tissue penetration in Wistar rats.The pharmacokinetic results indicate that the method may be easily used to investigate tissue distribution of clindamycin.The subcutaneous tissue penetration factor was 1.18 ± 0.05 indicating that clindamycin is distributed by diffusion. Abstract Clindamycin is widely used in antimicrobial prophylaxis to prevent surgical site infections. Adequate subcutaneous free tissue concentrations should reach therapeutic levels, which have to be maintained throughout the surgical procedure for antibiotic prophylaxis to be efficient. A method was developed and validated using high performance liquid chromatography coupled to a mass spectrometry to determine clindamycin concentrations in two biological matrices: plasma, for drug monitoring, and subcutaneous microdialysate, to determine free concentrations at the incision site. Gradient separation of clindamycin was carried out using a reverse phase C18 column eluted with a mixture of mobile phases (1% formic acid in water and 1% formic acid in acetonitrile). The monitored transitions were m/z 425.3 > 377.3 for clindamycin, and m/z 407 > 359 for lincomycin, used as the internal standard for plasma samples. Linearity was reached in the 0.5–100 &mgr;g/mL range for plasma and 25–1000 ng/mL for microdialysate samples. The method was selective, precise, and accurate, and was successfully employed in a preliminary pharmacokinetics study to investigate plasma and subcutaneous clindamycin penetration, determined by microdialysis, after intravenous administration of a 50 mg/kg dose to Wistar rats.

Effects of reduced oxygen availability on the vascular response and oxygen consumption of the activated human visual cortex.

To identify the impact of reduced oxygen availability on the evoked vascular response upon visual stimulation in the healthy human brain by magnetic resonance imaging (MRI).