Francisco González López

Lamotrigine analysis in blood and brain by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography assay was developed and validated to determine plasma and brain lamotrigine concentrations allowing pharmacokinetic-pharmacodynamic studies of this new antiepileptic drug in patients and laboratory animals. Lamotrigine and its internal standard were extracted, under alkaline conditions, from plasma and brain homogenate, into ethyl acetate; brain proteins were previously precipitated with trichloroacetic acid. The method was linear between 0.1 and 15.0 mg/l for plasma, with a detection limit of 0.008 mg/l, and between 0.1 and 5.0 mg/l for brain homogenate, with a detection limit of 0.023 mg/l. The method proved to be simple, useful and appropriate, not only for clinical and experimental research, but also for routine monitoring of lamotrigine concentrations in patients.

Development and validation of an HPLC method for vancomycin and its application to a pharmacokinetic study.

A rapid and simple method of high performance liquid chromatography with UV detection for the quantification of vancomycin in artificial perfusion fluid and lung tissue samples has been developed and validated. Chromatographic separation was carried out in a Nucleosil 120 C(18) 5 microm column (length, 15 cm; inner diameter, 0.4 cm) using a mixture of 0.05 M NH(4)H(2)PO(4) (pH 4)-acetonitrile (92:8, v/v) as the mobile phase at a flow rate of 1 mL/min, with UV detection at 220 nm. The method used for the vancomycin quantification showed linearity for concentration ranges of 0.1-2, 2-15 and 15-250 microg/mL, with r(2)=0.9985, 0.9996 and 0.9985, respectively. The limit of quantification of the method was 0.1 microg/mL and the coefficients of variation of the between- and within-day precision showed values between 0.6% and 7.0%. The retention time of vancomycin was 8.5 min. The method was used successfully to study the pharmacokinetics of vancomycin in isolated rat lung after its administration through the systemic and inhalatory routes.

Pulmonary versus systemic delivery of antibiotics : Comparison of vancomycin dispositions in the isolated rat lung

ABSTRACT Vancomycin dispositions in the respiratory system were compared after systemic and inhalatory administration under two respiratory conditions using the isolated-lung model. Inhalatory delivery led to much higher drug levels in pulmonary tissue and fluids. The respiratory pattern affects vancomycin disposition in the pulmonary system regardless of the administration route.

Encapsulation and in vitro evaluation of amikacin-loaded erythrocytes.

The aim of our present work was to establish the effect of the osmolality of the hypotonic buffer on the encapsulated amount and the in vitro properties of Amikacin-loaded erythrocytes. Amikacin was encapsulated in rat erythrocytes using a hypotonic dialysis method with hypotonic buffers of different osmolalities with mean values around 90 and 150 mOsm/kg. Morphological examination of the ghost erythrocytes was accomplished using scanning electron microscopy (SEM). The osmotic fragility of normal and loaded erythrocytes was tested using hypotonic solutions. Evaluation of the hematological parameters of the control and loaded erythrocytes was carried out using a hematology system analyzer. Amikacin release from loaded erythrocytes was tested in autologous plasma at 37°C over a 24-h period. The quantification of Amikacin in loaded erythrocytes and in autologous plasma was performed using an HPLC technique. A higher osmotic fragility of loaded erythrocytes was observed using a low osmolality buffer. Some hematological parameters showed statistically significant differences between the loaded erythrocytes obtained using two buffers of different osmolalities with respect to untreated erythrocytes. According to our results, Amikacin carrier erythrocytes obtained by hypotonic dialysis using a low osmolality buffer (90 mOsm/kg) should afford a good encapsulation yield, appropriate morphological properties, and sustained release in vitro.

DETERMINATION OF AMBROXOL HYDROCHLORIDE BY HPLC

A rapid, specific, and reliable technique has been developed for the determination of ambroxol hydrochloride in solution by high performance liquid chromatography. To do so, reverse phase liquid chromatography was employed, using methanol-0.01 M diammonium phosphate buffer, pH=6, (70:30 v/v) and a detector wavelength of 247 nm, the concentration range employed being 90–120 μg/mL. The technique was seen to have excellent linearity, accuracy, and intra- and inter-day precision for the concentration range studied, both when the response was measured by peak areas and when measured by peak heights.

Validation and clinical evaluation of a UHPLC method with fluorescence detector for plasma quantification of doxorubicin and doxorubicinol in haematological patients

A rapid and simple UHPLC-fluorescence detection method for the quantification of doxorubicin and its main metabolite, doxorubicinol, in human plasma has been developed. The method was also validated for its application in therapeutic drug monitoring, a clinical approach used in the optimization of oncologic treatments. Following a single protein precipitation step, chromatographic separation was achieved using a C18 column (50mm×2.10mm, particle size 1.7μm) at 50°C with a mobile phase consisting of water (containing 0.4% triethylamine and 0.4% orthophosphoric acid)/acetonitrile (77:23, v/v). Flow rate was 0.50mL/min and fluorescence detection with an excitation wavelength of 470nm and an emission wavelength of 548nm was used. The method met the specifications of linearity, selectivity, sensitivity, accuracy, precision and stability of the FDA and EMA guidelines for the validation of bioanalytical methods. Linearity for the drug (8-3000ng/mL) and the metabolite (3-150ng/mL) was observed (R(2)>0.992) and the maximum intra-day and inter-day precision coefficients of variation were less than 14% for both. The lower limits of quantification were 8 and 3ng/mL for doxorubicin and doxorubicinol, respectively. The method was successfully applied to the quantify plasma concentrations of doxorubicin and doxorubicinol in 33 patients diagnosed with haematological malignancies in which broad ranges for drug (8.3-2766.0ng/mL) and metabolite (4.8-104.9ng/mL) levels were measured adequately.

Determination of piperacillin in biological samples by HPLC

Abstract A rapid, specific and reliable technique has been developed for the determination of piperacillin in biological samples by High Performance Liquid Chromatography. To do so, reverse phase liquid chromatography was used, employing acetonitrile-phosphate buffer 0,1M and pH=6 as the mobile phase (20:80 v/v) and a detector wavelength of 254 nm. Concentrations ranged between 0,5 μg/mL and 200 μg/mL, divided into two calibrations: high concentrations, 15 μg/mL to 200 μg/mL, and low concentrations, 0,5 μg/mL to 15 μg/mL. The intraday and interday reproducibility of the analytical technique was studied by calculating the variation coefficient of 5 samples analyzed each day and on five consecutive days by ANOVA. The coefficients obtained were 4,02% and 7,11%, respectively, for concentrations ranging between 15–200 μg/mL and 5.71% and 7,54% for concentrations between 0,5 and 15 μg/mL.

Influence of the infusion rate on disposition of netilmicin in the isolated rat perfused kidney

A study of the disposition of netilmicin in the isolated rat kidney was carried out in order to establish the influence of the infusion rate on the drug profile in this tissue. A dose of 800 microg administered as a bolus injection or at infusion times of 5, 7.5 and 10 min, respectively, was injected through the afferent cannula into the isolated kidney. Analysis of outflow curves was carried out using different kinetic approaches. Comparison of statistical moments and derived parameters pointed to changes in the distribution process with the infusion rate. In contrast, elimination remained constant, since the extraction coefficient and relative area under the curve values did not change with the infusion rate, although the MTT (mean transit time) and distribution volume decreased for the longest infusion times. The UDF (unit disposition function) profiles were not superimposed for the different infusion rates and combined with the results of the kinetic analysis revealed that the behaviour of netilmicin in the isolated kidney depends on infusion rate. The apparent partition coefficients in renal cortex and medulla showed higher values for the slower perfusion rates. Yet, a progressive decrease in the absolute amount of netilmicin was predicted in the tubular epithelium compartment whereas the residence time tended to increase. The latter phenomenon could account for the higher aminoglycoside nephrotoxicity reported when these drugs are administered over longer infusion times.

Determination of bentazepam in plasma by high-performance liquid chromatography

Bentazepam (2,3-tetramethylene-4-phenyl-7-oxo-thiene), Fig. 1, is a derivative of thienodiazepine showing anxiolytic, tranquillizing, anticonvulsant, miorelaxing and sleep-inducing properties [ 11. Pre-clinical trials in volunteers have shown psychotropic activity for this drug such that the preparation may be considered as a tranquillizer with slight psychostimulatory properties. The drug also shows activity in combating symptoms of anxiety, restlessness, depression, difficulties in touch, sleep disorders [2] and in treatment of childhood anxiety [3]. Acute and chronic intolerance to the drug is rare at the clinical doses of 30-150 mg and is generally accompanied by side-effects similar to those displayed by several benzodiazepines. However these side-effects appear at doses greater than those normally used in clinical practice [4].

Optimización de una técnica de cromatografía líquida de alta eficacia para la determinación de lamotrigina en plasma humano

OBJECTIVE The purpose of this study was to optimise the HPLC-UV bio-analytical method currently used by the Salamanca University Clinical Hospital for determining lamotrigine plasma levels. MATERIAL AND METHODS The developed HPLC-UV analytic technique currently in use was shown to be linear, exact and precise, and suitable for use in routine monitoring of lamotrigine levels. The drawback of this method has always been the time required for analysing samples, so our aim was to improve on that elapsed time. That improvement involved using a different chromatographic column from the one used up until now. We replaced the column that was normally used (Kromasil-100C18-5 microm-15*0.4 cm with a LiChroCART-RP18e-3 microm-5.5*0.4 cm); in both cases, a liquid-liquid extraction was performed and the same sample extraction protocol was followed. RESULTS Both validation methods showed that the two column types are valid for routine lamotrigine monitoring. CONCLUSION The decrease in retention time, in addition to a lower quantification limit and better precision and accuracy parameters obtained with the LiChorCART column, suggest that this unit is ideal for use in clinical practice because it enables a large number of determinations to be performed in less time and the greater precision of LTG measurements.