Fluoxetine and norfluoxetine analysis by direct injection of human plasma in a column switching liquid chromatographic system.

A column switching LC method is presented for the analysis of fluoxetine (FLU) and norfluoxetine (NFLU) by direct injection of human plasma using a lab-made restricted access media (RAM) column. A RAM-BSA-octadecyl silica (C-18) column (40 mm x 4.6 mm, 10 microm) is evaluated in both backflush and foreflush elution modes and coupled with a C-18 lab-made (50 mm x 4.6 mm, 3 microm) analytical column in order to perform online sample preparation. Direct injection of 100 microL of plasma samples is possible with the developed approach. In addition, reduction of sample handling is obtained when compared with traditional liquid-liquid extraction (LLE) and SPE. The total analysis time is around 20 min. A LOQ of 15 ng/mL is achieved in a concentration range of 15-500 ng/mL, allowing the therapeutic drug monitoring of clinical samples. The precision values achieved are lower than 15% for all the evaluated points with adequate recovery and accuracy. Furthermore, no matrix interferences are found in the analysis and the proposed method shows to be an adequate alternative for analysis of FLU in plasma.

Analysis of tricyclic antidepressant drugs in plasma by means of solid-phase microextraction-liquid chromatography-mass spectrometry.

Solid-phase microextraction coupled to liquid chromatography and mass spectrometry (SPME-LC-MS) was used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. SPME was performed by direct extraction on a PDMS/DVB (60 microm) coated fiber, employing a stirring rate of 1200 rpm for 30 min, pH 11.0, and temperature of 30 degrees C. Drug desorption was carried out by exposing the fiber to the liquid chromatography mobile phase for 20 min, using a labmade SPME-LC interface at 50 degrees C. The main variables experimentally influencing LC-MS response were evaluated and mathematically modeled. A rational optimization with fewer experiments was achieved using a factorial design approach. The constructed empirical models were adjusted with 96-98% of explained deviation allowing an adequate data set comprehension. The chromatographic separation was realized using an RP-18 column (150 mm x 2.1 mm, 5 microm particles) and ammonium acetate buffer (0.01 mol/l, pH 5.50) : acetonitrile (50 : 50 v/v) as mobile phase. Low detection levels were achieved with electrospray interface (0.1 ng/ml). The developed method showed specificity, linearity, precision, and limit of quantification adequate to assay tricyclic antidepressant drugs in plasma.

Solid-phase microextraction-liquid chromatography (SPME-LC) determination of fluoxetine and norfluoxetine in plasma using a heated liquid flow through interface.

A simple and sensitive procedure using solid-phase microextraction coupled with high performance liquid chromatography (HPLC) to analyze fluoxetine (FLU) and its metabolite norfluoxetine (nor-FLU) in plasma samples was developed and validated. SPME conditions were optimized employing a factorial design. The sampling step was performed using a PDMS-DVB fiber and desorption was carried out in a novel homemade heated interface. Fluoxetine and norfluoxetine were analyzed by HPLC, using a C18 Phase Sep column (150mmx4.6mm, 3microm) packed "in house", and acetonitrile:acetate buffer 25mmoll(-1) with triethylamine 25mmoll(-1) pH 4.6 (70:30) as the mobile phase. The developed method has shown precision, linearity, specificity, and limit of quantification (LOQ) adequate to assay fluoxetine and norfluoxetine in plasma. Furthermore, the results obtained using the homemade interface has shown an improvement in the desorption process when compared with the results obtained using the off-line mode.

Optimization of the SPME parameters and its online coupling with HPLC for the analysis of tricyclic antidepressants in plasma samples.

Solid-phase microextraction (SPME)-liquid chromatography (LC) is used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. Extraction conditions are optimized using a 2(3) factorial design plus a central point to evaluate the influence of the time, temperature, and matrix pH. A Polydimethylsiloxane-divinylbenzene (60-mum film thickness) fiber is selected after the assessment of different types of coating. The chromatographic separation is realized using a C(18) column (150 x 4.6 mm, 5-microm particles), ammonium acetate buffer (0.05 mol/L, pH 5.50)-acetonitrile (55:45 v/v) with 0.1% of triethylamine as mobile phase and UV-vis detection at 214 nm. Among the factorial design conditions evaluated, the best results are obtained at a pH 11.0, temperature of 30 degrees C, and extraction time of 45 min. The proposed method, using a lab-made SPME-LC interface, allowed the determination of tricyclic antidepressants in in plasma at therapeutic concentration levels.

Automated microcolumn-switching system for drug analysis by direct injection of human plasma.

This study presents the application of a system that joins the known advantages of capillary liquid chromatography (e.g., higher concentration of the analytes and lower consumption of mobile phase) with those of column-switching using restricted access material (RAM) (sample clean up and extraction) to the analysis of fluoxetine in plasma samples. Automatically, the system loads the biological sample, while a RAM-BSA-C18 column (50 mm x 520 microm) excludes the macromolecules and focuses the analytes; afterwards, a second mobile phase elutes the analytes, in backflush mode, and provides the separation in a C18 analytical column (100 mm x 520 microm). We optimized the procedure for a total analysis time of 25 min. Using this approach the calibration curve shows r=0.998 with a linearity range from 20 to 500 ng ml(-1). Precision, calculated as relative standard deviation (RSD), was<20%. The developed miniaturized system showed to be adequate and attractive, demonstrating a large potential for sample preparation.

Development of an improved heated interface for coupling solid-phase microextraction to high-performance liquid chromatography.

The aim of the study described in this report has been the development and the evaluation of a new improved interface to be operated under continuous heating, for on-line coupling solid-phase microextraction (SPME) to high-performance liquid chromatography (HPLC). Heating is desirable to increase desorption rate and decrease carryover. The results obtained have been compared with that obtained by off-line desorption and online desorption without heating. The SPME-HPLC interface described here has an inner volume of 60 microL, fixation for infinite points and a novel leak less sealing system. When the heating system was used, the area values were almost 10-fold higher than that obtained using the off-line mode. The obtained chromatograms showed an increasing of the area and height of chromatographic peaks and proved the excellent performance and reproducibility of the interface developed in this work.