Column-switching Liquid Chromatographic System Research Articles

Determination of losartan, telmisartan, and valsartan by direct injection of human urine into a column-switching liquid chromatographic system with fluorescence detection

Column-switching high-performance liquid chromatographic (HPLC) method has been developed and validated for quantification of losartan, telmisartan, and valsartan in human urine. Urine samples were diluted on the extraction mobile phase (1:4, v/v) and a volume of 20 μL of this mixture were directly injected onto the HPLC system. The analytes were extracted from the matrix using an on-line solid-phase extraction procedure involving a precolumn packed with 25 μm C 18 alkyl-diol support (ADS), and a solution 2% methanol in 5 mM phosphate buffer (pH 3.8) at a flow-rate of 0.8 mL/min for isolation and preconcentration of losartan, telmisartan, and valsartan. The enriched analytes were back-flushed after, onto the analytical column with a mixture of 5 mM phosphate buffer (pH 3.8)–acetonitrile–methanol (65:20:15, v/v/v) at a flow-rate of 3.0 mL/min and detected by fluorescence at 259 and 399 nm as excitation and emission wavelength respectively. The separation of losartan, telmisartan, and valsartan was achieved on a Chromolith RP-18e monolithic column. The method provides extraction recoveries from spiked urine samples greater than 93%. Intra-day and inter-day precision were generally acceptable; the intra-day-assay C.V. was <3.5 for all compounds and the inter-day-assay C.V. was <3.7%. The estimated calibration range was 0.001–2.5 μg mL −1 with excellent coefficient of determination (>0.9981). The detection limits for losartan, telmisartan, and valsartan at a signal-to-noise ratio of 5:1 were 0.002, 0.0002 and 0.001 μg mL −1 when a sample volume of 20 μL was injected. The proposed method permitted the simultaneous determination of losartan, telmisartan, and valsartan in 8 min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to 12 samples/h. The developed column-switching method was successfully applied for the determination of these analytes in human urine samples of patients submitted at ARA-IIs therapy.

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.

Determination of melagatran in rabbit plasma by high-performance liquid chromatography with automated column switching.

Melagatran is an active thrombin inhibitor showing oral and parenteral bioavailability for antithrombotic therapy. A simple and convenient liquid chromatographic method has been developed and applied to the analysis of melagatran in rabbit plasma. The clean-up and separation of the sample solutions were performed by automated on-line column switching HPLC. The method validation shows the suitability of the column switching liquid chromatographic system for the quantitation of melagatran in biological fluids.

On-line coupling of microdialysis to packed capillary column liquid chromatography–tandem mass spectrometry demonstrated by measurement of free concentrations of ropivacaine and metabolite from spiked plasma samples

An on-line coupling of microdialysis to a packed capillary column switching liquid chromatographic system (0.2 mm I.D.) and mass spectrometric detection was developed. The microdialysates were collected in the loop of the first of three valves, coupled in direct series. A deuterated internal standard was added on-line by the middle valve and the third valve operated both a pre-column, for desalting of the physiological buffer used in the sampling procedure, and a separation column. The on-line system was used to study free concentrations of ropivacaine and its metabolite (PPX) in human plasma samples. The analytes were detected by electrospray ionization in a tandem mass spectrometer operating in multiple reaction monitoring mode. The free fractions of ropivacaine (200 n M total concentration) and PPX (20 n M total concentration) in spiked plasma samples were 12±3 and 47±5% (±standard deviation for day-to-day variations, n=3), respectively.

Effects of system peaks in a column-switching liquid chromatographic system for bioanalysis

A reversed-phase liquid chromatographic column-switching system was used for the determination of drugs and metabolites in blood plasma with a direct i