Biomedical subjects

S V Rahavendran

Publications and source records attributed to S V Rahavendran.

4 recordsLinked to original sources

PubMed1998

Visible diode laser induced fluorescence detection for capillary electrophoretic analysis of amantadine in human plasma following precolumn derivatization with Cy5.29.OSu.

Visible diode laser induced fluorescence (VDLIF) detection (620-700 nm) has become important in bioanalysis due to the increased sensitivity and selectivity that can be achieved in biological matrices. A selective and sensitive capillary electrophoretic method employing VDLIF detection has been developed for the analysis of amantadine in plasma. Amantadine was extracted from plasma into toluene under alkaline conditions and the residue was derivatized with the far-red label Cy5.29.OSu. The reaction mixture was dried under nitrogen, reconstituted and then injected onto a laboratory constructed capillary electrophoresis system equipped with a laboratory constructed visible diode laser detector temperature tuned to oscillate at 647.8 nm. The selectivity of the technique was evaluated by demonstrating a lack of interfering peaks in extracts of blank plasma. A calibration curve ranging from 1.8 to 461.1 ng ml(-1) was shown to be linear. The precision and accuracy of the assay (n = 6) were determined to be within 17% R.S.D. and 15% difference from the nominal concentration respectively. The limits of detection for unextracted amantadine and for amantadine from the extracted concentrate from plasma were determined to be 9.5 fmol and 115 amol respectively.

Amantadine↗

PubMed1997

Visible diode laser-induced fluorescence detection of phenylacetic acid in plasma derivatized with Nile blue and using precolumn phase transfer catalysis.

This study reports the application of Nile blue (NB), a farred oxazine label, as a precolumn derivatization reagent for the measurement of free levels of phenylacetic acid (PAA) in plasma. The measurement of PAA in psychiatric populations is important because it provides a marker for 2-phenylethylamine (PEA), which has been implicated in the pathogenesis of schizophrenia and major depression. PAA was derivatized with NB through an amide linkage in the presence of 2-chloro-1-methylpyridinium iodide (carboxylic acid activator, CMP) and triethylamine (base catalyst, TEA), respectively. The formation of the NB-PAA derivative was confirmed using normal phase and reversed phase thin-layer chromatography, reversed phase liquid chromatography, and electrospray mass spectrometry. The formation of the NB-PAA derivative was optimized using a sequential single factor approach. The optimal conditions for the formation and chromatographic separation of the derivative were determined to be 8.0 nmol/mL NB, 390 nmol/mL CMP, 2 mumol/mL TEA, a reaction time of 45 min, and a reaction temperature of 25 degrees C. This derivatization scheme was performed in a phase transfer catalysis mode that enabled the simultaneous extraction, preconcentration, and derivatization of the analyte in a single step. The limit of derivatization of PAA was determined to be 1.0 x 10(-9) M in phosphate-buffered saline, a PAA-free matrix. This derivatization was limited not by the kinetics of the reaction but by the chromatographic separation of the derivative from a side reaction product. The method was used to estimate endogenous free levels of PAA in human plasma samples. The levels of PAA in four sources of plasma were determined to be within 30-70 ng/mL using the method of standard addition and reflected levels that have been reported in the literature. The limit of detection of the derivative was determined to be 7.33 x 10(-11) M using a laboratory-constructed HPLC-VDLIF detector.

Catalysis↗

PubMed1996

An oxazine reagent for derivatization of carboxylic acid analytes suitable for liquid chromatographic detection using visible diode laser-induced fluorescence.

This study reports the use of Nile Blue, an oxazine dye, as a derivatization reagent that fluoresces in the far-red spectral region and is suitable for derivatization with carboxylic-acid-containing analytes. Model carboxylic acid analytes such as benzoic acid, acetic acid, phenylacetic acid and hexanoic acid have been reacted as acid chlorides to form Nile Blue derivatives. The synthesis product of the Nile Blue benzoic acid derivative was confirmed using electrospray-mass spectrometry, infrared spectrometry, 1H and 13C nuclear magnetic resonance, reversed phase liquid chromatography (RP-HPLC), normal phase-thin layer chromatography, and spectral characterization. The synthesized Nile Blue derivatives, separated from reaction by-products with RP-HPLC, all demonstrated an approximately 10-fold drop in molar absorptivity and relative quantum yield. In addition, a 40 nm increase in Stokes shift was observed. A portion of the fluorescence was regained through post-column ionization of the Nile Blue benzoic acid derivative at pH 12. A RP-HPLC limit of detection of 88.25 fmol on column has been reported with conventional fluorescence detection-post-column ionization of the Nile Blue benzoic acid derivative. A limit of detection of 1.99 fmol on column (3.98 x 10(-11) M) has been demonstrated for the Nile Blue benzoic acid derivative with the use of a laboratory-constructed visible diode laser fluorescence detector.

Carboxylic Acids↗

PubMed1993

Solid-state diode laser-induced fluorescence detection in high-performance liquid chromatography.

The need to measure ultralow levels of pharmaceuticals in biological matrices at femtogram and attogram levels presents a significant challenge to bioanalysts. Liquid chromatography has proven to be a versatile and valuable tool for separating analytes from complex biological matrices and fluorescence detection provides both the sensitivity and the selectivity required to measure femtogram and attogram levels of analytes. Solid-state diode lasers have been used primarily in printers, compact disc recorders, and bar code scanners but have recently been adapted for use as light sources for fluorescence detection. Excellent spectral features in the visible and near-infrared regions of the spectrum, together with low cost and ruggedness, make diode lasers an attractive alternative for use as light sources in analytical measurement. Biological matrices demonstrate minimal background signals in the far-red regions of the spectrum which diode lasers emit and diode lasers are among the most stable light sources available. These facts along with expected developments in labeling systems make the potential for use of diode lasers in LC-detection quite promising. This paper reviews characteristics of diode lasers, the properties of potential visible and near-infrared fluorescent probes, instrumental aspects of diode laser fluorometers, and future trends that can be expected in this exciting field of bioanalytical research.

Chromatography, High Pressure Liquid↗