Influence of mild and moderate hepatic impairment on axitinib pharmacokinetics.

OBJECTIVE: To evaluate the effects of hepatic impairment on the pharmacokinetics and safety of a single, oral axitinib dose in subjects with mild or moderate hepatic impairment. METHODS: In this phase I, open-label, parallel-group study, a total of 24 subjects with either normal hepatic function (n = 8) or with mild (n = 8) or moderate (n = 8) hepatic impairment were administered a single, oral dose of axitinib (5 mg). Blood samples were collected at intervals up to 144 h following dosing, and plasma pharmacokinetics and safety were assessed. Changes in axitinib plasma exposures in subjects with mild or moderate hepatic impairment were predicted using computer simulations and used to guide initial dosing in the clinical study. RESULTS: Axitinib exposure was similar in subjects with normal hepatic function and those with mild hepatic impairment, but approximately twofold higher in subjects with moderate hepatic impairment. Axitinib exposure weakly correlated with measures of hepatic function but was not affected by smoking status. Axitinib protein binding was similar in the three treatment groups. No significant treatment-related adverse events were reported. CONCLUSIONS: Compared with subjects with normal hepatic function, moderate hepatic impairment increased axitinib exposure, suggesting that the oral clearance of axitinib is altered in these subjects. In addition, these data indicate a possible need for a dose reduction in subjects who develop moderate or worse hepatic impairment during axitinib treatment. A single 5-mg dose of axitinib was well tolerated in subjects with mild or moderate hepatic impairment.

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.

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.

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.

Application of rhodamine 800 for reversed phase liquid chromatographic detection using visible diode laser-induced fluorescence.

This study reports the application of rhodamine 800, a far-red dye, suitable for excitation using visible diode laser-induced fluorescence (VDLIF) detection. A reagent synthesized from rhodamine 800 was evaluated as a precolumn reagent for derivatization with amino-containing analytes. The derivative of this reagent with primary amine analytes showed a loss of fluorescence. Rhodamine 800 was then applied as a mobile phase additive in the indirect mode for quantitation of valproic acid in plasma using reversed phase HPLC in combination with VDLIF detection. A visible diode laser (output power 8.50 mW) temperature-tuned to oscillate at 674.70 nm was used as a light source for a laboratory constructed HPLC fluorescence detector. A liquid/liquid extraction procedure was applied to human blank plasma. The selectivity of this method was validated by demonstration of a lack of interfering peaks in extracts of plasma (n = 3 sources). A calibration curve for valproic acid between 40 and 200 µg/mL was shown to be linear (r = 0.9932). The recoveries of valproic acid at concentrations of 50 and 100 µg/mL were evaluated and determined to be 73 and 72%, respectively. The precision and accuracy (n = 5) of the assay was within 7.0% RSD and 8.0% difference from the spiked concentration, respectively. The limits of detection (S/N = 3) for extracted and unextracted valproic acid were 15.0 and 11.54 µg/mL, respectively. The theoretical (C(lim)) and practical (C(det)) limits of detection in the detector flow cell for unextracted valproic acid at a S/N = 1 were found to be within 15%.

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.