RESUMEN
This paper describes the development of a novel on-line biosensor based on a fluorescently labeled human calmodulin (CaM), hCaM M124C-mBBr, immobilized on controlled-pore glass (CPG), for the analysis of trifluoroperazine (TFP); a phenothiazine drug in human urine samples. The device was automated by packing hCaM M124C-mBBr-CPG in a continuous-flow microcell connected to a monitoring system, composed of a bifurcated optical fiber coupled to a spectrofluorometer. Operating parameters of the on-line biosensor (flow rate, sample injection volume, and carrier solution and buffer pH) were studied and optimized. Under the optimal conditions, the biosensor provides a detection and a quantification limit of 0.24 and 0.52 µg mL(-1), respectively, and a dynamic range from 0.52 to 61.05 µg mL(-1) TFP (n = 5, correlation coefficient 0.998). The response time (t(100)) was shorter than 42 s (recovery time <4.5 min) and reproducibility and repeatability of the TFP measurements, within the linear response range, were lower than 1.4 and 2.7%, respectively. The device was successfully applied to the analysis of TFP in spiked human urine samples with recoveries ranging between 97 and 101% and with RSDs lower than 5.9%.
Asunto(s)
Técnicas Biosensibles/métodos , Calmodulina/química , Calmodulina/genética , Trifluoperazina/análisis , Técnicas Biosensibles/instrumentación , Calmodulina/metabolismo , Colorantes Fluorescentes/química , Humanos , Cinética , Mediciones Luminiscentes/instrumentación , Mediciones Luminiscentes/métodos , Unión Proteica , Trifluoperazina/orinaRESUMEN
INTRODUCTION: This study deals with a new methodology of on-line solid-phase extraction (SPE), derivatization reaction and spectrophotometric measurement utilizing sequential injection analysis (SIA) technique. The method was applied for the assay of trifluoperazine in human urine. METHODS: An SPE procedure was automated to perform sample clean-up, extraction and preconcentration into a homemade microcolumn connected to a SIA manifold. Unlike previous SIA methods, a simple SIA manifold included one syringe pump and one multi-position valve was constructed. This offered simplicity and rapidity to the proposed method (sampling frequency 11 sample h(-1)). Spectrophotometric measurement was based on a fast oxidation of trifluoperazine by Ce(IV) in sulfuric acidic media resulting in a spectrophotometrically detectable chromophore measured at wavelength 500 nm. The SPE procedure was optimized by the univariate method while the derivatization reaction and spectrophotometric measurement were optimized by multivariate methods. RESULTS: The method was linear in a range of 70-200 ng ml(-1) and accurate with a recovery of 92.7%. Relative standard deviation of repeatability (n=10 in a day) and intermediate precision (n=5 over a week) did not exceed 4.3% indicating satisfactory precision. The limits of detection and quantification were 18.2 and 55.2 ng ml(-1), respectively. The sensitivity of the method was improved by the preconcentration, the use of extended pathlength in the detection device and the optimization of absorbance measurement. The method is selective in the presence of chlordiazepoxide, which is sometimes taken in combination with trifluoperazine. DISCUSSION: The method is suitable for the application of overdose and therapeutic drug monitoring in human urine.