A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a BMS drug candidate and its acyl glucuronide (1-O-beta glucuronide) in rat plasma. A 50-microL aliquot of each plasma sample was fortified with acetonitrile containing the internal standard to precipitate proteins and extract the analytes of interest. After mixing and centrifugation, the supernatant from each sample was transferred to a 96-well plate and injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Phenomenex Luna C(18), 3 mm x 150 mm, 3 microm column. The mobile phase contained 0.075% formic acid in 70:30 (v/v) acetonitrile/water. Under the optimized chromatographic conditions, the BMS drug candidate and its acyl glucuronide were separated from its seven glucuronide positional isomers within 10 min. Resolution of the parent from all glucuronides and acyl glucuronide from its positional isomers was critical to avoid their interference with quantitation of parent or acyl glucuronide. Detection was by positive ion electrospray MS/MS on a Sciex API 4000. The standard curve, which ranged from 5 to 5000 ng/mL, was fitted to a 1/x(2) weighted quadratic regression model for both the BMS drug candidate and its acyl glucuronide. Whole blood and plasma stability experiments were conducted to establish the sample collection, storage, and processing conditions. The validation results demonstrated that this method was rugged and repeatable. The same methodology has also been used in mouse and human plasma for the determination of the BMS drug candidate and its acyl glucuronide.
This paper describes the design and construction of a thinlayer spectroelectrochemical cell with a long optical path length. This cuvette-based cell, which can be reproducibly filled by capillary action, facilitates the use of metal thinfilm electrodes such as those constructed on glass, silicon, and mica substrates. Electrochemical and spectroscopic data are presented that demonstrate the thin-layer behavior of the cell and other important performance characteristics (e.g., optical sensitivity, electrolysis time, and oxygen exclusion capability).Recent studies have exploited the advantages of thin-layer spectroelectrochemistry in explorations of a variety of heterogeneous and homogeneous electrochemical processes. In many instances, optical monitoring is accomplished by irradiating the solution confined in the thin-layer cavity with the propagation axis of the optical beam parallel to the electrode/solution interface. [8][9][10][11][12][21][22][23]25,33,34 The configuration of such long optical path length thin-layer spectroelectrochemical cells (LOPTLCs) yields path lengths of ∼1 cm. These path lengths result in an enhancement in sensitivity of ∼100-fold over that generally achieved using conventional thin-layer cells and optically transparent electrodes that have path lengths of ∼100 µm. A key feature of the LOPTLC is that the long optical path length and large electrode surface area to solution volume ratio provide ample optical sensitivity for the quantitative monitoring of interfacial processes such as adsorption, desorption, and electrocatalysis. [8][9][10][11]14,35 The work described herein stems from interest in applying the LOPTLC to our ongoing investigations of the electrode reactions of monolayers formed by the chemisorption of organosulfur compounds (i.e., alkanethiols, dialkyl disulfides, and dialkyl sulfides) at gold. 35,36 These monolayers are usually adsorbed at thin (200-300 nm) gold films coated onto fragile glass, silicon, and mica substrates by vapor deposition techniques. Many existing LOPTLC designs, however, employ pressure seals between the electrode and the cell for use with vacuum degasfilling procedures, [8][9][10][11][12]14,17,37 requiring mechanically strong electrodes constructed to critical size specifications. The purpose of this paper is to present the design and performance attributes of a LOPTLC that facilitates investigations of electrodes constructed by vapor deposition techniques and can be readily filled without using vacuum procedures. EXPERIMENTAL SECTIONReagents. Potassium ferrocyanide (Fisher), sodium perchlorate (Aldrich), sodium hydroxide (Aldrich), methanol (Fisher, HPLC grade), and ethanol (Quantum, punctilious grade) were used as received. Solutions were prepared using deionized water (Millipore).Electrode Preparation. Glass microscope slides were initially cut into substrates that were 0.99 cm wide and ∼3.5 cm long.
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