“…As reported by several published papers, PER and STP have native fluorescence [39–41]. This property is also presented by terbinafine that, together with its similar chemical structure (Figure 1) and physicochemical properties has a comparable chromatographic behavior to PER and STP, making it suitable for use as IS in this method.…”
We report a high-performance liquid chromatography method development able to simultaneously determine perampanel and stiripentol, two third-generation antiepileptics whose therapeutic spectrum can potentially be extended, in several mouse matrices. A salting-out assisted liquid-liquid extraction optimized by a design of experiments approach was adopted for sample preparations. Isopropanol and magnesium sulfate were the extraction solvent and salting-out agent, respectively. Both drugs and internal standard (terbinafine) were separated using a LiChroCART R Purospher Star column (C 18 , 55 × 4 mm; 3 μm) isocratically pumped with mobile phase [1% triethylamine in water (pH 2.5) and acetonitrile (53:47, v/v)] at 1 mL/min. Stiripentol and terbinafine were detected by fluorescence at 254/372 nm and perampanel at 275/430 nm. Good linearity was demonstrated for perampanel at 1-500 ng/mL range in brain, 2-2000 ng/mL in liver and 1-2000 ng/mL in plasma and kidney (r 2 ≥ 0.9922), and for stiripentol between 10 and 2000 ng/mL in brain and 10 and 20 000 ng/mL in the remaining matrices (r 2 ≥ 0.9917). Precision (CV ≤ 15%) and accuracy (bias ±15%) were also verified, with obtained recovery values consistent with those predicted by the experimental design. This method was applied in preliminary pharmacokinetic studies to quantify perampanel or stiripentol after oral administration to mice, showing to be a promising bioanalytical tool to support future nonclinical in vivo pharmacokinetic studies.
“…As reported by several published papers, PER and STP have native fluorescence [39–41]. This property is also presented by terbinafine that, together with its similar chemical structure (Figure 1) and physicochemical properties has a comparable chromatographic behavior to PER and STP, making it suitable for use as IS in this method.…”
We report a high-performance liquid chromatography method development able to simultaneously determine perampanel and stiripentol, two third-generation antiepileptics whose therapeutic spectrum can potentially be extended, in several mouse matrices. A salting-out assisted liquid-liquid extraction optimized by a design of experiments approach was adopted for sample preparations. Isopropanol and magnesium sulfate were the extraction solvent and salting-out agent, respectively. Both drugs and internal standard (terbinafine) were separated using a LiChroCART R Purospher Star column (C 18 , 55 × 4 mm; 3 μm) isocratically pumped with mobile phase [1% triethylamine in water (pH 2.5) and acetonitrile (53:47, v/v)] at 1 mL/min. Stiripentol and terbinafine were detected by fluorescence at 254/372 nm and perampanel at 275/430 nm. Good linearity was demonstrated for perampanel at 1-500 ng/mL range in brain, 2-2000 ng/mL in liver and 1-2000 ng/mL in plasma and kidney (r 2 ≥ 0.9922), and for stiripentol between 10 and 2000 ng/mL in brain and 10 and 20 000 ng/mL in the remaining matrices (r 2 ≥ 0.9917). Precision (CV ≤ 15%) and accuracy (bias ±15%) were also verified, with obtained recovery values consistent with those predicted by the experimental design. This method was applied in preliminary pharmacokinetic studies to quantify perampanel or stiripentol after oral administration to mice, showing to be a promising bioanalytical tool to support future nonclinical in vivo pharmacokinetic studies.
“… Structure and surface chemistry of supports for reversed‐phase HPLC. ( A ) Schematic presentation of surface chemistry of an end‐capped reversed‐phase C 8 silica‐based support (reprinted from Engelhardt et al, , with permission). ( B ) Structure of a polystyrene‐based reversed‐phase HPLC support (Courtesy of GE Healthcare)…”
Section: Strategic Planningmentioning
confidence: 99%
“…Ionizable silanol groups arise not only from inadequate end‐capping: they can also result from column aging or inappropriate storage of the silica‐based column (e.g., over a long period in water). Consequently, the selectivity of the column can be impaired (Engelhardt et al, ). The structure of a polystyrene‐based support is shown in Figure B.…”
Section: Strategic Planningmentioning
confidence: 99%
“…Consequently, the capacities of porous materials are significantly higher than those of nonporous silica gel– or polymer‐based supports. Nonporous chromatographic materials are also used for fast analytical high performance separations and for UHPRPLC of proteins (MacNair et al ; Engelhardt et al ).…”
Section: Strategic Planningmentioning
confidence: 99%
“…Mixed‐mode retention is caused by ionic interactions taking place between negatively charged silanol groups exposed on the surface of the silica medium and positively charged amino groups of proteins. These interactions occur if the silica‐based matrix is not (or is insufficiently) end‐capped, or during the aging process of the column (Engelhardt et al, and Fig. A).…”
Reversed-phase HPLC (RP-HPLC) is one of most important techniques for protein separations and the method of choice for peptide separation. RP-HPLC has been applied on the nano, micro, and analytical scale, and has also been scaled up for preparative purifications, to large industrial scale. Because of its compatibility with mass spectrometry, RP-HPLC is an indispensable tool in proteomic research. With modern instrumentation and columns, complex mixtures of peptides and proteins can be separated at attomolar levels for further analysis. In addition, preparative RP-HPLC is often used for large-scale purification of proteins. This unit provides protocols for packing and testing a column, protein separation by use of gradient or step elution, desalting of protein solutions, and separation of enzymatic digests before mass spectrometric analyses. A protocol is also provided for cleaning, regenerating, and storing reversed-phase chromatography columns.
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