Determination of Enantiomeric Purity of GSK962040 Based on Liquid Chromatography Using Chiral Stationary Phase Combined with a Pre-column Derivatization Procedure

A sensitive and accurate LC method was developed and further validated for the determination of enantiomeric purity of GSK962040. Before separation, a pre-column derivatization procedure was performed. Baseline separation with a resolution higher than 1.9 was accomplished within 15 min using a Chiralpak AD-H (250 × 4.6 mm; particle size 5 μm) column, with n-hexane: 2-propanol (85:15 v/v) as mobile phase at a flow rate of 1 mL min^?1. The eluted analytes were subsequently detected with a UV detector at 260 nm. The effects of mobile phase components and temperature on enantiomeric selectivity as well as resolution of enantiomers were thoroughly investigated. The calibration curves were plotted within the concentration range between 4 and 200 μg mL^?1 (n = 8), and recoveries between 98.15 and 101.48% were obtained, with relative standard deviation (RSD) lower than 1.42%. The LOD and LOQ for the Boc-GSK962040 were 1.23 and 4.15 μg mL^?1 and for its enantiomer were 1.38 and 4.76 μg mL^?1, respectively. The developed method was also evaluated and validated by analyzing bulk samples with different enantiomeric ratios of GSK962040. It was demonstrated that the method was accurate, robust and sensitive, and also had practical utilities for real analysis.     

Determination of AR-42 enantiomeric purity by HPLC on chiral stationary phase

A sensitive and accurate liquid chromatographic method for the determination of AR-42 enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 1.9 was accomplished within 10 min using a CHIRALPAK AD column (250 mm × 4.6 mm; particle size 5 μm) and n-hexane/2-propanol/diethylamine (75:25:0.1 v/v/v) as mobile phase at a flow rate of 1 mL min^?1. Eluted analytes were monitored by UV absorption at 260 nm. The effects of mobile phase components, temperature and flow rate on enantiomeric selectivity and resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 0.001 and 0.5 mg mL^?1 (n = 10), and the recoveries between 98.23 and 101.87% were obtained, with relative standard deviation lower than 1.31%. Limit of detection and limit of quantitation for AR-42 were 0.39 and 1.28 μg mL^?1 and for its enantiomer were 0.36 and 1.19 μg mL^?1, respectively. It was demonstrated that the developed method was accurate, robust and sensitive for the determination of enantiomeric purity of AR-42, especially for the analysis of bulk samples.     

Fluorescent detection of peptides and amino acids for capillary electrophoresis via on-line derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

An in-capillary derivatization of amino acids and peptides with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed for their subsequent capillary electrophoretic analysis with laser-induced fluorescence detection (λ _ex=488 nm). The in-capillary derivatization was achieved in zone-passing mode by introducing successive plugs of sample and NBD-F into a fused silica capillary previously equilibrated with an alkaline borate buffer. To prevent NBD-F hydrolysis and to achieve a reliable derivatization, NBD-F was prepared daily in absolute ethanol and a plug of absolute ethanol was introduced between the sample and NBD-F reagent plugs. Various parameters influencing the derivatization efficiency were investigated and the optimum conditions were as follows: background electrolyte (BGE), 20 mM borate buffer (pH 8.8); introduction time, 4 s for sample and 2 s for NBD-F; molar ratio of NBD-F/sample, above 215; temperature, 45 °C for amino acids and 35 °C for peptides; applied voltage, +15 kV. The validation of the in-capillary derivatization method under optimal conditions showed a good linearity between the heights of the derivative peaks and the concentrations of the amino acids. The intra-day relative standard deviations of the migration times and the peak heights were less than 1.3% and 4.6%, respectively. The efficient derivatization and separation of a mixture of valine, alanine, glutamic acid and aspartic acid were achieved using this technique. Peptides such as buccaline and β-protein fragment 1–42 could also be derivatized using the developed in-capillary derivatization procedure. In‑capillary derivatization and separation of amino acids with different concentrations. From the top to bottom the concentrations are 1.11×10⁻⁵ M, 5.55×10⁻⁶ M, 2.78×10⁻⁶ M, 6.95×10⁻⁷ M. for valine; 1.26×10⁻⁵ M, 6.30×10⁻⁶ M, 3.15×10⁻⁶ M, 7.88×10⁻⁷ M for alanine; 3.78×10⁻⁵ M, 1.89×10⁻⁵ M, 9.45×10⁻⁶ M, 2.36×10⁻⁶ M for glutamic acid;, 4.27×10⁻⁵ M, 2.14×10⁻⁵ M, 1.07×10⁻⁵ M, 2.68×10⁻⁶ M for aspartic acid. Experiment conditions: injection order: 4s for sample, 1s for absolute ethanol, and then 2s for 5.24×10⁻² M NBD‑F; BGE: 20 mM borate pH 8.77; Applied voltage: 15 kV.     

Determination of the Enantiomeric Purity of Trelagliptin by Pre-Column Derivatization and Liquid Chromatography on a Chiral Stationary Phase

A precise and sensitive LC method for determination of enantiomeric purity of trelagliptin has been developed and validated. Pre-column derivatization was performed before separation. Baseline separation with a resolution factor >2.5 was accomplished within 10 min by use of a Chiralpak AD column (250 × 4.6 mm; particle size 5 µm) and n-hexane–2-propanol (90:10 v/v) as mobile phase at a flow rate of 1 mL min⁻¹. Eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition and temperature on enantiomeric selectivity and on resolution of enantiomers were thoroughly investigated. Calibration curves were plotted within the concentration range 0.005–2 mg mL⁻¹ (n = 12), and recoveries between 98.23 and 101.34 % were obtained, with relative standard deviation (RSD) <1.39 %. LOD and LOQ for the trelagliptin derivative were 1.51 and 5.03 µg mL⁻¹; those for its enantiomer were 1.49 and 4.94 µg mL⁻¹, respectively. The method was evaluated and validated by analysis of bulk samples of trelagliptin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

     

Detection of Agmatine and Octopamine in Rat Brain and Human Plasma by Microchip Electrophoresis

A microchip electrophoresis method with laser induced fluorescence detection was developed for the detection of agmatine (Agm) and octopamine (Oct). The fluorescent derivatization reagent, fluorescein isothiocyanate was used for precolumn derivatization of Agm and Oct. The sodium dodecyl sulfate (SDS) micelles was employed as pseudostationary phase for the separation of Agm and Oct with other endogenous compounds exist in biological samples. Some parameters including buffer concentration, buffer pH, SDS concentration and separation voltage were investigated in detail. Under the optimum conditions, the separation and determination of Agm and Oct was performed within 40 s. The calibration curves were linear for both Agm and Oct over the concentration range of 1.0 × 10^?7 to 4.0 × 10^?5 M and 1.5 × 10^?7 to 4.5 × 10^?5 M, respectively. The detection limits of Agm and Oct (S/N = 3) are 5.0 × 10^?8 and 8.0 × 10^?8 M, respectively. These values make the method very suitable for the determination of Agm and Oct in rat brain tissue and human plasma as demonstrated in this paper.     

Simultaneous Determination of Levodopa and Its Metabolite in Human Blood by Capillary Electrophoresis with Laser‐induced Fluorescence Detection

A rapid, sensitive and reproducible method is described for the analysis of levodopa and its metabolite dopamine (DA) in human blood. The influence of carbidopa as the inhibitor againist the decarboxylase activity on the metabolism has been also studied. After derivatization in a dark pulsator for 12 h at room temperature, the fluorescein isothiocyanate (FITC) derivative of levodopa and other components were separated by capillary zone electrophoresis (CZE) within 13 min and detected with laser-induced fluorescence (LIF). Under the optimum analysis conditions, the linear range is 3.0×10^－8—4.0×10^－6 mol/L and 1.0×10^－8—2.0×10^－6 mol/L for levodopa and DA, respectively. The detection limits of levodopa and DA were 7.8×10^－9 mol/L (39.0 amol) and 3.1×10^－9 mol/L (15.5 amol), respectively. The method was successfully applied to monitoring the levodopa and DA in human blood after one took tablets orally.     

A validated chiral LC method for the enantiomeric separation of AT13148 on polysaccharide-based chiral stationary phase

A sensitive and accurate LC method for the determination of AT13148 enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 1.8 was accomplished within 15 min using a Chiralpak AD-H column (250 × 4.6 mm; particle size 5 μm) and n-hexane: 2-propanol: diethylamine (85:15:0.1, v/v) as mobile phase at a flow rate of 1 mL min^?1. Eluted analytes were monitored by UV absorption at 254 nm. The effects of mobile phase components, temperature and flow rate on enantiomeric selectivity and resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 7 and 500 μg mL^?1 (n = 11), and the recoveries between 98.24 and 100.99% were obtained, with relative standard deviation lower than 1.32%. LOD and LOQ for AT13148 were 2.46 and 7.38 μg mL^?1 and for its enantiomer were 2.54 and 7.49 μg mL^?1, respectively. It was demonstrated that the developed method was accurate, robust and sensitive for the determination of enantiomeric purity of AT13148, especially for the analysis of bulk samples.     

Enantioseparation of amino acids by micellar capillary electrophoresis using binary chiral selectors and determination of D-glutamic acid and D-aspartic acid in rice wine

A new chiral capillary electrophoresis (CE) analytical method for the determination of six pairs of amino acid enantiomer-derivatized precapillary with 9-fluorenylmethoxycarbonyl chloride (FMOC) was developed. CE separation parameters such as the pH of background electrolyte, the boric acid concentration, the addition of D-fructose and isopropanol, and the effect of new binary chiral selectors on the electropherograms were investigated. Precapillary and in-capillary derivatization was compared as well. The results showed that enantiomeric separations were obtained with enantio-resolution (Rs) ranged from 2.61 to 9.99 for the FMOC-derivatized amino acid enantiomers except alanine with Rs 1.06. Precapillary derivatization method provides overall better Rs for nearly all the targets except FMOC-aspartate with efficiency up to 1.309?×?10⁶ plates and limit of detection (LOD) down to 2.5?μM. This method was successfully applied to analyze the concentration of the D/L-glutamic acid and D/L-aspartate in seven rice wine samples.     

Voltammetric determination of 17α-ethinylestradiol hormone in supply dam using BDD electrode

In this work, a simple method for electroanalytical determination of 17α-ethinylestradiol (EE2) hormone in natural waters was developed using a boron-doped diamond electrode (BDD). The analyses were performed using square wave voltammetry and the parameters were optimized. The results showed a well-defined irreversible oxidation peak (BR buffer 0.1 mol L^?1, pH 8.0) at +0.65 V (vs. Ag/AgCl). The voltammetric results showed also that the oxidation process is controlled by adsorption of species and indicated that there are two electrons involved. The obtained analytical curves for 17α-ethinylestradiol presented good linearity in the concentration range 9.9?×?10^?7 to 5.2?×?10^?6 mol L^?1 in utlrapure water and 7.9?×?10^?7 to 5.2?×?10^?6 mol L^?1 in natural water samples (supply dam). Detection limits (DL) obtained were between 2.4?×?10^?7 and 7.5?×?10^?7 mol L^?1 and quantification limits (QL) between 7.9?×?10^?7 and 2.5?×?10^?6 mol L^?1. The recovery experiments showed values between 86 and 114 % for spiked samples thus indicating the applicability of the electroanalytical methodology to quantify 17α-ethinylestradiol directly in natural water of supply Dam (Billings Dam in Diadema-SP. Brazil), without any preconcentration or derivatization.     

Determination of intracellular glutathione and cysteine using HPLC with a monolithic column after derivatization with monobromobimane

An optimized high‐performance liquid chromatography (HPLC) method is used to show that, as myoblasts differentiate into multinucleated muscle fibers, there is a shift to a more oxidized cell redox state. The HPLC method incorporated derivatization with monobromobimane for the determination of the reduced (GSH) and oxidized (GSSG) forms of glutathione and the reduced (Cys) and oxidized (CysSS) forms of cysteine. The derivatization was optimized to improve the sensitivity of the approach; the limits of detection for glutathione and cysteine were 3 × 10^?8 and 5 × 10^?8 M , respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Selective Spectrofluorimetric Method and Uncertainty Calculation for the Determination of Camptothecin in the Presence of Irinotecan and Topotecan

A selective fluorimetric method was developed to quantify camptothecin (CPT) in irinotecan (CPT-11) and in topotecan (TPT) based anti-cancer drugs. Selectivity was achieved by experimental optimization and using synchronized scanning and second-order spectral derivatization. The limits of quantification for CPT were in the order of 1 × 10^?7 mol L^?1 with a linear response up to 1 × 10^?5 mol L^?1. The combined uncertainty associated with the CPT fluorescence measurement at a 5 × 10^?7 mol L^?1 level were 10.9% and 6% of the reference level, respectively, for TPT and CPT-11 based pharmaceutical formulations. The CPT recoveries of 95 ± 12% and 91 ± 5.6% (n = 3) were achieved, respectively, in samples containing CPT-11 and TPT.     

An Improved and Validated LC Method for Resolution of Panthenol

An improved LC method was developed and validated for determination of enantiomeric purity of panthenol in bulk drugs. The method is based on derivatization of panthenol with 3,5-dinitrobenzoyl chloride. Baseline separation with resolution >2.7 was achieved within 20 min on Kromasil CHI-DMB (250 × 4.6 mm) column using n-hexane:ethanol (95:5 v/v) as mobile phase at a flow rate of 1.5 mL min^?1. The analytes were detected by their UV absorbance at 265 nm. The effects of ethanol, 2-propanol and temperature on enantioselectivity and resolution of enantiomers were evaluated. The method was extensively validated and proved to be robust. The recoveries were between 98.3 and 101.4% with <1.6% relative standard deviation. The regression equations for the derivatives of d-panthenol and l-panthenol were y ₁ = 18.01x ₁ ? 32.56 (r ₁ ²  = 0.9984) and y ₂ = 17.855x ₂ ? 28.16 (r ₂ ²  = 0.9990), respectively. The LOD and LOQ for the derivative of d-panthenol were 10.6 and 37.4 μg mL^?1 and for the derivative of l-panthenol were 12.1 and 40 μg mL^?1, respectively. The improved method was found to be simple, rapid, and sensitive for the determination of enantiomeric purity of panthenol in bulk drugs.     

Differential-pulse polarography of amino acids in acetaldehyde-borax medium

A differential-pulse polarographic method for the determination of amino acids is reported, based on the formation of Schiff's base compounds in borax buffer solution (pH 10.10) containing acetaldehyde. The compounds are reduced at a mercury electrode with peak potentials of about ?1.5 V (vs. SCE) and well defined polarographic waves are obtained which can be used to determine amino acids in borax medium. The differential-pulse polarographic method was found to be the most sensitive and suitable for the determination of amino acids in the concentration range 1 × 10^?6–8 × 10^?4 M (lysine) and 2.8 × 10^?6–1 × 10^?3 M (arginine). The polarographic characteristics of these waves were studied by differential-pulse polarographic and cyclic voltammetric methods. The waves are ascribed to the reduction of the imido group in the Schiff's base compounds. The procedure was applied to the determination of lysine and arginine in foodstuffs and the total proteins in serum samples.     

A micellar electrokinetic chromatography approach using diastereomeric derivatization and a volatile surfactant for the enantioselective separation of selenomethionine

A MEKC methodology with UV detection was developed for the enantioselective separation of selenomethionine (SeMet). The use of (+)‐1‐(9‐fluorenyl)ethyl chloroformate (FLEC) as chiral derivatization reagent to form SeMet diastereomers enabled their subsequent separation using ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase. The effect of APFO concentration and pH, temperature, injection volume, and derivatization conditions (time and FLEC/SeMet ratio) were evaluated in order to select the best separation conditions. A chiral resolution of 4.4 for DL‐SeMet was achieved in less than 6 min using 100 mM APFO at pH 8.5 as electrophoretic buffer. Satisfactory results were obtained in terms of linearity, precision (RSD from 3.4 to 5.1% for migration times and from 1.8 to 4.6% for corrected peak areas), accuracy, and LODs (3.1 × 10^?6 M and 3.7 × 10^?6 M for d and l enantiomers, respectively). The method was successfully applied to the determination of l ‐SeMet in food supplements.     

Kinetics and mechanism of oxidation of glycine,alanine, and threonine by fluoride coordinated bismuth(V) in aqueous HClO4–HF medium

The kinetics of oxidation of amino acids viz. glycine, alanine, and threonine with bismuth(V) in HClO₄–HF medium have been studied. The kinetics of the oxidation of all these amino acids exhibit similar rate laws. The second-order rate constants were calculated to be 2.04 × 10^?2 dm³ mol^?1 and 2.72 × 10^?2 dm³ mol^?1 s^?1 for glycine and alanine, respectively, at 35°C and 5.9 × 10^?2 dm³ mol^?1 s^?1 for threonine at 25°C. All the possible reactive species of both bismuth(V) and amino acids have been discussed and a most probable kinetic model in each reaction has been envisaged. © 1994 John Wiley & Sons, Inc.     

Enantiospecific analysis of ibuprofen by high performance liquid chromatography: Determination of free and total drug enantiomer concentrations in serum and urine

Summary A reversed-phase high-performance liquid chromatographic (HPLC) assay, based on the indirect approach to enantiomeric analysis, for the determination of ibuprofen in human serum and urine has been developed. Following the addition of (R,S)-flurbiprofen, as internal standard, the enantiomers of ibuprofen were isolated from plasma and urine samples by liquid-liquid extraction at acidic pH. The enantiomers of flurbiprofen and ibuprofen were derivatized with (R)-1-(naphthen-1-yl)ethylamine, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole as coupling reagents, to yield the corresponding diastereoisomeric amides. Chromatographic resolution of the derivatives was achieved using a C₁₈ column (Waters Resolve C₁₈; 5 μm, 150×3.9 mm) using a mobile phase of phosphate buffer (pH 3.5, 0.01 M): acetonitrile (50∶50 v/v) at a flow rate of 1.5 mL min⁻¹ at ambient temperature. Quantification was carried out using a spectrofluorometer with excitation and emission wavelengths of 290 and 330 nm respectively. The use of a semimicrobore column (150×2.1 mm) containing the same stationary phase facilitated the analysis of the free drug enantiomer concentrations following equilibrium dialysis. The derivatization procedure was carried out as described above but with a reduction in the quantities of the reagents used in order to reduce the background noise in the chromatographic analysis. The HPLC methodology for the determination of free drug enantiomer concentrations was validated against a previously reported method employing the radiolabelled drug.     

A predictive-kinetic method for the quantitation of amino acids with ninhydrin

The development and evaluation of a predictive-kinetic method for quantifying amino acids based on reactions with ninhydrin are described. Conditions are developed for which reactions are pseudo-first-order in the amino acid. Absorbance vs. time data from the kinetic region of the reaction (1–3 half-lives) are fitted to a first-order model to predict the total absorbance change that would occur if the reaction were monitored to completion. Computed absorbance changes vary linearly with amino acid concentration between 1 × 10^?5 and 5 × 10^?5 mol l^?1. Results are virtually independent of changes in temperature (± 1° C) and ninhydrin concentration (± 3 × 10^?3 mol l^?1).     

Electrochemical Characterization of Gold 6‐Amino‐2‐mercaptobenzothiazole Self‐Assembled Monolayer for Dopamine Detection in Pharmaceutical Samples

A new sensor, gold‐6‐amino‐2‐mercaptobenzothiazole (6A2MBT), was fabricated via a self‐assembly procedure. Electrochemical properties of the monolayer were investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The modified electrode showed excellent antifouling property against the oxidation products of DA, allowed us to construct a dynamic calibration curve with two linear parts, 1.00×10^?6 to 3.72×10^?4 and 3.72×10^?4 to 6.42×10^?4 M DA, with correlation coefficients of 0.997 and 0.992 and a detection limit of 1.57×10^?7 M DA by using differential pulse voltammetry (DPV), respectively. Finally, the performance of the Au‐6A2MBT modified electrode was successfully tested for electrochemical detection of DA in a pharmaceutical sample.     

Etilefrine Plastic Membrane Electrodes Based on Individual and Mixed Ion-exchangers of Etilefrinium Phosphotungstate and Tetraphenylborate.

Abstract

Etilefrine hydrochloride (EfCl) selective PVC membrane electrodes based on Etilefrinium phosphotungstate (I), Etilefrinium tetraphenylborate (II) and a mixture of both (III) were prepared, The electrodes exhibited near Nernstian response over the concentration ranges 5.0 × 10^?6 - 1.0 × 10^?1, 6.3 × 10^?6 - 1.0 × 10^?1 and 6.3 × 10^?5 - 1.0 × 10^?1 M EfCl for electrodes I, II and III, respectively. The working pH ranges of electrodes I, II and III were 10 - 8.0, 10 - 7.5 and 10 - 7.5 and their isothermal coefficients were 0.00150, 0.00088 and 0.00072 V/°C, respectively. The electrodes showed good selectivity to EfCl with respect to many inorganic cations, sugars and amino acids. The standard additions method was used to determine EfCl in pure solutions and in pharmaceutical preparations.     

Spectrophotometric determination of lithium ion with the chromogenic crown ether, 2″,4″-dinitro-6″-trifluoromethylphenyl-4′-aminobenzo-14-crown-4

A spectrophotometric method of determining alkali metal ions with a chromogenice crown ether reagent was found to be more selective and sensitive than an ion-pairing method based on the same size of crown ether cavity. It is shown that in the ion-pairing method, the sensitivity toward lithium ion was 5.685 × 10^?4 absorbance/mg l^?1, with sodium interfering at 300 mg l^?1. The chromogenic crown ether, 2″,4″-dinitro-6″-trifluoromeethylphenyl-4′-aminobenzo-14-crown-4, was much superior to benzo-14-crown-4. The sensitivity of the chromogenic crown ether was 1.69 × 10^?3 absorbance/mg l^?1. This represents a three-fold increase in sensitivity and less reagent is needed (2 × 10^?4 M for the chromogenic method versus 1.4 × 10^?3 M for ion-pairing). Interference from sodium decreased to 3000 mg l^?1. The reagent was used to determine lithium ion in treated blood serum samples in both a batch and flow injection method and results were compared with data obtained with atomic absorption; excellent agreement was obtained in all cases.