Aqueous Sodium Phosphate Buffer Research Articles

Effect of Formulation and Process Parameters on the Disproportionation of Indomethacin Sodium in Buffered Lyophilized Formulations

(i) To investigate buffer salt crystallization and the consequent pH shifts during the freezing stage of the lyophilization of indomethacin sodium (IMCNa) in aqueous sodium phosphate buffer. (ii) To determine the effect of pH shift on the disproportionation of IMCNa in lyophilized formulations. Prelyophilization solutions containing IMCNa in sodium phosphate buffer, at initial buffer concentrations ranging from 10 to 100mM (pH7.0), and at IMCNa concentrations of 5, 10 & 15mg/ml, were investigated. Their phase behavior during cooling was monitored by low temperature X- ray diffractometry (XRD), differential scanning calorimetry (DSC) and pH measurements. The final lyophiles were characterized by infrared spectroscopy (IR) and XRD. Upon cooling to -25°C, pronounced pH shifts were observed only in IMCNa buffered solutions containing high initial buffer concentration (100mM), due to crystallization of Na2HPO4. 12H2O. In the final lyophiles, disproportionation of IMCNa to the free acid (IMC) was observed in systems with buffer concentrations ≥50mM, but not low buffer concentration (10mM). At intermediate buffer concentrations (35 & 20mM) the disproportionation depended on IMCNa concentration. The initial concentrations of both buffer and IMCNa influenced the buffer crystallization. During freeze drying, selective crystallization of a buffer component and the consequent pH shift can cause disproportionation of IMCNa. This can prolong the reconstitution time or retain particles of the poorly soluble free acid in the reconstituted solution.

Interactions of glycine with aqueous solutions of sodium phosphate buffer at T = (288.15, 293.15, 298.15, 303.15, 308.15, 310.15, 313.15, 318.15, 323.15 and 328.15) K: Volumetric and UV absorption studies

Interactions of glycine with sodium phosphate buffer have been investigated by volumetric and UV absorption studies. Densities have been measured for glycine in water of pH=(1.00, 7.40 and 13.40) and in 0.1M, 0.5M and 1M aqueous sodium phosphate buffer (NaPB) solutions as a function of concentration at T=(288.15 to 328.15) K and at pH=(1.00, 7.40 and 13.40) by using digital density meter. Apparent molar volumes (V∅) and partial molar volumes (V2o) obtained from these density data have been used to calculate partial molar volumes of transfer (∆trV2o) from water to aqueous sodium phosphate buffer solutions, which are positive for zwitterionic and deprotonated form of glycine. Interaction coefficients have been obtained from McMillan-Mayer's approach. Effect of temperature on these thermodynamic properties have been studied by determining the partial molar expansibilities (∂V2o/∂T)P at infinite dilution and their second order derivative (∂2V2o/∂T2)P at different temperatures and at pH=(1.00, 7.40 and 13.40). Hydration number (nH) of glycine at pH7.40 have been determined from the V2o data. The results have been rationalized in terms of various interactions taking place in these systems. Further, absorption spectra have also been recorded for the present system with the help of UV–visible spectrophotometer at 298.15K which is helpful in interpreting the possible molecular interactions between glycine and NaPB.

Influence of mixed electrolytes on the adsorption of lysozyme, PEG, and PEGylated lysozyme on a hydrophobic interaction chromatography resin.

In a recent work (Werner A and Hasse H in J Chromatogr A 2013;1315:135) the influence of mixed electrolytes on the adsorption of the macromolecules lysozyme, PEG and di-PEGylated lysozyme on a hydrophobic resin has been studied, but only at one overall ionic strength (3000mM). The present work, therefore, extends these studies to other ionic strengths (2400 and 2700mM), and explores the application of a model to predict the entire data set. The adsorbent is Toyopearl PPG-600M. The solvent is a 25mM aqueous sodium phosphate buffer at pH 7.0. The studied salts are sodium chloride, sodium sulfate, ammonium chloride and ammonium sulfate. Pure salts as well as binary and ternary mixtures of these salts with varying ratios of the amounts of the salts are studied at 25 °C. The loading of the adsorbent increases with increasing salt concentration for all macromolecules. Synergetic effects of the mixed electrolytes are observed. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1104-1115, 2017.

In Operando neutron Imaging of Enzymatic Electrochemical Cells

Enzymatic electrochemical cells (EECs) convert sugars, alcohols, and other fuels into electrical energy through electrochemical reactions catalyzed by naturally occurring enzymes. These devices typically consist of porous carbon electrodes, which may be separated by a polymer membrane. The porous electrodes in EECs are typically saturated with aqueous solutions to support electrochemical activity. The anode contains fuel within the liquid solution, while EECs often incorporate an air-breathing cathode. This work focuses on the characterization of the active anode assembled in a prototype three electrode cell developed to support investigation of the effects of varying environmental conditions on EEC performance. The aluminum three electrode cell reactor was designed and fabricated for in operando neutron imaging. This reactor consisted of two compartments for housing the working and counter electrodes. These halves of the reactor were separated by a Teflon gasket and filter paper. A three electrode cell arrangement was completed by embedding a silver reference electrode in the filter paper. Utilizing neutron tomography the active regions of the three electrode cell were mapped in the absence of the aqueous fuel solution. The reactor was then opened and wetted with a solution of 0.1 M glucose and 0.01 M hydroquinone in an aqueous sodium phosphate buffer. Cyclic voltammetry (CV) was performed to ensure proper function of the cell. Following CV in operando imaging was performed to generate radiographic representations of the anodic components prior to, during, and after discharge. Simultaneous chronoamperometry was performed to provide a connection between the dynamics of the aqueous fuel and buffer solutions and cell performance. Following discharge, tomographic scans were performed to allow for the observation of changes in the active regions of the half-cell. Following the initial measurements the three electrode cell was opened and flushed with fuel solution. The CV, in operando imaging, and tomographic measurements were then repeated. Increased aqueous solution content was found to improve the overall response of the system, yielding higher current in CV and a more consistent discharge behavior during chronoamperometry. Solution content is quantified based on tomographic scans and the related effects on performance are addressed. In general, the studies presented demonstrate a means of controlling test cell geometry to permit the study of EECs using neutron imaging techniques.

Unusual triskelion patterns and dye-labelled GUVs: consequences of the interaction of cholesterol-containing linear-hyperbranched block copolymers with phospholipids.

Cholesterol (Ch) linked to a linear-hyperbranched block copolymer composed of poly(ethylene glycol) (PEG) and poly(glycerol) (hbPG) was investigated for its membrane anchoring properties. Two polyether-based linear-hyperbranched block copolymers with and without a covalently attached rhodamine fluorescence label (Rho) were employed (Ch-PEG30-b-hbPG23 and Ch-PEG30-b-hbPG17-Rho). Compression isotherms of co-spread 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with the respective polymers were measured on the Langmuir trough and the morphology development of the liquid-condensed (LC) domains was studied by epi-fluorescence microscopy. LC domains were strongly deformed due to the localization of the polymers at the domain interface, indicating a line activity for both block copolymers. Simultaneously, it was observed that the presence of the fluorescence label significantly influences the domain morphology, the rhodamine labelled polymer showing higher line activity. Adsorption isotherms of the polymers to the water surface or to monolayers of DPPC and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), respectively, were collected. Again the rhodamine labelled polymer showed higher surface activity and a higher affinity for insertion into lipid monolayers, which was negligibly affected when the sub-phase was changed to aqueous sodium chloride solution or phosphate buffer. Calorimetric investigations in bulk confirmed the results found using tensiometry. Confocal laser scanning microscopy (CLSM) of giant unilamellar vesicles (GUVs) also confirmed the polymers' fast adsorption to and insertion into phospholipid membranes.

Experimental study and modeling of the influence of mixed electrolytes on adsorption of macromolecules on a hydrophobic resin

The influence of mixed electrolytes on the adsorption of macromolecules on a hydrophobic resin is explored. The macromolecules are native lysozyme, di-PEG-lysozyme-5kDa and pure PEG-6kDa, the adsorbent is Toyopearl PPG-600M, a mildly hydrophobic resin. The solvent is a 25mM aqueous sodium phosphate buffer at pH 7.0 containing additional salts with an overall ionic strength of 3000mM. The studied salts are sodium chloride, ammonium sulfate, sodium sulfate and ammonium chloride. Both pure salts as well as binary and ternary mixtures of these salts with varying ratios of the amounts of the salts are studied. For all solvents adsorption equilibrium isotherms are measured at 25°C. Mixing electrolytes can lead to synergetic effects, i.e. the adsorption in the mixed electrolyte system is sometimes distinctly larger than expected from the results of the single electrolyte systems. The single ions, not the salts, drive the adsorption. A mathematical model is developed, which describes the influence of the different ions and their cross-interactions on the studied adsorption process.

Application of phase optimized liquid chromatography to oligopeptide separations

Stationary phase optimized liquid chromatography (POPLC) has been applied to the separation of oligopeptides. The retention factors and theoretical plate numbers of 13 peptides were determined on five different stationary phases. Based on these values, an optimal stationary phase composition of 250 mm total length consisting of 3 segments of 20 mm octadecyl silica, 10 mm phenyl silica and 220 mm embedded polar octadecyl silica was calculated by the optimizer software. Good agreement between the calculated and experimental chromatograms was observed. In order to achieve short analysis time and baseline separation of all peptides gradient elution and optimization of the column temperature were performed. The optimized mobile phase conditions consisted of (A) acetonitrile and (B) 0.025 M aqueous sodium phosphate buffer, pH 3.0, operated at 10% A (0–12 min) followed by 10–50% A (12–32 min) at a flow rate of 0.5 mL min −1 and column temperature of 35 °C. Using these conditions all peptides could be separated in less than 30 min with good resolution and peak symmetry.

Capillary electrochromatographic separation of illicit drugs employing a cyano stationary phase

In this study, we present a capillary electrochromatographic method for separation of basic compounds of interest in forensic science (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyethylamphetamine, cocaine, codeine, heroin, morphine, and 6-monoacethylmorphine). Several analytical conditions were taken into account to completely separate in the same run the 10 drugs of abuse analyzed. Chromatographic retention, selectivity and efficiency were evaluated in dependence of the type of stationary phase (CN and RP-C 18 derivatized silica particles), mobile phase composition, buffer type and pH, sample injection. The optimum separation parameters were set up using a mixture of aqueous sodium phosphate buffer (pH 2.5)/acetonitrile (80/20, v/v) as the mobile phase, 10 kV and 20 °C as applied voltage and capillary temperature, respectively. Under these conditions all the studied analytes were baseline resolved within 20 min. The method performance was investigated in terms of precision, linearity, sensitivity and accuracy to demonstrate the applicability of the developed capillary electrochromatographic system to forensic analysis. Calibration curves provided a good linearity over a working range of 100–1200 ng/mL for all analytes. Limits of detection and quantification were in the range 5–12 ng/mL and 10–30 ng/mL, respectively. Then the method was applied to the analysis of a human urine sample spiked with a basic compounds’ mixture. Urine samples’ pre-treatment was carried out through a solid phase extraction (SPE) procedure on strong cation exchange (SCX) cartridges.

Peroxidase-Catalyzed Oligomerization of Ferulic Acid Esters

Valuable information about possible types of linkages, reaction mechanisms, and sequences for oxidative coupling of phenolic compounds in planta is available from in vitro model systems. Ferulate oligomers were generated in a system using ethyl ferulate, peroxidase, and hydrogen peroxide under various conditions. A molar ferulate/H2O2 ratio of 1:1, an ethanol level of 30% in an aqueous sodium phosphate buffer (pH 6.0), and a reaction time of 10 min were considered to be ideal to produce maximal proportions of ferulate trimers and tetramers from ethyl ferulate as starting material. The dominant trimer and tetramer were each isolated from the reaction mixture and identified as 8-O-4/8-5(cyclic)-dehydrotriferulic acid triethyl ester and 8-5(cyclic)/4-O-5/8-5(cyclic)-dehydrotetraferulic acid tetraethyl ester. The structure of the 8-O-4/8-5(cyclic)-dehydrotriferulic acid triethyl ester revealed that a third ferulate unit is bound to a preformed 8-O-4-diferulate dimer, a surprising reaction sequence considering the dominance of 8-5-coupled dimers among dehydrodiferulates in H2O2/peroxidase-based model reactions. As 4-O-5-coupling is not favored in the dimerization process of ferulates, the main tetramer isolated in this study is probably formed by 4-O-5-coupling of two preformed 8-5(cyclic)-diferulates, a logical step in analogy with reactions occurring in lignin biosynthesis.

Optimisation of the derivatization in cellulose‐type chiral selectors for enantioseparation by centrifugal partition chromatography

Cellulose was chemically modified with hydrophobic dodecanoyl groups followed by 3,5-dimethylphenylcarbamoyl substituents forming mixed ester/carbamate derivatives in order to improve the solubility in lipophilic solvents compared to the corresponding homosubstituted cellulose tris(3,5-dimethylphenylcarbamate). Two mixed derivatives of different degree of substitution were prepared, tested as chiral selectors (CSs) in counter-current chromatography (CCC) and compared to the homo-substituted derivative. Alternatively, cellulose tris(3,5-dichlorophenylcarbamate), was synthesised and tested with the same purpose. The racemic drugs pindolol and warfarin were used as test compounds. Biphasic organic/aqueous solvent systems, composed of methyl isobutyl ketone, t-butyl methyl ether or ethyl acetate and aqueous ammonium acetate or sodium phosphate buffer, were used. Centrifugal partition chromatography, a variety of CCC, in the classical elution mode and the pH-zone-refining displacement mode was applied. The enantioseparation of pindolol and warfarin was achieved in the latter conditions. The presence of dodecanoyl chains on the CS increased solubility in the organic solvents used. The selectivity of the mixed dodecanoyl/3,5-dimethylphenylcarbamoyl cellulose derivative with low degree of dodecanoyl groups (degree of substitution 0.3/degree of substitution 2.6, respectively) was similar to that of the homo-substituted derivative. Furthermore, the loading capacity for pindolol was increased by a factor of three compared to cellulose tris(3,5-dimethylphenylcarbamate). Nevertheless, the increasing degree of substitution with dodecanoyl groups on the CS, although improved solubility in the stationary phase, contributed negatively to the enantioselectivity, where warfarin was more affected than pindolol.

Challenges in the Analytical Method Development and Validation for an Unstable Active Pharmaceutical Ingredient

A sensitive high-performance liquid chromatography (HPLC) impurity profile method for the antibiotic ertapenem is developed and subsequently validated. The method utilizes an Inertsil phenyl column at ambient temperature, gradient elution with aqueous sodium phosphate buffer at pH 8, and acetonitrile as the mobile phase. The linearity, method precision, method ruggedness, limit of quantitation, and limit of detection of the impurity profile HPLC method are found to be satisfactory. The method is determined to be specific, as judged by resolving ertapenem from in-process impurities in crude samples and degradation products that arise from solid state thermal and light stress, acid, base, and oxidative stressed solutions. In addition, evidence is obtained by photodiode array detection studies that no degradate or impurity having a different UV spectrum coeluted with the major component in stressed or unstressed samples. The challenges during the development and validation of the method are discussed. The difficulties of analyzing an unstable active pharmaceutical ingredient (API) are addressed. Several major impurities/degradates of the API have very different UV response factors from the API. These impurities/degradates are synthesized or prepared by controlled degradation and the relative response factors are determined.

A Nickel Superoxide Dismutase Maquette That Reproduces the Spectroscopic and Functional Properties of the Metalloenzyme

Described herein is a nickel superoxide dismutase (NiSOD) maquette ([Ni(SOD(M1))]) based on the first 12 residues from the N-terminal sequence of Streptomyces coelicolor NiSOD. The apopeptide (SOD(M1)) was prepared by standard solid-phase Fmoc peptide synthesis. SOD(M1) will readily coordinate Ni(II) in a 1:1 ratio in slightly basic aqueous sodium phosphate buffer (0.1 M; pH = 7.2) forming a lightly colored beige/pink solution. Unlike NiSOD, which is isolated as a 1:1 mixture of oxidized (Ni(III)) and reduced (Ni(II)) forms, [Ni(SOD(M1))] can only be isolated in the Ni(II) oxidation state. The UV/vis, X-ray absorption, and CD spectra of [Ni(II)(SOD(M1))] correspond well with those reported for the reduced form of NiSOD. Despite the fact that [Ni(III)(SOD(M1))] is not isolable, [Ni(SOD(M1))] has an appropriate redox potential to act as an SOD (E(1/2) = 0.70(2) V vs Ag/AgCl) and in fact will catalytically disproportionate >40 000 equiv of KO(2).

Gelatin–alpha olefin sulfonate interactions studied by dynamic light scattering

Dynamic light scattering (DLS) measurements were performed to study the binding of anionic surfactant alpha olefin sulfonate (AOS) to gelatin chains at various NaCl concentrations at 30 °C in aqueous sodium phosphate buffer (pH=6.8) solutions. The surfactant concentration was varied from 0 to 80 mM and the NaCl concentrations chosen were 0.025, 0.05, and 0.1 M. AOS exhibited electrostatic binding to the positively charged sites of the polypeptide chain resulting in considerable reduction in its hydrodynamic radius up to critical micellar concentration (cmc=8 mM for no salt, 0.01 and 0.025 M, and 5 mM for 0.05 M and 2 mM for 0.1 M solutions). The correlation function revealed the presence of two types of structures above cmc; namely the micelles of AOS and gelatin–AOS micelle complexes. The micellar radii ( R m), the effective gelatin–surfactant complex radii ( R c), have been determined as a function of salt concentration. No critical aggregation concentration (cac) was observed. The inter-gelatin–surfactant complex ( k D 1 ) and inter-micellar interactions ( k D 2 ), were determined by fitting the concentration dependence of R m and R c to a virial expansion in reduced concentration ( c−cmc), which are compared. While k D 1 showed strong ionic strength dependence, k D 2 remained invariant of the same. The protein to surfactant binding ratio was found to be smaller than normal. Results have been discussed within the framework of the necklace-bead model of polymer–surfactant interactions.

Nanoscale Intermolecular Interactions between Human Serum Albumin and Alkanethiol Self-Assembled Monolayers

To study the molecular origins of hemocompatibility, the blood plasma protein human serum albumin (HSA) was covalently grafted to a nanosized probe tip at the end of a soft, microfabricated cantilever force transducer. The net force versus separation distance between the HSA-modified probe tip and three different model surfaces, including (1) gold; (2) a hydrophobic, CH3-terminated alkanethiol self-assembling monolayer (SAM); and (3) a hydrophilic, COO--terminated alkanethiol SAM in aqueous sodium phosphate buffer solution (PBS, ionic strength (IS) = 0.01 M, pH = 7.4), was recorded and compared to the values of various theoretical models. The approach interaction of the HSA probe tip on the COO--terminated SAM and Au substrates was found to be purely repulsive for D < 15 nm, nonlinear with decreasing separation distance, and consistent with electrostatic double layer repulsion. The approach interaction of the HSA probe tip on the CH3-terminated SAM substrate was found to be purely attractive, long range (D < 80 nm), nonlinear with decreasing separation distance, and much greater in magnitude and range than that known for van der Waals interactions between hydrocarbon SAMs terminated with hydrophilic chemical groups on Au. Large adhesive energies were observed for the HSA probe tip on both the CH3-terminated SAM and Au surfaces (≤−29 mN/m, −22 to −73kBT/protein), while smaller adhesive energies were observed on the COO--terminated SAM surface (≤ −4.9 mN/m, −5.3 to −12kBT/protein). It was shown that short-range adhesive contacts between the HSA chain segments and these surfaces give rise to energy dissipating mechanisms, such as HSA entropic molecular elasticity and enthalpic unfolding forces (deformation and rupture of noncovalent intramolecular bonds) and noncovalent bond rupture of the HSA chain segments adsorbed to the surface.

Investigation of guanidine hydrochloride induced unfolding of apolipoprotein A‐IMilano

A guanidine hydrochloride (GuHCl) induced unfolding study of apolipoprotein A‐IMilano (apo A‐IM) has been performed. The unfolding was followed by circular dichroism (CD) measurements at 222 nm and by isothermal titration (ITC) calorimetry at 25°C. In the ITC experiments enthalpies of transfer were determined for apo A‐IM from aqueous sodium phosphate buffer solution into solutions of different concentrations of GuHCl. The CD data and the ITC data give complementary and consistent results of the complex unfolding process. Analytical ultracentrifugation experiments were made on apo A‐IM in sodium phosphate buffer and in 0.6 M GuHCl, respectively. Analyses of the obtained sedimentation velocity data show that apo A‐IM is highly aggregated in sodium phosphate buffer. The aggregates are almost completely dissociated in 0.6 M GuHCl. Aggregation of the protein in sodium phosphate buffer solution induces an increase in α‐helical content. The loss of α‐helical secondary structural element of the protein upon dissociation of the aggregates destabilises the protein resulting in a low GuHCl concentration of unfolding, [GuHCl]m=1.1 M. The unfolded protein has a significant α‐helical content at the unfolded state. From ITC‐ and CD data we suggest that increased binding of GuHCl to the unfolded protein results in a disruption of the residual secondary structure.

The decomposition of peroxynitrite to nitroxyl anion (NO − ) and singlet oxygen in aqueous solution

The mechanism of decomposition of peroxynitrite (OONO − ) in aqueous sodium phosphate buffer solution at neutral pH was investigated. The OONO − was synthesized by directly reacting nitric oxide with superoxide anion at pH 13. The hypothesis was explored that OONO − , after protonation at pH 7.0 to HOONO, decomposes into 1 O 2 and HNO according to a spin-conserved unimolecular mechanism. Small aliquots of the concentrated alkaline OONO − solution were added to a buffer solution (final pH 7.0–7.2), and the formation of 1 O 2 and NO − in high yields was observed. The 1 O 2 generated was trapped as the transannular peroxide (DPAO 2 ) of 9,10-diphenylanthracene (DPA) dissolved in carbon tetrachloride. The nitroxyl anion (NO − ) formed from HNO (pKa 4.5) was trapped as nitrosylhemoglobin (HbNO) in an aqueous methemoglobin (MetHb) solution. In the presence of 25 mM sodium bicarbonate, which is known to accelerate the rate of decomposition of OONO − , the amount of singlet oxygen trapped was reduced by a factor of ≈2 whereas the yield of trapping of NO − by methemoglobin remained unaffected. Because NO 3 − is known to be the ultimate decomposition product of OONO − , these results suggest that the nitrate anion is not formed by a direct isomerization of OONO − , but by an indirect route originating from NO − .

The decomposition of peroxynitrite to nitroxyl anion (NO-) and singlet oxygen in aqueous solution

The mechanism of decomposition of peroxynitrite (OONO(-)) in aqueous sodium phosphate buffer solution at neutral pH was investigated. The OONO(-) was synthesized by directly reacting nitric oxide with superoxide anion at pH 13. The hypothesis was explored that OONO(-), after protonation at pH 7.0 to HOONO, decomposes into (1)O(2) and HNO according to a spin-conserved unimolecular mechanism. Small aliquots of the concentrated alkaline OONO(-) solution were added to a buffer solution (final pH 7.0-7.2), and the formation of (1)O(2) and NO(-) in high yields was observed. The (1)O(2) generated was trapped as the transannular peroxide (DPAO(2)) of 9, 10-diphenylanthracene (DPA) dissolved in carbon tetrachloride. The nitroxyl anion (NO-) formed from HNO (pKa 4.5) was trapped as nitrosylhemoglobin (HbNO) in an aqueous methemoglobin (MetHb) solution. In the presence of 25 mM sodium bicarbonate, which is known to accelerate the rate of decomposition of OONO(-), the amount of singlet oxygen trapped was reduced by a factor of approximately 2 whereas the yield of trapping of NO(-) by methemoglobin remained unaffected. Because NO(3)(-) is known to be the ultimate decomposition product of OONO(-), these results suggest that the nitrate anion is not formed by a direct isomerization of OONO(-), but by an indirect route originating from NO(-).

Capillary electrophoresis behavior of water-soluble anionic porphyrins in the presence of beta-cyclodextrin and its O-methylated derivatives.

The electrophoretic behavior of water-soluble anionic porphyrins, such as meso-tetrakis(4-carboxyphenyl) porphyrin (TCPP), meso-tetrakis(4-sulfonatophenyl) porphyrin (TSPP) and its zinc(II) and copper(II) complexes (ZnTSPP and CuTSPP, respectively) has been studied by capillary zone electrophoresis using fused-silica capillaries. The selectivity of the separation is strongly dependent on the type and concentration of betacyclodextrin (betaCD) or the O-methylated derivatives added to the background electrolyte. CuTSPP and TSPP can be separated using a pH 2.5 aqueous sodium phosphate buffer in the presence of 1 mM betaCD. Resolution is poorer or absent employing alkylated betaCDs, such as the heptakis (2,6-di-O-methyl)-beta-cyclodextrin or the heptakis(2,3,6-triO-methyl)-beta-cyclodextrin, as additives. On the other hand, separation of TSPP from its copper and zinc complexes has been achieved using a pH 7.0 aqueous sodium phosphate buffer, in the presence of 0.75 mM betaCD and 20% dimethyl sulfoxide (DMSO) as organic modifier. Under such conditions, the calibration curve for quantitative analysis of copper(II) was obtained. A rationale for the observed behavior will be presented and discussed on the basis of binding and protonation equilibria and a simple mathematical model.

High-performance liquid chromatographic analysis of the 2-chloroprocaine metabolite, diethylaminoethanol, in blood and serum

Background and Objectives. 2-Chloroprocaine is rapidly metabolized in the blood to yield 2-chloro-para-aminobenzoic acid (an inactive metabolite) and diethylaminoethanol (DEAE). DEAE possesses local anesthetic activity. The only reported assay for DEAE is a colorimetric method. Methods. Clinical samples of whole blood and serum were obtained from patients receiving stepped intravenous infusions of 3% 2-chloroprocaine. A high pH-dependent liquid-liquid extraction step with diethyl ether was used to eliminate interfering peaks in high-pressure liquid chromatography (HPLC) analysis. Separation and quantitation were performed using HPLC on a polymeric-reversed phase column with a mobile phase consisting of 10% or 20% acetonitrile (for whole blood or serum analysis, respectively) in 50 mm aqueous sodium phosphate buffer, pH = 11.50. The elution order of DEAE and its analogues was tested to interpret the HPLC separation mechanism. Results. Extraction recovery of DEAE from whole blood was 67 ± 13.5%, from serum, 71 ± 12.2%, and from water, 75 ± 2.9%. The high pH value of the mobile phase resulted in sharp, well-resolved peaks with run times of approximately 8 minutes using 20% acetonitrile. The lower limit of detection was 5 ng/mL of DEAE from a 1-mL sample. The elution order of DEAE and its analogues indicated that separation was based on the hydrophobicity of the analytes rather than polar group interactions occurring with silica-based stationary phase. Conclusions. A new, simple and rapid HPLC method for extraction and measurement of DEAE in whole blood or serum samples is reported here.

Solution and Photoproperties of N-(2-Hydroxypropyl)methacrylamide Copolymer−Meso-chlorin e6 Conjugates

The solution properties of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing various numbers of meso-chlorin e6 monoethylenediamine (Mce6) molecules attached to the copolymer via either glycine (G) or tetrapeptide (GFLG) side chains were studied. Dynamic light scattering, spectroscopic, fluorescence quenching, and time-resolved fluorescence techniques were used. The photosensitizing efficiencies of the various derivatives were also examined. Reactions were measured in aqueous sodium phosphate buffer (SPB) and ethanol. The dynamic light scattering data indicate that the intermolecular aggregation of Mce6 species within the HPMA copolymer conjugates is not important at the conjugate concentration measured (5 × 10-4 g/mL). However, intramolecular aggregation of the hydrophobic Mce6 moieties does occur and was studied using absorption and fluorescence techniques. The degree of intramolecular aggregation was decreased by the addition of detergents or ethanol to the SPB solutions. The cationic detergent, CTAB, strongly enhanced the fluorescence of the copolymer conjugates due to its efficient electrostatic interactions with the negatively charged Mce6 species. It also significantly increased the relative quantum yield of oxygen uptake during the copolymer conjugate-sensitized photooxidation of furfuryl alcohol. The observed iodide quenching of copolymer conjugate fluorescence implies that hydrophobic domains of aggregated Mce6 moieties may exist in SPB solutions of the conjugates. The time-resolved fluorescence decay measurements showed that about 15% of the Mce6 species were aggregated in SPB solutions of those copolymer conjugates with the highest Mce6 content. There was a low degree of aggregation of free Mce6 molecules in SPB solutions at the concentrations used.