ABSORPTION AND PHOTOCHEMISTRY OF SENSORY RHODOPSIN—I: pH EFFECTS

Pyranine (8-hydroxyl-1,3,6-pyrene-trisulfonate) was used as a pH-probe to test whether there is a light-induced proton release to the bulk phase during the photochemical reaction cycle of sensory rhodopsin-I (SR-I). We conclude that the retinylidene Schiff-base proton is retained by SR-I-containing envelope vesicles during the SR-I photocycle under the conditions described here. Bacteriorhodopsin containing vesicles were used as a control to show that light-induced proton release can be observed under identical data acquisition parameters as those used for SR-I-containing vesicles. In addition, the effects of extravesicular pH on the absorption maximum (lambda max) and the SR-I photocycle were studied. SR-I properties are insensitive to pH in the range approximately 3 to approximately 8 with lambda max remaining at 587 nm. The lambda max shifts to 565 nm below pH 3.0 and to 552 nm at pH 10.8 with an apparent pKa of 8.5. Flash-induced absorbance changes of SR-I are described under neutral, alkaline and acidic conditions. The neutral, alkaline and acid SR-I forms each undergo similar photoreactions producing long-lived (> 500 ms decay half-time) blue-shifted intermediates. The UV/near-UV absorption of the photoproducts from neutral and alkaline SR-I indicate a deprotonated Schiff base, whereas acid SR-I produces a species with lambda max > 460 nm indicative of a protonated Schiff base.     

Study of ph and substrate effects on room-temperature phosphorimetry of some indolecarboxylic acids

Room-temperature phosphorescence (RTP) of eight indolecarboxylic acids has been investigated under different pH conditions, and on several ion-exchange filter papers. RTP excitation and emission wavelengths do not change significantly with pH. The largest RTP signals are obtained from neutral solutions adsorbed on DE-81 anion-exchange filter paper, while alkaline (pH approximately 13) solutions on S & S 903 filter paper treated with DTPA (diethylenetriaminepenta-acetic acid) give stronger signals than neutral or acidic (pH approximately 1.6) solutions on the same substrate. The existence of several hydrogen-bonding interactions between the various ionic indolecarboxylic acid species and the substrates is discussed. Heavy-atom enhancement factors ranging between 4 and 550, according to the compound, are obtained with iodide. Absolute limits of detection between 100 and 500 pg demonstrate the usefulness of RTP for determination of indolecarboxylic acids at the trace level.     

Phanquinone as a suitable derivatization reagent in micellar electrokinetic chromatography and HPLC analysis of amino acids

Phanquinone (chemically: 4,7-phenanthroline-5,6-dione) was applied as an original precolumn derivatization reagent for amino acids followed by separation using MEKC with UV detection (240 nm). The derivatization reaction was carried out at 68 degrees C in the presence of aqueous phosphate buffer (pH 8.0) and it was found to be complete after 30 min. Twelve derivatized standard amino acids were separated in about 22 min under MEKC conditions using sodium cholate (250 mM) as the surfactant in phosphate buffer (20 mM, pH 9.0). The developed method was validated for the analysis of D,L-phosphoserine (D,L-p-Ser) and L-glutamine (L-Gln); good linearity (r > 0.999) was achieved in the calibration range of 0.25-2.5 micromol/mL. The sensitivity of the MEKC method (LOD 0.1 micromol/mL; LOQ 0.25 micromol/mL, RSD% <5.0%, n = 3) was found to be adequate for quantitation of amino acids in pharmaceuticals. Quantitative applications of the validated MEKC method were carried out by the analysis of commercially available oral polyaminoacid formulations (tablets and extemporaneous solutions) containing L-Gln and D,L-p-Ser; the obtained results were found to be in agreement with those from a validated reference RP-HPLC method.     

Isolation and characterization of degradation products of citalopram and process-related impurities using RP-HPLC

A reversed-phase high-performance liquid chromatographic method for simultaneous separation and determination of citalopram hydrobromide and its process impurities in bulk drugs and pharmaceutical formulations was developed. The separation was accomplished on an Inertsil ODS 3V (250x4.6 mm; particle size 5 mum) column using 0.3% diethylamine (pH = 4.70) and methanol/acetonitrile (55:45 v/v) as mobile phase in a gradient elution mode. The eluents were monitored by a photodiode array detector set at 225 nm. The chromatographic behavior of all the related substances was examined under variable conditions of different solvents, buffer concentrations, and pH. The method was validated in terms of accuracy, precision, and linearity. The method could be of use not only for rapid and routine evaluation of the quality of citalopram in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations. Three unknown impurities were consistently observed during the analysis of different batches of citalopram. Forced degradation of citalopram was carried out under thermal, photo, acidic, alkaline, and peroxide conditions. The degradation products and unknown impurities were isolated and characterized by ESI-MS/MS, (1)H NMR, and FT-IR spectroscopy.     

A validated high-performance liquid chromatographic method for the determination of moclobemide and its two metabolites in human plasma and application to pharmacokinetic studies

A rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with ultraviolet detection has been developed for the determination of moclobemide and its metabolites, p-chloro-N-(-2-morpholinoethyl)benzamide N'-oxide (Ro 12-5637) and p-chloro-N-[2-(3-oxomorpholino)ethyl]-benzamide (Ro 12-8095), in human plasma. The assay was performed after single liquid-liquid extraction with dichloromethane at alkaline pH using phenacetin as the internal standard. Chromatographic separation was performed on a C(18) column using a mixture of acetonitrile and water (25:75, v/v), adjusted to pH 2.7 with ortho-phosphoric acid, as mobile phase. Spectrophotometric detection was performed at 239 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The quantification limit for moclobemide and Ro 12-8095 was 10 ng/mL, and for Ro 12-5637 was 30 ng/mL. Linearity of the method was confirmed for the range 20-2500 ng/mL for moclobemide (r = 0.9998), 20-1750 ng/mL for Ro 12-8095 (r = 0.9996) and 30-350 ng/mL for Ro 12-5637 (r = 0.9991). Moreover, within-day and between-day precisions and accuracies of the method were established. The described method was successfully applied in pharmacokinetic studies of parent drug and its two metabolites after a single oral administration of 150 mg of moclobemide to 20 healthy volunteers.     

Determination of saccharides in biological materials by high-performance anion-exchange chromatography with pulsed amperometric detection

High-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) under alkaline conditions (pH 9-13) separates aminosaccharides, neutral saccharides and glycuronic acids based upon their molecular size, saccharide composition and glycosidic linkages. Carbohydrates were extracted by utilizing 0.5 M H2SO4 (neutral monosaccharides), 0.25 M H2SO4 coupled with enzyme catalysis (glycuronic acids) and 3 M H2SO4 (aminosaccharides). Solid-phase extraction with strong cation and strong anion resins was used to partition the cationic aminosaccharides and anionic glycuronic acids and to deionize acid extracts for neutral saccharides. Separation was conducted on a medium-capacity anion-exchange column (36 mequiv.) utilizing sodium hydroxide (5-200 mM and sodium acetate (0-250 mM) as the mobile phase. The saccharides were detected by oxidation at a gold working electrode with triple-pulsed amperometry. HPAEC-PAD was found superior to high-performance liquid chromatography with refractive index (RI) detection for neutral monosaccharides and aminosaccharides and to low-wavelength UV detection for glycuronic acids in terms of resolution and sensitivity. HPAEC-PAD was not subject to interferences as was the case for low UV detection (210 nm) or RI analyses and was highly selective for mono- and aminosaccharides and glycuronic acids. The use of HPAEC-PAD was applied for the determination of the saccharide composition of organic materials (plant residues, animal wastes and sewage sludge), microbial polymers and soil.     

Surfaces designed for charge reversal

We have created surfaces which switch from cationic at pH < 3 to anionic at pH > 5, by attaching aminodicarboxylic acid units to silica and gold substrates. Charge reversal was demonstrated by monitoring the adsorption of cationic dyes (methylene blue and a tetracationic porphyrin) and an anionic sulfonated porphyrin, at a range of pH using UV-vis absorption and reflection spectroscopy. The cationic dyes bind under neutral conditions (pH 5-7) and are released at pH 1-4, whereas the anionic dye binds under acidic conditions (pH 1-4) and is released at pH 5-7. Gold surfaces were functionalized with two different amphoteric disulfides with short (CH(2))(2) and long (CH(2))(10)CONH(CH(2))(6) linkers; the longer disulfide gave surfaces exhibiting charge reversal in a narrower pH range. Adsorption is much faster on the functionalized gold (t(1/2) = 62 s) than on functionalized silica (t(1/2) = 6900 s), but the final extents of coverage on both surface are similar, for a given dye at a given pH, with maximal coverages of around 2 molecules nm(-)(2). These charge-reversal processes are reversible and can be repeatedly cycled by changing the pH. We have also created surfaces which undergo irreversible proton-triggered charge switching, using a carbamate-linked thiol carboxylic acid which cleaves in acid. These surfaces are versatile new tools for controlling electrostatic self-assembly at surfaces.     

Optimization and validation of a stability-indicating RP-HPLC method for determination of azithromycin and its related compounds

A validated stability-indicating HPLC method was developed for the analysis of azithromycin (AZ) and its related compounds in raw materials, capsule, and suspension using an Xterra RP C18 column at 50 degrees C with UV detection at 215 nm. Isocratic elution was employed using the mobile phase 14 mM disodium hydrogen phosphate (pH 10.5, adjusted by 1 M NaOH)-methanol-acetonitrile-tetrahydrofuran (40.0 + 30.0 + 30.0 + 0.1, v/v/v/v). AZ and 14 of its related compounds were separated and quantified. The described method was linear over the range of 2-1800 microg/mL AZ with (r = 0.9999). The stability of AZ was studied under accelerated acidic, alkaline, and oxidative conditions. The proposed method was used to investigate the kinetics of acidic and alkaline hydrolysis process of AZ at different temperatures, and the apparent pseudo first-order rate constant, half-life, and activation energy were calculated. The major peak detected from the degradation of AZ in alkaline and acidic conditions was decladinosylazithromycine, while azithromycin N-oxide was detected from the oxidative degradation. Long-term stability studies for capsule and oral suspension were carried out. The proposed stability-indicating method was completely validated according to the U.S. Food and Drug Administration requirements.     

Separation and determination of chiral composition in penicillamine tablets by capillary electrophoresis in a broad pH range

A chiral capillary electrophoretic method with nearly full pH window was explored for the separation and determination of dl-penicillamine. A facile one-pot labeling technique was coupled in the method for introduction of chromophore and charge groups onto the analytes to facilitate the electromigration and sensitive detection. By using simply a cost-effective neutral β-cyclodextrin as chiral selector, baseline separation of the dl-penicillamine was achieved from pH 2.0 to over pH 10. Quantification of standard d- and l-penicillamines was demonstrated by taking pH 4.5, 7.4, and 9.7 as the representatives of acidic, neutral, and basic conditions. The working curves were constructed between peak area and concentration, having linear ranges of 8.56-8.56 × 10(2) μg/mL for pH 4.5 and 8.56-1.71 × 10(3) μg/mL for pH 7.4 and 9.7, with correlation coefficients all better than 0.999. The limit of detection (S/N = 3) was 2.58 μg/mL in acidic and neutral conditions or 1.41 μg/mL in basic condition. The method was further validated by assaying the commercial penicillamine tablets, applicable to quantification of the effective enantiomer and the trace impurity of l-penicillamine at a content of down to 0.2, 0.6, and 2.0% for pH 9.7, 4.5, and 7.4, respectively. The recovery determined by spiking technique was in a range from 93.1 to 105 %. The method is easily extendable to the analysis of other chiral amines or amino acids.     

HPLC–PDA identification and resolution of rufinamide forced degradation impurities: A congregated chemometric expedite optimization coupled with factorials and desirability

Rufinamide is used presently to treat Lenaux–Gastaut syndrome. A full factorial design and desirability approach was investigated for the optimization of hydrolytic stress via response surface curves (RSCs). The degradation impurities were identified and resolved using reversed-phase high-performance liquid chromatography (RP-HPLC) on the Qualisil® BDS C₈ column. Acetonitrile–water (29:71, v/v) was optimized for the mobile phase and used at a flow rate of 1.0 ml/min with detection at a wavelength of 230 nm. Rufinamide showed appreciable susceptibility to hydrolysis under acidic and alkaline stress, and substantial degradation in the neutral condition. It degraded much less under oxidative stress. Exposure towards thermal and photolytic stress conditions indicated appreciable stability. The developed method was subjected to validation as per the recommendations of the International Conference on Harmonization. The proposed method showed no influence from the excipients and the degradation products. As well as good precision and accuracy in determination, the method showed a linear response between 2 and 12 μg ml⁻¹. The method was extended for determination in a human plasma sample, which resulted in excellent recovery without interference from matrix effects. The combined use of desirability and design for the optimization of acidic and alkaline hydrolytic stress led to simple and rapid analysis.     

Reversed Phase Chromatographic Analysis of 13 Amino Acids in Honey Samples

A simple method using reversed phase high-performance liquid chromatography (RP-HPLC) was developed for the simultaneous analysis of 13 amino acids. Amino acids were pre-column derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl) before analysis by RP-HPLC. Experimental parameters affecting the derivatization and chromatographic separation were investigated. Amino acids were derivatized with FMOC-Cl under alkaline condition in 0.1 mol/L borate buffer pH 10.0 at room temperature. The FMOC-amino acid derivatives were separated on an Atlantis C18 column under the gradient elution of 0.05 % trifluoroacetic acid and acetonitrile and UV detection at 265 nm. Linear ranges were 0.2–100.0 μg/mL with the correlation coefficients greater than 0.992. Limits of detection and limits of quantitation were in the range of 0.05–2.0 and 0.2–5.0 µg/L, respectively. The intra-day precision (n = 3) of retention time was less than 1 %, while for the peak area was less than 4 %. The inter-day precision (n = 3 × 3) of retention time was less than 2 % and the peak area was less than 8 %. This method was applied in honey samples and the results showed that proline is the major amino acids in honey samples.

     

High performance liquid chromatography and capillary electrophoresis determination of sanguinarine in biological matrices

HPLC and CE methods were employed to determine the quaternary benzo[c]phenanthridine alkaloid sanguinarine in biological matrices (rat hepatocytes, human gingival fibroblasts, feed, porcine faeces, body fluids and tissues). HPLC was carried out on a C₁₈ column using gradient elution and ion pairing techniques with 1‐heptanesulfonic acid as ion pairing agent under acidic conditions. The detection limit for fluorimetric detection at λ_ex = 327 nm and λ_em = 577 nm was 3 nM sanguinarine. CE analyses were performed in 50 mM phosphate‐Na buffer pH 2.5, with 150 mM SDS used for pre‐concentration by the sweeping effect. This experimental configuration allows injecting the total capillary length with sanguinarine sample. The detection limit for UV detection at 285 nm was 12 nM. Both methods are suitable for analysing submicromolar quantities of sanguinarine in biological materials. The HPLC method is more sensitive than CE because it uses fluorescence detection.     

氨基苯甲酸衍生化高效液相色谱法 分析多糖中的单糖及糖醛酸组成

衍生反相离子对色谱法同时分离检测多糖中单糖及糖醛酸组成的方法,筛选出适合于p-AMBA糖衍生物分离的色谱柱,考察了流动相组成对9种单糖和两种糖醛酸的p-AMBA衍生化产物的保留值及分离的影响,优化了反应温度和反应时间等衍生化条件,并应用优化的分析方法测定了螺旋藻中的单糖和糖醛酸的组成。采用紫外检测时,方法的检出限为 (2.55～13.4)×107mol/L;采用荧光检测时,方法的检出限为(3.38～176)×108 mol/L。     

High-performance liquid chromatography-mass spectrometry of an osteocalcin derivative.

High-performance liquid chromatography (HPLC) was combined on-line with electrospray ionization mass spectrometry (ESI-MS) for structural analysis of a synthetic osteocalcin derivative and its degradation products. Initial determination of amino acid sequence of the synthetic peptide was performed after tryptic degradation. Hydrolytic degradation of the osteocalcin derivative was studied under different pH conditions: pH 2, pH 7 and pH 10 at 60 degrees C up to 20 h. According the HPLC-ESI-MS results, the chemical stability was dependent on pH. Two major degradation products and a number of other fragments were obtained in acidic solution, whereas the osteocalcin molecule was rather stable in neutral and alkaline conditions.     

The effect of uranium speciation on the removal of gross alpha activity from acid mine drainage using anion exchange resin

The study was conducted to understand, (a) the effect of pH on the speciation of uranium in acid mine drainage and (b) determine the effect of uranium speciation on the removal of gross alpha activity from acid mine drainage. Speciation modeling predicted a dominance of neutral and positively charged species under acidic and neutral conditions. Negative species become dominant from pH 8. The effectiveness of the anionic exchange resin to remove gross alpha activity was lower under acidic conditions (pH < 6) compared to alkaline conditions (pH ≥ 8).

     

Developing a novel ratiometric fluorescent probe based on ESIPT for the detection of pH changes in living cells

As an important parameter of intracellular metabolism, pH plays important roles in maintaining normal physiological processes. The abnormal pH could cause disorder of cell function which may cause neurological diseases. Herein, we present two novel ratiometric fluorescent probes to detect pH changes. The probes employed 2-(2′-hydroxyphenyl)benzothiazole as fluorescent platform, and displayed desirable fluorescence response to pH on the basis of excited state intramolecular proton transfer (ESIPT) process. The probe BtyC-1 showed green fluorescence at 546 nm under acidic conditions, while it displayed strong blue fluorescence at 473 nm and weak green fluorescence at 546 nm under alkaline conditions. Biological experiments demonstrated that the probe BtyC-1 could be successfully applied for the ratiometric imaging of cellular pH and the NH₄Cl-induced pH changes in living cells.     

A novel technique for on-capillary preconcentration of anionic compounds applied to the trace analysis of rapamycin in human blood by capillary electrophoresis

A capillary electrophoretic (CE) method with UV detection at 278 nm has been developed for analysis of the immunosuppressant rapamycin (sirolimus) in human blood at low microg.L(-1) levels. Separation has been achieved in an acidic carrier electrolyte containing sodium dodecyl sulfate and 20% v/v methanol. For sample cleanup and preconcentration, both an off-line solid-phase extraction step using a silica-based reversed-phase material and a newly developed on-capillary focusing technique have been employed. The subsequent treatment of rapamycin under alkaline conditions leads to a cleavage of the lacton bond of the molecule, generating a negatively charged carboxylic group which allows electrokinetic injection into the CE instrument. During the injection process, the negatively charged analyte migrates into an acidic carrier electrolyte, so that it becomes neutral due to protonation and is focused at the capillary inlet. Injection times of 300 s at -7.5 kV could be applied without band-broadening. Results for real samples indicated that the method is fully suited for routine applications and may be an attractive alternative to established liquid chromatographic techniques.     

High-voltage contactless conductivity detection of underivatized amino acids in capillary electrophoresis

High-voltage contactless conductivity detection of underivatized amino acids in both acidic and basic media is demonstrated. The suitability of different acidic buffer solutions at pH values of about 2.5 was investigated with 12 amino acids. Lactic acid as background electrolyte gave the best results in terms of detection limits for arginine, lysine and histidine, which were approximately 2 x 10(-7), 3 x 10(-7) and 4 x 10(-7) M, respectively. However, the sensitivity for other species was not quite as good and the detection limits in the order of 0.5-1 x 10 (-5) M. The use of basic conditions at pH 10-11 generally led to more stable baselines and more consistent sensitivities. A range of 20 amino acids was investigated with alkaline buffers and detection limits were typically about 10(-6) M. Urine and beer samples were analyzed. Nine and eleven amino acids could be identified, respectively.     

FLUORESCENCE AND PHOTOCHEMISTRY OF OLIGOCYTIDYLIC AND POLYCYTIDYLIC ACIDS IN AQUEOUS SOLUTION

Abstract —In addition to the monomer-like fluorescence, a long-wavelength emission (Λ^max_em= 410 nm) has been detected in the dinucleoside 5'-5' pyrophosphate (CppC) at room temperature. This emission looks very similar to that previously reported for the acidic forms of Poly C (Poly C. Poly C⁺ and Poly I. Poly C. Poly C⁺). Only the monomer-like emission (Λ^max_em= 330 nm) can be detected in neutral Poly C, acidic CppC, and the neutral or protonated forms of the dinucleoside phosphate CpC.A correlation between the room temperature fluorescence of oligo and polycytidylic acids and their photochemical behaviour is found. Irradiation of all the polymeric samples at both neutral and acid pH results in the formation of minor photoproducts. They have been characterized by their absorbance (in the range 300–400 nm) and their fluorescence spectra. The same product is obtained in all cases where the monomer-like fluorescence only is detected. Distinct products are formed in neutral CppC and in the acidic Poly C forms.
The results are discussed with respect to the conformation of the oligo and polycytidylic acids and possible relationships between the 410–420 nm emission and adduct formation. An excimer is proposed as a common, intermediate excited state in both radiative deactivation and adduct formation in neutral CppC and the acidic Poly C forms.     

Gallium(III) complexes of DOTA and DOTA-monoamide: kinetic and thermodynamic studies

Given the practical advantages of the (68)Ga isotope in positron emission tomography applications, gallium complexes are gaining increasing importance in biomedical imaging. However, the strong tendency of Ga(3+) to hydrolyze and the slow formation and very high stability of macrocyclic complexes altogether render Ga(3+) coordination chemistry difficult and explain why stability and kinetic data on Ga(3+) complexes are rather scarce. Here we report solution and solid-state studies of Ga(3+) complexes formed with the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, (DOTA)(4-), and its mono(n-butylamide) derivative, (DO3AM(Bu))(3-). Thermodynamic stability constants, log K(GaDOTA) = 26.05 and log K(GaDO3AM(Bu)) = 24.64, were determined by out-of-cell pH-potentiometric titrations. Due to the very slow formation and dissociation of the complexes, equilibration times of up to ～4 weeks were necessary. The kinetics of complex dissociation were followed by (71)Ga NMR under both acidic and alkaline conditions. The GaDOTA complex is significantly more inert (τ(1/2) ～12.2 d at pH = 0 and τ(1/2) ～6.2 h at pH = 10) than the GaDO3AM(Bu) analogue (τ(1/2) ～2.7 d at pH = 0 and τ(1/2) ～0.7 h at pH = 10). Nevertheless, the kinetic inertness of both chelates is extremely high and approves the application of Ga(3+) complexes of such DOTA-like ligands in molecular imaging. The solid-state structure of the GaDOTA complex, crystallized from a strongly acidic solution (pH < 1), evidenced a diprotonated form with protons localized on the free carboxylate pendants.