Accelerated dissolution testing for controlled release microspheres using the flow-through dissolution apparatus.

Theophylline controlled release capsules (THEO-24 CR) were used as a model system to evaluate accelerated dissolution tests for process and quality control and formulation development of controlled release formulations. Dissolution test acceleration was provided by increasing temperature, pH, flow rate, or adding surfactant. Electron microscope studies on the theophylline microspheres subsequent to each experiment showed that at pH values of 6.6 and 7.6 the microspheres remained intact, but at pH 8.6 they showed deterioration. As temperature was increased from 37-57 degrees C, no change in microsphere integrity was noted. Increased flow rate also showed no detrimental effect on integrity. The effect of increased temperature was determined to be the statistically significant variable.

The binary eutectic of NSAIDS and two-phase liquid system for enhanced membrane permeation.

The eutectic properties of binary mixtures of some nonsteroidal anti-inflammatory drugs (NSAIDs) with ibuprofen were studied using differential scanning calorimetry (DSC) and phase equilibrium diagrams. The melting points of selected NSAIDs were significantly depressed due to binary eutectic formation with ibuprofen. Ketoprofen and ibuprofen were selected to study the effect of eutectic formation on membrane permeation using Franz diffusion cells and snake skin as the model membrane. The presence of aqueous isopropyl alcohol (IPA) was necessary to completely transform the solid drugs into an oily state at ambient temperature. As much as the 99.6% of ibuprofen and the 88.8% of ketoprofen added were found in the oily phase of the two-phase liquid system formed when aqueous IPA was added to the eutectic mixture. Due to the high drug concentration in the oily phase, and maximum thermodynamic activity, the two-phase liquid system showed enhanced membrane permeation rates of ibuprofen (37.5 microg/cm2/hr) and ketoprofen (33.4 microg/cm2/hr) compared to other reference preparations used.

Nonsuperoxide lucigenin-enhanced chemiluminescence from phospholipids and human saphenous veins.

The use of lucigenin-enhanced chemiluminescence (CL) for the detection of superoxide (O2.-) has grown in popularity due to an increased demand for a simple and specific system capable of measuring superoxide. In this study we report a lucigenin-CL signal emanating from human saphenous veins (SV) that was not inhibited by superoxide dismutase (SOD) and lasted for more than 24 h. A larger CL-signal with similar properties was produced by saphenous veins that had been dehydrated. A similar, non-SOD-inhibitable lucigenin-CL was also produced with a variety of phospholipids and phosphatidic acid. The chemical moiety responsible for the phospholipid CL is oxygen dependent but remains unidentified because a variety of lipids and phosphate containing species failed to produce such a signal. These results suggest that the use of lucigenin as a specific CL enhancer for O2.- must be clearly discriminated with a specific O2.- inhibitor when used in biological systems.

Chemiluminescent detection of artemisinin. Novel endoperoxide analysis using luminol without hydrogen peroxide.

A novel method for artemisinin quantitation employing high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection in the absence of hydrogen peroxide (H2O2), is reported. After elution from the HPLC column, artemisinin is combined with an alkaline solution of hematin and luminol. The resulting CL signal is detected by use of a spectrofluorometer with the excitation lamp disabled, and is proportional to artemisinin concentration. The CL method was optimized and applied to the analysis of artemisinin in spiked human serum. CL in the absence of H2O2 or other known oxidizing species is remarkable since such oxidizers are usually required to produce CL from luminol under alkaline conditions. Artemisinin, a naturally occurring sesquiterpene, is one of several natural products that contain an endoperoxide functional group. Since H2O2 is not needed in the analysis, the endoperoxide moiety on artemisinin is implicated as a contributing source of superoxide radicals required for the light-producing reaction with luminol.

Analysis of chlortetracycline by high performance liquid chromatography with postcolumn alkaline-induced fluorescence detection.

A high performance liquid chromatographic method for the analysis of chlortetracycline (CTC) using postcolumn fluorescence detection has been developed. After chromatographic separation of CTC on a polystyrene-divinylbenzene copolymer column, a highly fluorescent derivative isochlortetracycline (iso-CTC) was formed postcolumn in an on-line reaction coil with the addition of 25% NaOH (w/v). Chromatographic separation was achieved on a PRP-1 column, 15 cm x 4.6 mm, with 27:73 acetonitrile:0.2% perchloric acid (v/v), at 1.0 mL/min. Fluorescence derivatization was achieved by the on-line addition of 25% NaOH (w/v), at a flow rate of 0.2 mL/min, into the column eluant in a post-column reaction coil. The reaction coil was 9 m of teflon (1/16 in o.d., 0.3 mm i.d.) knitted into a six-sided coil. The fluorescent derivative was detected at lambda ex 355 nm and lambda em > 389 nm. Using this method after a simple sample cleanup, CTC can be detected in milk at 0.04 micrograms/mL, which is comparable to that obtained by microbiological assays. The detection method was linear between 0.02 micrograms/mL and 4 micrograms/mL. Because of the chromatographic separation, the method is more selective than microbiological assays and more sensitive than ultraviolet detection. With the chromatographic system described, the keto tautomeric forms of CTC and 4-epi-CTC are separated in a system which minimizes their formation on-column. In acidic aqueous organic solutions, the keto tautomer of CTC is the only product formed to any significant amount.

Use of stop-flow oxalate ester chemiluminescence as a means to determine conditions for high-performance liquid chromatography chemiluminescence detection of retinoids using normal-phase chromatography.

Stop-flow chemiluminescence (CL) has been used to determine the conditions necessary for the oxalate ester CL detection of selected retinoids after separation by normal-phase HPLC. Also, the detection of the selected retinoids by bis(2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen peroxide (H2O2) chemiluminescence and fluorescence (FL) are compared. Stop-flow CL was performed on a prototype uit from High-Tech Scientific, Ltd (Salisbury, UK). Detection limits were determined for retinyl palmitate, retinyl acetate, retinol, etretinate, acitretin and tretinoin, after separation on a YMC PVA-sil, 25 cm x 4.6 mm column using hexane-tetrahydrofuran-acetic acid (75:25:0.01, v/v/v) as eluent at 1.5 ml min-1. A Schoeffel 970 detector was used for both CL and FL detection. Detection by FL was determined with lambda ex 355 nm and lambda em greater than 418 nm. CL detection was performed with the deuterium lamp off and no emission filter. CL was induced by mixing the reagents post-column. Pump A--hexane--THF--AcOH (75:25:0.01, v/v/v) at 1.5 ml min-1, pump B--70 mM TCPO in THF at 0.5 ml min-1, pump C--THF--50% aq H2O2 (75:25, v/v) 1.0 mg ml-1 imidazole at 1.0 ml min-1.

Tobacco smoke xenobiotic compound appearance in mothers' milk after involuntary smoke exposures. I. Nicotine and cotinine.

In the development of the extraction procedure for the analysis of nicotine and cotinine from a single breast milk sample, nicotine and cotinine were detected in 3 of 10 nonsmoking mothers' milk samples. Interviews of these women revealed the presence of nonsmoking husbands and households but of tobacco smoke exposures during the working day. Clinically these levels may be considered inconsequential in regard to the threat to the mother and nursing infant but may be important in studies designed to monitor tobacco smoke xenobiotic compound appearance in breast milk.

Improved and rapid high-performance liquid chromatographic assay for 13-cis-retinoic acid or all-trans-retinoic acid.

A rapid, specific, and sensitive reversed-phase high-performance liquid chromatographic (HPLC) assay for the quantitative determination of all-trans-retinoic acid (I) or 13-cis-retinoic acid (II) in rat serum without extraction of lyophilization is described. Chromatographic separation from retinol, serum components, and retinol acetate standard was achieved on octadecylsilane-coated particles with acetonitrile-1% ammonium acetate as th eluent. Serum samples (100 microliter) containing as little as 10 ng of retinoid were analyzed. Serum level profiles of rats dosed with the retinoids demonstrated the utility of the assay and indicated elimination half-lives of 0.58 and 0.92 hr for I and II, respectively.

Ionization constants and fluorescence excitation and emission spectra of the isomeric benzo[a]pyrene phenols.

The acid dissociation constants, for the ground and lowest excited singlet states of the benzo[a]pyrene phenols, are reported, and correlated with current thought on carcinogen activation. The corrected fluorescence excitation and emission spectra of these compounds and their anions are recorded. The shifts caused by ring hydroxylation of the parent compound, and the relative spectral band intensities for each phenol are compared to those of pyrene, in a brief assessment of spectral transition polarization in the phenols.

Intramolecular hydrogen-bonding in beta adrenergic blocking agents.

The acid dissociation constants for nine beta-adrenergic blocking agents have been determined. Their unusually low pK(a), values are explained as being due to the formation of an intramolecular hydrogen bond, between the terminal amino group and the beta-hydroxyl group on the alkanolamine side-chain which is common to this class of drugs.

Characterization of pharmacologically important prototropic species derived from a pyridinemethanol antimalarial by electronic absorption and fluorescence spectroscopy.

Variations of the absorption and fluorescence spectra of the experimental antimalarial drug, alpha-dibutylaminomethyl-2,6-bis(p-trifluoromethylphenyl)-4-pyridinemethanol, were investigated throughout the pH region in concentrated sulfuric acid media and in n-hexane. The predominant prototropic species at physiological pH is the singly charged cation. In the pH 6--12 region, the structured fluorescence of the monocation is quenched with the concomitant appearance of a diffuse, long wavelength emission while the corresponding absorption spectra shift only slightly to longer wavelengths. Furthermore, the dibutylamino group exhibits an unusually low basicity. This behavior is explained as due to the formation of an intramolecular hydrogen bond in the neutral molecule in the ground and lowest excited singlet states. A similar intramolecular hydrogen bond in the monocation is not spectroscopically visible.

Binding of 3-aminoacridinium and 7-aminoquinolinium monocations to double-stranded DNA: evidence for nonlinear binding isotherm in intercalative region.

The complexations of the singly charged cations of 3-aminoacridine and 7-aminoquinoline by double-stranded, calf thymus DNA were studied by electronic absorption spectrophotometry. At high ratios of total DNA phosphate concentration to total probe concentration (the region associated with intercalative binding), four DNA phosphate units are associated with each bound monocation. However, a binding isotherm based on four DNA phosphate groups in a single binding site does not yield a reproducible equilibrium constant for the binding process. Rather, the sequestration of the monocations by two binding sites, each containing two DNA phosphate units, yields a satisfactory equilibrium expression. This result suggests that the intercalative mode of binding by DNA is not a simple one-step process, which is in agreement with previous kinetic studies. In the region of low DNA phosphate concentration to monocation concentration (the external binding region), the binding is described adequately by a linear isotherm.

Confirmation of iminoacridan structure of singly protonated 9-aminoacridine.

The occurrence of the second absorption band of singly protonated 9-aminoacridine, which arises from an acridinium ring-localized transition, at anomalously short wavelength indicates the disruption of the aromaticity of the central ring of the singly charged cation. The shifting of the two longest wavelength absorption bands and the fluorescence band of 10-methyl-9-aminoacridine monocation, a model of singly protonated 9-aminoacridine which is constrained to dissociate from the exocyclic nitrogen atom, to shorter wavelengths, upon dissociation, indicates the imine-like nature of the 10-methyl derivative. Thes observations support the conclusion that the predominant site of positive charge in singly protonated 9-aminoacridine is the exocyclic nitrogen atom, even though the heterocyclic nitrogen atom is the site of protonation of the neutral molecule.

Electronic spectra and electronic structures of some antimicrobials derived from proflavine.

The shifts in the absorption and fluorescence spectra of 3-aminoacridine, proflavine, acridine orange, and acridine yellow were employed to show that the singly charged cations, the predominant species at biological pH, exist in the ground state in the amino form. In the lowest excited singlet state, however, the monocations of the diaminoacridines have the imino structure, a conclusion supported by the relative ground- and excited-state pKa values of the reactions of the monocation with H-+. The ground-state amino structure has its positive charge concentrated at the heterocyclic nitrogen atom, a fact that is of primary importance in determining the geometry of binding to DNA.