High-speed liquid chromatographic analysis of dapsone and related compounds.

A nonaqueous solvent absorptive support system and an aqueous solvent reversed-phase support liquid chromatographic system for analysis of dapsone and related compounds were investigated. The absorptive support system was more suitable for analysis of dapsone in raw materials, formulations, and tissue residues. The suitability was judged by the relative selectivity, efficiency, precision, and sensitivity of the systems. The adsorptive support system was used for the analysis of trace amounts of raw material impurities and dapsone metabolites. Coupling fluorometric detection to the chromatographic system yielded a 10-pg on-column detection limit for dapsone; the UV detection limit was 250 pg.

High-performance liquid chromatographic determination of pralidoxime chloride and its major decomposition products in injectable solutions.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of pralidoxime chloride (I) and its major decomposition products in an injectable formulation is described. I and its decomposition products were detected and quantitated by their UV absorbances at 270 nm, after being separated from related compounds and formulation excipients on a reverse-phase C-18 column using a mobile phase consisting of 52% acetonitrile and 48% of an aqueous solution containing 0.005 M phosphoric acid and 0.001 M tetraethylammonium chloride. The major decomposition products of I in the injectable formulation were identified by their retention times and stop-flow spectroscopy as 2-carboxy-N-methylpyridinium chloride, N-methyl-2-pyridone, 2-carbamoyl-N-methylpyridinium chloride, 2-hydroxymethyl-N-methylpyridinium chloride, and 2-cyano-N-methylpyridinium chloride. A substance of unknown identity also was detected in degraded solutions of I. Stop-flow spectroscopy, employing the spectral discrimination technique, showed that the method is specific for I. Recovery of I from a spiked placebo formulation averaged 99.9%. The accuracy of the method was also demonstrated for the decomposition products over a range of concentrations representing 1-50% decomposition. Replicate determinations of I in degraded solutions gave coefficients of variation of 1.0 and 1.5%, while the precision of determining the decomposition products range from 1.3 to 6.5%. Regression lines with correlation coefficients greater than 0.9999 were obtained for I and its decomposition products, and solutions of these compounds were shown to be stable in the mobile phase for several days. Results for I by the HPLC and USP procedures are compared.

Quantitative determination of isosorbide dinitrate and two metabolites in plasma.

A GLC method for the determination of plasma isosorbide dinitrate and two metabolites, isosorbide 2-mononitrate and isosorbide 5-mononitrate, is described. The three substances are extracted from alkalinized plasma with ether. Quantitation is effected by electron-capture detection after GLC separation with a 30% SE-30 column. The unusually heavy liquid phase loading is necessary to eliminate irreversible adsorption on the solid support. The electron-capture detector provides excellent sensitivity and specificity because of the electronegative nature of the nitric ester. The method was used to study the blood levels of isosorbide dinitrate and two metabolites in four beagle dogs after single oral doses of 40 mg of isosorbide dinitrate and in two human volunteers after a sublingual dose of 10 mg of isosorbide dinitrate.

Steady-state urinary excretion method for determining bioequivalence of conjugated estrogen products.

The steady-state excretion of conjugated estrogens in the urine of postmenopausal women dosed with conjugated estrogens tablets was studied using a modification of a previously published method. The procedure was used to quantitate the estrogens both before and during conjugated estrogens replacement therapy. The method, which is relatively specific, involves enzyme hydrolysis of urine samples a number of classical extraction and purification steps, and analysis of the silylated estrogens on a 2.7-m, 1.7% diethylene glycol succinate column using flame-ionization detection. The results indicate that steady-state urinary estrogen excretion levels were obtained within 17 days of dosing. Furthermore, the urinary estrogen excretion profile was significantly different from the composition of the estrogens in the dosage form.

Gas-liquid chromatographic determination of isosorbide dinitrate in tablets.

A gas-liquid chromatographic (GLC) method is described for determining isosorbide dinitrate (ISDN) in tablets. ISDN is extracted from tablet excipients by a 2-phase system composed of water-ethyl ether, and detected and quantitated with a flame ionization detector after separation from the internal standard (glyceryl tributyrate) on a 3% OV-210 column. Ten replicate assays on 2 different batches of tablets, each containing 5 mg ISDN, gave coefficients of variation of 1.16 and 1.48%, respectively. Comparison of results obtained for tablets containing 5, 10, and 40 mg ISDN, and for diluted isosorbide dinitrate raw material containing 25% ISDN, showed good correlation among the GLC, USP colorimetric, and USP polarographic procedures. Assay of synthetic mixtures containing ISDN, isosorbide-2-mononitrate, and isosorbide-5-mononitrate demonstrated that the GLC procedure is specific for ISDN, whereas the USP polarographic procedure is subject to interference from the mononitrates.

Determination of hydralazine in tablets by gas chromatography.

A description is given of a gas chromatographic method for the determination of hydralizine in various tablet formulations based on the quantitative reaction of hydralazine with 2,4-pentanedione to yield 1-(3,5-dimethylpyrazole)phthalazine. The stable hydralazine derivative formed is extracted from aqueous solution and chromatographed resulting in a precise and specific determination of hydralazine which correlates well with the U.S.P. XIX titrimetric procedure.

Nonaqueous reverse phase liquid chromatographic determination of vitamin D2 in multivitamin tablets, using vitamin D3 as internal standard.

The technique of nonaqueous reverse phase chromatography has been successfully applied to the separation of lipophilic vitamins D2 and D3. Separation is achieved with a Zorbax-ODS column and a mobile phase of 10% methanol in acetonitrile. A predetermined amount of vitamin D3 internal standard is added to a portion of a powered tablet sample, which is then extracted and partitioned with a mixture of dimethyl sulfoxide, water, methanol, and hexane. A portion of the hexane extract is concentrated, and purified by passing it through a disposable silica cartridge to remove interfering materials. The method should also be applicable to formulations containing vitamin D3 where vitamin D2 can then be used as the internal standard. A system suitability test is included in the method.

Gas-solid chromatographic determination of oxygen in various pharmaceutical products.

A method is described for the gas chromatographic determination of oxygen at the low part per million level in both aqueous systems and nonaqueous systems such as creams, oils, and waxes. This method offers advantages over other popular assay procedures such as the Winkler and polarographic methods. The purging technique developed can be applied to any liquid or solid with a melting point of 75 degrees C or less. Oxygen purged from the sample is passed through a molecular sieve 5A column where it is separated from nitrogen. Peaks obtained are compared to similarly prepared oxygen standards. The method is simple enough to be used routinely and up to 15 samples can be analyzed per day.

Determination of degradation products and impurities of amoxicillin capsules using ternary gradient elution high-performance liquid chromatography.

A specific high-performance liquid chromatography method has been developed for the determination of amoxicillin penicilloic acids (PA), p-hydroxyphenylglycine (p-HPG), 6-aminopenicillanic acid (6-APA) and unidentified materials in amoxicillin capsules. The accuracy of the measurements was demonstrated by the standard addition technique on actual capsule samples. Within-day and between-day precision studies gave coefficients of variation of 3.15 and 1.70% for PA, 4.75 and 3.81% for p-HPG, and 5.59 and 32.23% for 6-APA, respectively. Typical calibration and expanded linearity of response curves for these components show no curvature over the range of interest. The detection limits for PA, p-HPG and 6-APA are 0.7, 0.2 and 0.6 micrograms/ml of sample extract and are considered adequate for the intended use of the method. These levels correspond to 0.35, 0.10, 0.30 mg per 500-mg Amoxil capsule, respectively.

Trace analysis of the MIF analogue pareptide in blood plasma by high-performance liquid chromatography and short-wavelength excitation fluorometry.

A high-performance liquid chromatographic procedure was developed and applied to analysis of the pharmacologically active MIF analogue pareptide in human plasma. The procedure involves formation of a fluorescent 7-chloro-4-nitrobenzyl-2-oxa-1,3-diazole (NBD-Cl) pareptide derivative followed by separation of the NBD derivative from plasma components on a 30-cm microparticle octadecylsilane bonded column. The separated derivative was quantitated using a short-wavelength excitation fluorometric detector. The detection limit of pareptide in plasma samples was 5 ng or 17 pmoles per ml of plasma. In the absence of plasma, the corresponding on-column detection limit was 0.5 pmoles.