Use of capillary electrophoresis for the characterization of human serum albumin heterogeneity.

Human serum albumin (HSA) is used in large amounts as an excipient in many biopharmaceutical formulation to prevent loss of the active ingredient through adsorption and/or degradation. Traditionally, iso-electric focusing has been used to demonstrate charge heterogeneity in HSA preparations. In an effort to develop new methods for the analysis of formulation components, a capillary zone electrophoresis method was developed for the analysis of HSA. Under initial separation conditions using untreated silica capillaries and 20 mM sodium phosphate, pH 6.0 as electrophoretic buffer, HSA migrated as a single peak. Addition of 1,4-diaminobutane allowed separation of several components which could be further resolved by varying the buffer pH. Optimal separation conditions were attained at 5 mM 1,4-diaminobutane and pH 8.5. The reproducibility of the separation conditions was verified by using capillaries from a different manufacturer. A comparative analysis of HSA preparations from different manufacturers provided evidence that the method may be used to qualitatively differentiate individual preparations. The analysis of rhEPO formulations, composed largely of HSA, showed levels of heterogeneity comparable to that of HSA preparations. Electrospray ionisation mass spectrometry (ESA-MS) was used as an independent method to confirm the heterogeneous nature of HSA.

Fenofibrate raw materials: HPLC methods for assay and purity and an NMR method for purity.

HPLC methods for drug content and HPLC and NMR methods for related compounds in fenofibrate raw materials were developed. The HPLC methods resolved 11 known and six unknown impurities from the drug. The HPLC system was comprised of a Waters Symmetry ODS column (100 x 4.6 mm, 3.5 microm), a mobile phase consisting of acetonitrile water trifluoroacetic acid 700/300/l (v/v/v) at a flow rate of 1 ml min(-1). and a UV detector set at 280 nm. Minimum quantifiable amounts were about 0.1% for three of the compounds and less than 0.05% for the other eight. Individual impurities in 14 raw materials ranged from trace levels to 0.25%, and total impurities from 0.04 to 0.53% (w/w). Six unknown impurities were detected by HPLC, all at levels below 0.10%, assuming the same relative response as fenofibrate. An NMR method for related compounds was also developed and it was suitable for 12 known and several unknown impurities. It requires an NMR of 400 MHz, or greater, field strength. Individual impurities in the raw materials analyzed ranged from trace levels to 0.24%, and total impurities from trace levels to 0.59%. Several lots contained small amounts of unknown impurities at trace levels. Three lots, all from the same manufacturer, contained an unknown impurity, not detectable by HPLC, which was not present in the other raw materials. It was estimated to be present at a level greater than 0.2%. The results for related compounds by the two techniques were consistent. The main differences stem from the low sensitivity of the HPLC method for some of the related compounds at 280 nm, or from the higher limits of quantitation by the NMR method for several other impurities using the conditions specified. A fifteenth raw material was not homogeneous in its content of impurity VI, a synthetic intermediate and possible degradation product. The HPLC/MS results provided information on the peak purity (number of components) for minor HPLC peaks, as well as structural data such as the molecular ions and diagnostic fragment ions. The HPLC/MS results showed that there were five unknown drug related impurities, for which there were no standards available. Results for the assay of 15 raw materials by HPLC were within the range 98.5-101.5%.

Microbial transformation of 3,4-methylenedioxy-N-methylamphetamine and 3,4-methylenedioxyamphetamine.

The biotransformation of 3,4-methylenedioxy-N-methylarnphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) was examined in the fungus Cunninghamella echinulata. In addition to the reported mammalian metabolites (MDA, 3,4-methylenedioxybenzyl methyl ketoxime, 3,4-methylenedioxybenzyl methyl ketone) and the parent substrate, there were six novel metabolites detected. N-Acetyl-3,4-methylenedioxyamphetamine (NAcMDA) was unequivocally identified and three unidentified metabolites related to NAcMDA were also detected. N-Acetyl-3,4-methylenedioxy-1-phenyl-1-hydroxy-2-aminopropane was tentatively identified as a metabolite of MDMA. The only metabolite of MDA identified was NAcMDA. Two metabolites related to MDA remain unidentified.Key words: Cunninghamella, amphetamine, biotransformation, 3,4-methylenedioxy-N-methylamphetamine, 3,4-methylenedioxyamphetamine.

Preparative liquid chromatographic separation of the oligomers of nonoxynol-9 and their characterization by 1H-NMR and mass spectroscopy.

Conditions are described for the preparative LC separation of a bulk nonoxynol-9 material into 16 components. 1H-NMR analyses of these fractions provided evidence for all but the first of the components to be oligomers of nonylphenoxypolyethoxyethanol (nonoxynol) with n-values for (OCH2CH2)n ranging consecutively from 3 to 17, corresponding to the LC fractions 2-16. Mass spectral analysis of the separated LC fractions confirmed the oligomeric sizes deduced from 1H-NMR spectral data, and provided EI fragmentation information for these oligomeric substances.

Identification of 3-O-methyl-alpha-methyldopamine as a urinary metabolite of 3,4-methylenedioxyamphetamine in dog and monkey.

3-O-Methyl-alpha-methyldopamine has been separated by gas-liquid chromatography (GC) as a metabolite of MDA in the urine of dog and monkey. The metabolite was identified as its mono- and di-trifluoroacetyl derivatives by comparison of their GC and GC-mass spectral properties with those of synthetic compounds. The amount of metabolite increased on hydrolyzing the urine from dosed dogs and monkeys with a preparation containing beta-glucuronidase and sulfatase.