Carbohydrate metabolism in erythrocytes of copper deficient rats.

Dietary copper deficiency is known to adversely affect the circulatory system of fructose-fed rats. Part of the problem may lie in the effect of copper deficiency on intermediary metabolism. To test this, weanling male Long-Evans rats were fed for 4 or 8 weeks on sucrose-based diets containing low or adequate copper content. Copper deficient rats had significantly lower plasma and tissue copper as well as lower plasma copper, zinc-superoxide dismutase activity. Copper deficient rats also had a significantly higher heart:body weight ratio when compared to pair-fed controls. Direct measurement of glycolysis and pentose phosphate pathway flux in erythrocytes using (13)C NMR showed no differences in carbon flux from glucose or fructose to pyruvate but a significantly higher flux through the lactate dehydrogenase locus in copper deficient rats (approximately 1.3 times, average of glucose and glucose + fructose measurements). Copper-deficient animals had significantly higher erythrocyte concentrations of glucose, fructose, glyceraldehyde 3-phosphate and NAD(+). Liver metabolite levels were also affected by copper deficiency being elevated in glycogen and fructose 1-phosphate content. The results show small changes in carbohydrate metabolism of copper deficient rats.

Temperature regulation of glucose metabolism in red blood cells of the freeze-tolerant wood frog.

The low-temperature metabolism of erythrocytes from the freeze-tolerant frog Rana sylvatica was investigated by (13)C and (31)P NMR spectroscopy. Erythrocytes readily took up high concentrations of the natural cryoprotectant, glucose, at both high (12 and 17 degrees C) and low (4 degrees C) temperatures but glucose was apparently not metabolized at 4 degrees C. Strong inhibition of glucose catabolism at low temperature would facilitate the maintenance of the very high concentrations of glucose (approximately 200 mM) that are accumulated to provide cryoprotection during freezing in wood frogs. Analysis of (13)C labeling of glycolytic intermediates at 4 degrees C showed mixing of label primarily in hexose (fructose) and hexose phosphate (glucose 6-phosphate, fructose 6-phosphate) pools but little label incorporation into triose phosphate intermediates. These data are consistent with a profound low-temperature-induced inhibition of phosphofructokinase (PFK). Investigations into potential PFK control mechanisms were undertaken. (31)P NMR analysis showed that the intracellular pH of erythrocytes increased from 7.0 to 7.3 as temperature decreased from 17 to 4 degrees C in a manner consistent with alphastat regulation. This change is exactly opposite to that expected if overall PFK activity was regulated by changes in cellular pH since PFK is less active at lower pH values in vitro. Other factors must, therefore, operate to regulate PFK at lower temperatures.

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%.

Multinuclear NMR (1H, 13C and 19F) spectroscopic re-examination of the solvolytic behaviour of flurazepam dihydrochloride.

The dissolution of reference and archival samples of flurazepam dihydrochloride (2) was studied in DMSO-d6 and in D2O by 1H-, 13C- and 19F-NMR spectroscopy to identify and distinguish solvated species of the parent drug (2), the "benzophenone" (4) and glycine (5) hydrochloride degradation products. In DMSO-d6, for most samples, only the ring intact form (2) could be detected by 13C-NMR whereas the inherently greater sensitivity of 19F-NMR allowed detection of initial trace amounts (< 1%) of the open-ring form (3). 19F-NMR spectroscopy also afforded the best means of quantifying the various entities in solution, including the increase towards equilibrium levels of the open-ring entity and detection/quantitation of a new equilibrium species, possibly the cis/trans rotamer of the open-chain entity (3). Various chemical shifts for flurazepam dihydrochloride and USP flurazepam related reference standards C and F are reported for DMSO-d6 solutions. The bases for 1H- and 19F-NMR assay of DMSO-d6 solutions of (2) for (4) are discussed with comparative data. The solvation characteristics of (2) in D2O at 0 and 27 degrees C were found to be too complex to follow by 13C-NMR; however, 19F-NMR studies at these temperatures permitted one to clearly discern that no additional formation of entity (4) occurred beyond whatever initial levels were present in degraded samples while the open-ring entity (3) was observed to increase to an equilibrium level of 56% over 24 h at 27 degrees C. Dissolution in D2O at either 0 or 27 degrees C does not contribute to solvolytic degradation of (2) to (4) over 24 h.

Chiral identification and determination of ephedrine, pseudoephedrine, methamphetamine and methcathinone by gas chromatography and nuclear magnetic resonance.

The enantiomers of the related substances methamphetamine, ephedrine, pseudoephedrine and methcathinone were determined by both gas chromatography after derivatization and by nuclear magnetic resonance using a chiral solvating agent. For GC the substances were derivatized with (R)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid (MTPA) to give diasteromeric derivatives. Resolution (baseline) of at least 1.6 was obtained between all derivatives. NMR determination of the enantiomers was conducted in a chiral environment by the addition of the chiral solvating agent, (R)-(+)-1,1'-bi-2-naphthol, to NMR solutions of the substances. Racemization of methcathinone was demonstrated to be facile by exposure to alkaline solutions for varying periods of time. Enantiomeric ratios of some products derived from the oxidation of ephedrine were determined.

Utilizing nuclear magnetic resonance (NMR) spectroscopy for assessing nadolol racemate composition.

NMR methods were developed for the determination of the racemate composition in nadolol raw materials. With high-field instruments (400 MHz or greater) the racemate ratio may be determined by the relative heights of the t-butyl peaks, which are well enough resolved for this determination. For lower field spectrometers, the t-butyl peaks are not resolved. An NMR method has been developed which involves preparation of the tribenzoate derivative of the drug. Seven lots of nadolol raw material, as well as several standards, were analysed for their racemate content. Three lots of raw material did not meet the USP limits of 40-60% for racemate A. Of these, two were granular in appearance and were found to vary markedly in racemate composition in successive analyses. The results for all the materials of uniform content agree very well with those from the HPLC method, as well as for the USP IR method using the absorbance at the corrected wavelength.

Mouse hepatic metabolites of ketoconazole: isolation and structure elucidation.

Oxidation, cleavage and degradation of the imidazole and piperazine rings, O-dealkylation, and aromatic hydroxylation are the reported pathways of ketoconazole (KC) metabolism. Metabolites were examined in hepatic extracts from male Swiss Webster mice treated with KC (350 mg kg-1 po x 7 days) in a 0.25% gum tragacanth suspension at 10 ml kg-1. Livers were collected 24 h after the last dose and stored at -70 degrees C. A mixture of chloroform/methanol extracts of liver homogenates were dried under vacuum and methanol extracts of the residue were chromatographed by a series of preparative and analytical HPLC techniques. Structure assignments were made by NMR and MS/MS techniques. It was demonstrated that KC was biotransformed to a number of products. Nine were isolated and seven identified as exclusive products of the biotransformation of the 1-acetylpiperazine moiety of KC. This substituent was biotransformed to the following: piperazine (de-N-acetyl ketoconazole, DAKC), N-carbamylpiperazine, N-formylpiperazine, 2,3-piperazinedione, 2-formamidoethylamine, ethylenediamine and amine. The 1H-NMR and MS data suggested that the remaining two metabolites were products resulting from the oxidation of the imidazole ring.

Vibrational and NMR spectroscopic study of aged flurazepam mono- and dihydrochloride salts for content identity.

Archival samples of flurazepam monohydrochloride and "hydrochloride" (i.e., the dihydrochloride) were examined by Fourier transform infrared and Raman spectroscopy to determine evidence of degradation during storage for 13-15 years. No degradation of the three different batches of monohydrochloride salts was detected, but various degrees of degradation of the eight specimens of flurazepam hydrochloride diprotonated salts were indicated by enhanced intensities (IR 1635, 1509, 1226; Raman 1636, 1408, 1149 cm-1) and new features (IR 1742, 943, 755; Raman 1554, 837, 742 cm-1). All of these features, except the 1742 cm-1 IR band, were attributed to the presence of the hydrolysis product 5-chloro-2-[[2-(diethylamino)ethyl]amino]-2'-fluorobenzophenone hydrochloride whereas the 1742 cm-1 band was attributed to glycine hydrochloride, the other hydrolytic moiety. The flurazepam hydrochloride samples were also examined in deuterated dimethyl sulfoxide solution by proton nuclear magnetic resonance (1H-NMR) spectroscopy to verify the presence of the degradation products and to estimate the levels of degradation (approximately 3-36%) of the drug. IR and Raman spectra of the "benzophenone" hydrochloride in the "fingerprint" region are compared with two samples of flurazepam dihydrochloride (slightly and highly degraded) and their features discussed. Vibrational assignments are made and discussed for the observed IR and Raman wavenumbers for the "benzophenone" hydrochloride.

Donation reactions among autologous donors.

Studies of risk factors associated with reactions among autologous blood donors have been limited. Therefore, 2091 autologous and 4737 homologous donations were examined. Donors at greatest risk for reaction were autologous donors who had reactions at first donation; among 45 who made repeat donations for the same surgery, 17 (38%) had repeat reactions. The group least likely to experience reactions were the autologous donors greater than or equal to 66 years old; they experienced a 1.9 greater than or equal to percent (6/310) incidence of reactions. More reactions were seen in both autologous and homologous donors in the categories of first-time donor, female gender, decreasing age, and lower weight. Multiple logistic regression analysis showed that all of these variables were independent predictors of donor reaction, with first-time donation (odds ratio, 2.4) and female gender (odds ratio, 1.9) being the strongest predictors of reaction. Donor room personnel should be alerted that autologous donors who react at first donation are very likely to react at subsequent donations. Contrary to common concern, elderly autologous donors are least likely to have reactions.

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.

Gas chromatographic method for solvent residues in drug raw materials.

A gas chromatographic (GC) method for screening drug raw materials, soluble in aqueous media, for volatile solvent residues has been developed. After dissolution, separate portions of the drug are each separately extracted with n-octane, toluene, and ether and injected into a chromatograph equipped with a porous polymer column and a flame ionization detector. The range of extractant polarities provides chromatograms which, taken together, are free of interfering peaks from 0 to approximately 20 min. Peaks due to solvent residues in the drug are identified by retention time with confirmation of identity by GC-MS.

Drug quality assessment methods. I. Gas-liquid chromatographic assay and identification of seven barbiturates.

A gas-liquid chromatographic (GLC) procedure has been developed for the assay and identification of amobarbital, butabarbital, heptabarbital, mephobarbital, pentobarbital, phenobarbital, and secobarbital in single component capsule, elixir, injectable, suppository, and tablet formulations. After extraction into chloroform from an acidified aqueous mixture of the product, the drug is eluted isothermally from a methylphenylsilicone GLC column at 210 or 240 degrees C and quantitated relative to thiamylal internal standard. Results were in good agreement with those obtained using pharmacopeial assay methods. The method is suitable for the rapid assessment of commercial formulations.

Routine quality evaluation of benzodiazepine drugs to USP-NF specifications.

A GLC procedure was developed for the evaluation of diazepam, chlordiazepoxide, and flurazepam formulations to USP-NF specifications for drug content, content uniformity, impurities, and identity by retention times and peak areas. The polyimide column, instrument zone temperatures, gas flows, internal standard solution, extraction solvent, and auxiliary equipment were the same for each drug. No derivatization of the samples was required. The GLC assay values (mean of 10 individual dosage units) for diazepam and flurazepam products were in good agreement with the results obtained by the pharmacopeial composite assays. With chlordiazepoxide capsules, when the levels of the two pharmacopeial impurities determined by GLC were added to the GLC assay results (mean of 10), athe aggregate values were consistent wit the drug content results found by the nonspecific USP method. The procedure can be made sensitive to impurity levels of approximately 0.01% for 2-amino-5-chlorobenzophenone and to approximately 0.2% for 7-chloro-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one 4-oxide. With the equipment used, the estimated potential outputs in lots per working day for a complete quality profile (drug content, content uniformity, purity, and identity) were seven for chlordiazepoxide if no impurity test was required, five if such a test was required, eight for diazepam, and seven for flurazepam.

Estimation of the degree of crystallinity in digoxin by X-ray and infrared methods.

The X-ray procedure for estimation of the degree of crystallinity in digoxin is based upon measurement of the total X-ray scattering and the scattering from crystalline regions of the drug. The infrared procedures are based upon measurement of the peak height ratios, 1775/1618 and 3095/1618 cm-1. Correlation between results obtained by the two methods is good. These methods are of value in the physico-chemical characterization of digoxin, particularly as the properties may be altered by comminution.

Model for clinical social work practice in a health care facility.

A model of clinical social work practice for direct patient service in a health care setting, providing clarity of role and function, was a side benefit of social service department's addressing the accountability issue. This document has provided the structure for a recording and reporting scheme to demonstrate what social service performs, what it accomplishes, and in what quantity. The model has secondary usages for staff education, student training, and definition of programs for administrative purposes.