Identification by HSQC and quantification by qHNMR innovate pharmaceutical amino acid analysis.

This study introduces a new NMR-based methodology for identification (ID) and quantification (purity, strength) assays of widely used amino acids. A detailed analysis of four amino acids and their available salts was performed with both a high-field (600 MHz) and a benchtop (60 MHz) NMR instrument. To assess sensitivity constraints, samples for 1H NMR analysis were initially prepared using only 10 mg of analyte and 1 mg of maleic acid (MA) as an internal calibrant (IC) and secondary chemical shift reference. The characteristic dispersion of the peak patterns indicating the presence or absence of a counterion (mostly chloride) was conserved at both high and low-field strength instruments, showing that the underlying NMR spectroscopic parameters, i.e., chemical shifts and coupling constants, are independent of the magnetic field strength. However, as the verbal descriptions of 1H NMR spectra are challenging in the context of reference materials and pharmaceutical monographs, an alternative method for the identification (ID) of amino acids is proposed that uses 13C NMR patterns from multiplicity-edited HSQC (ed-HSQC), which are both compound-specific and straightforward to document. For ed-HSQC measurements, the sample amount was increased to 30 mg of the analyte and several acquisition parameters were tested, including t1 increments used in the pulse program, number of scans, and repetition time. Excellent congruence with deviations <0.1 ppm was achieved for the 13C chemical shifts from 1D 13C NMR spectra (150 MHz) vs. those extracted from ed-HSQC (15 MHz traces). Finally, all samples of amino acid candidate reference materials were quantified by 1H qNMR (abs-qHNMR) at both 600 and 60 MHz. At high field, both IC and relative quantitations were performed, however, with the low-field instrument, only the IC method was used. The results showed that the analyzed reference material candidates were generally highly pure compounds. To achieve adequately low levels of uncertainty for such high-purity materials, the sample amounts were increased to 100 mg of analytes and 10 mg of the IC and replicates were analyzed for selected amino acids.

Chemical shift MR imaging methods for the quantification of transcatheter lipiodol delivery to the liver: preclinical feasibility studies in a rodent model.

PURPOSE: To demonstrate the feasibility of using chemical shift magnetic resonance (MR) imaging fat-water separation methods for quantitative estimation of transcatheter lipiodol delivery to liver tissues. MATERIALS AND METHODS: Studies were performed in accordance with institutional Animal Care and Use Committee guidelines. Proton nuclear MR spectroscopy was first performed to identify lipiodol spectral peaks and relative amplitudes. Next, phantoms were constructed with increasing lipiodol-water volume fractions. A multiecho chemical shift-based fat-water separation method was used to quantify lipiodol concentration within each phantom. Six rats served as controls; 18 rats underwent catheterization with digital subtraction angiography guidance for intraportal infusion of a 15%, 30%, or 50% by volume lipiodol-saline mixture. MR imaging measurements were used to quantify lipiodol delivery to each rat liver. Lipiodol concentration maps were reconstructed by using both single-peak and multipeak chemical shift models. Intraclass and Spearman correlation coefficients were calculated for statistical comparison of MR imaging-based lipiodol concentration and volume measurements to reference standards (known lipiodol phantom compositions and the infused lipiodol dose during rat studies). RESULTS: Both single-peak and multipeak measurements were well correlated to phantom lipiodol concentrations (r(2) > 0.99). Lipiodol volume measurements were progressively and significantly higher when comparing between animals receiving different doses (P < .05 for each comparison). MR imaging-based lipiodol volume measurements strongly correlated with infused dose (intraclass correlation coefficients > 0.93, P < .001) with both single- and multipeak approaches. CONCLUSION: Chemical shift MR imaging fat-water separation methods can be used for quantitative measurements of lipiodol delivery to liver tissues.

A sex-specific metabolite identified in a marine invertebrate utilizing phosphorus-31 nuclear magnetic resonance.

Hormone level differences are generally accepted as the primary cause for sexual dimorphism in animal and human development. Levels of low molecular weight metabolites also differ between men and women in circulating amino acids, lipids and carbohydrates and within brain tissue. While investigating the metabolism of blue crab tissues using Phosphorus-31 Nuclear Magnetic Resonance, we discovered that only the male blue crab (Callinectes sapidus) contained a phosphorus compound with a chemical shift well separated from the expected phosphate compounds. Spectra obtained from male gills were readily differentiated from female gill spectra. Analysis from six years of data from male and female crabs documented that the sex-specificity of this metabolite was normal for this species. Microscopic analysis of male and female gills found no differences in their gill anatomy or the presence of parasites or bacteria that might produce this phosphorus compound. Analysis of a rare gynandromorph blue crab (laterally, half male and half female) proved that this sex-specificity was an intrinsic biochemical process and was not caused by any variations in the diet or habitat of male versus female crabs. The existence of a sex-specific metabolite is a previously unrecognized, but potentially significant biochemical phenomenon. An entire enzyme system has been synthesized and activated only in one sex. Unless blue crabs are a unique species, sex-specific metabolites are likely to be present in other animals. Would the presence or absence of a sex-specific metabolite affect an animal's development, anatomy and biochemistry?

Limnophilaspiroketone, a highly oxygenated phenolic derivative from Limnophila geoffrayi.

A highly oxygenated phenolic spiroketone, limnophilaspiroketone (1), was isolated from the aerial parts of Limnophila geoffrayi collected in Thailand. The structure of 1 was determined based on spectroscopic data interpretation. This novel isolate, obtained as a major secondary metabolite constituent, was verified as a racemate using the Mosher ester method.

An evaluation of preparation methods and storage conditions of tribromoethanol.

This reports the in vitro portion of a study designed to establish guidelines for the preparation, storage, and use of tribromoethanol (TBE). We evaluated: 1) the purity of TBE powder from three suppliers; 2) nine methods of preparation of a 25-mg/ml (working) solution for formation of particulates and breakdown products; 3) formation of particulates and breakdown products and pH change in 1-g/ml (stock) solutions and working solutions stored under four conditions (25 degrees C and 5 degrees C in light and in dark); and 4) stock and working solutions of TBE that caused lethal effects in mice. These objectives were met by using nuclear magnetic resonance spectroscopy, gas chromatography-mass spectroscopy, particle-size and turbidity analyses, and pH strips. TBE powder from three suppliers varied in purity. No significant differences in breakdown product formation, particle size, or turbidity were noted between the nine preparation methods evaluated. Stock solutions and the working solution stored at 5 degrees C in the dark maintained a pH of 6.5 to 7.0, whereas the pH dropped for all other working solutions. A low level of dibromoacetaldehyde (DBA), a potential breakdown product reported to cause toxic effects, was detectable in all newly prepared solutions. Regardless of the storage condition or pH, DBA concentration did not increase measurably in any of the solutions after 8 weeks. The stock and working solutions that demonstrated lethal effects in mice had a pH of 6.5 and did not differ notably from newly prepared, non-lethal solutions, when evaluated for DBA. A decrease in pH could not be correlated to an increase in DBA or potential lethality, as suggested in the literature. The toxicity associated with the lethal TBE in this study appears to be a result of a chemical reaction or breakdown product that has not yet been reported.