Analysis of norethindrone in plasma by high-performance liquid chromatography

The lack of specificity of some radioimmunological assays for the determination of norethindrone has been reported. This paper describes a high performance liquid chromatographic (HPLC) method that is sufficiently sensitive and specific for the determination of plasma samples containing 2 ng/ml norethindrone. Plasma samples were collected from 8 human volunteers. The solvents used for the mobile phase were methanol, HPLC grade acetonitrile, HPLC grade, and double glass-distilled water. The HPLC (Waters Associates, Milford, Massachusetts) was used at a nominal attenuation of .005 absorbance units full scale (AUFS) and fed into a 5 mV recorder, giving an overall response of 0.0025 AUFS. The HPLC was also fitted with an ultraviolet detector (254 nm). The organic solvent extract from plasma is chromatographed on a reversed phase column using the HPLC. Tritiated norethindrone was added to 2.0 ml of plasma containing from 2 to 20 ng/ml of norethindrone and was extracted using the procedure as described. The organic extract was then transferred into a scintillation vial and evaporated to dryness. A Beckman LS 150 scintillation counter with an automatic quench correction device was used to determine radioactivity. The results show that the extraction efficiency and reproducibility are comparable at plasma concentrations ranging between 2-20 ng/ml. No interfering compounds (metabolites and endogenous substances) were extracted using HPLC. Data analysis of a number of spiked plasma samples ranging from 2 ng/ml-20 ng/ml suggests the accuracy and precision of the method. In another experiment, 40 mg of norethindrone was dissolved in ethanolic saline and orally administered in a mini-pig. The plasma norethindrone concentration was readily detected. The experiments illustrate the stronger specificity of the new method compared to reported radioimmunoassays.     

Identification of alpha-galactosyl epitope mimetics through rapid generation and screening of C-linked glycopeptide library

A general methodology has been established for rapid generation and screening of combinatorial glycopeptide library and subsequent mass spectrometric sequencing to identify the mimetics of Galalpha(1,3)Gal epitopes. Using this approach, several active glycopeptide sequences were recognized and found to inhibit the binding of human natural anti-Gal antibodies with comparable IC(50)s to synthetic Galalpha(1,3)Gal oligosaccharides. The most active glycopeptides detected from the library included Gal-Tyr-Trp-Arg-Tyr, Gal-Thr-Trp-Arg-Tyr, and Gal-Arg-Trp-Arg-Tyr. These glycopeptides showed higher affinities to anti-Gal antibodies than known Galalpha(1,3)Gal peptide mimetics, such as DAHWESWL and SSLRGF. Our results suggest that, by combining a peptide sequence (the "functional" mimic part) with a terminal alpha-linked galactose moiety (the "structural" mimic part), the resulting glycopeptide could be a very good Galalpha(1,3)Gal mimetic. Analysis of these active glycopeptides provided first-hand information regarding the binding site of anti-Gal antibodies to facilitate the structurally based design of more potent and stable inhibitors.     

Interaction of angiotensin peptides and zinc metal ions probed by electrospray ionization mass spectrometry

Electrospray ionization-tandem mass spectrometry experiments were used to provide evidence regarding the sites of interactions between zinc metal ions and angiotensin peptides. The electrospray ionization mass spectra of histidine-containing human angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) in the presence of zinc show abundant multiply charged ions for the zinc-attached peptide [M + aZn²⁺ +(c ? 2a)H⁺]^c+, where a = 1, 2 and c is charge. From collisionally activated dissociation experiments, with both low energy (triple quadrupole mass spectrometry) and high energy collisions (linked scan at constant B/E with a double focusing instrument) of the [M + Zn]²⁺ and [M + Zn + H]³⁺ ions for angiotensin II, a [b ₆ + Zn]²⁺ species is produced as the most abundant product ion, suggesting that the zinc interaction site is in the vicinity of the His⁶ residue. Additionally, tandem mass spectra from the zinc-attached ions for angiotensin I show abundant [b ₆ + Zn]²⁺ and [b ₉ + Zn]²⁺ products, providing evidence that both His⁶ and His⁹ are involved in zinc coordination.     

Solvent-dependent assembly of terphenyl- and quaterphenyldithiol on gold and gallium arsenide

The assembly of terphenyldithiol (TPDT) and quaterphenyldithiol (QPDT) on gold and gallium arsenide from ethanol (EtOH), tetrahydrofuran (THF), and solutions consisting of both solvents has been characterized by near-edge X-ray absorption fine structure spectroscopy. The surface coverage and the average orientation of both TPDT and QPDT on gold are solvent-independent. These molecules readily form monolayers on gold with an ensemble-average backbone tilt of 30 degrees +/- 3 degrees from the substrate normal. In sharp contrast, the assembly of TPDT and QPDT on gallium arsenide is extremely solvent-sensitive. At high ethanol fractions, both molecules form monolayers with an ensemble-average orientation that is indistinguishable from those on gold substrates. At low ethanol fractions and in pure THF, however, these molecules are disordered on gallium arsenide and the surface coverage is poor.     

Polymer crystallization in 25-nm spheres

Crystallization within the discrete spheres of a block copolymer mesophase was studied by time-resolved x-ray scattering. The cubic packing of microdomains, established by self-assembly in the melt, is preserved throughout crystallization by strong interblock segregation even though the amorphous matrix block is well above its glass transition temperature. Homogeneous nucleation within each sphere yields isothermal crystallizations which follow first-order kinetics, contrasting with the sigmoidal kinetics normally exhibited in the quiescent crystallization of bulk polymers.     

Molecular orientation of thiourea chemisorbed on copper and silver surfaces

Surface-enhanced Raman spectroscopy has been used to study chemisorbed thiourea complexes on Cu and Ag electrodes. It was found that the thiourea molecule was bonded via its S atom to the Cu and Ag ad-atoms on the substrates.