Reliable Analysis of the Interaction between Specific Ligands and Immobilized Beta-2-Adrenoceptor by Adsorption Energy Distribution.

Although a comparatively robust method, immobilized protein-based techniques have displayed limited precision and inconsistent results due to a lack of strategy for the accurate selection of drug adsorption models on the protein surface. We generated the adsorption data of three drugs on immobilized beta-2-adrenoceptor (ß2-AR) by frontal affinity chromatography-mass spectrometry (FAC-MS) and site-specific competitive FAC-MS. Using adsorption energy distribution (AED) calculations, we achieved the best adsorption models for the binding of salbutamol, terbutaline, and pseudoephedrine to immobilized ß2-AR. The Langmuir model proved to be desirable for describing the adsorptions of salbutamol and terbutaline on immobilized ß2-AR, while the bi-Langmuir model was favorable to characterize the adsorption of pseudoephedrine on the receptor. Relying on the accurate determination of association constants, we presented an efficient approach for ß2-AR ligand screening based on the loss of breakthrough time of an indicator drug caused by the inclusion of competitive drugs in the mobile phase. We concluded that the current strategy enables the reliable and accurate analysis of G protein-coupled receptor (GPCR)-drug interaction. The percentage change in the breakthrough time for drugs can provide useful information for estimating their binding affinity to the receptor. This approach builds a powerful platform for high-throughput ligand screening.

Interlaboratory study of free cyanide methods compared to total cyanide measurements and the effect of preservation with sodium hydroxide for secondary- and tertiary-treated waste water samples.

Several methods exist for the measurement of cyanide levels in treated wastewater,typically requiring preservation of the sample with sodium hydroxide to minimize loss of hydrogen cyanide gas (HCN). Recent reports have shown that cyanide levels may increase with chlorination or preservation. In this study, three flow injection analysis methods involving colorimetric and amperometric detection were compared within one laboratory, as well as across separate laboratories and equipment. Split wastewater samples from eight facilities and three different sampling periods were tested. An interlaboratory confidence interval of 3.5 ppb was calculated compared with the intralaboratory reporting limit of 2 ppb. The results show that free cyanide measurements are not statistically different than total cyanide levels. An artificial increase in cyanide level is observed with all methods for preserved samples relative to nonpreserved samples, with an average increase of 2.3 ppb. The possible loss of cyanide without preservation is shown to be statistically insignificant if properly stored up to 48 hours. The cyanide increase with preservation is further substantiated with the method of standard additions and is not a matrix interference. The increase appears to be correlated with the amount of cyanide observed without preservation, which appears to be greater in those facilities that disinfect their wastewater with chlorine, followed by dechlorination with sodium bisulfite.

Thermodynamic characterization of separations on alkaline-stable silica-based C18 columns: why basic solutes may have better capacity and peak performance at higher pH.

A systematic study was made to explain the large improvements in separation performance and capacity of basic compounds at alkaline conditions. The adsorption of three probe components was investigated on four alkaline-stable silica-based C18 columns at three different pH-levels: 3, 7 and 11. The probes were 3-phenyl-1-propanol (neutral), 2-phenylbutyric acid (acidic) and metoprolol (basic). Adsorption isotherms were acquired over a broad concentration range, in order to detect both high and low energy sites. Before the choice of the proper adsorption isotherm model, the adsorption energy distribution (AED) was calculated yielding the number of different kinds of interaction sites between the solute and the stationary phase. The neutral probe was entirely unaffected by pH and its AED was unimodal (one site) indicating homogenous adsorption. For the acidic probe the interactions were unimodal at pH 3 where the probe is uncharged and at least bimodal (two sites) at pH 7 and 11 where the probe is charged. For the basic probe, the interactions were heterogeneous at both pH 3 and 11. The equilibrium constants of the high and low energy sites were different by a factor of 55-100 at pH 3 and only 6-7 at pH 11. The difference in saturation capacities between the two sites was much smaller at pH 11 where 20% of the total capacity is from the high energy site, as compared to pH 3 where the high energy site was only 2-5% of the total capacity. This explains why peaks of amines (basic solutes) tail at low pH while their peaks are symmetrical at alkaline pH. The Langmuir model fit the unimodal data and the bi-Langmuir model fit the bimodal AED data. The calculated band profiles based on these parameters agreed excellently with the experimental data. The electrostatic-modified Langmuir, on the other hand, did not describe this adsorption process well.

Postnatal manganese exposure attenuates cocaine-induced locomotor activity and reduces dopamine transporters in adult male rats.

In the present study, we examined whether exposing rats to manganese (Mn) during the preweanling period would affect basal or cocaine-induced locomotor activity in adulthood and reduce the number of striatal dopamine transporter binding sites. On postnatal day (PD) 1-21, rats were given oral supplements of vehicle or Mn chloride (250 or 750 microg/day). Striatal Mn and iron (Fe) accumulation as well as serum Fe levels were measured on PD 14, PD 21, and PD 90. Throughout the dosing period, rats were evaluated on standard measures of sensory and motor development. During adulthood, the basal and cocaine-induced locomotor activity of vehicle- and Mn-exposed rats was assessed using automated testing chambers. After completion of behavioral testing, striatal dopamine transporter binding sites were measured using [(3)H]GBR 12935. Results showed that early Mn exposure enhanced striatal Mn accumulation on PD 14 and PD 21, while depressing serum Fe levels on PD 21. Exposure to Mn on PD 1-21 did not affect striatal or serum Mn or Fe levels on PD 90. During the second postnatal week, Mn-exposed rat pups performed more poorly than controls on a negative geotaxis task, however basal motor activity of preweanling rat pups was not affected by Mn treatment. When tested in adulthood, basal locomotor activity of vehicle- and Mn-exposed rats also did not differ. In contrast, adult rats previously exposed to 750 microg/day Mn showed an enhanced locomotor response when challenged with 10 mg/kg cocaine. A different pattern of results occurred after treatment with a higher dose of the psychostimulant, because Mn-exposed rats showed an attenuated locomotor response when given 20 mg/kg cocaine. Importantly, Mn-exposed rats exhibited long-term reductions in striatal dopamine transporter binding sites. Considered together, these results indicate that postnatal Mn exposure has long-term behavioral and neurochemical effects that can persist into adulthood.

Heterogeneous adsorption of 1-indanol on cellulose tribenzoate and adsorption energy distribution of the two enantiomers.

The distributions of the adsorption energies (AED) of two enantiomers, (R)-1- indanol and (S)-1-indanol, on a chiral stationary phase were measured and the results are discussed. The chiral phase used is made of cellulose tribenzoate coated on porous silica. The AEDs were determined using the expectation maximization method, a numerical method that uses directly the raw experimental isotherm data, inverts this set of data into an AED, and introduces no arbitrary information in the calculation. However, it uses the Langmuir equation as the local isotherm. The experimental data fit very well to the bi-Langmuir isotherm model for the more retained enantiomer. Our results show that the AEDs of these two enantiomers have no energy modes that would be identical (same mean energy, mode profile, and mode area), in contrast to numerous cases previously studied, e.g., that of the beta-blockers on a Cel7A column. This indicates a significantly different retention mechanism.

Analysis of active sites and heterogeneity in commercial reversed-phase octadecylsilanated silica with numerically calculated sorption distributions.

Sorption isotherms spanning six orders of magnitude of pyridine concentration in a 60:40 methanol:water mobile phase adjusted to pH 5 were obtained with the frontal analysis method on three ODS stationary phases: Zorbax Pro-10/150, Vydac 218TPB10, and YMC 120AS10. The data was fit to a heterogeneous Langmuir model in which the association constant, K, is continuously distributed over a finite range of values. The results indicate a small degree of secondary adsorption for all three phases as a separate peak in K-space at higher values of K than the primary hydrophobic partitioning, and additional adsorption at even higher K values for the Zorbax and to a much smaller degree the YMC phase. Integration of the distributions yields the amount of sorption at each of the modeled sites. The results correlate with information known about the synthesis of these phases and the degree of band tailing in elution experiments at these conditions.

Determination of single component isotherms and affinity energy distribution by chromatography.

Adsorption isotherm data were acquired by frontal analysis (FA) and large sample-size band profiles were recorded for phenol and caffeine. For both compounds, the isotherm data fit well to the Langmuir, Toth, and Bi-Langmuir models of adsorption. The Langmuir model must be dismissed because it does not predict accurately the overloaded band profiles. However, profiles calculated using the unimodal Toth and the bimodal Bi-Langmuir models are indistinguishable. The expectation-maximization procedure was used to calculate directly the affinity energy distribution (AED) from the raw FA data points. For both compounds, the AED converges to a bimodal distribution at high numbers of iterations. This result, which shows the high sensitivity of the EM method, suggest that the Bi-Langmuir model makes better physical sense than the Toth model. This model also permits a detailed investigation of the properties of active sites, a feature often evoked in chromatography but so far rarely the topic of a quantitative investigation.