Application of main component fraction collection method for purification of compound libraries.

In order to support high-throughput library purification, a novel UV triggered fraction collection method was developed in which a maximum-seeking-algorithm-driven, six-port valve collects the largest chromatographic peak. This straightforward strategy achieves the one sample-one fraction approach, thus resulting in a simpler and less error prone workup procedure. The effectiveness of this main component fraction collection method will be illustrated here by the results of the purification of compound libraries (altogether 6086 compounds, having an averaged success rate of 79.4%). Advanced applications, where the desired component differs from the main component, will also be discussed.

UV-triggered main-component fraction collection method and its application for high-throughput chromatographic purification of combinatorial libraries.

A maximum-seeking, algorithm-driven fraction collection method was developed to support high-throughput chromatographic purification, which provides new possibilities for off-line high-performance liquid chromatography mass spectroscopy (HPLC/MS) quality control experiments. The method is based on manipulation of a six-port valve that is installed downstream from the UV detector and equipped with a fraction collector loop. The detector signal is monitored by a programmable microcontroller that controls the state of the fraction collector valve. After detecting a chromatographic peak, the appropriate fraction is stored in the collector loop. The height of the next peak is compared to the previous one (using a maximum-seeking algorithm) and, depending on the result, the collected fraction is or is not exchanged with the new one. At the end of the run, the stored UV main component is pumped into the external fraction vial. This configuration was used for chromatographic purification of large compound libraries (the results of the purification of 5324 compounds are reported here), as well as for high-throughput off-line HPLC quality control experiments, where the collected main component fractions of an analytical-scale separation were subjected to further mass spectrometric molecular weight verification.

Metalloporphyrin catalyzed oxidation of N-hydroxyguanidines: a biomimetic model for the H2O2-dependent activity of nitric oxide synthase.

A chemical model for the H2O2 promoted oxidation by nitric oxide synthase (NOS) has been developed. Biomimetic oxidations were carried out using H2O2 and tetrakis(perfluorophenyl)porphyrinato-iron(III) chloride (FeTPPF20) as a catalyst. Similarly to NOS our model system produces Ndelta-cyanoornithine, citrulline and NO from NOHA and did not oxidize arginine itself. Based on these results we propose a peroxide shunt to be involved in the catalytic cycle of NOS. To the best of our knowledge this is the first chemical system that semiquantitatively mimics NOS activity.

Chemical models of cytochrome P450 catalyzed insecticide metabolism. Application to the oxidative metabolism of carbamate insecticides.

Cytochrome P450 (CP450) catalyzed oxidative metabolism of carbofuran (1), carbaryl (2), and pirimicarb (3) has been modeled using biomimetic oxidations catalyzed by iron(III) tetraarylporphyrins. Oxidation products of 1 were identified by comparison of HPLC retention times measured under standardized conditions for metabolites synthesized and characterized by (1)H and (13)C NMR spectroscopy. Comparison of product distributions to in vivo metabolic profiles revealed that the H(2)O(2)/meso-tetrakis(pentafluorophenyl)porphyrin iron(III) chloride [Fe(TF(20)PP)] system mimics the action of insect CP450s against carbofuran. The effectiveness of this system was further demonstrated by the biomimetic oxidation of other carbamate insecticides (2 and 3) monitored by HPLC/electrospray MS. The predictive power of this biomimetic model was compared to that of knowledge-based expert systems. Although similar models were recently applied in pharmaceutical research, the usefulness of this approach has first been demonstrated for the prediction of metabolic profiles of agrochemicals.

Application of overpressured layer chromatography combined with digital autoradiography and mass spectrometry in the study of deramciclane metabolism.

Overpressured layer chromatography was combined with the highly sensitive and rapid digital autoradiography (DAR) and mass spectrometry to separate, detect, and identify 3H- and 14C-labeled deramciclane metabolites in different biological matrixes. Several minor and major metabolites were separated from plasma and urine samples. The radioactive metabolites localized by DAR were scraped from the thin-layer chromatographic plate and transferred to a mass spectrometer for structure identification. Several metabolites were isolated and characterized, including hydroxy-N-desmethyl deramciclane, which is described in detail. The combination of techniques is efficient and has good sensitivity: about 2 micrograms metabolite from a biological matrix was isolated and identified this way.

Analysis of purine and pyrimidine bases, nucleosides and deoxynucleosides in brain microsamples (microdialysates and micropunches) and cerebrospinal fluid.

A new chromatographic method is reported for the synchronous analysis of endogenous purine and pyrimidine bases, ribonucleosides, and deoxyribonucleosides in brain samples. An optimized gradient chromatography system with a cooled reversed-phase column allows the detection of these compounds in very low concentrations in microsamples (microdialysates and micropunches). Chromatographic peaks were identified via the retention times of known standards, with detection at two wavelengths, and also by electrospray tandem mass spectrometry, which permits the identification of certain compounds at extremely low concentrations. The method was tested on in vivo brain microdialysis samples, micropunch tissue sample and cerebrospinal fluid of rats. Extracellular concentrations of pyrimidine metabolites in brain samples and of various purine metabolites in thalamic samples are reported here first. A comparison of the results on microdialysis and cerebrospinal fluid samples suggests that the analysis of cerebrospinal fluid provides limited information on the local extracellular concentrations of these compounds. Basic dialysis experiments revealed temporarily stable baseline levels one hour after implantation of the microdialysis probes. An elevated potassium concentration in the perfusion solution caused increases in the extracellular levels of adenosine and its metabolites, and of guanosine and the pyrimidine nucleoside uridine.

[Investigation of the impurity profile of nifedipine by LC/MS]. / A nifedipin hatóanyag szennyezésprofiljának LC/MS vizsgálata.

The manufacture-characteristic impurity profile of nifedipine drug substance was studied by HPLC and on-line LC/MS coupling. Using a new gradient HPLC procedure and MS detection, spectroscopic evidences for the chemical structure of some previously unknown minor impurities (all under 0.1%) were obtained.