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In the field of pharmaceutical and biomedical analysis of peptides, a rapid on-line detection and identification for a methodology have been required for the discovery of new biological active products. In this study, a high-speed counter-current chromatography with electrospray mass spectrometry (HSCCC/ESI-MS) was developed for the on-line detection and purification of polypeptide antibiotics of enramycin-A and -B. The analytes were purified on HSCCC model CCC-1000 (multi-layer coil planet centrifuge) with a volatile solvent of two-phase system composed of n-butanol/hexane/0.05% aqueous trifluoroacetic acid solution (43/7/50, V/V/V), and detected on an LCMS-2010EV quadrupole mass spectrometer fitted with an ESI source system in positive ionization following scan mode (m/z 100-2000). The HSCCC/ESI-MS peaks indicated that enramycin-A (major m/z 786 [M+3H](3+) and minor m/z 1179 [M+2H](2+)) and enramycin-B (major m/z 791 [M+3H](3+) and minor m/z 1185 [M+2H](2+)) have the peak resolution value of 2.9 from 15mg of loaded enramycin powder. The HSCCC collected amounts of the peak fractions were additionally 4.3mg (enramycin-A), and 5.9mg (enramycin-B), respectively. These purified substances were analyzed by LC/ESI-MS with scan positive mode. Based on the LC/ESI-MS chromatograms and spectra of the fractions, enramycin-A and -B were estimated to be over 95% purity. The overall results indicate that this approach of HSCCC/ESI-MS is a powerful technique for the purification and identification of bioactive peptides., (Copyright 2009 Elsevier B.V. All rights reserved.)

Turbulent Flow Chromatography (TFC) is a powerful approach for on-line extraction in bioanalytical studies. It improves sensitivity and reduces sample preparation time, two factors that are of primary importance in drug discovery. In this paper the application of the ARIA system to the analytical support of in vivo pharmacokinetics (PK) and in vitro drug metabolism studies is described, with an emphasis in high throughput optimization. For PK studies, a comparison between acetonitrile plasma protein precipitation (APPP) and TFC was carried out. Our optimized TFC methodology gave better S/N ratios and lower limit of quantification (LOQ) than conventional procedures. A robust and high throughput analytical method to support hepatocyte metabolic stability screening of new chemical entities was developed by hyphenation of TFC with mass spectrometry. An in-loop dilution injection procedure was implemented to overcome one of the main issues when using TFC, that is the early elution of hydrophilic compounds that renders low recoveries. A comparison between off-line solid phase extraction (SPE) and TFC was also carried out, and recovery, sensitivity (LOQ), matrix effect and robustness were evaluated. The use of two parallel columns in the configuration of the system provided a further increase of the throughput., (Copyright 2009 Elsevier B.V. All rights reserved.)

Chitosan and its derivatives are an important group of polymers used extensively in pharmaceutics. Thus, their physicochemical properties are of considerable interest and need to be characterized carefully. The most important feature to determine is their molecular weight and molecular weight distribution while the molecular weight of polymers plays an important role as pharmaceutical excipients. In this study, the feasibility of using asymmetrical flow field-flow-fractionation (AF4) connected online to a multi-angle light scattering (MALS) detector to measure the molecular weight of chitosans, trimethyl chitosans were studied and compared with the results from some traditional measurement methods. It was found that the influence of trimethyl chitosan synthesis process on the resulting molecular weight decrease depends on the initial molecular weight of chitosan. Significant molecular weight decrease was observed when chitosan molecular weight was larger than 100 kDa. In contrast, the influence was marginal when the molecular weight of chitosan was less than 50 kDa. The AF4-MALS was found to be a suitable method for the characterization of pure chitosans and trimethyl chitosans.

Screening analysis that aims at rapidly distinguishing new molecules in the presence of a large number of known compounds becomes increasingly important in the fields of drug metabolite profiling and nature product investigation. In the past decade, online-coupled liquid chromatography-nuclear magnetic resonance spectroscopy-mass spectrometry (LC-NMR-MS) has emerged as a powerful tool for the detection and identification of known and, more important, emerging compounds in complex clinical, pharmaceutical samples and nature product extracts, due to the complementary information provided by the two detectors for unambiguous structure elucidation. This review discusses the practical conditions under which LC-NMR-MS is suitable as a routine tool for unknown analysis, as well as the fundamental concepts and their advantage aspects. Particular attention is paid to its major operating parameters that include the instrumental configurations, working modes, NMR probe improvement and LC mobile phase selection. Finally, the recent applications of LC-NMR-MS to clinical metabolite and nature product analysis are summarized which have shown the benefit of this promising hyphenated technique.

Membrane pervaporation is presented as a method for on-line concentration and monitoring of trace analytes in a simulated pharmaceutical process stream. Pervaporation involves the selective transport of volatile organics across a membrane and into a gas stream. Experiments were carried out using a polar solvent-permeable Nafion membrane and several model pharmaceutical compounds. Solvent reductions greater than 90% and enrichment factors in excess of 7.9 were observed. Residence time and temperature were found to be important operating parameters. Interaction with membrane bound sulfonic acid residues resulted in the loss of reactive analytes such as 1,2-diphenylhydrazine. The concentrated stream was monitored using HPLC and UV/vis detection. Method detection limits were 0.5-1.2 microg/mL and the relative standard deviation for six repeat injections was 3.9-6.2%.

A new, simple and fully automated liquid chromatographic (LC) method with UV detection has been developed for the direct determination of atropine in plasma. Sample clean-up was based on the use of cation exchange restricted access material (RAM) in a pre-column, coupled to LC by means of a column switching system. After direct injection of a 200 microl-volume of plasma sample, the biological matrix was washed out for 10 min using a washing liquid composed of 2 mM lithium perchlorate adjusted to pH 3.0 and methanol (97:3; v/v). By rotation of the switching valve, atropine was then eluted in the back-flush mode for 2 min and transferred to the analytical column packed with octadecyl silica by the LC mobile phase constituted of a mixture of acetonitrile and potassium phosphate buffer (pH 3.0; 50 mM) containing 2 mM sodium heptanesulfonate (16:84; v/v). The UV detection was performed at 220 nm. The method was validated according to a new approach based on accuracy profile over a concentration range from 25 ng/ml, corresponding to the limit of quantitation, to 1000 ng/ml. The method was then applied for the determination of atropine in plasma after intravenous administration to hospitalised patients.