The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.
Introduction. This article provides a tentative justification of use of soft contact lenses as a carrier of ophthalmological solutions to eye tissues.Aim. The aim of the research is to develop the composition of the ophthalmic transport system for the treatment of glaucoma. Also, the proposed manufacturing algorithms were proposed for drug loading ophthalmological solutions.Materials and methods. Preparation of sodium hyaluronate solution was carried out based on the methodology described in EP 7.0 [01/2011:1472] (Sodium hyaluronate).Results and discussion. The conducted investigation with the purpose to justify a concentration of sodium hyaluronate helped to determine its concentration that gives the viscosity level as close to the upper limit stated in Russian Pharmacopoeia as possible. Buffer solution was chosen with criteria to use as less components in ophthalmological solution as possible. Also, it was found out that the more components are added to the viscous solution, the more viscosity level is decreased. Thus, initial viscosity of solution containing sodium hyaluronate was 149.59 mm2/s, and after addition of active pharmaceutical substance it dropped down to 88.49 mm2/s and 81.36 mm2/s for model solution number 1 and 2, respectively. After addition of citric acid and disodium hydrophosphate, the viscosity of model solution was found to be 78.11 mm2/s and 75.28 mm2/s for model solution number 1 and 2, respectively.Conclusion. As a result of the studies, two alternative model compositions of an ophthalmic solution for the saturation of soft contact lenses for the treatment of glaucoma were proposed. The choice of active pharmaceutical substances, and excipients has been justified. Technological procedure of preparation of model solution was described and explained, where the most attention was paid on dissolving of sodium hyaluronate in purified water.
Aim of this study is the theoretical justification and experimental confirmation of new approach to description of equilibrium state in extraction system vegetal raw materialextractant. In framework of this study the milled raw material was used containing the particles sized 0,1-0,5 mm: roots and rhyzomes of Glycyrrhiza glabra or uralensis, leaves of Uvaurzi, leaves of Eucalyptus viminalis, flowers of Helichrysum arenarium, flowers of Calendula officinalis and etc. The process of equilibrium establishment was studied at temperature levels such as 4, 20, 40 and 60±1ºС, wherein the method of simple maceration was implemented for 24 hours of decoction. Interphase distribution of BAS was studied using the proportion of raw material weight / extractant volume as 1:5, 1:10, 1:20, 1:40. The watery ethanol solution of 70 or 80±1 % vol. was chosen as an extractant for the purpose of this study. Assay test of BAS in raw material was carried out with the help of RP HPLC instrumentation method. Experimental points for all the studied BAS from various types of vegetal raw materials are well-approximated by regression lines built up in theoretically predicted coordinates, which is confirmed by the high value of determination coefficient R²≥0.99. The new approach has been proposed to description of equilibrium state in extraction system vegetal raw material-extractant, which is based on the promotion of hypothesis, according to which the mechanism of interphase molecular distribution of BAS in extraction system is explained and described by classic Boltzmann distribution for discrete values of molecular energy (or Fermi-Dirac quantum distribution). The developed and experimentally confirmed mathematical model was based on the promoted hypothesis, and it successfully described the relation of the main parameters of extraction system, which allows to develop the rational technology of tincture or extracts manufacturing.
Sample preparation involving the cleavage of proteins into peptides is the first critical step for successful bottom-up proteomics and protein analyses. Time- and labor-intensiveness are among the bottlenecks of the commonly used methods for protein sample preparation. Here, we report a fast online method for postinjection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7–8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.
В статье представлен обзор литературной информации и нормативной документации, описывающей методики качественного и количественного определения латанопроста, бетаксолола гидрохлорида, а также определения латанопроста в комбинированных офтальмологических лекарственных формах. Выявлено, что преобладающим методом идентификации бетаксолола гидрохлорида является ИК спектрофотометрия и реакция на хлориды. Для латанопроста применим метод ВЭЖХ и УФ спектрофотометрии. Обе субстанции количественно определяются преимущественно методами ВЭЖХ.
Sample preparation involving the cleavage of proteins in peptides is the first critical step for successful bottom-up proteomics analyses. The main limitations to commonly used methods for protein sample preparation are time- and labor-intensiveness. Here we report a fast online method for post-injection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7-8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the chemical method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.
The development of an ophthalmic therapeutic system includes research on the spatial structure of soft contact lens polymers and the study of the processes of saturation and release of medicinal substances from them. This allows you to determine the methods of saturation of contact lenses with medicinal agents and will open up new opportunities in the treatment of ophthalmological diseases. The purpose of this preliminary fragment of large-scale research was to study the surface structure of soft contact lenses made of various polymers. The following polymers were used in the work: Nelfilcon A, Hilafilcon B, Nezofilcon A, Etafilcon A, Lotrafilcon B. The following pharmaceutical substances were used: Brimonidine Tartrate, Betaxolol Hydrochloride, Pyridoxine Hydrochloride. The surface structure of soft contact lenses was studied using atomic force microscopy. Each material under study has a different surface character, which together with the differences in pore properties determines its individuality. Based on this, it should be assumed that the surface of soft contact lenses affects the possibility of their potential use as a means of delivering drug agent molecules to the eye tissues. In all cases of soft contact saturation, the highest absorption capacity was demonstrated by Hilafilcon B and Etafilcon A with a similar surface.
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