structural phase characterization
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Radioimmunoconjugates represent a promising class of therapeutics and diagnostics. The characterization of intermediate chelator-antibody products, i.e., without the radionuclide, is frequently omitted, bringing significant uncertainty in the radioimmunoconjugate preparation. In the present study, we explored the utility of reversed-phase (RPLC) and hydrophilic interaction (HILIC) liquid chromatography with UV detection to characterize ramucirumab stochastically conjugated with p-SCN-Bn-CHX-A"-DTPA chelator (shortly DTPA). The conjugation was well reflected in RPLC chromatograms, while chromatograms from HILIC were significantly less informative. RPLC analyses at the intact level confirmed that the conjugation resulted in a heterogeneous mixture of modified ramucirumab. Moreover, the RPLC of DTPA-ramucirumab confirmed heterogeneous conjugation of all subunits. The peptide mapping did not reveal substantial changes after the conjugation, indicating that most parts of ramucirumab molecules remained unmodified and that the DTPA chelator was bound to various sites. Eventually, the RPLC method for analysis of intact ramucirumab was successfully applied to online monitoring of conjugation reaction in 1 h intervals for a total of 24 h synthesis, which readily reflected the structural changes of ramucirumab in the form of retention time shift by 0.21 min and increase in peak width by 0.22 min. The results were obtained in real-time, practically under 10 min per monitoring cycle. To the best of our knowledge, our study represents the first evaluation of RPLC and HILIC to assess the quality of intermediates during the on-site preparation of radioimmunoconjugates prior to radiolabeling.
The morphology, composition, and selectivity of a silica-based monolithic stationary phase, grafted by a layer of trioctyl(3/4-vinylbenzyl)phosphonium chloride ([P888VBn]Cl), is presented. The results of elemental analysis confirmed that the prepared stationary phase contains 38.8 at.% of silicon, 60.2 at.% of carbon, and 1.0 at.% of phosphorus. Capillary columns (150 × 0.1 mm) for liquid chromatography were evaluated using alkylbenzenes, monosubstituted benzenes, polyaromatic compounds, substituted benzene regioisomers, and aromatic carboxylic and amino acids. The prepared ionic liquid (IL)-based stationary phase exhibits hydrophobic, hydrophilic, and electrostatic interactions, as confirmed by experiments on the evaluation of the effect of the mobile phase composition (content of acetonitrile and ammonium formate) on the isocratic chromatographic separation. Thus, the IL-based capillary column demonstrates a unique separation selectivity compared to Phenyl-, C8-, and C18-stationary phases, and high efficiency for an expanding number of structurally diverse compounds.
A sustained effort to maximize the therapeutic effect of newly discovered active pharmaceutical ingredients (APIs) leads to the search for and development of advanced drug formulations. In this regard, a range of multicomponent and nanostructured systems that often combine the properties of solid and liquid materials have been developed. Besides the sophisticated supramolecular synthesis the development of these systems also requires in-depth view into their local architecture at atomic-resolution level. As these materials naturally exist at the borderline between the solid and liquid phases, the high-quality diffraction data are inherently unavailable. Therefore the structural description of these materials requires development of novel and highly efficient strategies. The aim of all this process is formulation of computation-experimental procedures allowing for precise characterization of the complex pharmaceutical systems including composite solids, nanocrystalline systems as well as partially ordered materials. In this regard, NMR crystallography belongs among the most successful approaches. In this contribution we report our recent achievements in characterizing atomic-resolution structure of complex pharmaceutical solids such as peptide derivatives of boronic acid, hybrid organic-inorganic liquisolid drug delivery systems, polymer-drug solid dispersions and mucoadhesive buccal films.
- Klíčová slova
- krystalové struktury,
- MeSH
- algináty chemie MeSH
- Aspirin chemie MeSH
- ciklopirox chemie MeSH
- farmaceutická technologie klasifikace MeSH
- krystalografie * metody MeSH
- léčivé přípravky MeSH
- magnetická rezonanční spektroskopie * metody MeSH
- nanomedicína dějiny metody MeSH
- polymery aplikace a dávkování chemie MeSH
- sloučeniny boru chemie MeSH
- Publikační typ
- práce podpořená grantem MeSH
Analysis of glycosylation is challenging due to micro- and macro-heterogeneity of the protein attachment. A combination of LC with MS/MS is one of the most powerful tools for glycopeptide analysis. In this work, we show the effect of various monosaccharide units on the retention time of glycopeptides. Retention behavior of several glycoforms of six peptides obtained from tryptic digest of haptoglobin, hemopexin, and sex hormone-binding globulin was studied on a reversed phase chromatographic column. We observed reduction of the retention time with increasing number of monosaccharide units of glycans attached to the same peptide backbone. Fucosylation of larger glycans provides less significant retention time shift than for smaller ones. Retention times of glycopeptides were expressed as relative retention times. These relative retention times were used for calculation of upper and lower limits of glycopeptide retention time windows under the reversed phase conditions. We then demonstrated on the case of a glycopeptide of haptoglobin that the predicted retention time window boosts confidence of identification and minimizes false-positive identification. Relative retention time, as a qualitative parameter, is expected to improve LC-MS/MS characterization of glycopeptides.
- MeSH
- chromatografie s reverzní fází metody MeSH
- glykopeptidy krev chemie metabolismus MeSH
- glykosylace MeSH
- lidé MeSH
- nanotechnologie metody MeSH
- peptidové fragmenty analýza chemie metabolismus MeSH
- proteomika MeSH
- senzitivita a specificita MeSH
- trypsin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Coiled coils are a common structural motif in many natural proteins that can also be utilized in the design and preparation of drug delivery systems for the noncovalent connection of two macromolecules. In this work, two different pairs of peptides forming coiled coil hetero-oligomers were designed, synthesized, and characterized. While the peptide sequences (VAALEKE)4 and (VAALKEK)4 predominantly form coiled coil heterodimers with randomly orientated peptide chains, (IAALESE)2-IAALESKIAALESE and IAALKSKIAALKSE-(IAALKSK)2 tend to form higher hetero-oligomers with an antiparallel orientation of their peptide chains. The associative behavior of these peptides was studied in aqueous solutions using circular dichroism spectroscopy, size-exclusion chromatography, isothermal titration calorimetry and sedimentation analyses. The orientation of the peptide chains in the coiled coil heterodimers was assessed using fluorescence spectroscopy with fluorescence resonance energy transfer labels attached to the ends of the peptides. The formation of the heterodimer can be used as a general method for the selective noncovalent conjugation of a specific targeting moiety with various drug carrier systems; this process involves simple self-assembly in a physiological solution before drug administration. The preparation of targeted macromolecular therapeutics consisting of a synthetic polymer drug carrier and a recombinant protein targeting ligand is discussed.
The acute destructive pneumonias (ADP) occupy up to 80% of the total number of pneumonias. They require constant improvement of treatment strategy. Nowadays the use of surfactants is a part of most treatment protocols. The aim was to study the features of the solid phase bronchoalveolar lavage in children with the ADPs in the dynamics of complex treatment with exogenous surfactant. Material and methods: We examined 39 patients of contaminated surgery. We identified 2 groups of patients. The patients of first group (n=27) had pulmonary pleural form of ADP, the second group (n=12) had pulmonary form of ADP. All patients got classical treatment and the earlier draining of pleural cavity. We used as an antiseptic reamberin 1.5% by 10 ml/kg and endobronchially injected exogenous surfactant Bl in dose12 mg/kg body weight a day, 6 mg/kg every 12 hours. All the children were made a bronchoscopy to obtain BAL to study the crystallization properties. The solid phase of BAL was studied by method of cuneal dehydration. Results: All facies before treatment were divided into two groups according to classification of facies of biological fluids. Only the facies of the second and the third types were detected there. It was revealed that the sizes of the zones of the facies were different in the comparison groups before treatment and after. And the level of crystalline structures and amorphous aggregates were different in the groups with different degrees of inflammation. Conclusion: So, we can assume that the change in surfactant system is characterized by changes in the morphological structure of solids phases of BAL. And the morphological structure of BAL depends on the chemical composition of BAL.
- MeSH
- akutní nemoc MeSH
- bronchoalveolární laváž MeSH
- bronchoalveolární lavážní tekutina * cytologie MeSH
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- plicní surfaktanty * aplikace a dávkování terapeutické užití MeSH
- pneumonie * farmakoterapie MeSH
- předškolní dítě MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- předškolní dítě MeSH
Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002-2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H](+) and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H](-) ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MS( n ) analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well.
Cardiolipins (1,3-bis(sn-3'-phosphatidyl)-sn-glycerol) (CLs) are widespread in many organisms, from bacteria to higher green plants and mammals. CLs were observed in Gram-positive bacterium of the genus Kocuria, brewer's yeast Saccharomyces, the green alga Chlamydomonas, spinach and beef heart. A mixture of molecular species of CLs was obtained from total lipids by hydrophilic interaction liquid chromatography (HILIC), and these were further separated and identified by reversed phase LC/MS with negative tandem electrospray ionization. The majority of CLs molecular species from each organism were cleaved using phospholipase C from Bacillus cereus. This phospholipase cleaves CLs into 1,2-diglycerols and phosphatidylglycerol 3-phosphates, which were then separated. After CLs cleavage, diacylglycerols such as sn-1,2-diacyl-3-acetyl-glycerols (i.e., triacylglycerols) were separated and identified by chiral chromatography/MS-positive tandem ESI. Significant differences in the composition of the molecular species between the 3-(3-sn-phosphatidyl) and 1-(3-sn-phosphatidyl) moieties of CLs were found in all organisms tested. Molecular species of CLs that contained four different fatty acids were identified in all five samples, and CLs containing very long chain fatty acids were identified in yeast. In addition, CLs containing both enantiomers (at the sn-2 carbon) were present in the bacterium tested. These findings were further supported by data already published in GenBank where, in the same family - Micrococcaceae - both enzymes responsible for chirality in the sn-2 position, glycerol-3-phosphate and glycerol-1-phosphate dehydrogenases, were present.
- MeSH
- chemická frakcionace MeSH
- Chlamydomonas reinhardtii chemie MeSH
- chromatografie kapalinová metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- hydrolýza MeSH
- kardiolipiny chemie MeSH
- mastné kyseliny analýza MeSH
- skot MeSH
- stereoizomerie MeSH
- triglyceridy chemie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
A new separation and quantification method using ultra high-performance liquid chromatography (UHPLC) with UV detection was developed for the detection of sibiromycin in fermentation broth of Streptosporangium sibiricum. The solid phase extraction method based on cation-exchange was employed to pre-concentrate and purify fermentation broth containing sibiromycin prior to UHPLC analysis. The whole assay was validated and showed a linear range of detector response for the quantification of sibiromycin in a concentration from 3.9 to 250.0 μg mL⁻¹, with correlation coefficient of 0.999 and recoveries ranging from 71.66±3.55% to 74.76±5.18%. Method limit of quantification of the assay was determined as 0.18 μg mL⁻¹ and was verified with resulting RSD of 9.6% and accuracy of 97.6%. The developed assay was used to determine the sibiromycin production in 12 different fermentation broths. Moreover, several natural sibiromycin analogues/derivatives were described with pilot characterization using off-line mass spectrometry: the previously described dihydro-sibiromycin (DH-sibiromycin) and tentative bis-glycosyl forms of sibiromycin and its dihydro-analogue.
- MeSH
- Actinomycetales metabolismus MeSH
- aminoglykosidy analýza chemie metabolismus MeSH
- extrakce na pevné fázi MeSH
- fermentace MeSH
- hmotnostní spektrometrie metody MeSH
- kalibrace MeSH
- kultivační média speciální chemie MeSH
- lineární modely MeSH
- molekulární struktura MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- stabilita léku MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
