Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography–mass spectrometry

Peptide quantification by liquid chromatography–mass spectrometry (LC–MS) combines the high resolving power of reversed-phase (RP) chromatography with the excellent selectivity and sensitivity of mass spectrometric detection. On the basis of comprehensive practical experience in the analysis of small molecules, pharmaceutical research is developing technologies for analysis of a growing number of peptidic drug candidates. This article is a detailed review of procedures based on LC–MS techniques for quantitative determination of peptides. With the focus on pharmaceutical applications several technologies for sample preparation, various aspects of peptide chromatography, important characteristics of ESI–MS, selectivity of MS-detection modes, the large variability of internal standards, and modern instrumentation are discussed. The demand for reliable, robust, sensitive, and accurate methods is discussed using numerous examples from the literature, complemented by experiments and results from our laboratory.     

The fluorescence of biliverdin dimethyl ester

Freshly prepared solutions of biliverdin dimethyl ester ( 2 ) in ethanol showed fluorescence maxima at 710 and 770 nm [Φ_F = 1.1. 10^?4 (room temperature) and 5.0 10^?4 (77 K)]. The maxima of monoprotonated 2 at 77 K were shifted to 725 and 806 nm and the quantum yield was increased to 2.6. 10^?2. This acid effect was reversible by neutralization with base. When a neutral solution was kept standing in the dark at room temperature, or when an acidic solution was neutralized by base, an additional fluorescence maximum at 500 nm with a mirror image excitation spectrum with λ_max = 470 nm developed, which disappeared on addition of acid and which is attributed to a chemical change of 2 .     

UV/Vis-spektroskopische Untersuchungen an Cyclosilanderivaten

UV/Vis-spectra of Cyclosilanes (SiX ₂) _n withX=Cl, Br, I, OMe andn=4, 5, 6 are recorded and interpreted qualitatively. The spectra exhibit increasing influence of the substituents on the Si-Si-bond system within the rings in the order Cl OMe Br I. At the low energy side of the spectra weak absorption bands, possibly due to intramolecular charge transfer transitions, appear. The strong inductive effect of the electronegative substituents is documented by a marked increase of the first IP's compared to permethylcyclosilanes.

     

Zur Stereochemie der Vitamin D3-Epoxide Röntgenstrukturanalyse einer 5,6-7,8-Diepoxidverbindung

Epoxidation of vitamin D₃ with benzonitrile ¦H₂O₂¦ KHCO₃ (Payne's reagent) yields exclusively the 5,6-mono-oxirane2. Further epoxidation with the same reagent gives the 5,6-7,8-bisepoxide3 a. Its stereochemistry and therefore the stereochemistry of2 was established by single crystal X-ray analysis of thep-Brbenzoate3b. Thereby for the first time a directive effect of a homoallylic hydroxyl group during thePayne-epoxidation was established.

Herrn Prof.Mihailovic (Universität Belgrad) mit den besten Wünschen zum 60. Geburtstag gewidmet.     

Practical asymmetric enzymatic reduction through discovery of a dehydrogenase-compatible biphasic reaction media

[reaction: see text] An enzyme-compatible biphasic reaction media for the asymmetric biocatalytic reduction of ketones with in situ cofactor regeneration has been developed. In this biphasic reaction media, which is advantageous for reactions at higher substrate concentrations, both enzymes (alcohol dehydrogenase and FDH from Candida boidinii) remain stable. The reductions with poorly water-soluble ketones were carried out at substrate concentrations of 10-200 mM, and the optically active (S)-alcohols were formed with moderate to good conversions and with up to >99% ee.     

New aspects of the protonation of biliverdins

Biliverdins undergo appreciable self association in acidic solutions especially if solvents like benzene or chloroform are employed. The population of aggregates further depends on the concentration of both the acid and the solute. In chloroform and benzene solutions at high acidity a doubly protonated species is formed. This is concluded from a combinatory evaluation of the CD and electronic absorption spectra of chiral optically active biliverdins (1–6) in benzene, chloroform, and ethanol solutions containing trifluoroacetic acid or hydrochloric acid. The aggregates formed at medium acid concentrations exhibit largeCotton effects in the long-wavelength absorption bands and thus dominate the CD spectra. Similarly, agrregates are the main contributors to theCotton effects of achiral, protonated biliverdins in (S)-(–)-ethyl lactate (SICD) at usual concentrations. The consequence and relevance of these findings with regard to the recent literature is briefly discussed.Dedicated to Prof. Dr.Karl Schlögl on occasion of his 60^th birthday.     

Volume and compressibility changes of complex formation between 18-Crown-6 and NaCl,KCl, and CsCl in water

The apparent molal volumes and compressibilities of NaCl, KCl, and CsCl in mixtures of 18-Crown-6 and water have been calculated from density and speed-of-sound measurements at 25°C. The partial molal volumes and compressibilities of the salts when all cations have formed complexes with 18-Crown-6 molecules have been evaluated. The sign and magnitude of the volume and compressibility changes of complex formation strongly suggest that the charge of the cation becomes very effectively screened by the crown ether.     

Prerequisites for peptidomic analysis of blood samples: II. Analysis of human plasma after oral glucose challenge -- a proof of concept

Mass spectrometric plasma analysis for biomarker discovery has become an exploratory focus in proteomic research: the challenges of analyzing plasma samples by mass spectrometry have become apparent not only since the human proteome organization (HUPO) has put much emphasis on the human plasma proteome. This work demonstrates fundamental proteomic research to reveal sensitivity and quantification capabilities of our Peptidomics technologies by detecting distinct changes in plasma peptide composition in samples after challenging healthy volunteers with orally administered glucose. Differential Peptide Display (DPD) is a technique for peptidomics studies to compare peptides from distinct biological samples. Mass spectrometry (MS) is used as a qualitative and quantitative analysis tool without previous trypsin digestion or labeling of the samples. Circulating peptides (< 15 kDa) were extracted from 1.3 mL plasma samples and the extracts separated by liquid chromatography into 96 fractions. Each fraction was subjected to MALDI MS, and mass spectra of all fractions were combined resulting in a 2D-display of > 2,000 peptides from each sample. Endogenous peptides that responded to oral glucose challenge were detected by DPD of pre-and post-challenge plasma samples from 16 healthy volunteers and subsequently identified by nESI-qTOF MS. Two of the 15 MS peaks that were significantly modulated by glucose challenge were subsequently identified as insulin and C-peptide. These results were validated by using immunoassays for insulin and C-peptide. This paper serves as a proof of principle for proteomic biomarker discovery down to the pM concentration range by using small amounts of human plasma.     

Synthesis and optical properties of (a)chiral terpyridine-ruthenium complexes

Chiral terpyridine ligands have been synthesized and characterized. By applying Ru(III)/Ru(II) chemistry, symmetrical as well as asymmetrical bis-terpyridine ruthenium(II) complexes were obtained. These materials were fully characterized and their optical properties investigated. While the chiral metal complexes revealed no Cotton effect in good solvents such as chloroform, CD-measurements in dodecane showed an effect in both ligand and MLCT regions, suggesting chirality transfer from the lateral alkyl chains to the complex core. This behavior points to the formation of supramolecular aggregates in dodecane. Furthermore, the analogous achiral ligand and its corresponding ruthenium(II) complexes were prepared.