Christian Neusüß

A robust approach for the analysis of peptides in the low femtomole range by capillary electrophoresis-tandem mass spectrometry

A capillary electrophoresis‐tandem mass spectrometry (CE‐MS/MS) approach has been developed for routine application in proteomic studies. Robustness of the coupling is achieved by using a standard coaxial sheath‐flow sprayer. Thereby, greater stability than nanoelectrospray ionization‐mass spectrometry coupling of sheathless capillary electrophoresis or nanoliquid chromatography (nano‐LC) is achieved, resulting in stable operation for several weeks and unattended overnight sequences. The applied sheath flow is reduced to 1–2 νL/min in order to increase sensitivity. Standard peptides and those of digests of standard proteins and gel‐separated proteins can be detected in the low femtomole range (full scan and MS/MS). Detection limits are found to be as low as 500 amol. Low femtomole amounts are required for unequivocal identification by MS/MS experiments in the ion trap and subsequent database search. By applying a simple pH‐mediated stacking the concentration sensitivity can be lowered to some tens of fmol/νL (nM), depending on capillary size. This sensitivity is close to published values for sheathless CE‐MS and nano‐LC‐MS, respectively (a comparison to reference values is presented). Moreover, with capillaries of about 50 cm in length separations in less than 10 min are possible resulting in a throughput of up to four analyses per hour. This is a factor of 4–12 times faster than nano‐LC separation, being the state‐of‐the‐art techniques for proteomic studies.

Guidelines for reporting the use of capillary electrophoresis in proteomics

1Department of Comparative Molecular Medicine, School of Veterinary Science, The University of Liverpool, Liverpool, UK. 2Manchester Centre for Integrative Systems Biology, Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, UK. 3Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, UK. 4National Institute for Biological Standards and Guidelines for reporting the use of column chromatography in proteomics

Capillary electrophoresis–mass spectrometry as a new approach to analyze neonicotinoid insecticides

This paper represents the first report of a capillary electrophoresis (CE) method compatible with mass spectrometry (MS) detection for simultaneously analyzing seven neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam). Different variables affecting CE separation (buffer concentration, pH, applied voltage and injection time) and MS detection (electrospray parameters) were studied. Low limits of detection (LOD) and quantification (LOQ) were achieved for all analytes, ranging from 1.0 to 2.3μg/L, and from 3.5 to 7.2μg/L, respectively. In addition, the proposed method showed itself to be linear in the range from LOQ to 1000μg/L and to be precise, as the relative standard deviations of the migration times were lower than 4% in all cases. Finally, the proposed CE-MS method was applied to assess the efficacy of a beeswax cleaning treatment with oxalic acid to remove residues of three of the most commonly used neonicotinoids (clothianidin, imidacloprid and thiamethoxam), use of which has recently been restricted by the European Union.

Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection.

Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein‐bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED‐induced fluorescence with limit of detections (LODs 0.5–3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1–14 amol/cell) and FAD (2.2–17.0 amol/cell) were the predominant flavins, while FMN (0.46–3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools.

Fast capillary electrophoresis coupled with time-of-flight mass spectrometry under separation conditions of high electrical field strengths.

An experimental approach is presented that enables very fast capillary electrophoretic separations in conjunction with time-of-flight mass spectrometry. Field strengths exceeding 1 kV cm(-1) have been applied for separations of model analytes resulting in migration times on the timescale of seconds.

Alignment of laser-induced fluorescence and mass spectrometric detection traces using electrophoretic mobility scaling in CE-LIF-MS of labeled N-glycans

The combination of optical detection techniques like photometry (UV) or laser‐induced fluorescence (LIF) with mass spectrometry for capillary electrophoresis offers advantages, both for later use of stand‐alone CE‐UV or CE‐LIF systems and for combined CE‐UV‐MS or CE‐LIF‐MS analysis. Faster method development is enabled, the identification of analytes is facilitated, and it allows christian the optical detection scheme to be used for more precise quantification. However, shortcomings of current methodology and equipment hindered the broader use of such detection combinations mainly due to the long distance between the detection points (at least 20 cm). Large shifts in migration times and changes in resolution are visible between the detection traces hindering their straightforward comparison. We present here novel equipment for a robust coupling of CE‐LIF‐MS with the shortest possible distance between detection points (12 cm) determined by the length of the electrospray needle. In addition, we encourage the use of a normalization of detection traces using a scale of effective electrophoretic mobility to obtain the same x‐scale for both detection traces. As an example, the proposed methodology is applied to a mixture of labeled as well as non‐labeled N‐glycans.

Raman spectroscopy and capillary zone electrophoresis for the analysis of degradation processes in commercial effervescent tablets containing acetylsalicylic acid and ascorbic acid

HIGHLIGHTSDegradation of effervescent tablets at high humidity by Raman spectroscopy and CZE.Comparison of two analytical techniques for the characterization of pharmaceuticals.Quantification of active pharmaceutical ingredients and degradation products by CZE.Fast detection of degradation of effervescent tablets by Raman spectroscopy and PCA.Quantification of mono‐ and diacetylated ascorbic acid in effervescent tablets by CZE. ABSTRACT In order to ensure the stability of pharmaceutical products appropriate manufacturing and storage conditions are required. In general, the degradation of active pharmaceutical ingredients (APIs) and subsequent formation of degradation products affect the pharmaceutical quality. Thus, a fast and effective detection and characterization of these substances is mandatory. Here, the applicability of Raman spectroscopy and CZE for the characterization of the degradation of effervescent tablets containing acetylsalicylic acid (ASA) and ascorbic acid (AA) was evaluated. Therefore, a degradation study was performed analyzing tablets from two different manufacturers at varying conditions (relative humidity (RH) 33%, 52% and 75% at 30 °C). Raman spectroscopy combined with principal component analysis could be successfully applied for the fast and easy discrimination of non‐degraded and degraded effervescent tablets after a storage period of approximately 24 h (RH 52%). Nevertheless, a clear identification or quantification of APIs and degradation products within the analyzed tablets was not possible, i.a. due to missing reference materials. CZE‐UV enabled the quantification of the APIs (ASA, AA) and related degradation products (salicylic acid (SA); semi‐quantitative also mono‐ and diacetylated AA) within the complex tablet mixtures. The higher the RH, the faster the degradation of ASA and AA as well as the formation of the degradation products. Mono‐ and diacetylated AA are major primary degradation products of AA for the applied effervescent tablets. A significant degradation of the APIs was detected earlier by CZE (6–12 h, RH 52%) than by Raman spectroscopy. Summarized, Raman spectroscopy is well‐suited as quick test to detect degradation of these tablets and CZE can be utilized for further detailed characterization and quantification of specific APIs and related degradation products.

Isomerization and epimerization of the aspartyl tetrapeptide Ala-Phe-Asp-GlyOH at pH 10-A CE study.

Isomerization and enantiomerization of Asp in the tetrapeptide Ala‐Phe‐Asp‐GlyOH are studied at pH 10 and 80°C as well as 25°C. CE‐MS allowed the distinction between α‐Asp and β‐Asp linkages in degradation products based on the ratio of the b and y fragment ions. Besides isomerization and enantiomerization of Asp, enantiomerization of Ala and Phe was also observed at both temperatures by chiral amino acid HPLC analysis using Marfeys reagent for derivatization. The rate of enantiomerization of the amino acids proceeded in the order Asp > Ala > Phe. The CE assay was validated with respect to linearity, LOQ, LOD, and precision and employed to characterize the time course of the degradation of the tetrapeptide upon incubation in borate buffer, pH 10. Isomerization to β‐Asp peptides was identified as the major degradation reaction. The configuration of Asp or Ala affected the half‐life of the starting peptide to a minor extent but did not influence the distribution of the individual products under equilibrium conditions at 80°C. Degradation at 25°C proceeded very slowly so that the equilibrium was not reached after 245 days.