Characterization of different poly(2‐oxazoline) block copolymers by MALDI‐TOF MS/MS and ESI‐Q‐TOF MS/MS

A complete library of poly(2‐oxazoline) block copolymers was synthesized via cationic ring opening polymerization for the characterization by two different soft ionization techniques, namely matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF MS). In addition, a detailed characterization was performed by tandem MS to gain more structural information about the block copolymer composition and its fragmentation behavior. The fragmentation of the poly(2‐oxazoline) block copolymers revealed the desired polymer structure and possible side reactions, which could be explained by different mechanisms, like 1,4‐ethylene or hydrogen elimination and the McLafferty +1 rearrangement. Polymers with aryl side groups showed less fragmentation due to their higher stability compared to polymers with alkyl side groups. These insights represent a further step toward the construction of a library with fragments and their fragmentation pathways for synthetic polymers, following the successful examples in proteomics. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

MALDI‐TOF MS Coupled with Collision‐Induced Dissociation (CID) Measurements of Poly(methyl methacrylate)

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) was chosen for an in‐detail analysis of poly(methyl methacrylate) (PMMA) in order to determine the possible fragmentation mechanism with the help of collision‐induced dissociation (CID). All experiments were performed on a well‐defined PMMA standard and were optimized for sample preparation and measurement conditions of both MS and MS/MS. In order to investigate the fragmentation pathways, two parent peaks—both charged with sodium (m/z = 1 625.9 and 2 226.2 Da, respectively)—were selected, thus permitting the examination of possible cleavages, and reaction pathways. For both chosen peaks, the MALDI‐TOF MS/MS spectra revealed four fragmentation series that could be explained by single or multiple main chain scissions and secondary reactions of the PMMA side groups. According to the molar mass of the fragments, a loss or migration of the side group to the end of the free radical, followed by a β‐scission, was favored. These insights are the first steps toward the construction of a library with fragments and fragmentation pathways, complementary to proteomics libraries, in order to obtain fast and automated identification of substances.

     

An approach to differentiating between multi‐ and monolayers using MALDI‐TOF MS

Analysis and confirmation of monolayer film thickness on metal oxide surfaces has proven to be challenging. XPS and AFM have been used to investigate the monolayer formation. However, these techniques are difficult to access and/or determine the composition of the organic molecules on the surfaces. Here we demonstrate the ability of MALDI‐TOF to characterize long alkyl chain phosphonic acid molecules in thin films on titanium, iron and stainless steel. These systems are known to be stable, strongly adhered films. The thin films were characterized by IR, AFM, contact angle measurements and the results were confirmed by MALDI‐TOF. Moreover, the MALDI‐TOF was used to differentiate between mono‐ and multilayers on planar surfaces. Copyright © 2007 John Wiley & Sons, Ltd.     

Applicability of MALDI‐TOF MS for determination of quinolone residues in fish

MALDI‐TOF MS approach for determination of six quinolones residues in fillets of pangasius (Pangasionodon hypophthalmus) was studied, considering that is a very sensitive analytical technique with simple and high‐throughput operation, contributing to knowledge regarding application of this technique to the determination of small‐molecular‐weight organic compound residues in foods. LIFT‐MS/MS showed to be a successful approach to identify the presence of all quinolone residues in the fish fillet, at their respective MRL level. This study opens an important field of research for the development of simple and high‐throughput bioanalytical screening methods for the determination of veterinary drug residues in foods.     

Determination of block size in poly(ethylene oxide)‐b‐polystyrene block copolymers by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Characterization of block size in poly(ethylene oxide)‐b‐poly(styrene) (PEO‐b‐PS) block copolymers could be achieved by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) after scission of the macromolecules into their constituent blocks. The performed hydrolytic cleavage was demonstrated to specifically occur on the targeted ester function in the junction group, yielding two homopolymers consisting of the constitutive initial blocks. This approach allows the use of well‐established MALDI protocols for a complete copolymer characterization while circumventing difficulties inherent to amphiphilic macromolecule ionization. Although the labile end‐group in PS homopolymer was modified by the MALDI process, PS block size could be determined from MS data since polymer chains were shown to remain intact during ionization. This methodology has been validated for a PEO‐b‐PS sample series, with two PEO of number average molecular weight (M_n) of 2000 and 5000 g mol^?1 and M_n(PS) ranging from 4000 to 21,000 g mol^?1. Weight average molecular weight (M_w), and thus polydispersity index, could also be reached for each segment and were consistent with values obtained by size exclusion chromatography. This approach is particularly valuable in the case of amphiphilic copolymers for which M_n values as determined by liquid state nuclear magnetic resonance might be affected by micelle formation. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3380–3390, 2009     

A MALDI‐TOF MS study of lanthanide(III)‐cored poly(phenylenevinylene) dendrimers

An extensive study by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry (MS) of some first‐generation and second‐generation lanthanide(III)‐cored poly(phenylenevinylene) dendrimers is described. The complexes were obtained by self‐assembly of suitably functionalized carboxylate dendrons around the lanthanide ion (La³⁺, Er³⁺). Fourier transform infrared (FT‐IR) spectroscopy gave reasonable evidence for the proposed structures. However, MS was used to ascertain unequivocally the complex formation. The most reliable results were found in the negative reflector mode, using 2‐[(2E)‐3‐(4‐tert‐butylphenyl)‐2‐methylprop‐2‐enylidene]malononitrile (DCTB) as matrix. Well‐defined and highly resolved base peaks corresponding to negative ions of [Gn₄La]^? and [Gn₄Er]^? were found in all cases, with an excellent match between the theoretical and observed isotope distributions. However, the 3 : 1 stoichiometry used in the synthesis guarantees an empirical formula Gn₃Ln for the complexes. Copyright © 2008 John Wiley & Sons, Ltd.     

Evaluation of temperature,excipients impact on the primary structure of Ganirelix in an injectable formulation,and comparison with Orgalutran® using MALDI‐TOF/TOF‐MS

Ganirelix is a linear polypeptide consisting of covalently bonded 10 amino acid residues. The amino acid sequence in a peptide determines the properties of the molecule. The slightest change in the primary structure (amino acid sequence) of therapeutic peptides can significantly impact its safety, efficacy, and immunogenicity. Hence, the primary structure analysis of therapeutic peptides is regarded as a critical quality attribute (CQA). A vast array of analytical techniques can be used to capture the primary structure of the peptide. In this study, we applied matrix‐assisted laser desorption ionization (MALDI)/tandem time of flight mass spectroscopic (TOF/TOF MS) method to demonstrate the primary structure of Ganirelix in an injectable formulation. The apparent monoisotopic molecular mass of Ganirelix is 1,568.9 Da. The attained primary amino acid sequence of Ganirelix in temperature‐stressed generic product matched with the theoretical sequence and showed homology with those of the reference listed drug (RLD).     

Brushless and controlled microphase separation of lamellar polystyrene‐b‐polyethylene oxide thin films for block copolymer nanolithography

Creating perpendicular alignment in lamellar block copolymer (BCP) systems has considerable industrial and commercial significance, most importantly for generating nanowire structures in electronic devices. In general, these lamellar systems require careful interface engineering to obtain vertical orientation of the blocks. To avoid the strong preferential adsorption of one block to either the substrate or the polymer/air interface, the surface must be “neutralized” by chemical brushes or external forces, for example, solvent fields. Reported here is a stepwise thermo/solvent annealing process allowing the formation of perpendicular domains of polystyrene‐b‐polyethylene oxide lamellar structures while avoiding brush or other surface modifications. This BCP has a relatively small minimum feature size and can be used to generate substrate patterns for use in fabrication of nanowire electronic device structures. © 2012 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2012     

High‐resolution MALDI‐TOF MS study on analysis of low‐molecular‐weight products from photo‐oxidation of poly(3‐hexylthiophene)

High‐resolution matrix‐assisted laser desorption/ionization (MALDI) time‐of‐flight mass spectrometry (TOF MS) was used for the analysis of the low‐molecular‐weight products from the photo‐oxidation of poly(3‐hexylthiophene) (P3HT) in solution and thin film. Eight new peak series were observed in the low‐mass range of the mass spectra of the products degraded in solution, and the formulas of the eight components were determined from the accurate mass. From SEC/MALDI‐TOF MS, two components were identified as the degraded products, and the other six components were derived from the fragmentation of the degraded products during the MALDI process. A mechanism for the formation of these components was proposed on the basis of the results of MALDI‐TOF MS. For the thin film degradation, a part of products in the solution degradation were observed, which supports that the oxidation of P3HT in solution and thin film proceeded in the same mechanism. This study shows that high‐resolution MALDI‐TOF MS is effective for the analysis of the low‐molecular‐weight products from P3HT photo‐oxidation and expected to be feasible for the degradation analyses of other polymers. Copyright © 2015 John Wiley & Sons, Ltd.     

RAFT synthesis of a water‐soluble triblock copolymer of poly(styrenesulfonate)‐b‐poly(ethylene glycol)‐b‐poly(styrenesulfonate) using a macromolecular chain transfer agent in aqueous solution

Triblock copolymers of poly(styrenesulfonate)‐b‐poly(ethylene glycol)‐b‐poly(styrenesulfonate) with narrow molecular weight distribution (M_w/M_n = 1.28–1.40) and well‐defined structure have been synthesized in aqueous solution at 70 °C via reversible addition‐fragmentation chain transfer polymerization. Poly(ethylene glycol) (PEG) capped with 4‐cyanopentanoic acid dithiobenzoate end groups was used as the macro chain transfer agent (PEG macro‐CTA) for sole monomer sodium 4‐styrenesulfonate. The reaction was controllable and displayed living polymerization characteristics and the triblock copolymer had designed molecular weight. The reaction rate depended strongly on the CTA and initiator concentration ratio [CTA]₀/[ACPA]₀: an increase in [CTA]₀/[ACPA]₀ from 1.0 to 5.0 slowed down the polymerization rate and improved the molecular weight distribution with a prolonged induction time. The polymerization proceeded, following first‐order kinetics when [CTA]₀/[ACPA]₀ = 2.5 and 5.0. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 3698–3706, 2007     

Comparison of MALDI‐TOF mass spectra with microsatellite length polymorphisms in Candida albicans

Candida albicans is the most frequent yeast involved in human infections. Its population structure can be divided into several genetic clades, some of which have been associated with antifungal susceptibility. Therefore, detecting and monitoring fungal clones in a routine laboratory setting would be a major epidemiological advance. Matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectra results are now widely used as bar codes to identify microorganisms in clinical microbiology laboratories. This study aimed at testing MALDI‐TOF mass spectra bar codes to identify clades among a set of C. albicans isolates. Accordingly, 102 clinical strains were genotyped using 10 microsatellite markers and analyzed via MALDI‐TOF mass spectrometry. The mass spectra were compared with a reference spectral library including 33 well‐characterized collection strains, using a Microflex^TM system and Biotyper^TM software, to test the capacity of the spectrum of a given isolate to match with the reference mass spectrum of an isolate from the same genetic clade. Despite high confidence species identification, the spectra failed to significantly match with the corresponding clade (p = 0.74). This was confirmed with the MALDI‐TOF spectra similarity dendrogram, in which the strains were dispersed irrespective of their genetic clade. Various attempts to improve intra‐clade spectra recognition were unsuccessful. In conclusion, MALDI‐TOF mass spectra bar code analysis failed to reliably recognize genetically related C. albicans isolates. Further studies are warranted to develop alternative MALDI‐TOF mass spectra analytical approaches to identify and monitor C. albicans clades in the routine clinical laboratory. Copyright © 2015 John Wiley & Sons, Ltd.     

Molecular weight analysis of water‐soluble poly(phenylene ethynylene)s using MALDI‐TOF MS

Molecular weights of seven poly(phenylene ethynylene)‐based water‐soluble conjugated polyelectrolytes (CPEs) obtained through Sonogashira coupling are determined by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). A standard sample preparation protocol is developed to characterize the seven CPEs using 2,5‐dihydroxybenzoic acid as the matrix (M) and AgTFA as the cationization reagent (CR). High‐quality MALDI mass spectra are obtained at volume mixing ratios (CPE/M/CR) of 5/5/1 for anionic polymers (P1–P4) and 5/50/1 for cationic polymers (P5–P7). Molecular weight, molecular weight distribution, and end‐group information are analyzed. The effects of molecular weight of CPEs on optical and quenching properties are also studied. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 2537–2543     

Applications of MALDI‐TOF MS to large‐scale human mtDNA population‐based studies

Analysis of the mitochondrial DNA variation in populations is commonly carried out in many fields of biomedical research. We propose the analysis of mitochondrial DNA coding region SNP (mtSNP) variation to a high level of phylogenetic resolution based on MALDI‐TOF MS. The African phylogeny has been chosen to test the applicability of the technique but any other part of the worldwide phylogeny (or any other mtSNP panel) could be equally suitable for MALDI‐TOF MS genotyping. SNP selection thus aimed to fully cover all the mtSNPs defining major and minor branches of the known African tree, including, macro‐haplogroup L, and haplogroups M1, and U6. A total of 230 mtSNPs were finally selected. We used tests samples collected from two different African locations, namely, Mozambique and Chad Basin. Different internal genotyping controls and other indirect approaches (e.g. phylogenetic checking coupled with automatic sequencing) were used in order to evaluate the reproducibility of the technique, which resulted to be 100% using samples previously subjected to whole genome amplification. The advantages of the MALDI‐TOF MS are also discussed in comparison with other popular methods such as minisequencing, highlighting its high‐throughput nature, which is particularly suitable for case–control medical studies, forensic databasing or population and anthropological studies.     

MALDI‐TOF MS investigation of the unconventional termination of living polyoxazoline with ammonia

Poly(2‐oxazoline) (POx) based materials have experienced renewed interest, due to their biocompatibility and broad functional diversity. Although research pertaining to cationic ring opening polymerization (CROP) of 2‐oxazoline derivatives spans upwards of 5 decades, modern characterization techniques, specifically Matrix Assisted Laser‐Desorption Ionization—Time of Flight Mass Spectrometry (MALDI‐TOF MS), were not available during early studies to aid in identifying polymer end‐groups. Through careful synthesis and analytical characterization, evidence supports an alternative mode of nucleophilic attack during ammonia termination, with nucleophilic attack occurring at a different site on the terminal oxazolinium than where polymer propagation occurs. Furthermore, investigations employing targeted end‐group modification, in conjunction with MALDI and NMR analysis, determined the structure of the resultant terminal group was a hydroxyethylamino end group. Based on the observed data, a mechanistic explanation for the observed 2‐oxazoline ring‐opening termination is proposed. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 1303–1312     

MALDI‐TOF mass spectrometry,an efficient technique for in situ detection and characterization of actinomycins

An extensive study of actinomycins was performed using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐TOF MS). Actinomycins represent a well‐known family of peptidolactone chromopeptides with potent cytostatic and antibiotic properties. Using five well‐characterized streptomycete strains, we introduced MALDI‐TOF MS as an efficient technique for rapid in situ detection of actinomycins in surface extracts of cells picked from agar plates. By this procedure, actinomycin complexes can be investigated with high sensitivity and accuracy in a minimum of time. These studies were complemented by mass spectrometric investigation of actinomycins obtained from culture filtrate extracts and purified by high‐performance liquid chromatography to detect yet unknown actinomycin species. By feeding experiments, C‐demethyl‐actinomycins from Streptomyces chrysomallus and Streptomyces parvulus as well as hemi‐actinomycins from Streptomyces antibioticus lacking one of the two pentapeptide lactone rings were isolated and characterized as novel variants for structure–activity relationship studies. Structural characterization of the investigated actinomycins was performed by post source decay MALDI‐TOF MS. The specific features of the fragmentation patterns of the protonated and cationized forms of selected actinomycins were investigated in detail. Copyright © 2014 John Wiley & Sons, Ltd.     

Direct screening of herbal blends for new synthetic cannabinoids by MALDI‐TOF MS

Since 2004, a number of herbal blends containing different synthetic compounds mimicking the pharmacological activity of cannabinoids and displaying a high toxicological potential have appeared in the market. Their availability is mainly based on the so‐called “e‐commerce”, being sold as legal alternatives to cannabis and cannabis derivatives. Although highly selective, sensitive, accurate, and quantitative methods based on GC–MS and LC–MS are available, they lack simplicity, rapidity, versatility and throughput, which are required for product monitoring. In this context, matrix‐assisted laser desorption ionization‐time of flight mass spectrometry (MALDI‐TOF MS) offers a simple and rapid operation with high throughput. Thus, the aim of the present work was to develop a MALDI‐TOF MS method for the rapid qualitative direct analysis of herbal blend preparations for synthetic cannabinoids to be used as front screening of confiscated clandestine preparations. The sample preparation was limited to herbal blend leaves finely grinding in a mortar and loading onto the MALDI plate followed by addition of 2 µl of the matrix/surfactant mixture [α‐cyano‐4‐hydroxy‐cinnamic acid/cetyltrimethylammonium bromide (CTAB)]. After drying, the sample plate was introduced into the ion source for analysis. MALDI‐TOF conditions were as follows: mass spectra were analyzed in the range m/z 150–550 by averaging the data from 50 laser shots and using an accelerating voltage of 20 kV. The described method was successfully applied to the screening of 31 commercial herbal blends, previously analyzed by GC–MS. Among the samples analyzed, 21 contained synthetic cannabinoids (namely JWH‐018, JWH‐073, JWH‐081, JWH‐250, JWH‐210, JWH‐019, and AM‐694). All the results were in agreement with GC–MS, which was used as the reference technique. Copyright © 2012 John Wiley & Sons, Ltd.     

Direct identification of trypanosomatids by matrix‐assisted laser desorption ionization–time of flight mass spectrometry (DIT MALDI‐TOF MS)

Accurate and rapid determination of trypanosomatids is essential in epidemiological surveillance and therapeutic studies. Matrix‐assisted laser desorption ionization/time of flight mass spectrometry (MALDI‐TOF MS) has been shown to be a useful and powerful technique to identify bacteria, fungi, metazoa and human intact cells with applications in clinical settings. Here, we developed and optimized a MALDI‐TOF MS method to profile trypanosomatids. trypanosomatid cells were deposited on a MALDI target plate followed by addition of matrix solution. The plate was then subjected to MALDI‐TOF MS measurement to create reference mass spectra library and unknown samples were identified by pattern matching using the BioTyper software tool. Several m/z peaks reproducibly and uniquely identified trypanosomatids species showing the potentials of direct identification of trypanosomatids by MALDI‐TOF MS. Moreover, this method discriminated different life stages of Trypanosoma cruzi, epimastigote and bloodstream trypomastigote and Trypanosoma brucei, procyclic and bloodstream. T. cruzi Discrete Typing Units (DTUs) were also discriminated in three clades. However, it was not possible to achieve enough resolution and software‐assisted identification at the strain level. Overall, this study shows the importance of MALDI‐TOF MS for the direct identification of trypanosomatids and opens new avenues for mass spectrometry‐based detection of parasites in biofluids. Copyright © 2016 John Wiley & Sons, Ltd.     

Identification and determination of the major constituents in Kai‐Xin‐San by UPLC‐Q/TOF MS and UFLC‐MS/MS method

In order to have overall chemical material information of Kai‐Xin‐San (KXS), the reliable ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometer (UHPLC–Q‐TOF‐MS) and ultra‐fast liquid chromatography mass spectrometer (UFLC‐MS/MS) methods were developed for the identification and determination of the major constituents in KXS. Moreover, the UHPLC–Q‐TOF‐MS method was also applied to screen for multiple absorbed components in rat plasma after oral administration of KXS. The UHPLC–Q‐TOF‐MS method was achieved on Agilent 6520 Q‐TOF mass and operated in the negative ion mode. Good separation was performed on a ZORBAX Eclipse Plus C18 column with a gradient elution at a flow rate of 0.2 ml/min. A total of 92 compounds in KXS were identified or tentatively characterized based on their exact molecular weights, fragmentation patterns, and literature data. A total of 26 compounds including 23 prototype components and three metabolites were identified in rat plasma after oral administration of KXS. Then, 16 major bioactive constituents were chosen as the benchmark substances to evaluate the quality of KXS. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple‐reaction monitoring mode(MRM). The analysis was completed with a gradient elution at a flow rate of 0.4 ml/min within 35 min. The simple and fast method was validated and showed good linearity, precision, and recovery. Furthermore, the method was successful applied for the determination of 16 compounds in KXS. All results would provide essential data for identification and quality control of active chemical constituents in KXS. Copyright © 2016 John Wiley & Sons, Ltd.     

Impact of updating the MALDI‐TOF MS database on the identification of nontuberculous mycobacteria

Conventional identification of mycobacteria species is slow, laborious and has low discriminatory power. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has proved highly effective for identifying conventional bacteria, and it may also be useful for identifying mycobacteria. The aim of this study was to evaluate and compare MALDI‐TOF MS with currently recommended molecular methods for the identification of nontuberculous mycobacteria (NTM), applying Mycobacteria Libraries v3.0 (ML3.0) and v2.0 (ML2.0). A total of 240 clinical isolates of 41 NTM species grown on solid media were analysed: 132 isolates of slow‐growing mycobacteria and 108 of rapid‐growing mycobacteria. MALDI‐TOF MS, using ML3.0, identified 192 (80%) NTM isolates with a score ≥1.7, encompassing 35 (85.4%) different species, that is, 17 (7.1%; p  = 0.0863) isolates and 15 (36.6%; p  = 0.0339) species more than currently recommended molecular techniques (polymerase chain reaction reverse hybridization). All these isolates were correctly identified according to molecular identification methods. The application of ML3.0 also identified 15 (6.2%) NTM isolates more than ML2.0 (p  < 0.01). The scores obtained with MALDI‐TOF MS using ML3.0 (mean score: 1.960) were higher in 147 (61.2%) isolates than when using ML2.0 (mean score: 1.797; p  < 0.01). Three of the species analysed were not included in either database, so they were not recognized by this system. In conclusion, MALDI‐TOF MS identified more isolates and species than the recommended polymerase chain reaction reverse hybridization assays. Although the new ML3.0 is not the definitive database, it yielded better results than ML2.0. This shows that the updating of the MALDI‐TOF MS database plays an essential role in mycobacterial identification. Copyright © 2017 John Wiley & Sons, Ltd.