Bartłomiej Witkowski

Analysis of α-acyloxyhydroperoxy aldehydes with electrospray ionization–tandem mass spectrometry (ESI-MSn)

A series of α-acyloxyhydroperoxy aldehydes was analyzed with direct infusion electrospray ionization tandem mass spectrometry (ESI/MS(n)) as well as liquid chromatography coupled with the mass spectrometry (LC/MS). Standards of α-acyloxyhydroperoxy aldehydes were prepared by liquid-phase ozonolysis of cyclohexene in the presence of carboxylic acids. Stabilized Criegee intermediate (SCI), a by-product of the ozone attack on the cyclohexene double bond, reacted with the selected carboxylic acids (SCI scavengers) leading to the formation of α-acyloxyhydroperoxy aldehydes. Ionization conditions were optimized. [M + H](+) ions were not formed in ESI; consequently, α-acyloxyhydroperoxy aldehydes were identified as their ammonia adducts for the first time. On the other hand, atmospheric-pressure chemical ionization has led to decomposition of the compounds of interest. Analysis of the mass spectra (MS(2) and MS(3)) of the [M + NH(4)](+) ions allowed recognizing the fragmentation pathways, common for all of the compounds under study. In order to get detailed insights into the fragmentation mechanism, a number of isotopically labeled analogs were also studied. To confirm that the fragmentation mechanism allows predicting the mass spectrum of different α-acyloxyhydroperoxy aldehydes, ozonolysis of α-pinene, a very important secondary organic aerosol precursor, was carried out. Spectra of the two ammonium cationized α-acyloxyhydroperoxy aldehydes prepared with α-pinene, cis-pinonic acid as well as pinic acid were predicted very accurately. Possible applications of the method developed for the analysis of α-acyloxyhydroperoxy aldehydes in SOA samples, as well as other compounds containing hydroperoxide moiety are discussed.

Proteinaceous binders identification in the works of art using ion-pairing free reversed-phase liquid chromatography coupled with tandem mass spectrometry

A simple, fast and reliable procedure for the proteinaceous binders identification in the works of art samples is presented. The procedure consisted of ammonia extraction in order to suppress pigment interferences, acidic hydrolysis and quantification of underivatized amino acids using reversed phase liquid chromatography coupled with electrospray tandem mass spectrometry (RP-LC–ESI-MS/MS). Fourteen underivatized amino acids were quantified without the addition of ion-pairing agents (IPA) using the multiple reaction monitoring (MRM) mode. The chromatographic separation was optimized by testing three C18 columns and three different eluent compositions. The optimal chromatographic resolution and the ionization efficiency were achieved with Symmetry C18 column and the eluent consisting of water with methanol. The amino acids composition of the proteins commonly found in the paint binding media, eggs, casein and animal glues, was determined. The procedure was tested using a set of naturally aged samples. Calculated detection and quantification limits indicated that the method is suitable for the analysis of protein binders in the paint micro-samples. Animal glues, casein and eggs were identified in the samples from 18th and 19th century paintings by Jacek Malczewski, while eggs and casein were detected in the mural painting samples from the 13th century UNESCO-listed church of Yemrehanna Krestos. The LC/MS/MS based method of the protein binders identification described here can be used as an alternative for the approach based on the gas chromatography coupled with mass spectrometry (GC/MS).

Analysis of latex protein content by liquid chromatography coupled with tandem mass spectrometry (HPLC/MS/MS)

In this work, the protein content of latex gloves and c-serum isolated from ammonia stabilized Hevea brasiliensis latex rubber concentrate was studied using a novel analytical protocol utilizing HPLC-ESI/MS/MS. Latex proteins were hydrolyzed to their corresponding building blocks and the resulting amino acid mixture was subsequently quantified by HPLC-ESI/MS/MS. Fifteen underivatized amino acids were separated with a hydrophilic interaction column (ZIC-HILIC) and the sample protein content was calculated. By utilizing a very specific and selective detection method (selected reaction monitoring mode) it was possible to minimize interferences from the sample matrix. For the natural rubber latex concentrate c-serum, the proteins were precipitated with trichloroacetic acid (TCA) and extracted with ammonia solution in water before hydrolysis. This procedure eliminated a large portion of c-serum rubber which interfered with the hydrolysis reaction. A c-serum protein concentration of 11.68 ± 0.43 mg ml−1 was obtained. The highest value available in the literature is 7.62 ± 0.28 mg ml−1 – these results prove the high efficiency of the developed analytical protocol. Data obtained for the c-serum were used to estimate the efficiency of different protein extraction methods from the latex gloves. Thus, the results obtained in this work were used to link the protein content of the raw material with the protein content of the vulcanized latex gloves for the first time. Afterwards, the optimized protocol was used to evaluate the performance of different protein removal methods, utilized during latex glove manufacturing. Thus, a practical application of the developed method is presented.

Identification of proteins, drying oils, waxes and resins in the works of art micro-samples by chromatographic and mass spectrometric techniques

Simplified method for simultaneous identification of proteins, drying oils, waxes, and resins in the works-of-art samples was developed. Liquid chromatography with mass spectrometry and gas chromatography with mass spectrometry were used to identify natural materials most frequently encountered in historical paintings. Protein binders were extracted with ammonia and purified using miniaturized solid-phase microextraction (Omix tips) to efficiently suppress matrix interferences. Zwitterionic stationary phase was used for separation of 16 underivatized amino acids analysis with hydrophilic interaction liquid chromatography that was subsequently quantified with liquid chromatography with mass spectrometry. Gas chromatography with mass spectrometry was used to analyze drying oils, waxes, and resins after one-step saponification/transmethylation with (m-trifluoromethylphenyl)trimethylammonium hydroxide (Meth-Prep II). While the drawback of this reagent is low reactivity towards hydroxyl groups, sample pretreatment was much simpler as compared to the other methods. Fatty acids derivatization with the Meth-Prep II reagent was compared with their silylation using N,O-bis(trimethylsilyl) trifluoroacetamide/trimethylchlorosilane mixture. It was concluded that fatty acids analysis as their methyl esters instead of trimethylsilyl esters had a minor impact on the method sensitivity. The developed method was used to analyze samples from 16th and 17th century historical paintings.

Kinetics of limonene SOA oxidation in the aqueous phase

Twenty semivolatile organic compounds that contribute to limonene secondary organic aerosol (SOA) were synthesized in the flow-tube reactor. Kinetics of the aqueous-phase oxidation of the synthesized compounds by hydroxyl radicals (OH) and ozone (O3) were investigated at 298 ± 2 K using the relative rate method. Oxidized organic compounds identified as the major components of limonene SOA were quantified with liquid chromatography coupled to the electrospray ionization and quadrupole tandem mass spectrometry (LC-ESI/MS/MS). The bimolecular rate coefficients measured for the oxidation products of limonene are kOH = 2-5 × 109 M-1 s-1 for saturated and kOH = 1-2 × 1010 M-1 s-1 for unsaturated compounds. Ozonolysis reaction bimolecular rate coefficients obtained for the unsaturated compounds in the aqueous phase are between 2 and 6 × 104 M-1 s-1. The results obtained in this work also indicate that oxidation of limonene carboxylic acids by OH was about a factor of 2 slower for the carboxylate ions than for the protonated acids while the opposite was true for the ozonolysis. The data acquired provided new insights into kinetics of the limonene SOA processing in the aqueous phase. Ozonolysis of limonene SOA also increased the concentration of dimers, most likely due to reactions of the stabilized Criegee intermediates with the other, stable products. These results indicate that aqueous-phase oxidation of limonene SOA by OH and O3 will be relevant in clouds, fogs, and wet aerosols.

Limononic Acid Oxidation by Hydroxyl Radicals and Ozone in the Aqueous Phase

Kinetics and mechanism of limononic acid (3-isopropenyl-6-oxoheptanoic acid, LA) oxidation by hydroxyl radicals (OH) and ozone (O3) were studied in the aqueous phase at 298 ± 2 K. These reactions were investigated using liquid chromatography coupled to the electrospray ionization and quadrupole tandem mass spectrometry (LC-ESI/MS/MS). The rate coefficients determined for LA + OH reaction were: 1.3 ± 0.3 × 1010 M-1 s-1 at pH = 2 and 5.7 ± 0.6 × 109 M-1 s-1 at pH = 10. The rate coefficient determined for LA ozonolysis was 4.2 ± 0.2 × 104 M-1 s-1 at pH = 2. The calculated Henrys law constant (H) for LA was ca. 6.3 × 106 M × atm-1, thereby indicating that in fogs and clouds with LWC = 0.3-0.5 g × m-3 LA will reside entirely in the aqueous phase. Calculated atmospheric lifetimes due to reaction with OH and O3 strongly indicate that aqueous-phase oxidation can be important for LA under realistic atmospheric conditions. Under acidic conditions, the aqueous-phase oxidation of LA by OH will dominate over reaction with O3, whereas the opposite is more likely when pH ≥ 4.5. The aqueous-phase oxidation of LA produced keto-limononic acid and a number of low-volatility products, such as hydroperoxy-LA and α-hydroxyhydroperoxides.

cis-Pinonic Acid Oxidation by Hydroxyl Radicals in the Aqueous Phase under Acidic and Basic Conditions: Kinetics and Mechanism

Aqueous-phase oxidation of cis-pinonic acid (CPA) by hydroxyl radicals (OH) was studied using a relative rate technique under acidic and basic conditions. Liquid chromatography (LC) coupled to the negative electrospray ionization (ESI) quadrupole tandem mass spectrometry (MS/MS) was used to monitor the concentrations of CPA and reference compounds. The measured second order reaction rate coefficients of CPA with OH were: 3.6 ± 0.3 × 109 M-1 s-1 (pH 2) and 3.0 ± 0.3 × 109 M-1 s-1 (pH 10) - combined uncertainties are 2σ. These results indicated that the lifetimes of CPA in the atmosphere are most likely independent from the aqueous-phase pH. LC-ESI/MS/MS was also used to tentatively identify the CPA oxidation products. Formation of carboxylic acids with molecular weight (MW) 216 Da (most likely C10H16O5) and MW 214 Da (C10H14O5) was confirmed with LC-ESI/MS/MS. When the initial CPA concentration was increased from 0.3 to 10 mM, formation of additional products was observed with MW 188, 200, 204, and 232 Da. Hydroperoxy, hydroxyl and carbonyl-substituted CPA derivatives were tentatively identified among the products. Similar products were formed by the CPA oxidation by OH in the gas-phase, at the air-water interface as well as in the solid phase (dry film). Formation of the stable adduct of CPA and H2O2 was also observed when the reaction mixture was evaporated to dryness and redissolved in water. Acquired mass spectrometric data argues against formation of oligomers.