Electrospray Mass Spectrometry Detection Research Articles

Application of dispersive liquid–liquid extraction followed by rapid and direct mass spectrometry analysis to evaluate parabens in high salinity water sample

As an emerging marine pollutant, parabens have attracted more and more attention due to their widespread existence and potential toxicity in living organisms. In this work, a sensitive and accurate ultrasound-assisted liquid–liquid extraction (LLE) method, followed by a combination of nanoscale electrospray mass spectrometry detection technique, was proposed to determine the trace parabens in high salinity water sample. The method used only one organic solvent for the extraction and ionization of the target in simulated seawater samples. The extraction/ionization of salts is restrained due to their microsolubility or even insolubility in the extraction solvent (also spray solvent), which greatly enhanced the signal intensity of the target analytes and achieved desalination in the mass spectrometry. Several parameters that affect the extraction and ionization efficiencies were investigated. Under the optimal conditions (extraction solvent, 800 µL ethyl acetate; volume ratio of sample to organic phase, 6:1; extraction time, 7 min; nano-tube, a-nanotube; ionization voltage, 2.3 kV), the proposed method was effectively implemented for the detection of five parabens in different real water samples (seawater water and domestic water). The results showed that the limits of detection ranged between 0.21 and 1.11 ng mL−1. In addition, satisfactory accuracy and precision were obtained in real seawater samples for all parabens. This study demonstrated that the combination of LLE and nanoscale electrospray technology is suitable for detecting analytes in high salinity such as seawater.

High-speed countercurrent chromatography with offline detection by electrospray mass spectrometry and nuclear magnetic resonance detection as a tool to resolve complex mixtures: A practical approach using Coffea arabica leaf extract.

Many secondary metabolites isolated from plants have been described in the literature owing to their important biological properties and possible pharmacological applications. However, the identification of compounds present in complex plant extracts has remained a great scientific challenge, is often laborious, and requires a long research time with high financial cost. The aim of this study was to develop a method that allows the identification of secondary metabolites in plant extracts with a high degree of confidence in a short period of time. In this study, an ethanolic extract of Coffea arabica leaves was used to validate the proposed method. Countercurrent chromatography was chosen as the initial step for extraction fractionation using gradient elution. Resulting fractions presented a variation of compounds concentrations, allowing for statistical total correlation spectroscopy (STOCSY) calculations between liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and NMR across fractions. The proposed method allowed the identification of 57 compounds. Of the annotated compounds, 20 were previously described in the literature for the species and 37 were reported for the first time. Among the inedited compounds, we identified flavonoids, alkaloids, phenolic acids, coumarins, and terpenes. The proposed method presents itself as a valid alternative for the study of complex extracts in an effective, fast, and reliable way that can be reproduced in the study of other extracts.

Coupling microchip pressure regulators with chipHPLC as a step toward fully portable analysis system

Herein the coupling of a miniaturized, nanoliter scaled, pressure regulator (chipPR), and a chipHPLC device is introduced. The active temperature based flow control of the chipPR is able to generate rapid pressure changes and therefore enables on-chip pinched injection and flow gradients with reduced instrumental effort and minimal dead volumes. The functionality of the chipPR empowered chipHLPC device was demonstrated with high-speed HPLC-separations applying fluorescence and electrospray mass spectrometry (ESI-MS) detection. The system shows excellent long-term stability of chromatography integrity (retention times with RSD of 0.44–0.91%) due to the integration of a PID feedback regulation. This first chip-based HPLC device equipped with chipPRs enables precise flow control with significantly reduced technical effort compared to the state-of-the-art.

High-throughput analysis of polyethoxylated tallow amine homologs in citrus using a modified QuEChERS-HILIC-MS method.

A new method is described based on ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC) with electrospray mass spectrometry detection for comprehensive quantitative analysis of 66 polyethoxylated tallow amine (POE-tallowamine) homologs in citrus. Efficient separation, reduced band broadening, and high sensitivity were achieved by employing an acetonitrile-aqueous solution containing a 10 mM ammonium formate gradient on a hydrophilic interaction chromatography (HILIC) column with a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method. The quantitative accuracy and precision of the method were improved by the use of matrix-matched calibration standards. At spiked levels of (50 + 250) μg/kg, (200 + 1000) μg/kg, and (500 + 2500) μg/kg POE-5 and POE-15 (1:5), the average recoveries of the POE-tallowamine homologs ranged from 71.9 to 112%, with RSDs < 16.6%. The limits of detection (LODs) and limits of quantification (LOQs) for POE-tallowamine homologs were 0.01-2.57 and 0.03-8.58 μg/kg, respectively. The method was successfully applied to determine POE-tallowamine in citrus samples from typical Chinese regions in 2021. POE-tallowamine was detected in all 54 samples, and the highest concentration (143 μg/kg) of POE-tallowamine was found in Jelly orange from Zhejiang Province, which might indicate a higher usage and demand of glyphosate herbicides in Zhejiang.

Combining high-speed countercurrent chromatography three-phase solvent system with electrospray ionization-mass spectrometry and nuclear magnetic resonance to profile the unconventional food plant Syzygium malaccense

Syzygium malaccense (L.) Merr. & L.M. Perry is a native tree to Malaysia, but also occurs in other tropical regions of the world, including Brazil. The increasing interest in the consumption of its leaves motivated the investigation of compounds of the plant. Metabolite profiling of S. malaccense leaves was achieved by high-speed countercurrent chromatography (HSCCC) fractionation coupled off-line to electrospray mass-spectrometry (ESI-MS) detection and nuclear magnetic resonance (NMR) analysis. The ethanolic leaf extract was submitted to HSCCC using a three-phase solvent system (TPSS) composed by n-hexane – ethyl acetate – acetonitrile – H2O (2:1:1:1, v/v). The stepwise gradient elution was employed due to the extract's chemical complexity. HSCCC fractions were further analyzed by ESI-MS/MS using a flow injection experiment and by NMR acquiring 1H, HSQC and HMBC spectra. MS based dereplication was achieved by comparing acquired data to those available in public and commercial databases. Results were also correlated to previously isolated compounds described for the Syzygium genus. This process led to the annotation of 90 compounds. The NMR data provided structural confirmation and substitution patterns for some of them. Extract chemical composition is characterized by having flavonoids, benzoic acids, hydroxycinnamic acids, quinic acids, hydrolizable tannins, fatty acids, anacardic acids and others primary metabolites. Most of these compounds were described for the first time in the plant. This approach greatly facilitates phytochemical analysis and could be applied to improve metabolite discovery in other studies.

Application of betacyanins pigments from Alternanthera brasiliana as yogurt colorant

Betacyanins are betalain derivatives showing a red-violet color suitable as a natural food colorant. An aqueous extract rich in betacyanin pigments and phenolic compounds from Alternanthera brasiliana was characterized using a high-performance liquid chromatography system with diode array and electrospray mass spectrometry detection. The major betacyanins (amaranthine, isoamaranthine, betanin and isobetanin, celosianin II and isocelosianin II) were identified and quantified. Eleven peaks were analyzed for phenolic compounds grouped into hydroxycinnamic acids and flavones. Different plant extract concentrations were added to freshly prepared set type yogurts (350, 700, and 1400 μL/80 g yogurt). Color properties in CIELAB space, pH, total phenolic content, and ferric reducing antioxidant power were analyzed on yogurts during storage time. The evolution of pigments and polyphenolic compounds during cold storage was studied by HPLC-ESI-QTOF-MS screening.The extracted color showed to be stable at the yogurt pH range. When added to yogurts, a natural berry-like color was achieved. These results provide a natural plant pigment-based alternative for application in food products, being the first time that A. brasiliana is used as a colorant in a dairy product. Besides color, polyphenols input turns Alternanthera brasiliana extract into a desirable functional food ingredient.

Acylsugar diversity in the resin glycosides from Ipomoea tricolor seeds as chemosensitizers in breast cancer cells

Mexican morning glory, Ipomoea tricolor Cav., is native to America and widely cultivated as an ornamental plant. High performance liquid chromatography profiling coupled with off-line electrospray mass spectrometry detection was applied to identify novel acylsugar from the CHCl3-soluble resin glycoside from seeds. Dereplication of known tricolorins A-J included the recording of mass values for ions [M + Na]+ and [M – H]−, in addition to comparison of retention times. Recycling HPLC was used for the purification of novel tricolorins K-M. NMR analysis revealed acylation at the C-2 and C-4 positions on the third pyranose unit of the tetrasaccharides by short chain aliphatic acyl groups. Acylsugar diversity results from variations in the esterification of sugar cores. Tricolorin A at a concentration of 25 μg/ml exerted a strong potentiation of vinblastine susceptibility in multidrug-resistant human breast carcinoma cells with a reversal factor of 2164-fold.

Qualitative and quantitative drug residue analyses: Chlortetracycline in white-tailed deer (Odocoileus virginianus) and supermarket meat by liquid chromatography tandem-mass spectrometry

Chlortetracycline is (CTC) is a tetracycline antibiotic which is being in the white-tailed deer industry to improve production and animal health. In this paper, we present a method for determining chlortetracycline residues in edible white-tailed deer tissues, using liquid chromatography with heated electrospray ionization and mass spectrometry detection.The procedure involved extraction with EDTA-McIlvaine buffer at pH 4.0, followed by solid-phase extraction cleanup using a hydrophilic-lipophilic balance (HLB) cartridge. The liquid chromatography analysis was performed with heated electrospray ionization and mass spectrometry detection. The limit of quantification for the method was 2.7 ng/g and limit of detection was 0.8 ng/g. The recovery values were >78.5% for muscle, 65.1% for kidney, 63.1% for liver. Mean tissue residue concentration of chlortetracycline and it's epimer, 4-epi chlortetracycline (4-epi-CTC) at 10-day withdrawal period for kidney, liver, muscle was 122.8, 44.7 and 26.7 ng/g, respectively. Chlortetracycline tissue residue concentration at 45-day withdrawal period for kidney, liver, muscle was 19.2, 28.9 and 10.7 ng/g, respectively. Mean tissue concentration of CTC was less than the established maximum residual limit (MRL) values for bovine tissues. We have validated and successfully applied this method in the qualitative and quantification of chlortetracycline in white-tailed deer tissue samples.

Betalains and phenolic compounds of leaves and stems of Alternanthera brasiliana and Alternanthera tenella

Betacyanins and phenolic compounds from acetonitrile:acidified water extracts of Alternanthera brasiliana and Alternanthera tenella were characterized and quantified using a high-performance liquid chromatography system coupled with diode array and electrospray mass spectrometry detection. Four betacyanins (amaranthine, isoamaranthine, betanin and isobetanin) were tentatively identified and quantified. Twenty eight phenolic compounds of four different families (hydroxybenzoic and hydroxycinnamic acids, flavones and flavonols) were separated and characterized on the basis of their accurate MS and MS/MS information out of which ten compounds were confirmed by authentic standards. These plant species could be considered as an especially rich source of natural bioactive compounds and potential food colorants. A. brasiliana showed the highest betacyanin and polyphenols content (89μg/g and 35,243μg/g, respectively). Among polyphenols, flavonols were the more abundant (kaempferol-glucoside, kaempferol-rutinoside and kaempferol-rhamnosyl-rhamnosyl-glycoside). Meanwhile, A. tenella showed a different polyphenols profile with flavones as major compounds (glucopyranosil-vitexin and vitexin). As a novelty, pentosyl-vitexin and pentosyl-isovitexin were detected for the first time in Alternanthera plants. Both A. brasiliana and A. tenella leaves showed high total polyphenol content and in vitro antioxidant activity (FRAP). These results provide an analytical base concerning the phenolic and betalains composition and the antioxidant properties of two members of the promising Alternanthera gender, for subsequent applications, such as functional food ingredients.

Phytochemical screening by LC-MS and LC-PDA of ethanolic extracts from the fruits of Kigelia africana (Lam.) Benth

Kigelia africana is a tree native to Africa, with a local employment in numerous fields, ranging from traditional medicine to cosmetics and religious rituals. Parts of the plant generally used are stem bark, fruits, roots and leaves. The fruits, which have a singular ‘sausage’ shape, are widely exploited by local folk, in particular for applications/products involving genito-urinary apparatus of both human genders. The scope of this work was to make a consistent chemical investigation on this plant species, in order to clarify and increase the information at present available in literature. To this aim, ethanolic extracts of K. africana fruits were analysed by high-performance liquid chromatography with photodiode array (HPLC-PDA) and electrospray-mass spectrometry (HPLC-ESI-MS) detection, revealing the presence of polyphenols and iridoids. The two detection systems used along with standard co-injection and comparison with previous reports, led to the identification and quantification of six phenolic compounds and three iridoids.

Aqueous Photochemistry of Glyoxylic Acid.

Aerosols affect climate change, the energy balance of the atmosphere, and public health due to their variable chemical composition, size, and shape. While the formation of secondary organic aerosols (SOA) from gas phase precursors is relatively well understood, studying aqueous chemical reactions contributing to the total SOA budget is the current focus of major attention. Field measurements have revealed that mono-, di-, and oxo-carboxylic acids are abundant species present in SOA and atmospheric waters. This work explores the fate of one of these 2-oxocarboxylic acids, glyoxylic acid, which can photogenerate reactive species under solar irradiation. Additionally, the dark thermal aging of photoproducts is studied by UV-visible and fluorescence spectroscopies to reveal that the optical properties are altered by the glyoxal produced. The optical properties display periodicity in the time domain of the UV-visible spectrum of chromophores with absorption enhancement (thermochromism) or loss (photobleaching) during nighttime and daytime cycles, respectively. During irradiation, excited state glyoxylic acid can undergo α-cleavage or participate in hydrogen abstractions. The use of (13)C nuclear magnetic resonance spectroscopy (NMR) analysis shows that glyoxal is an important intermediate produced during direct photolysis. Glyoxal quickly reaches a quasi-steady state as confirmed by UHPLC-MS analysis of its corresponding (E) and (Z) 2,4-dinitrophenylhydrazones. The homolytic cleavage of glyoxylic acid is proposed as a fundamental step for the production of glyoxal. Both carbon oxides, CO2(g) and CO(g) evolving to the gas-phase, are quantified by FTIR spectroscopy. Finally, formic acid, oxalic acid, and tartaric acid photoproducts are identified by ion chromatography (IC) with conductivity and electrospray (ESI) mass spectrometry (MS) detection and (1)H NMR spectroscopy. A reaction mechanism is proposed based on all experimental observations.

Determination of gluconate in nuclear waste by high-performance liquid chromatography: comparison of pulsed amperometric detection and electrospray mass spectrometry detection

The measurement of gluconate is required in the framework of nuclear waste management. Liquid chromatography coupled with amperometric detection proved to be insufficiently selective to quantify gluconate in radioactive effluents due to the complexity of the matrix. An analytical method based on a desalting step and quantification with liquid chromatography coupled to mass spectrometry was therefore developed. As gluconate may be present as several chelates, speciation simulation studies enabled to determine the prevailing chelate form depending on experimental conditions and thus to optimize sample preparation. The analytical method was successfully applied to real nuclear effluents, with a limit of quantification of 2.6 × 10−5 mol L−1.

HPLC‐DAD‐ESI/MS Identification of Light Harvesting and Light Screening Pigments in the Lake Sediments at Edmonson Point

The composition of sedimentary pigments in the Antarctic lake at Edmonson Point has been investigated and compared with the aim to provide a useful analytical method for pigments separation and identification, providing reference data for future assessment of possible changes in environmental conditions. Reversed phase high performance liquid chromatography (HPLC) with electrospray-mass spectrometry (ESI-MS) detection and diode array detection (DAD) has been used to identify light screening and light harvesting pigments. The results are discussed in terms of local environmental conditions.

Determination of ethylenediaminetetraacetic acid in nuclear waste by high-performance liquid chromatography coupled with electrospray mass spectrometry

EDTA is a chelating agent that has been used in decontamination processes. Its quantification is required for nuclear waste management because it affects the mobility of radionuclides and metals in environment and, thus, can harm the safety of the storage. Ion-pair chromatography coupled with electrospray mass spectrometry detection is a convenient method for quantitative analysis of EDTA but EDTA should be present as a single anionic chelate form. However, radioactive liquid wastes contain high concentrations of heavy metals and salts and consequently, EDTA is present as several chelates. Speciation studies were carried out to choose a metal cation to be added in excess to the solution to obtain a major chelate form. Fe is the predominant cation and Fe(III)–EDTA is thermodynamically favored but these speciation studies showed that ferric hydroxide precipitated above pH 2. Consequently, it was not possible to quantify EDTA as Fe(III)–EDTA complex. Therefore, Ni2+ was chosen but its use implied pretreatment with a base of the solution to eliminate Fe. Deuterated EDTA was used as tracer in order to validate the whole procedure, from the treatment with a base to the final analysis by HPLC–ESI-MS. This analytical method was successfully applied for EDTA quantification in two real effluents resulting from a nuclear liquid waste process. A recovery rate between 60 and 80% was obtained. The limit of detection of this method was determined at 34×10−9molL−1.

Multiclass analysis of antibacterial residues in milk using RP-liquid chromatography with photodiode array and fluorescence detection and tandem mass spectrometer confirmation

A simplified procedure for simultaneous quantification of ceftiofur (CEF), fluoroquinolone (FQ) and sulfonamide (SA) antibacterials in bovine milk was developed. The reverse-phase liquid chromatography (RP-LC) multiclass method for analysis of eleven distinct compounds, from three antibacterial classes, was validated in line with Commission Decision 2002/657/EC. Confirmation of the analytes identities was performed by electrospray mass spectrometry detection. The analytes were extracted from milk matrix by liquid–liquid extraction with acidified ultrapure water and directly analyzed in the chromatograph. The SA compounds were pre-column derivatized with fluorescamine for fluorescence detection. The method provided good results regarding the analytical parameters of linearity, selectivity, sensitivity, precision, recovery, decision limit (CCα), detection capability (CCβ), limit of detection (LOD), limit of quantification (LOQ), stability and robustness. Analytes were extracted by liquid–liquid extraction in the fortified matrix and the compounds identity was confirmed by their precursor ion and fragments through tandem mass spectrometry analysis. Additionally, milk samples from two state capitals in the South Region of Brazil were analyzed by both the quantitative and confirmatory methods. The validation process showed correlation coefficients (r2) greater than 0.98 for all the analytes, with recovery rates up to 98% for all the studied drugs. LOD and LOQ limits ranged from 8.0 to 20.0ngmL−1 and 10.0 to 32.0ngmL−1, demonstrating good specificity of the method. The intra-day and inter-day precisions for all the analytes were below or equal to 7.40 and 10.13, respectively. The studied antibacterials were not detected in milk samples. The developed method represents an efficient alternative for multi-residue analysis in milk, being suitable and especially viable for monitoring in developing countries.

An efficient hydrophilic interaction liquid chromatography separation of 7 phospholipid classes based on a diol column

A hydrophilic interaction liquid chromatography (HILIC) - ion trap mass spectrometry method was developed for separation of a wide range of phospholipids. A diol column which is often used with normal phase chromatography was adapted to separate different phospholipid classes in HILIC mode using a mobile phase system consisting of acetonitrile, water, ammonium formate and formic acid. An efficient between-class separation of seven phospholipid classes including phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinostol, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine was successfully achieved within 14 min using a gradient elution which starts with 90% of organic solvent and ends with 70% of organic solvent. 53 mM formic acid (in both organic phase and aqueous phase) and 60mM ammonium formate (only in aqueous phase) were used as mobile phase modifier. The relatively high amount of ammonium formate was essential to obtain well-shaped peaks of each phospholipid class, especially phosphatidylserines; actually, no negative effect due to ammonium formate was observed for electrospray-mass spectrometry detection in real-life samples. Good chromatographic separation between different lipid classes was obtained (Rs, from 0.73 to 4.97) and well-shaped peaks (tailing factor, from 0.98 to 1.20) were obtained. The developed method was fully validated and the satisfactory performance characteristics such as linearity (R(2), 0.990-0.999), retention time stability (RSD<1%), within day repeatability (RSD, 5-13%), between day variation (RSD, 7-14%) and recoveries (99.6-115.5%) indicated the gradient HILIC method was appropriate for profiling of plasma phospholipids. The method was successfully applied to separate phospholipids extracts from human plasma, mouse plasma and rat plasma.

Probing Protein Surface with a Solvent Mimetic Carbene Coupled to Detection by Mass Spectrometry

Much knowledge into protein folding, ligand binding, and complex formation can be derived from the examination of the nature and size of the accessible surface area (SASA) of the polypeptide chain, a key parameter in protein science not directly measurable in an experimental fashion. To this end, an ideal chemical approach should aim at exerting solvent mimicry and achieving minimal selectivity to probe the protein surface regardless of its chemical nature. The choice of the photoreagent diazirine to fulfill these goals arises from its size comparable to water and from being a convenient source of the extremely reactive methylene carbene (:CH(2)). The ensuing methylation depends primarily on the solvent accessibility of the polypeptide chain, turning it into a valuable signal to address experimentally the measurement of SASA in proteins. The superb sensitivity and high resolution of modern mass spectrometry techniques allows us to derive a quantitative signal proportional to the extent of modification (EM) of the sample. Thus, diazirine labeling coupled to electrospray mass spectrometry (ESI-MS) detection can shed light on conformational features of the native as well as non-native states, not easily addressable by other methods. Enzymatic fragmentation of the polypeptide chain at the level of small peptides allows us to locate the covalent tag along the amino acid sequence, therefore enabling the construction of a map of solvent accessibility. Moreover, by subsequent MS/MS analysis of peptides, we demonstrate here the feasibility of attaining amino acid resolution in defining the target sites.

Hydrogen/deuterium exchange mass spectrometry and optical spectroscopy as complementary tools for studying the structure and dynamics of a membrane protein

The hydrophobic nature of membrane proteins poses considerable challenges for the application of traditional analytical techniques. Hydrogen/deuterium exchange (HDX) with electrospray mass spectrometry (ESI-MS) detection is a potentially very powerful approach for investigating the structure and dynamics of membrane proteins, but thus far this technique has been applied mostly to soluble species. By using ESI-MS in conjunction with low temperature size exclusion chromatography (SEC) the current study explores the global HDX behavior of the integral membrane protein bacteriorhodopsin (BR) under various conditions. The experiments are complemented by UV–vis absorption measurements that report on the environment of the protein's retinal chromophore and fluorescence spectroscopy which probes the extent of resonance energy transfer between Trp residues and retinal. In agreement with previous reports, our HDX data imply that amide hydrogens in the transmembrane helices of native BR are highly protected, whereas most sites in solvent-exposed loops are readily exchangeable. Low temperature SEC/ESI-MS allows the labile Schiff-base linkage between protein and retinal to be completely preserved, such that the HDX properties of co-existing protein populations can be monitored individually. Solubilization of BR in sodium dodecyl sulfate (SDS) induces major structural changes and hydrolytic loss of retinal. The SDS-denatured protein appears to retain a significant degree of stable helical structure (around 25%, compared to the native state value of 74%). Bicelle-mediated refolding of SDS-denatured bacterioopsin in the presence of free retinal regenerates a native-like BR conformation with a yield close to 0.9. The HDX/SEC/ESI-MS approach developed in this work should be applicable to other membrane protein systems as well.

Stability of Th(IV) polymers measured by electrospray mass spectrometry and laser-induced breakdown detection

The formation of polynuclear thorium hydroxide complexes in acidic aqueous solutions is observed by electrospray mass spectrometry. Size, number of hydroxide ligands and charge of all complexes in solution are identified independently. By repeated measurements over a time span of up to 418 days the stability of the polymers is observed. At concentrations above [Th(IV)]tot = 10 mM pentamers and dimers are the dominant stable species. In contrast, the fractions of pentamers initially present at [Th(IV)]tot = 0.2 mM decrease within less than 4 weeks, correlated with the formation of nm-sized colloids observed by LIBD indicating an Ostwald-ripening like process.

Interaction of thimerosal with proteins—ethylmercuryadduct formation of human serum albumin and β-lactoglobulin A

The interaction of thimerosal, an ethylmercury-containing bactericide and fungicide used as preservative in vaccines and other drugs, with free thiols in proteins has been investigated using gradient reversed phase liquid chromatography (LC) with inductively coupled plasma mass spectrometry (ICP-MS) and electrospray mass spectrometry (ESI-MS) detection. As model proteins, β-lactoglobulin A (18.4 kDa) from bovine milk and human serum albumin (66.5 kDa) have been used. Physiological conditions upon an intravenous injection of thimerosal-containing drugs were mimicked. The formation of ethylmercury–protein adducts was proved and the identification of the binding site of ethylmercury, a free thiol residue in the peptide T13 was achieved after tryptic digestion of β-lactoglobulin A.