Migration time correction for dual pressure capillary electrophoresis in semi-targeted metabolomics study.

Migration time fluctuation strongly affects peak alignment and identification of unknown compounds, making migration time correction an essential step in capillary electrophoresis (CE)-based metabolomics. To obtain more reliable information, metabolites with different apparent mobilities are analyzed by tandem mass spectrometry. Applying a small pressure is a common practice for reducing the analysis time of anions in a positive mode CE, known as the pressure-assisted CE. However, applying pressure may reduce the separation efficiency and can be undesirable for cation analysis. A simple way to address this issue is to increase the pressure after a certain time, during the separation. We term this practice as dual pressure CE. However, changing the pressure during the CE separation complicates migration time correction. Previous migration time correction methods were established based on a consistent electroosmotic flow and a constant pressure-driven bulk-flow velocity. We proposed a new correction method to support the peak alignment when dual pressure CE is used. A Python-based script was developed to implement dual pressure CE migration time correction for semi-targeted metabolomics study performed by a multiple reaction monitoring-based method. This script can help select suitable endogenous metabolites as correction markers, perform migration time correction, and conduct peak alignment. A case study showed that migration time precision of 156 metabolites in 32 samples can be improved from 4.8 to 11.4%RSD (relative standard deviation) to less than 1.8%RSD.

Quantitative characterization of human oncogene promoter G-quadruplex DNA-ligand interactions using a combination of mass spectrometry and capillary electrophoresis.

Human c-KIT oncogene is known to regulate cell growth and proliferation, and thus, acts as a probable target in the treatment of gastrointestinal tumors (GIST). To identify small molecule ligands which can specifically bind with the G-quadruplex (G4) in the c-KIT promoter region as potential antitumor agents, we propose the combination of electrospray ionization-mass spectrometry (ESI-MS), capillary electrophoresis frontal analysis (CE-FA), and Taylor dispersion analysis (TDA) to accurately investigate the G4/ligands binding properties. First, ESI-MS was used for initial screening of natural products (NPs). CE-FA was then used to calculate specific binding constants and the stoichiometry of the native state binding pair in solution. Next, TDA, a micro-capillary flow technique was used to examine the effect of the ligand binding on the diffusivity and particle size of the c-KIT G4. Two of the screened NPs, scopolamine butylbromide (L1) and isorhamnetin-3-O-neohesperidoside (L3), were found to specifically bind to the c-KIT G4 with binding constants of around 104 M-1 and 1:1 stoichiometry in a free solution. TDA data showed that ligand binding (both L1 and L3) induced the c-KIT strands to fold into a tightly structured G4 with a decreased hydrodynamic radius. These ligands have the potential to be drug candidates for the regulation of c-KIT gene transcription by stabilizing the G4 structure. This methodology not only increased the speed of analysis but also improved its accuracy and specificity compared with the conventional binding approaches.

Rapid fingerprint analysis for herbal polysaccharides using direct analysis in real-time ionization mass spectrometry.

RATIONALE: Herbal polysaccharides have various potential medicinal values. Development of reliable analytical method for the fingerprint analysis of polysaccharides is critical for their quality assessment, origin identification, and authenticity evaluation. METHODS: Mechanochemical extraction (MCE) was used to extract polysaccharide components from different herbal species. Intact polysaccharides were then directly analyzed by direct analysis in real-time mass spectrometry (DART-MS). Standard addition method with isotope-labeled internal standard was used to quantify polysaccharide amounts directly from liquid extract. Multivariate data analysis was further conducted for species classification. RESULTS: The intact and large polysaccharides were decomposed into small fragment ions less than m/z 350 instantaneously using DART ion source. Different polysaccharides showed distinguished fingerprint DART-MS spectra using both individual and mixed herbal species. The liquid supernatant from MCE was validated to be used as direct sample for DART-MS analysis. Quantitation was successfully achieved for polysaccharide contents in Dendrobium officinale from different locations. CONCLUSIONS: A rapid fingerprint protocol in combination of MCE and DART-MS for herbal polysaccharides was developed. The whole process could be accomplished within a few minutes, from raw materials to final spectra, without requirements of pre-digestion and additional sample purification.

Complementary Methods for de Novo Monoclonal Antibody Sequencing to Achieve Complete Sequence Coverage.

Mass spectrometry is a powerful tool for de novo sequencing of novel proteins. Recent efforts in this area have mainly focused on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Here, we present an alternative method, capillary electrophoresis tandem mass spectrometry (CE-MS/MS), for sequencing novel monoclonal antibodies. Using less than 200 ng in total of tryptic digest sample in a triplicated measurement, CE-MS/MS with pH-mediated focusing successfully sequenced mAb infliximab with 100% sequence coverage and 100% accuracy for the light chain and 96% coverage and 93% accuracy for the heavy chain. It was also demonstrated that CE-MS/MS gives comparable results, and in some cases, even better results, as compared to LC-MS/MS when used as a standalone technique. A combined workflow using both CE-MS/MS and LC-MS/MS was also used to sequence a novel antibody, anti-CD-176, resulting in the first proposed sequence for this mAb.

Bottom-up proteomics of envelope proteins extracted from spinach chloroplast via high organic content CE-MS.

A high organic content CE-MS/MS (HOCE-MS/MS) method was developed for the proteomic analysis of envelope proteins extracted from spinach leaves. Separation was performed in a 1-m long hydroxypropyl cellulose coated capillary, using 8% (v/v) formic acid in 70% (v/v) methanol and 22% water as the BGE. A flow-through microvial interface was used to couple the CE system with an Orbitrap Fusion Lumos mass spectrometer, and field-amplified sample stacking was used to improve the concentration sensitivity. Using this optimized method, 3579 peptides and 1141 proteins were identified using the Proteome Discoverer software with a 1% false discovery rate at the protein level. Relative to conventional aqueous CE, HOCE-MS did a better job of discovering hydrophobic peptides and provided more peptide and protein identifications. Relative to nano-LC-MS, it achieved comparable peptide and protein identification performance and detected peptides not identified by LC-MS: of the full set of peptides identified using the two techniques, 19% were identified only using HOCE-MS. It also outperformed nano-LC-MS with respect to the detection of low molecular weight peptides.

Dynamic pH barrage junction focusing of amino acids, peptides, and digested monoclonal antibodies in capillary electrophoresis-mass spectrometry.

Dynamic pH barrage junction focusing in CE enables effective signal enhancement, quantitative capture efficiencies, and straightforward optimization. The method is a technical variant of dynamic pH junction focusing. CE separation with dynamic pH barrage junction focusing is compatible with both optical and mass spectrometric detection. We developed a CE-MS/MS method using hydrophilic polyethyleneimine-coated capillaries and validated it for the qualitative analysis of amino acids, peptides, and tryptic peptides of digested monoclonal antibodies. The S/N of extracted ion electropherograms of zwitterionic analytes were enhanced by approximately two orders of magnitude with a tradeoff of a shortened separation window. Online focusing improved the MS signal intensity of a diluted antibody digest, enabling more precursor ions to be analyzed with subsequent tandem mass spectrometric identification. It also broadened the concentration range of protein digest samples for which adequate sequence coverage data can be obtained. With only 0.9 ng of digested infliximab sample loaded into the capillary, 76% and 100% sequence coverage was realized for antibody heavy and light chains, respectively, after online focusing. Full coverage was achieved with 9 ng of injected digest.

Application of multisegment injection on quantification of creatinine and standard addition analysis of urinary 5-hydroxyindoleacetic acid simultaneously with creatinine normalization.

In this paper, the development of a simple dilute-and-shoot method for quantifying urinary creatinine by CE-ESI-MS was described. The creatinine analysis time was about 7 min/sample by conventional single injection (SI) method and can be significantly reduced to less than 2 min/sample with multi-segment injection (MSI). In addition, the standard addition analysis of 5-hydroxyindole-3-acetic acid (5-HIAA) and creatinine normalization was performed within one run by the MSI technique, and the total analysis time was 14-min faster compared to the SI method for analyzing the same set of samples. The uses of isotopic and non-isotopic internal standards (ISs) were compared. Creatinine-(methyl-13 C) and 5-hydroxyindole-4,6,7-D3 -3-acetic-D2 acid (5-HIAA-D5 ) used as isotopic ISs can provide both accurate and precise results. In contrast, 1,5,5-trimethylhydantoin (1,5,5-TH) used as the non-isotopic IS for creatinine may cause a bias of over 13% in SI method and even worse when the MSI technique was used. Another compound, 2-methyl-3-indoleacetic acid (2-MIAA), was determined not suitable for MSI analysis of 5-HIAA due to endogenous interferences despite its acceptable performance in conventional methods of analysis.

RawTools: Rapid and Dynamic Interrogation of Orbitrap Data Files for Mass Spectrometer System Management.

Optimizing the quality of proteomics data collected from a mass spectrometer (MS) requires careful selection of acquisition parameters and proper assessment of instrument performance. Software tools capable of extracting a broad set of information from raw files, including meta, scan, quantification, and identification data, are needed to provide guidance for MS system management. In this work, direct extraction and utilization of these data is demonstrated using RawTools, a standalone tool for extracting meta and scan data directly from raw MS files generated on Thermo Orbitrap instruments. RawTools generates summarized and detailed plain text outputs after parsing individual raw files, including scan rates and durations, duty cycle characteristics, precursor and reporter ion quantification, and chromatography performance. RawTools also contains a diagnostic module that includes an optional "preview" database search for facilitating informed decision-making related to optimization of MS performance based on a variety of metrics. RawTools has been developed in C# and utilizes the Thermo RawFileReader library and thus can process raw MS files with high speed and high efficiency on all major operating systems (Windows, MacOS, Linux). To demonstrate the utility of RawTools, the extraction of meta and scan data from both individual and large collections of raw MS files was carried out to identify problematic characteristics of instrument performance. Taken together, the combined rich feature-set of RawTools with the capability for interrogation of MS and experiment performance makes this software a valuable tool for proteomics researchers.

Differential Hydrogen/Deuterium Exchange during Proteoform Separation Enables Characterization of Conformational Differences between Coexisting Protein States.

Characterization of structural differences between coexisting conformational states of protein is difficult with conventional biophysical techniques. Hydrogen/deuterium exchange (HDX) coupled with top-down mass spectrometry (MS) allows different conformers to be deuterated to different extents and distinguished through gas-phase separation based on molecular weight distributions prior to determination of deuteration levels at local sites for each isolated conformer. However, application of this strategy to complex systems is hampered by the interference from conformers with only minor differences in overall deuteration levels. In this work, we performed differential HDX while the different conformers were separated according to their differing charge to size ratios in capillary electrophoresis. Mixtures of holo- and apo-myoglobin (Mb) and disulfide isomers of lysozyme (Lyz) were characterized in a conformer-specific fashion using this strategy, followed by conformation interrogation for the sequentially eluted 2H-labeled species in real-time using top-down MS. Under mildly denaturing conditions that minimize the charge difference, disulfide isomers of Lyz were differentially labeled with 2H during separation based on their disulfide-dependent sizes. The resulting differences in deuteration pattern between these isomers are in line with their difference in covalent structural constraints set by the disulfide patterns. Under physiologically relevant conditions, we identified the segments undergoing conformational changes of Mb in the absence of the heme group by comparing the deuteration patterns of holo- and apo-Mb.

Quantitative analysis of microcystin variants by capillary electrophoresis mass spectrometry with dynamic pH barrage junction focusing.

Dynamic pH junction is an online focusing method in CE based on the electrophoretic mobility difference of analytes in the sample matrix and the background electrolyte. An advantage of this method over the conventional CE is that the sensitivity can be significantly improved. By injecting a long sample plug in the capillary and focusing the analytes at the pH boundary between the background electrolyte and sample matrix, the LOD can be improved by 10-100 folds. The dynamic pH junction method can be easily coupled with ESI-MS. In this work, we used this method for the analysis of microcystins (MCs). The detection limits and dynamic ranges were studied. The separation was optimized by adjusting the injection time, and concentrations and pH values of the background electrolyte. The optimization of analyte focusing leads to enhanced detection response compared to conventional injections, achieving 200-400 fold higher averaged peak heights for four microcystin (MC) variants. More importantly, this method was successfully used for the quantitative analysis of microcystins (MCs) in crude algae samples from natural water bodies, making it promising for practical applications.

Parsing and Quantification of Raw Orbitrap Mass Spectrometer Data Using RawQuant.

Effective analysis of protein samples by mass spectrometry (MS) requires careful selection and optimization of a range of experimental parameters. As the output from the primary detection device, the "raw" MS data file can be used to gauge the success of a given sample analysis. However, the closed-source nature of the standard raw MS file can complicate effective parsing of the data contained within. To ease and increase the range of analyses possible, the RawQuant tool was developed to enable parsing of raw MS files derived from Thermo Orbitrap instruments to yield meta and scan data in an openly readable text format. RawQuant can be commanded to export user-friendly files containing MS1, MS2, and MS3 metadata as well as matrices of quantification values based on isobaric tagging approaches. In this study, the utility of RawQuant is demonstrated in several scenarios: (1) reanalysis of shotgun proteomics data for the identification of the human proteome, (2) reanalysis of experiments utilizing isobaric tagging for whole-proteome quantification, and (3) analysis of a novel bacterial proteome and synthetic peptide mixture for assessing quantification accuracy when using isobaric tags. Together, these analyses successfully demonstrate RawQuant for the efficient parsing and quantification of data from raw Thermo Orbitrap MS files acquired in a range of common proteomics experiments. In addition, the individual analyses using RawQuant highlights parametric considerations in the different experimental sets and suggests targetable areas to improve depth of coverage in identification-focused studies and quantification accuracy when using isobaric tags.

Nonaqueous capillary electrophoresis mass spectrometry method for determining highly hydrophobic peptides.

A nonaqueous capillary electrophoresis mass spectrometry (NACE-MS) method was developed to separate and determine highly hydrophobic temporin peptides. The nonaqueous background electrolyte solution was a mixture of 20% acetonitrile, 78% methanol and 2% formic acid, with 20 mM ammonium formate. The separation of six peptides was completed within 12 min. The CE system was connected to a triple quadrupole mass spectrometer operating in MRM mode using a chemical modifier solution of 2 mM ammonium formate in ethanol with the flow through microvial interface. The mass spectrometer offered a second dimension of separation for peptides having identical migration times but different structures. The new method represents the first system capable of reliably determining hydrophobic peptides without using reversed phase liquid chromatography mass spectrometry.

Pressure-assisted capillary electrophoresis frontal analysis for faster binding constant determination.

Adding external pressure during the process of capillary electrophoresis usually add to the band broadening, especially if the pressure induced flow is significant. The resolution is normally negatively affected in pressure-assisted capillary electrophoresis (PACE). Frontal analysis (FA), however, can potentially benefit from using an external pressure while avoiding the drawbacks in other modes of CE. In this work, possible impact from the external pressure was simulated by COMSOL Multiphysics®. Under a typical CE-FA set-up, it was found that the detected concentrations of analyte will not be significantly affected by an external pressure less than 5 psi. Besides, the measured ligand concentration in PACE-FA was also not affected by common variables (molecular diffusion coefficient (10-8 to 10-11 m2 /s), capillary length etc). To provide an experimental proof, PACE-FA is used to study the binding interactions between hydroxypropyl ß-cyclodextrin (HP-ß-CD) and small ligand molecules. Taking the HP-ß-CD /benzoate pair as an example, the binding constants determined by CE-FA (18.3 ± 0.8 M-1 ) and PACE-FA (16.5 ± 0.5 M-1 ) are found to be similar. Based on the experimental results, it is concluded that PACE-FA can reduce the time of binding analysis while maintaining the accuracy of the measurements.

Characterization of Athabasca lean oil sands and mixed surficial materials: Comparison of capillary electrophoresis/low-resolution mass spectrometry and high-resolution mass spectrometry.

RATIONALE: Oil sands mining in Alberta, Canada, requires removal and stockpiling of considerable volumes of near-surface overburden material. This overburden includes lean oil sands (LOS) which cannot be processed economically but contain sparingly soluble petroleum hydrocarbons and naphthenic acids, which can leach into environmental waters. In order to measure and track the leaching of dissolved constituents and distinguish industrially derived organics from naturally occurring organics in local waters, practical methods were developed for characterizing multiple sources of contaminated water leakage. METHODS: Capillary electrophoresis/positive-ion electrospray ionization low-resolution time-of-flight mass spectrometry (CE/LRMS), high-resolution negative-ion electrospray ionization Orbitrap mass spectrometry (HRMS) and conventional gas chromatography/flame ionization detection (GC/FID) were used to characterize porewater samples collected from within Athabasca LOS and mixed surficial materials. GC/FID was used to measure total petroleum hydrocarbon and HRMS was used to measure total naphthenic acid fraction components (NAFCs). HRMS and CE/LRMS were used to characterize samples according to source. RESULTS: The amounts of total petroleum hydrocarbon in each sample as measured by GC/FID ranged from 0.1 to 15.1 mg/L while the amounts of NAFCs as measured by HRMS ranged from 5.3 to 82.3 mg/L. Factors analysis (FA) on HRMS data visually demonstrated clustering according to sample source and was correlated to molecular formula. LRMS coupled to capillary electrophoresis separation (CE/LRMS) provides important information on NAFC isomers by adding analyte migration time data to m/z and peak intensity. CONCLUSIONS: Differences in measured amounts of total petroleum hydrocarbons by GC/FID and NAFCs by HRMS indicate that the two methods provide complementary information about the nature of dissolved organic species in a soil or water leachate samples. NAFC molecule class Ox Sy is a possible tracer for LOS seepage. CE/LRMS provides complementary information and is a feasible and practical option for source evaluation of NAFCs in water.

Deducing disulfide patterns of cysteine-rich proteins using signature fragments produced by top-down mass spectrometry.

Direct mapping of protein disulfide patterns using top-down mass spectrometry (MS) is often hampered by inadequate fragmentation at the disulfide-enclosing region, and insufficient structural information provided by the fragments. Here we used electron-transfer/high energy collision dissociation (EThcD) to improve the fragmentation efficiency, and developed strategies that minimize the false positive identification of fragments and deconvolute the signals representing specific modifications made to the disulfide-cleavage-induced fragments. We observed clear correlations between unique modification (attachment or removal of H or SH) patterns and the number of disulfide bonds that enclose the corresponding region. Using the characteristic signature fragments, we in part localized the Cys-bridging sites in disulfide-scrambled lysozymes, and reduced the number of putative disulfide patterns from 104 to 6. The results demonstrated the feasibility of direct analysis of complex disulfide patterns using top-down MS.

Electroosmotic Flow Dispersion of Large Molecules in Electrokinetic Migration.

It is a common knowledge that large molecules have small diffusion coefficients. Therefore, it was thought that these molecules should generate very narrow peaks in electropherograms, because longitudinal diffusion was thought to be the only factor that contributes to band broadening in electrophoretic separation systems in capillaries and microfluidics. However, it is also common to observe that large molecules such as proteins and polymers often produce wider peaks than most small molecules. It is demonstrated in this work that dispersion of analytes during electrokinetic migration is also the results of Taylor dispersion. Similar to conventional Taylor dispersion, peak broadening is more pronounced for particles with smaller diffusion coefficients. The theoretical description of band broadening caused by electroosmotic flow dispersion (EOFD) and the experimental verification of this phenomenon are presented in this paper. The dispersion of various sized molecules, from small peptides to large proteins, in capillary electrophoresis is used as examples in this study.

Quantitative Nonaqueous Capillary Electrophoresis-Mass Spectrometry Method for Determining Active Ingredients in Plant Extracts.

Nonaqueous capillary electrophoresis (NACE) is very well suited for online coupling with mass spectrometry due to the relatively high volatility and low surface tension of most organic solvents. Here we present a quantitative NACE-ESI-MS/MS method for separating and determining physcion, chrysophanol, and aloe-emodin in rhubarb. Dantron was used as an internal standard to ensure accuracy and reproducibility in quantitative analyses. Parameters including the pH, background electrolyte (BGE) composition, flow-through microvial chemical modifier solution composition, and modifier solution flow rate were carefully optimized. The developed method was validated by assessing its precision, LODs, and linear range. The contents of physcion, chrysophanol, and aloe-emodin in rhubarb were determined to be 0.22%, 1.0%, and 0.17%, respectively.

Mobility-based correction for accurate determination of binding constants by capillary electrophoresis-frontal analysis.

Capillary electrophoresis frontal analysis (CE-FA) can be used to determine binding affinity of molecular interactions. However, its current data processing method mandate specific requirement on the mobilities of the binding pair in order to obtain accurate binding constants. This work shows that significant errors are resulted when the mobilities of the interacting species do not meet these requirements. Therefore, the applicability of CE-FA in many real word applications becomes questionable. An electrophoretic mobility-based correction method is developed in this work based on the flux of each species. A simulation program and a pair of model compounds are used to verify the new equations and evaluate the effectiveness of this method. Ibuprofen and hydroxypropyl-ß-cyclodextrinare used to demonstrate the differences in the obtained binding constant by CE-FA when different calculation methods are used, and the results are compared with those obtained by affinity capillary electrophoresis (ACE). The results suggest that CE-FA, with the mobility-based correction method, can be a generally applicable method for a much wider range of applications.

Characterization of dicarboxylic naphthenic acid fraction compounds utilizing amide derivatization: Proof of concept.

RATIONALE: The characterization of naphthenic acid fraction compounds (NAFCs) in oil sands process affected water (OSPW) is of interest for both toxicology studies and regulatory reasons. Previous studies utilizing authentic standards have identified dicarboxylic naphthenic acids using two-dimensional gas chromatography hyphenated to time-of-flight mass spectrometry (GC × GC/TOFMS). The selective derivatization of hydroxyl groups has also recently aided in the characterization of oxy-NAFCs, and indirectly the characterization of dicarboxylic NAFCs. However, there has been no previous report of derivatization being used to directly aid in the standard-free characterization of NAFCs with multiple carboxylic acid functional groups. Herein we present proof-of-concept for the characterization of dicarboxylic NAFCs utilizing amide derivatization. METHODS: Carboxylic acid groups in OSPW extract and in a dicarboxylic acidstandard were derivatized to amides using a previously described method. The derivatized extract and derivatized standard were analyzed by direct-injection positive-mode electrospray ionization ((+)ESI) high-resolution mass spectrometry (HRMS), and the underivatized extract was analyzed by (-)ESI MS. Tandem mass spectrometry (MS/MS) was carried out on selected ions of the derivatized standard and derivatized OSPW. Data analysis was carried out using the Python programming language. RESULTS: The distribution of monocarboxylic NAFCs observed in the amide-derivatized OSPW sample by (+)ESI-MS was generally similar to that seen in underivatized OSPW by (-)ESI-MS. The dicarboxylic acid standard shows evidence of being doubly derivatized, although the second derivatization appears to be inefficient. Furthermore, a spectrum of potential diacid NAFCs is presented, identified by both charge state and derivatization mass. Interference due to the presence of multiple derivatization products is noted, but can be eliminated using on-line separation or an isotopically labelled derivatization reagent. CONCLUSIONS: Proof of concept for the characterization of dicarboxylic NAFCs utilizing amide derivatization is demonstrated. Furthermore, (+)ESI-HRMS of the derivatized monocarboxylic NAFCS yields similar information to (-)ESI-MS analysis of underivatized NAFCs, with the benefit of added selectivity for carboxylic acid species and the characterization of diacids.

A preliminary study on the effects of lanthanum (III) on plant vitronectin-like protein and its toxicological basis.

Vitronectin-like protein (VN) is widely found outside plant plasma membranes. The VN molecular surface contains a large number of active groups that combine strongly with rare earth elements (REEs), which means that VN is a preferential binding target for REEs exhibiting their toxic effects, but the toxicological mechanism remains unknown. This study used transmission electron microscopy, circular dichroism, fluorescence spectrometry, ultraviolet-visible spectroscopy, X-ray photoelectron spectroscopy, and calculational chemistry (homology modeling, molecular dynamics simulation and quantum chemical calculation) to preliminarily investigate the effect of lanthanum [La(III)] as an REE, on the structure of VN and its toxicological mechanism. The results showed that low-concentration La(III) could cause micro-interference to the VN molecular structure through weak interactions, such as electrostatic attraction. High-concentration La(III) formed stable complexes with VN, which changed the average binding energy and electron cloud density of VN, loosened the molecular structure and increased the disorder of VN molecule. The results of building a 3D model of VN and simulating the interaction between La(III) and VN using calculational chemistry showed that La(H2O)73+ in solution could coordinately bind to the carboxyl-/carbonyl-O groups in the negatively charged areas on the VN molecular surface. Furthermore, one or more strong H-bonds were formed to enhance the stability of the La(H2O)73+-VN complexes. In summary, low La(III) concentrations could cause micro-interference to the VN molecular structure, whereas high La(III) concentrations could coordinately bind to VN to form stable La-VN complexes, which destroyed the molecular structure of VN; thus the toxicological basis by which La(III) exhibits its toxic effects is its binding to VN.