Analysis of Colubroidea snake venoms by liquid chromatography with mass spectrometry: evolutionary and toxinological implications

The evolution of the venomous function of snakes and the diversification of the toxins has been of tremendous research interest and considerable debate. It has become recently evident that the evolution of the toxins in the advanced snakes (Colubroidea) predated the evolution of the advanced, front-fanged delivery mechanisms. Historically, the venoms of snakes lacking front-fanged venom-delivery systems (conventionally grouped into the paraphyletic family Colubridae) have been largely neglected. In this study we used liquid chromatography with mass spectrometry (LC/MS) to analyze a large number of venoms from a wide array of species representing the major advanced snake clades Atractaspididae, Colubrinae, Elapidae, Homalopsinae, Natricinae, Psammophiinae, Pseudoxyrhophiinae, Xenodontinae, and Viperidae. We also present the first sequences of toxins from Azemiops feae as well as additional toxin sequences from the Colubrinae. The large body of data on molecular masses and retention times thus assembled demonstrates a hitherto unsuspected diversity of toxins in all lineages, having implications ranging from clinical management of envenomings to venom evolution to the use of isolated toxins as leads for drug design and development. Although definitive assignment of a toxin to a protein family can only be done through demonstrated structural studies such as N-terminal sequencing, the molecular mass data complemented by LC retention information, presented here, do permit formulation of reasonable hypotheses concerning snake venom evolution and potential clinical effects to a degree not possible till now, and some hypotheses of this kind are proposed here. The data will also be useful in biodiscovery.     

Electrospray ionization mass spectrometry fingerprinting of propolis

Crude ethanolic extracts of propolis, a natural resin, have been directly analysed using electrospray ionization mass (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) in the negative ion mode. European, North American and African samples have been analyzed, but emphasis has been given to Brazilian propolis which displays diverse and region-dependent chemical composition. ESI-MS provides characteristic fingerprint mass spectra, with propolis samples being divided into well-defined groups directly related to their geographical origins. Chemometric multivariate analysis statistically demonstrates the reliability of the ESI-MS fingerprinting method for propolis. On-line ESI-MS/MS tandem mass spectrometry of characteristic [M - H](-) ion markers provides an additional dimension of fingerprinting selectivity, while structurally characterizing the ESI-MS marker components of propolis. By comparison with standards, eight such markers have been identified: para-coumaric acid, 3-methoxy-4-hydroxycinnamaldehyde, 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran, 3-prenyl-4-hydroxycinnamic acid, chrysin, pinocembrin, 3,5-diprenyl-4-hydroxycinnamic acid and dicaffeoylquinic acid. The negative mode ESI-MS fingerprinting method is capable of discerning distinct composition patterns to typify, to screen the sample origin and to reveal characteristic details of the more polar and acidic chemical components of propolis samples from different regions of the world.     

Electrospray ionization mass spectrometry fingerprinting of beer

After just simple degassing, dilution, pH adjustment and direct flow injection, characteristic fingerprint spectra of beer samples have been obtained by fast (few seconds) electrospray ionization mass spectrometry (ESI-MS) analysis in both the negative and positive ion modes. A total of 29 samples belonging to the two main beer types (lagers and ales) and several beer subtypes from USA, Europe and Brazil could be clearly divided into three groups both by simple visual inspection of their ESI(+)-MS and ESI(-)-MS fingerprints as well as by chemometric treatment of the MS data. Diagnostic ions with contrasting relative abundances in both the positive and negative ion modes allow classification of beers into three major types: P = pale (light) colored (pilsener, pale ale), D = dark colored (bock, stout, porter, mild ale) and M = malt beer. For M beers, samples of a dark and artificially sweetened caramel beer produced in Brazil and known as Malzbiers were used. ESI-MS/MS on these diagnostic beer cations and anions, most of which are characterized as arising from ionization of simple sugars, oligosaccharides, and iso-alpha-acids, yield characteristic tandem mass spectra adding a second and optional MS dimension for improved selectivity for beer characterization by fingerprinting. Direct ESI-MS or ESI-MS/MS analysis can therefore provide fast and reliable fingerprinting characterization of beers, distinguishing between types with different chemical compositions. Other unusual polar components, impurities or additives, as well as fermentation defects or degradation products, could eventually be detected, making the technique promising for beer quality control.     

Poly(amic acid) and polyimide characterization using gas chromatography/mass spectrometry and particle beam liquid chromatography/mass spectrometry

A number of polymers were hydrolyzed in NH₄OH and studied using gas chromatography/ mass spectrometry (GC/MS) and particle beam liquid chromatography/mass spectrometry (particle beam LC/MS) techniques. The polymers studied in this report were as follows: BPDA-PDA, BPDA-PDA-ODA, BPDA-PDA-GFDA, PMDA-ODA, and BTDA-APB. Some of the polymer samples were hydrolyzed in both their acid and imide forms to see if any mass spectrometric differences could be detected. ln all cases, the acid and imide spectra looked the same. GC/MS was unable to determine either the amine or acid portion of these polymers via a direct injection of the sample, but when the samples were first extracted with diethyl ether and this ether extract was injected into the chromatograph, the amine portion of the polymers was readily detected. The acid portion was, again, not detected in either the sample or the ether extract. The particle beam was able to detect both the amine and acid monomeric units in the nonextracted sample.     

Electrospray photoionization (ESPI) liquid chromatography/mass spectrometry for the simultaneous analysis of cyclodextrin and pharmaceuticals and their binding interactions

We report on the use of a multimode electrospray ionization/atmospheric pressure photoionization source (ESI/APPI or ESPI for short) with liquid chromatography/mass spectrometry (LC/MS) to measure all components of a mixed-polarity liquid sample containing: (1) low-polarity component (hormone, pharmaceutical or sterol), (2) polar component (cyclodextrin substrate), and (3) bound polar complex. The ESPI source has several advantages over both single ESI and multimode electrospray ionization/chemical ionization (ESCI) analysis, including an enhanced bound-complex detection and better performance at lower solvent flow rates. Relative binding constants are determined with (i) ESI mode, resulting in relative R(ESI-MS) values, and (ii) both ESI and APPI modes, providing relative K(D) values. We find that low molecular-substitution (Ms) values of cyclodextrin, i.e., Ms = 0.4, preferentially bind to the low-polarity compounds tested. This investigation is intended to demonstrate the feasibility of ESPI as an additional tool for investigating mixed-polarity binding systems, providing mass-specific data for all solution components, both polar and non-polar.     

Electrospray ionization mass spectrometry fingerprinting of perfumes: Rapid classification and counterfeit detection

A fast procedure to classify perfumes and identify counterfeit samples is described. Dilution of a few microL of the sample in a 1:1 methanol/water solution is followed by detection of its major polar components via direct infusion electrospray ionization mass spectrometry (ESI-MS) in the positive ion mode. As proof-of-principle cases, three famous brands of perfumes were used. The ESI+-MS fingerprints of authentic samples were very characteristic, showing distinctive sets of polar markers for each sample. Principal component analysis (PCA) placed samples of the three perfume brands in well-defined groups. Counterfeit samples were also clearly detected owing to contrasting ESI-MS fingerprints, with PCA placing these samples far away from the authentic samples.     

Quantitative liquid chromatography/time-of-flight mass spectrometry

Over the last decade, time-of-flight (TOF) instruments have increasingly been used as quantitation tools. In addition, because of their high resolving power, they can be used for verification of empirical formulas. Historically, TOF instruments have had limited quantitation capabilities because of their narrow dynamic range. However, recent advances have improved these limitations. This review covers the rationale for using TOF for LC detection, and describes the many methods currently in the literature for the quantitation of pharmaceuticals, environmental pollutants, explosives and many phytochemicals.     

Protein open-access liquid chromatography/mass spectrometry

Each year increasing numbers of proteins are submitted for routine characterization by liquid chromatography/mass spectrometry (LC/MS). This paper reports a solution that transforms routine LC/MS analysis of proteins into a fully automated process that significantly reduces analyst intervention. The solution developed, protein open-access (OA) LC/MS, consists of web-enabled sample submission and registration, automated data processing, data interpretation, and report generation. Sample submissions and results are recorded in a LIMS that utilizes an Oracle database. The protein sequence is captured during the sample submission process, stored in the database, and utilized to determine the theoretical protein molecular weight. This calculated mass is used to set the parameters for transformation of the mass-to-charge spectra to the mass domain and evaluate the presence or absence of the desired protein. Three protein OA-LC/MS instruments have been deployed in our facility to support protein characterization, purification, and modification efforts.     

Two-dimensional liquid chromatography/mass spectrometry/mass spectrometry separation of water-soluble metabolites

Off-line two-dimensional liquid chromatography with tandem mass spectrometry detection (2D-LC/MS-MS) was used to separate a set of metabolomic species. Water-soluble metabolites were extracted from Escherichia coli and Saccharomyces cerevisae cultures and were immediately analyzed using strong cation exchange (SCX)-hydrophilic interaction chromatography (HILIC). Metabolite mixtures are well-suited for multidimensional chromatography as the range of components varies widely with respect to polarity and chemical makeup. Some currently used methods employ two different separations for the detection of positively and negatively ionized metabolites by mass spectrometry. Here we developed a single set of chromatographic conditions for both ionization modes and were able to detect a total of 141 extracted metabolite species, with an overall peak capacity of ca. 2500. We show that a single two-dimensional separation method is sufficient and practical when a pair or more of unidimensional separations are used in metabolomics.     

Determination of chlormequat and mepiquat in foods by liquid chromatography/mass spectrometry or liquid chromatography/tandem mass spectrometry: interlaboratory study

Interlaboratory validation studies have been performed on 2 methods for the determination of chlormequat (CLQ) and mepiquat (MPQ). Both methods used identical extraction procedures and stable isotope internal standardization but differed in the use of liquid chromatography/mass spectrometry (LC/MS) or LC/tandem mass spectrometry (LC/MS/MS) for the determination, the amount of internal standard used, and the expected limit of detection. After addition of deuterated internal standards, CLQ and MPQ were extracted with methanol-water and determined by LC//MS or LC/MS/MS with positive electrospray ionization. Eight European laboratories participated in the LC/MS method study, analyzing mushroom, pear, wheat flour, and fruit puree with residues of CLQ in the range 0.040-1.19 mg/kg and of MPQ in the range 0.041-0.39 mg/kg. For CLQ, the Horwitz ratio (HoRat) values for individual test materials/levels were in the range 0.85-1.13 with a mean of 1.00, showing good method performance. For MPQ, the Ho values for mushroom, pear (both levels), and wheat flour were in the range 0.83-0.94, again indicating good method performance. For the determination of MPQ in infant food (fruit puree) at 0.041 mg/kg, the Ho was 1.7 when a value of 0 reported by one participant was excluded. In the LC/MS/MS study, in which 11 laboratories participated, a separate sample set was analyzed with residues of CLQ in the range 0.007-1.03 mg/kg and of MPQ in the range 0.008-0.72 mg/kg. Ho values for CLQ were in the range 0.27-1.36 and for MPQ in the range 0.51-2.10, all corresponding to acceptable method performance.     

Electrospray ionization mass spectrometry fingerprinting of whisky: immediate proof of origin and authenticity

Authentic samples of whisky produced in Scotland and USA and counterfeit whisky samples commercialized in Brazil have been directly submitted to electrospray ionization mass spectrometry (ESI-MS) analysis in both the negative and positive ion modes to assess the potential of this technique for simple and rapid quality control and proof of authenticity of whisky samples. ESI in the negative ion mode yields the most characteristic whisky fingerprinting mass spectra in just a few seconds by direct infusion of the samples, detecting the most polar or acidic components of each sample in their deprotonated anionic forms. No pre-treatment of the sample, such as extraction or derivatization or even dilution, is required. The analysis of the ESI(-)-MS data both by simple visual inspection but more particularly by chemometric data treatment enables separation of the whisky samples into three unequivocally distinct groups: Scotch, American and counterfeit whisky, whereas single malt and blended Scotch whiskies are also distinguished to some extent. As indicated by ESI-MS/MS analysis, the diagnostic anions are simple sugars, disaccharides and phenolic compounds. Direct infusion ESI-MS therefore provides immediate chemical fingerprinting of whisky samples for type, origin and quality control, as demonstrated herein for American, Scottish and counterfeit samples, whereas ESI-MS/MS analysis of diagnostic ions adds a second dimension of fingerprinting characterization when improved selectivity is desired.     

High performance liquid chromatography —Electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) for the analysis of heterocyclic aromatic amines (HAA)

Summary Sensitive and selective detection of the sixteen most abundant heterocyclic aromatic amines (HAA) has been achieved by application of high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) in combination with selected reaction monitoring (SRM). Detection limits between 0.1 and 50 ng mL^–1 were established by use of HAA model solutions.     

Comparative chemical fingerprinting of Oroxylum indicum and Scutellaria baicalensis using liquid chromatography and mass spectrometry

Introduction: Identification of Oroxylum indicum and Scutellaria baicalensis provides an interesting challenge in selection of biomarker compound to be used in routine analysis. Both plants have similar phytochemical profile and are rich sources of flavones and flavone glycosides. The objective of this study was to prepare the chemical fingerprinting of O. indicum bark and S. baicalensis roots using the liquid chromatography and mass spectroscopy in single chromatographic method. Materials and methods: Extracts prepared using various solvent systems (methanol, aqueous methanol, chloroform, hexane, and water) of both plants were analyzed using C18 reverse phase column with solvent system containing acetonitrile and 0.1% formic acid. Major flavonoids were identified based on mass spectra, fragmentation pattern, and UV spectra. Results: In this article, well-resolved high-performance liquid chromatographic (HPLC) separation in both plant extracts was obtained and chemical fingerprints for both plant extracts were established and flavonoids present (baicalin, oroxylin A-7-O-glucuronide, chrysin-7-O-glucuronide, baicalein, chrysin, oroxylin-A, wogonin, skullcap flavone II) were identified as possible biomarkers. Conclusion: Mass spectrometry coupled with HPLC can be a tool for fingerprinting of various natural products used in dietary supplement industry. The fingerprint developed in the article can be used for quality evaluation as well as identifying possible adulteration of extracts of both the plants.     

Electrospray ionization mass spectrometry of 5-methyl-2'-deoxycytidine and its determination in urine by liquid chromatography/electrospray ionization tandem mass spectrometry.

The behaviour of 5-methyl-2'-deoxycytidine (m(5)dCyd, claimed to be a potential marker for leukaemia) during the electrospray process was studied. In particular, considerations concerning the effect of solution chemistry (e.g. analyte concentration, pH, etc.) on electrospray ionization mass spectra were drawn. Furthermore, a procedure using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) for the determination of urinary m(5)dCyd is described. The method is simple, sensitive and highly specific. The pre-treatment procedure gave an average recovery of 79% (relative standard deviation (RSD) of 3%). Method performance was evaluated on spiked urine samples covering the concentration range from 50 ng/mL to 10 microgram/mL, the same as that of an existing inhibition ELISA method. Contrary to findings based on this immunoassay technique, urinary m(5)dCyd in healthy individuals was not detectable and did not increase in the presence of the malignant disease.     

High-throughput chiral liquid chromatography/tandem mass spectrometry

Chiral liquid chromatography is a well-established area of bioanalytical chemistry and is often used during the processes of drug discovery and development. The development and use of a chiral drug require the understanding of the pharmacokinetic characteristics of each of the enantiomers, including potential differences in their absorption, distribution, metabolism, and excretion. Chromatographic techniques coupled to atmospheric pressure ionization-tandem mass spectrometry have shown potential as sensitive and robust tools in the quantitative and qualitative determination of enantiomers in biologic fluids and tissue extracts. However, development of a chiral liquid chromatography method requires time-consuming procedures that are devised empirically. Clearly, there is an incentive to design chromatographic approaches that are easy to use, compatible with mass spectrometry ionization interface conditions, exhibit relatively short run times without compromising sensitivity, and offer a broad analyte specificity. For these reasons, the present paper explores the feasibility of the bonded macrocyclic glycopeptide phases (teicoplanin and vancomycin) for analysis by chiral liquid chromatography/tandem mass spectrometry. Ritalinic acid, pindolol, fluoxetine, oxazepam, propranolol, terbutaline, metoprolol, and nicardipine were tested in this study. Furthermore, an example of a simultaneous chiral LC/MS/MS detection (chromatographic run time approximately 10 min) of four pharmaceutical products resulting in baseline resolutions of all four pairs of enantiomers is presented. Methanol, an MS-compatible mobile phase, was utilized in all the experiments.     

Characterization of explosives by liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry using electrospray ionization and parent-ion scanning techniques

Analytical techniques for the detection of small amounts of explosives (in the picogram range) are now involved in various application. Some of them concern soil, water and air monitoring in order to face environmental problems related to improper handling procedures either in stocking or in wasting of the explosive products. Other areas are strictly related to forensic analysis of samples coming either from explosion areas where the matrix is various (metal, glass, wood, scraps), or from explosives transportation related to international terrorism. Generally speaking, for these applications the bulk of the matrix seriously interferes in the detection of the explosive analyte, which is usually present at trace levels. Unfortunately, despite some improvements, analytical techniques developed up today in this domain are still faced to two main constraints: the introduction of new products with unanticipated chemico-physical properties and the requirement of a routine and fast analytical method which can handle any matrix with a minimal clean-up and performing a sensitivity compatible either with the ever-decreasing demanded detection limit and with the ever-decreasing available specimen amount. These requirements can be fulfilled now by the new LC-MS and LC-MSMS techniques: mass spectrometry (MS) is likely an universal detector but even specific, especially when implemented in tandem MS (MSMS); LC is by far the most suitable technique to handle such a kind of compounds. Moreover, of a particular concern are some explosives which are reported to be thermally stable but difficult to dissolve. Some of the experiments on characterization of explosives [Octagen (HMX), Ethyleneglycol dinitrate (EGDN), Exogen (RDX), Propanetriol trinitrate (NG), Trinitrotoluene (TNT), N-Methyl-N-tetranitrobenzenamine (TETRYL), Dintrotoluene (DNT), Bis-(nitrooxy-methyl) propanediol dinitrate (PETN), Hexanitrostilbene (HNS), Triazido-trinitrobenzene (TNTAB), Tetranitro-acridone (TENAC), Hexa-nitrodiphenylamine (HEXYL), Nitroguanidine (NQ)] by LC-MS and LC-MSMS with the API-IonSpray source and using the Parent-Scan technique are presented.     

Study on the photostability of guaiazulene by high-performance liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

The photostability of guaiazulene (1,4-dimethyl-7-isopropylazulene; GA), a natural azulenic compound used in cosmetic and health-care products, as well as in pharmaceutical preparations, was investigated in solution (methanol, ethanol, acetonitrile), by different techniques: gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry and UV detection (LC/APCI-MS and HPLC/UV). A solar simulator (xenon-arc lamp) was used as UV-A radiation source. The study involved: monitoring compound decomposition, identifying products of photodegradation (PPs), assessing the role of oxygen and evaluating the kinetics of the process. Minor PPs are volatile compounds and were characterized by GC/MS, while oligomeric polyoxygenated compounds, tentatively characterized on the basis of MS and MS/MS spectra, were found to be the main photoproducts. The photodegradation was found to be enhanced by the presence of oxygen; nevertheless, determination of the singlet oxygen quantum yield for GA gave a lower value than that for the reference standard Rose Bengal. The obtained results and the developed stability-indicating methods (GC/MS and LC/MS) are of interest for stability studies and/or quality control purposes of GA as raw material or cosmetic products.     

Electrospray ionization mass spectrometry coupled to liquid chromatography for detection of cisplatin and its hydrated complexes

Electrospray ionization mass spectrometry (ESI-MS) coupled to liquid chromatography (LC) has been applied to investigate cisplatin and its hydrated complexes. Three hydrolysis products were identified following incubation of cisplatin in aqueous solution: monohydrated species, dihydrated species and the hydroxo-bridged dimer, each of which exhibited characteristic mass spectrometric behavior. The technique was employed to investigate the time- and pH-dependent hydrolysis of cisplatin in aqueous solution. The results have demonstrated that LC/ESI-MS is a powerful technique for the analysis of Pt(II) complexes and for monitoring their hydrolysis reactions.