Ionic liquid matrix for direct UV-MALDI-TOF-MS analysis of dermatan sulfate and chondroitin sulfate oligosaccharides

Polyanionic oligosaccharides such as dermatan sulfate (DS) and chondroitin sulfate (CS) exhibit poor ionization efficiencies and tend to undergo thermal fragmentation through the loss of SO(3) under conventional ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) conditions. A new ionic liquid matrix (ILM), a guanidinium salt of alpha-cyano-4-hydroxycinnamic acid, facilitates direct UV-MALDI mass spectrometric (MS) analysis of underivatized DS and CS oligosaccharides up to a decasaccharide in their common form as sodium salts. The resulting mass spectra show very low extent of fragmentation through an SO(3) loss. The new ILM is suitable for MALDI-MS analysis of mixtures containing oligosaccharides with different numbers of sulfo groups.     

Online reverse phase-high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry separation and characterization of heparan sulfate, heparin, and low-molecular weight-heparin disaccharides derivatized with 2-aminoacridone

A high-resolution online reverse-phase-high-performance liquid chromatography (RP-HPLC)-fluorescence detector (Fd)-electrospray ionization-mass spectrometry (ESI-MS) separation and structural characterization of disaccharides prepared from heparin (Hep), heparan sulfate (HS), and various low-molecular-weight (LMW)-Hep using heparin lyases and derivatization with 2-aminoacridone (AMAC) are described. A total of 12 commercially available Hep/HS-derived unsaturated disaccharides were separated and unambiguously identified on the basis of their retention times and mass spectra. The constituent disaccharides of various samples, including unfractionated Hep/HS, fast-moving and slow-moving Hep components, and several marketed products, were characterized. Furthermore, for the first time, the saturated trisulfated disaccharide belonging to the nonreducing end of Heps was detected as being approximately 2% in unfractionated samples and ~15-21% in LMW-Heps prepared by nitrous acid depolymerization. No desalting of the commercial products prior to enzymatic digestion or prepurification steps to eliminate any excess of AMAC reagent or interference from proteins, peptides, and other sample impurities before RP-HPLC-Fd-ESI-MS injection were necessary. This method has applicability for the rapid differentiation of pharmaceutical Heps and LMW-Heps prepared by means of different depolymerization processes and for compositional analysis of small amounts of samples derived from biological sources by using the highly sensitive fluorescence detector.     

Compositional analysis and quantification of heparin and heparan sulfate by electrospray ionization ion trap mass spectrometry

A new method using a combination of electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MSn) was developed for the identification and quantitative analysis of eight heparan sulfate (HS)- and heparin-derived delta-disaccharides obtained by enzymatic depolymerization. The compositional analysis of nonisomeric disaccharide constituents of heparin/HS was achieved from full-scan MS1 spectra using an internal standard and a calculated response factor for each disaccharide. Diagnostic product ions from MSn spectra of isomeric disaccharides were used for the quantitative analysis of the relative amounts of each of the isomers in mixtures. The protocol was validated using several quality control samples and showed satisfactory accuracy and precision. The analysis is rapid, accurate, and uses no purification or separation steps prior to analysis by MS, thus reducing sample consumption and analysis time of traditional methods. Using this quantitative analysis procedure, percentages of disaccharide compositions for heparins from porcine and bovine intestinal mucosa and heparan sulfate from bovine kidney were determined.     

Two-layer sample preparation method for MALDI mass spectrometric analysis of protein and peptide samples containing sodium dodecyl sulfate

Sodium dodecyl sulfate (SDS) is widely used in protein sample workup. However, many mass spectrometric methods cannot tolerate the presence of this strong surfactant in a protein sample. We present a practical and robust technique based on a two-layer matrix/sample deposition method for the analysis of protein and peptide samples containing SDS by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The two-layer method involves the deposition of a mixture of sample and matrix on top of a thin layer of matrix crystals. It was found that for SDS-containing samples, the intensity of the MALDI signals can be affected by the conditions of sample preparation: on-probe washing, choice of matrix, deposition method, solvent system, and protein-to-SDS ratio. However, we found that, under appropriate conditions, the two-layer method gave reliable MALDI signals for samples with levels of SDS up to approximately 1%. The applications of this method are demonstrated for MALDI analysis of hydrophobic membrane proteins as well as bacterial extracts. We envision that this two-layer method capable of handling impure samples including those containing SDS will play an important role in protein molecular weight analysis as well as in proteome identification by MALDI-MS and MS/MS.     

Simultaneous sample preparation and species-specific isotope dilution mass spectrometry analysis of monomethylmercury and tributyltin in a certified oyster tissue

A rapid, accurate, sensitive, and simple method for simultaneous speciation analysis of mercury and tin in biological samples has been developed. Integrated simultaneous sample preparation for tin and mercury species includes open focused microwave extraction and derivatization via ethylation. Capillary gas chromatography-inductively plasma mass spectrometry (CGC-ICPMS) conditions and parameters affecting the analytical performance were carefully optimized both for species-specific isotope dilution analysis of MMHg and TBT and for conventional analysis of MBT and DBT201Hg-enriched monomethylmercury and 117Sn-enriched tributyltin were used for species-specific isotope dilution mass spectrometry (SIDMS) analysis. As important, accurate isotope dilution analysis requires equilibration between the spike and the analyte to achieve successful analytical procedures. Since the spike stabilization and solubilization are the most critical and time-consuming steps in isotope dilution analysis, different spiking procedures were tested. Simultaneous microwave-assisted spike stabilization and solubilization can be achieved within less than 5 min. This study originally introduces a method for the simultaneous speciation and isotope dilution of mercury and tin in biological tissues. The sample throughput of the procedure was drastically reduced by fastening sample preparation and GC separation steps. The accuracy of the method was tested by both external calibration analysis and species-specific isotope dilution analysis using the first biological reference material certified for multielemental speciation (oyster tissue, CRM 710, IRMM). The results obtained demonstrate that isotope dilution analysis is a powerful method allowing the simultaneous speciation of TBT and MMHg with high precision and excellent accuracy. Analytical problems related to low recovery during sample preparation are thus minimized by SIDMS. In addition, a rapid procedure allows us to establish a performant routine method using CGC-ICPMS technique.     

Determination of 16 phthalate metabolites in urine using automated sample preparation and on-line preconcentration/high-performance liquid chromatography/tandem mass spectrometry

We developed an on-line solid-phase extraction (SPE) method, coupled with isotope dilution high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) and with automated sample preparation, to simultaneously quantify 16 phthalate metabolites in human urine. The method requires a silica-based monolithic column for the initial preconcentration of the phthalate metabolites from the urine and a silica-based conventional analytical column for the chromatographic separation of the analytes of interest. It uses small amounts of urine (100 microL), is sensitive (limits of detection range from 0.11 to 0.90 ng/mL), accurate (spiked recoveries are approximately 100%), and precise (the inter- and intraday coefficients of variation are <10%). The method is not labor intensive, and, because pretreatment of the urine samples was performed automatically using an HPLC autosampler, involves minimal sample handling, thus minimizing exposure to hazardous chemicals. The method was validated on spiked, pooled urine samples and on urine samples from 43 adults with no known exposure to phthalates. The high sensitivity and high throughput (HPLC run time, including the preconcentration step, is 27 min) of this analytical method combined with the ease of use and effective automated sample preparation procedure make it suitable for large epidemiological studies to evaluate the prevalence of human exposure to phthalates.     

Spectroscopic characterization of ethyl xanthate oxidation products and analysis by ion interaction chromatography

An ion interaction chromatographic separation method, coupled with UV spectroscopic detection, has been developed for the analysis of ethyl xanthate (O-ethyl dithiocarbonate) and its oxidative decomposition products in mineral flotation systems. The effects of the ion-pairing agent (tetrabutylammonium ion), pH modifier (phosphoric acid), and organic modifier (acetonitrile) in the eluant upon the retention characteristics of the ethyl xanthate oxidation products have been determined. The optimized separation procedure has been successfully applied to the analysis of ethyl xanthate and its oxidation products in a nickel-iron sulfide mineral suspension containing a number of other anionic species, including cyanide complexes of nickel and iron, as well as sulfur-oxy anions. The ethyl xanthate oxidation products investigated in this study have been isolated as pure compounds and characterized by UV-visible, FT-IR, and NMR spectroscopies. The UV-visible and FT-IR spectroscopic properties of these species are discussed in terms of the chemical modifications of the thiocarbonate group.     

Fractionation and solvent-free MALDI-MS analysis of polymers using liquid adsorption chromatography at critical conditions in combination with a multisample on-target homogenization/transfer sample preparation method

A solvent-free homogenization/transfer matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) method is described for the preparation and precise transfer of up to 100 samples simultaneously on a single MALDI plate. This method is demonstrated using a poly(ethylene oxide) (PEO) mixture consisting of different molecular weights (500-6000) and end groups (PEO, dimethoxy-PEO, monomethoxy monomethacrylate-PEO, and dimethacrylate-PEO) that was fractionated using liquid adsorption chromatography at critical conditions. Off-line fractionation is performed prior to the on-target homogenization/transfer solvent-free sample preparation and MALDI mass analysis. The miniaturization of the solvent-free MALDI approach allowed analysis of less than 2 microg per PEO component per fraction corresponding to approximately 200 pmol for PEO 6000. The amounts of polymer sample used for LC separation and the quality of the MS results are equivalent to the "dry spray" method; however, three times more fractions were collected and analyzed with the newly developed hyphenated approach. The off-line method eliminates optimization of, for example, spray conditions or spreading of organic solvents on the MALDI plate that occurs with droplet deposition methods. The widespread applications of MALDI make this solvent-free, multisample method particularly important as it expands the capabilities for obtaining mass measurements with great efficiencies in areas with increased sample numbers. In addition, the solvent-free method is well suited for automated MALDI analysis as it virtually eliminates the "dead-spot" phenomenon.     

Speciation analysis of gadolinium-based MRI contrast agents in blood plasma by hydrophilic interaction chromatography/electrospray mass spectrometry

The first analytical method for simultaneous speciation analysis of five of the most important gadolinium-based magnetic resonance imaging (MRI) contrast agents in blood plasma samples was developed. Gd-DTPA (Magnevist), Gd-BT-DO3A (Gadovist), Gd-DOTA (Dotarem), Gd-DTPA-BMA (Omniscan), and Gd-BOPTA (Multihance) were separated by hydrophilic interaction liquid chromatography (HILIC) and detected with electrospray mass spectrometry (ESI-MS). Spiking experiments of blank plasma with Magnevist and Gadovist were performed to determine the analytical figures of merit and the recovery rates. The limits of detection ranged from 1 x 10 (-7) to 1 x 10 (-6) mol/L depending on the ionization properties of the individual compounds, and limits of quantification ranged from 5 x 10 (-7) to 5 x 10 (-6) mol/L. The linear concentration range comprised 2 orders of magnitude. With application of this method, blood plasma samples of 10 healthy volunteers, with Magnevist or Gadovist medication, were analyzed for Gd-DTPA and Gd-BT-DO3A, respectively. The obtained results were successfully validated with inductively coupled plasma-optical emission spectroscopy (ICP-OES).     

Collection, storage, and filtration of in vivo study samples using 96-well filter plates to facilitate automated sample preparation and LC/MS/MS analysis

The benefits of high-throughput bioanalysis within the pharmaceutical industry are well established. One of the most significant bottlenecks in bioanalysis is transferring in vivo-generated study samples from their collection tubes during sample preparation and extraction. In most cases, the plasma samples must be stored frozen prior to analysis, and the freeze/thaw (F/T) process introduces thrombin clots that are capable of plugging pipets and automated liquid-transfer systems. A new approach to dealing with this problem involves the use of Ansys Captiva 96-well 20-microm polypropylene filter plates to collect, store frozen, and filter plasma samples prior to bioanalysis. The samples are collected from the test subjects, and the corresponding plasma samples are placed directly into the wells of the filter plate. Two Duoseal (patent pending) covers are used to seal the top and bottom of the plate, and the plate is stored at down to -70 degrees C. Prior to sample analysis, the seals are removed and the plate is placed in a 96-well SPE manifold. As the plasma thaws, it passes (by gravity or mild vacuum) through the polypropylene filter into a 96-well collection plate. A multichannel pipet or automated liquid-transfer system is used to transfer sample aliquots without fear of plugging. A significant advantage of this approach is that, unlike other methods, issues related to incomplete pipetting are virtually eliminated. The entire process is rapid since thawing and filtering take place simultaneously, and if a second F/T cycle is required for reanalysis, it is not necessary to refilter the samples (additional clotting was not observed after three F/T cycles). This technique was tested using monkey, rat, and dog plasma and sodium heparin and EDTA anticoagulants. To assess the possibility of nonspecific binding to the polypropylene filter, a variety of drug candidates from diverse drug classes were studied. Validation data generated for two Lilly compounds from distinct classes, before and after filtering, are presented in this paper as practical examples of this technique. While LC/MS/MS is the primary method of bioanalysis in our laboratory, the technique presented in this paper is applicable to other forms of detection as well.     

Quantitative and qualitative determination of estrogen sulfates in human urine by liquid chromatography/tandem mass spectrometry using 96-well technology.

A sensitive and robust method to determine five estrogen sulfates in human urine has been developed employing high-throughput solid-phase extraction with 96-well technology, and HPLC coupled with negative turbo ion spray tandem mass spectrometry in the selected reaction monitoring mode. The five estrogen sulfates determined include three major endogenous estrogen sulfates in the human, estrone 3-sulfate (E1-3S), estriol 3-sulfate (E3-3S), and 17 beta-estradiol 3-sulfate (E2-3S), and two biochemical synthetic estrogen sulfates, 17 beta-estradiol 17-sulfate (E2-17S) and 17 beta-estradiol 3,17-disulfate (E2-3,17S). For E2-3,17S, E3-3S, and E2-17S, external standard calibration was used for quantitation, and for the remaining two compounds, internal standard calibration using a stable isotopic labeled internal standard was employed. A total of 96 samples may be prepared with 96-well C18 extraction disk plate techniques performed by a robot within 25 min including the time for evaporation of solvent. The lower level of quantitation (LOQ) for these estrogen sulfates in human urine was determined at 0.2 ng/mL based on 100-microL aliquots of human urine using the optimum tuning parameters for each individual selected precursor ion/product ion transition. The assay was validated with a linear concentration range of 0.2-200 ng/mL, and the interassay accuracy, intraassay precision, and interassay precision do not exceed 8.6%, 12%, and 12%, respectively, by analysis of quality control samples at five concentration levels including the LOQ of 0.2 ng/mL, from four 96-well plates. The target endogenous test articles were qualitatively determined by comparing the full-scan LC/MS/MS mass spectra and retention time in test samples and reference standards. The LOQ is significantly improved compared to previous reports for the targeted compounds using LC/MS/MS. The described simple and automated sample preparation procedure recovered 91% of the target compounds. A total of 192 samples can be analyzed within 1 day (22 h). The method can measure the endogenous estrogen sulfates in urine from both gravid and nongravid subjects.     

Combinatorial peptide ligand library treatment followed by a dual-enzyme, dual-activation approach on a nanoflow liquid chromatography/orbitrap/electron transfer dissociation system for comprehensive analysis of swine plasma proteome

The plasma proteome holds enormous clinical potential, yet an in-depth analysis of the plasma proteome remains a daunting challenge due to its high complexity and the extremely wide dynamic range in protein concentrations. Furthermore, existing antibody-based approaches for depleting high-abundance proteins are not adaptable to the analysis of the animal plasma proteome, which is often essential for experimental pathology/pharmacology. Here we describe a highly comprehensive method for the investigation of the animal plasma proteome which employs an optimized combinatorial peptide ligand library (CPLL) treatment to reduce the protein concentration dynamic range and a dual-enzyme, dual-activation strategy to achieve high proteomic coverage. The CPLL treatment enriched the lower abundance proteins by >100-fold when the samples were loaded in moderately denaturing conditions with multiple loading-washing cycles. The native and the CPLL-treated plasma were digested in parallel by two enzymes (trypsin and GluC) carrying orthogonal specificities. By performing this differential proteolysis, the proteome coverage is improved where peptides produced by only one enzyme are poorly detectable. Digests were fractionated with high-resolution strong cation exchange chromatography and then resolved on a long, heated nano liquid chromatography column. MS analysis was performed on a linear triple quadrupole/orbitrap with two complementary activation methods (collisionally induced dissociation (CID) and electron transfer dissociation). We applied this optimized strategy to investigate the plasma proteome from swine, a prominent animal model for cardiovascular diseases (CVDs). This large-scale analysis results in identification of a total of 3421 unique proteins, spanning a concentration range of 9-10 orders of magnitude. The proteins were identified under a set of commonly accepted criteria, including a precursor mass error of <15 ppm, Xcorr cutoffs, and ≥2 unique peptides at a peptide probability of ≥95% and a protein probability of ≥99%, and the peptide false-positive rate of the data set was 1.8% as estimated by searching the reversed database. CPLL treatment resulted in 55% more identified proteins over those from native plasma; moreover, compared with using only trypsin and CID, the dual-enzyme/activation approach enabled the identification of 2.6-fold more proteins and substantially higher sequence coverage for most individual proteins. Further analysis revealed 657 proteins as significantly associated with CVDs (p < 0.05), which constitute five CVD-related pathways. This study represents the first in-depth investigation of a nonhuman plasma proteome, and the strategy developed here is adaptable to the comprehensive analysis of other highly complex proteomes.     

Determination of deamidation artifacts introduced by sample preparation using (18)o-labeling and tandem mass spectrometry analysis

The sites and levels of Asn deamidation in proteins are often determined by LC-MS analysis of peptides obtained from enzymatic digestion. However, deamidation that occurs during sample preparation steps results in overestimation of the original level of deamidation. The inherent deamidation and those introduced by sample preparation can be differentiated by preparing samples in (18)O water. When using H(2)(18)O, the formation of isoAsp and Asp by Asn deamidation during sample preparation results in a molecular weight increase of 3 Da due to the incorporation of the (18)O atom to the side chains of isoAsp or Asp; in contrast, inherent deamidation only results in a molecular weight increase of 1 Da. In addition, up to two (18)O atoms can also be incorporated into the peptide C-terminal carboxyl group during enzymatic digestion. Therefore, the 2 Da molecular weight difference at the deamidation sites can only be used to differentiate deamidation that occurs prior to or during sample preparation under conditions that a fixed number of (18)O atoms are incorporated into the peptide C-terminal carboxyl groups. Otherwise, it is challenging to apply this procedure because of the resulting complicated isotopic distributions. Here, a new procedure of using (18)O-water for sample preparation coupled with tandem mass spectrometry (MS/MS) was established to calculate the deamidation artifacts. In this method, b ions were used for the calculation of Asn deamidation that occurred prior to or during sample preparation, which eliminated the complicated factor of various number of (18)O-atoms to the peptide carboxyl groups. This procedure has the potential to be applied under the general peptide mapping conditions.     

Simultaneous measurement of urinary bisphenol A and alkylphenols by automated solid-phase extractive derivatization gas chromatography/mass spectrometry

Bisphenol A (BPA) and alkylphenols (APs) are widely used industrial chemicals. BPA is used to manufacture polycarbonate plastic and epoxy resins; APs are used to make alkylphenol ethoxylates, common nonionic surfactants. BPA and APs can leach into the environment during industrial production and after degradation of the polycarbonate plastics and nonionic surfactants. Environmental exposure to these phenolic compounds has been associated with adverse reproductive and developmental effects in wildlife. We developed a sensitive and robust method for measuring BPA and six APs; 3-tert-butylphenol, 4-tert-butylphenol, 4-n-octylphenol, 4-tert-octylphenol, 4-n-nonylphenol, and technical-grade nonylphenol in urine. The method is based on the use of automated solid-phase extraction (SPE) coupled to isotope dilution-gas chromatography/mass spectrometry (GC/MS). During the automated SPE process, the phenols are both extracted from the urine matrix and derivatized, using pentafluorobenzyl bromide, on commercially available styrene-divinylbenzene copolymer-based SPE cartridges. After elution from the SPE column, the derivatized phenols in the SPE eluate are analyzed by GC/MS. The method, validated on spiked pooled urine samples and on urine samples from exposed persons, has limits of detection of approximately 0.1 ng in 1 mL of urine.     

Solid-phase extraction procedure of polar benzene- and naphthalenesulfonates in industrial effluents followed by unequivocal determination with ion-pair chromatography/electrospray-mass spectrometry

Highly water soluble benzene- and naphthalenesulfonates are widely used in the chemical, pharmaceutical, tannery, paper, and textile industries. In this work, Isolute ENV+ polystyrene divinylbenzene sorbent was used for the enrichment of 14 benzene- and naphthalenesulfonates from industrial wastewaters. The elution step was performed by adding 1 mL of water containing ion-pair reagent (5 mM of triethylamine (TEA) and 5 mM of acetic acid at pH 6.5) and 9 mL of methanol at 1 mL/min. The most relevant contribution was the use of ion-pair liquid chromatography followed by an orthogonal electrospray interface coupled to mass spectrometry in the negative ionization mode with postcolumn addition at 0.2 mL/min of methanol in combination with a volatile substance (triethylamine) as an ion-pair reagent. [M-H](-) ion was the base peak using low fragmentor voltages of 80 V with the electrospray interface. Significant fragmentation of the quasimolecular [M-H](-) ion occurs at high fragmentor voltage, producing [M-SO(2)H](-), [M-SO(3)H](-), and [SO(3)](?)(-) as diagnostic ions. Collision-induced dissociation of the parent ions for the benzene- and naphthalenesulfonates studied gave the [SO(3)](?)(-) fragment ion common to sulfonated compounds. At high fragmentor voltages of 150 V, [M-SO(3)H](-) ion is more abundant and therefore has a larger peak than the [M-H](-) peaks for 1,5-naphthalenedisulfonate, 2,6-naphthalenedisulfonate, 1-hydroxy-3,6-naphthalenedisulfonate, 2-hydroxy-3,6-naphthalenedisulfonate, and 2-amino-1,5-naphthalenedisulfonate. Recoveries were higher than 70%, with relative standard deviations between 1.3 and 10.7% with the exception of two naphthalenesulfonate compounds that had recoveries between 26 and 41%. Limits of detection (signal-to-noise ratio, 3) ranging from 0.6 pg to 0.13 ng (0.03-6.48 μg/L) were achieved when 150 mL of groundwater was processed. The aromatic sulfonates 3-nitrobenzenesulfonate, 4-methylbenzenesulfonate, 4-chlorobenzenesulfonate, 1-hydroxy-4-naphthalenosulfonate, 1-amino-6-naphthalenosulfonate, 1-amino-7-naphthalenosulfonate, and 1-naphthalenesulfonate and the linear alkyl benzenesulfonates C(10)-LAS and C(11)-LAS were unequivocally identified and quantitatively determined in μg/L, in wastewater samples from wastewater treatment plants and textile and tannery industries. 2-Naphthalenesulfonate was found as a major pollutant at mg/L concentration levels.     

Simultaneous derivatization and quantification of the nitric oxide metabolites nitrite and nitrate in biological fluids by gas chromatography/mass spectrometry

Simultaneous quantification of nitrite and nitrate, the major oxidative metabolites of L-arginine-derived nitric oxide (NO), in biological fluids by GC or GC/MS methods is currently impossible. The separate analysis of these anions is associated with severe methodological problems. Therefore, a GC/MS method was developed which allows, for the first time, simultaneous quantification of nitrite and nitrate in various biological fluids. The method involves a single derivatization procedure, by which endogenous nitrite and nitrate and their externally added 15N-labeled analogues are simultaneously converted in aqueous acetone by pentafluorobenzyl bromide to the nitro and nitric acid ester pentafluorobenzyl derivatives, respectively, and a single GC/MS analysis. Nitrite and nitrate concentrations measured in plasma and urine of humans by this method correlated excellently with those from quantification of nitrite and nitrate in these matrixes using a previously reported GC/MS method that, however, requires reduction of nitrate to nitrite. Also, the present method enables discrimination between S-nitro- and S-nitroso-glutathione, which have identical chromatographic and spectrophotometric properties. The method is very useful to routinely study metabolism and reactions of NO and its metabolites in vitro and in vivo. It is accurate, interference-free, sensitive-50 fmol of [15N]-nitrite and [15N]nitrate were detected at signal-to-noise ratios of 870:1 and 95:1, respectively-and should be a reference method for nitrite and nitrate measurements.