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.     

Ultraperformance ion-pair liquid chromatography coupled to electrospray time-of-flight mass spectrometry for compositional profiling and quantification of heparin and heparan sulfate

Heparin and heparan sulfate (HS) are important pharmaceutical targets because they bind a large number of proteins, including growth factors and cytokines, mediating many biological processes. Because of their biological significance and complexity, there is a need for development of rapid and sensitive analytical techniques for the characterization and compositional analysis of heparin and HS at the disaccharide level, as well as for the structure elucidation of larger glycosaminoglycan (GAG) sequences important for protein binding. In this work, we present a rapid method for analysis of disaccharide composition using reversed-phase ion-pairing ultraperformance liquid chromatography coupled with electrospray time-of-flight mass spectrometry ((RPIP)-UPLC-MS). Heparin disaccharide standards were eluted in less than 5 min. The method was used to determine the constituents of GAGs from unfractionated heparin/HS from various bovine and porcine tissues, and the results were compared with literature values.     

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.     

Measurement of individual particle atomic composition by aerosol mass spectrometry

Advances in instrumentation used for particle compositional analysis have enabled real-time identification and classification of individual particles. However, precise quantitation of individual particle compositions has been elusive. Here, we demonstrate that real-time quantitative single-particle compositional analysis is possible. This is illustrated for individual particles of sodium chloride (70 nm), ammonium sulfate (70 nm), and silica (40-2000 nm) using a real-time aerosol mass spectrometer. Atomic fractions for major components (> 1% concentration) of individual multicomponent particles have been measured within +/-20% accuracy. Trace element detection limits of 20 ppm are also demonstrated when individual particle compositions are ensemble averaged.     

Evaluation of metal-ions containing sludges in the preparation of black inorganic pigments

Inorganic pigments were prepared from industrial wastes: galvanizing sludges resulting from Cr/Ni plating processes (S, G, and T) and a sludge generated from steel wiredraw process that is Fe-rich (F). These industrial wastes were characterized in order to determine the main compositional variations and discover their influence on the colour characteristics of pigments for glazes and ceramics. The toxic character was also investigated and established. Attempting to form the black spinel structure, several combinations of sludges were prepared and then calcined at 1000 °C. XRD and microscopy analysis confirmed the presence of nichromite for compositions with higher Ni amounts, while trevorite was detected in iron-rich formulations. The combination of S and F sludges (SF compositions) generates pigments with higher black colorimetric quality, which is similar to, and sometimes better than, a commercial black pigment. Pigments containing GF and TF sludges develop brown hues on glazes and on porcelain stoneware bodies, being this effect more evident upon industrial firing trials. The addition of minor amounts of cobalt or manganese enhances the black coloration, and might adjust some formulation deviations.     

Nanoparticulate PdZn--pathways towards the synthetic control of nanosurface properties

This paper reports an in-depth structural investigation of PdZn nanoparticulates prepared over an entire compositional range. By using a combination of HRTEM, ICP-OES, EDX and XPS alongside PXRD, we are able to show how a liquid-type reduction process can be exploited to target different PdZn bimetallic structures while maintaining reproducibly narrow particle size distributions and average particle diameters of approximately 3 nm. Samples have been further analyzed by quantitative phase analysis of the Rietveld refined diffraction data, providing indications as to how variations in specific surface compositions are obtained when Zn is used as the alloying metal. The influence of nanolattice strain is investigated by geometric analysis of TEM data. Results suggest, in conjunction with previously published catalytic data, how different compositions of this specific bimetallic system may be exploited in catalytic processes to control substrate/product affinity. We thus demonstrate a new and simplified approach to PdZn bimetallics, which may offer novel perspectives for applications in industrial catalysis.     

Development and evaluation of a reference measurement procedure for the determination of estradiol-17beta in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry

Estradiol is the most potent natural estrogen and is derived from the ovaries. Its concentration in blood is measured to determine ovarian function. A reference measurement procedure for estradiol in serum involving isotope-dilution coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. A deuterated estradiol (estradiol-d3) was used as an internal standard. The estradiol and its internal standard were extracted from serum matrix using solid-phase extractions and derivatized with dansyl chloride prior to reversed-phase LC/MS/MS. The accuracy of the measurement was evaluated by a comparison of results of this reference method on lyophilized human serum reference materials for estradiol [Certified Reference Materials (CRMs) 576, 577, and 578] with the certified values determined by gas chromatography/mass spectrometry (GC/MS) reference methods and by a recovery study for the added estradiol. The results of this method for estradiol agreed with the certified values within the uncertainty of the measurements for the three CRMs. The recovery of the added estradiol ranged from 100.7 to 101.8%. This method was applied to the determination of estradiol in frozen serum samples from three individual female donors. Excellent reproducibility was obtained with within-set coefficient of variations (CVs) ranging from 0.6 to 2.2% and between-set CVs ranging from 0.2 to 0.4%. Excellent linearity was also obtained, with correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) ranging from 0.998 to 1.000. The detection limit at a signal-to-noise ratio of approximately 3 was 0.6 pg of estradiol (or 1 ng/L, as expressed as a concentration). This well-characterized LC/MS/MS method for serum estradiol, which demonstrates good accuracy and precision, low susceptibility to interferences, and comparability with GC/MS reference methods, qualifies as a reference measurement procedure that can be used to provide an accuracy base to which routine methods for estradiol can be compared and that will serve as a standard of higher order for measurement traceability.     

Compositional variation of photoluminescence from Mn doped MgAl2O4 spinel

Spinel (MgAl₂O₄) crystals doped with 1.0% Mn have been grown by floating zone (FZ) technique with various Mg compositions, x = MgO/Al₂O₃, from 0.2 to 1.0. Compositional variations of photoluminescence are evaluated for a fluorescence thermometer application using crystals grown. Strong photoluminescence (PL) peak is observed at λ from 512 to 520 nm from the crystals grown from compositions, x, from 0.3 to 1.0. Peak wavelength of PL increases linearly from 512 to 520 nm with x. Weak PL peaking at λ = 750 nm is also observed from the specimens. Compositional variations of PL are considered to be due to the variation of crystal field surrounding the Mn²⁺ ions. The variation of crystal field strength agrees with the compositional variation of lattice constant.     

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS(1) spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS(2) and MS(3) spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods.     

Simultaneous quantification of homocysteine and folate in human serum or plasma using liquid chromatography/tandem mass spectrometry

A unified extraction and quantification procedure based on stable isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the simultaneous determination of total homocysteine and folate (5-methyltetrahydrofolic acid and folic acid) levels in human serum and plasma. This is the first report documenting the simultaneous extraction and quantification of these structurally dissimilar analytes. Analytes are quantitatively isolated from samples (500 microL) prior to LC/MS/MS analysis using a two-step stabilization process combined with C18 solid-phase extraction. The method exhibits excellent linearity over 4 orders of magnitude for each analyte. Measurement repeatability (RSD, N = 2) ranged from 0.3% to 3% for all analytes over 1 day of analysis. Total method variability (RSD, N = 6) ranged from 0.7% to 10% for all analytes over three independent days of analysis. The accuracy and practical applicability of the method were demonstrated by applying the method to the quantitative determination of each analyte in a new NIST serum Standard Reference Material (NIST SRM 1955 Homocysteine and Folate in Frozen Human Serum) and in a small subset of normal donor plasma samples.     

Nonstoichiometry in line compounds

Phases having compositional fields narrower than about 1 at.% and appearing as lines in binary phase diagrams often are assumed to have properties independent of composition. That such an assumption can be seriously in error is illustrated by recent measurements on pairs of samples prepared to have compositions at opposing phase boundaries. Two microscopic properties, lattice locations of highly dilute solutes and diffusional jump frequencies, were studied, respectively, through measurement of static and dynamic nuclear quadrupole interactions using the method of perturbed angular correlation of gamma rays (PAC). At opposing boundary compositions, PAC probe atoms have been observed to occupy different lattice sites or to have jump frequencies differing by a factor of 100. Such gross differences suggest measurements of other properties of line compounds should be made on pairs of samples having the opposing boundary compositions in order to avoid inconsistent or unreproducible results.     

Glycoblotting-assisted O-glycomics: ammonium carbamate allows for highly efficient o-glycan release from glycoproteins

Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hydrazide-polymers/solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing O-glycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers.     

Comprehensive solid-phase extraction method for persistent organic pollutants. Validation and application to the analysis of persistent chlorinated pesticides

The Centers for Disease Control and Prevention (CDC) is involved in many epidemiological studies regarding the measurement of chlorinated pesticides and polychlorinated biphenyls in specimens obtained from humans. In addition to these commonly determined analytes, there is a need to include additional persistent organic pollutants (POPs) in our analyses, which further stresses the analyses because sample volumes remain small. Thus, a single method of analysis for all POPs in human serum is needed. CDC has recently developed a semiautomated and comprehensive solid-phase extraction method for POPs. The method is comprehensive since it was optimized for the extraction of many different POP compound classes. We then developed a purification and fractionation scheme that allows (a) separation of different compound classes by particular functionalities and (b) purification of those fractions to remove coextracted interferences. This paper describes the first step in the semiautomated comprehensive extraction and multiple fractionation method developed by CDC for monitoring POPs. In this paper, we validate the analysis of the persistent chlorinated pesticides, a compound class difficult to examine because of their structural diversity, in human plasma. The method was validated against an existing CDC method by using a spiked quality-control serum pool. The concentrations determined for all analytes using both methods were within 2%-14% relative standard deviations. A multilevel (i.e., 3-4 point) matrix spike showed good linearity for the analytes tested (r2 = 0.978-0.999). The method was then applied to 40-year-old archived plasma samples for the quantitative analysis of selected chlorinated pesticides. Mean recoveries of the 13C-labeled internal quantification standards ranged from 64% to 123% for the 11 monitored pesticides. The overall method proved to be robust by handling old coagulated plasma samples. It allowed faster throughput of samples than our previous methods and provided cleaner samples with less frequent interferences or background as analyzed by high-resolution mass spectrometry. The method represents a preliminary step in establishing an automated, comprehensive multiresidue analysis method for POPs in human serum.     

Non–steady state solidification of superconducting oxides

The non–steady state solidification of RE123 (RE = Y, Nd) superconductive oxides was investigated by using an undercooling growth method. In both the Y– and Nd–systems, RE123 crystals grew steadily in the initial growth stage, but the growth rates decreased gradually in the later stage under the constant growth temperature. In the case of Nd–system, the substitution ratio of Hd/Ba in the grown Nd123 crystal was found to be gradually changed. The liquid compositions revealed that the liquid composition was close to the ternary equilibrium point after the growth rates decreased. This non-steady growth of Nd123 crystal is intrinsically caused since the composition of grown Nd123 solid solution is not on the 422–123 line and the residual liquid composition gradually shifts to the ternary equilibrium point to compensate for the mass balance during 123 growth. On the other hand, in the case of the Y–system, the evaluation of the volume fraction of Y211 particles showed that the volume fractions in the grown crystal were lower than those expected from the initial composition. On the contrary, those in liquid were higher and had similar valuesirrespective of the initial compositions and growth conditions. This accumulation of 211 phase particles near the 123/liquid interface would reduce the diffusion of the solute elements, which have the partition ratio of less than unity, away from the 123 interface. Therefore, such a large volumefraction would enhance the compositional shift close to the growth interface and cause locally a final transient to decrease the growth rate of the 123 crystal, if there is a compositional shift from the line connecting 123 and 211 compositions.     

Canine cancer screening via ultraviolet absorbance and fluorescence spectroscopy of serum proteins

A cost-effective optical cancer screening and monitoring technique was demonstrated in a pilot study of canine serum samples and was patented for commercialization. Compared to conventional blood chemistry analysis methods, more accurate estimations of the concentrations of albumin, globulins, and hemoglobin in serum were obtained by fitting the near UV absorbance and photoluminescence spectra of diluted serum as a linear combination of component reference spectra. Tracking these serum proteins over the course of treatment helped to monitor patient immune response to carcinoma and therapy. For cancer screening, 70% of dogs with clinical presentation of cancer displayed suppressed serum hemoglobin levels (below 20 mg/dL) in combination with atypical serum protein compositions, that is, albumin levels outside of a safe range (from 4 to 8 g/dL) and globulin levels above or below a more normal range (from 1.7 to 3.7 g/dL). Of the dogs that met these criteria, only 20% were given a false positive label by this cancer screening test.     

Non-steady state solidification of superconducting oxides

The non-steady state solidification of RE123 (RE=Y, Nd) superconductive oxides was investigated by using an undercooling growth method. In both the Y- and Nd-systems, RE123 crystals grew steadily in the initial growth stage, but the growth rates decreased gradually in the later stage under the constant growth temperature. In the case of Nd-system, the substitution ratio of Hd/Ba in the grown Nd123 crystal was found to be gradually changed. The liquid compositions revealed that the liquid composition was close to the ternary equilibrium point after the growth rates decreased. This non-steady growth of Nd123 crystal is intrinsically caused since the composition of grown Nd123 solid solution is not on the 422-123 line and the residual liquid composition gradually shifts to the ternary equilibrium point to compensate for the mass balance during 123 growth. On the other hand, in the case of the Y-system, the evaluation of the volume fraction of Y211 particles showed that the volume fractions in the grown crystal were lower than those expected from the initial composition. On the contrary, those in liquid were higher and had similar values irrespective of the initial compositions and growth conditions. This accumulation of 211 phase particles near the 123/liquid interface would reduce the diffusion of the solute elements, which have the partition ratio of less than unity, away from the 123 interface. Therefore, such a large volume fraction would enhance the compositional shift close to the growth interface and cause locally a final transient to decrease the growth rate of the 123 crystal, if there is a compositional shift from the line connecting 123 and 211 compositions.     

Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry

The development of a quantification method for monoclonal antibodies in serum has been accomplished by high-performance liquid chromatography multiple reactions monitoring mass spectrometry. A human monoclonal antibody (HmAb) was used as the model protein for method development and validation. A peptide from the CDR3-region of its heavy chain was selected and used for quantifying the entire mAb. This signature peptide served as a template for the internal standard. Prior to mass spectrometric analysis approximately 50% of the total serum protein content was removed by albumin depletion. The accuracy of the method ranged between 99 and 112% in cynomolgus monkey serum. The intra-assay coefficient of variation (CV) was lower than 4% at 4 microg/mL and 200 microg/mL HmAb (n = 3). The CV at 400 microg/mL corresponded to 9% (n = 3). In addition, the interassay variation was investigated in a male cynomolgus serum pool and in a female cynomolgus serum pool. The CV for the male cynomolgus pool at 4 microg/mL HmAb was 7% (n = 3). The CV obtained from the female pool was 8% (n = 3), at 4 microg/mL. The dynamic range of the method was 3 orders of magnitude. After albumin depletion of 25 microL of serum, a lowest limit of quantification of 2 microg/mL HmAb was reached in both human and cynomolgus monkey samples.     

Modelling Young’s modulus for porous bones with microstructural variation and anisotropy

A structural model with three compositional phases and two levels of hierarchical organization is proposed for predicting Young’s modulus of porous bones with microstructural variations and anisotropy based on their geometric similarity to metal foams. It has been shown that the proposed single model provides predictions of Young’s modulus with high accuracy up to ±30% for cortical and cancellous bones compared with measured data from the literature. In addition, the conversion of the solid bone shape from “Plate-like” to “Rod-like” at a porosity of 70% or higher (BV/TV 30% or lower)—verified by observations—can be predicted using the proposed model.     

Synthesis and microstructural characterization of Ce1-xTb(x)O2-delta (0 < or = x < or = 1) nano-powders

Ce1-xTb(x)O2-delta nano-powders have been successfully synthesized by using the ammonium carbonate coprecipitation method in an entire compositional range of 0 < or = x < or = 1 by adjusting the preparation conditions. Studies of X-ray diffraction (XRD) and transmission electron microscopy (TEM) showed that the powders with different compositions mainly consist of fluorite structure. In addition, a small amount of secondary phase was observed in the powders with x > or = 0.7. TEM observation indicated that the secondary phase could have a superstructure formed by a structural modulation of the fluorite structure.     

3D Printing in alloy design to improve biocompatibility in metallic implants

3D Printing (3DP) or additive manufacturing (AM) enables parts with complex shapes, design flexibility, and customization opportunities for defect specific patient-matched implants. 3DP or AM also offers a design platform that can be used to innovate novel alloys for application-specific compositional modifications. In medical applications, the biological response from a host tissue depends on a biomaterial's structural and compositional properties in the physiological environment. Application of 3DP can pave the way towards manufacturing innovative metallic implants, combining structural variations at different length scales and tailored compositions designed for specific biological responses. This study shows how 3DP can be used to design metallic alloys for orthopedic and dental applications with improved biocompatibility using in vitro and in vivo studies. Titanium (Ti) and its alloys are used extensively in biomedical devices due to excellent fatigue and corrosion resistance and good strength to weight ratio. However, Ti alloys' in vivo biological response is poor due to its bioinert surface. Different coatings and surface modification techniques are currently being used to improve the biocompatibility of Ti implants. We focused our efforts on improving Ti's biocompatibility via a combination of tantalum (Ta) chemistry in Ti, the addition of designed micro-porosity, and nanoscale surface modification to enhance both in vitro cytocompatibility and early stage in vivo osseointegration, which was studied in rat and rabbit distal femur models.