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Analytical and Bioanalytical Chemistry (v.399, #2)

New developments in the characterization of heparin and its impurities by Cynthia K. Larive (pp. 525-526).

is Professor of Analytical Chemistry at the University of California Riverside. She has an active research program involving bioanalytical and environmental analytical applications of NMR Spectroscopy (for more information see http://www.chem.ucr.edu.faculty/larive/larive.html ). Professor Larive is also active in curricular reform and the promotion of undergraduate research. She is editor-in-chief and principal investigator of the Analytical Sciences Digital Library ( http://www.asdlib.org ), an Internet-based resource for instructors, students, and practitioners of analytical chemistry. This digital library is a collection of peer-reviewed web sites on topics including pedagogical approaches, analytical techniques, applications, and classroom resources.

New developments in the characterization of heparin and its impurities by Cynthia K. Larive (pp. 525-526).

Analysis and characterization of heparin impurities by Szabolcs Beni; John F. K. Limtiaco; Cynthia K. Larive (pp. 527-539).

This review discusses recent developments in analytical methods available for the sensitive separation, detection and structural characterization of heparin contaminants. The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007–2008 spawned a global crisis resulting in extensive revisions to the pharmacopeia monographs on heparin and prompting the FDA to recommend the development of additional physicochemical methods for the analysis of heparin purity. The analytical chemistry community quickly responded to this challenge, developing a wide variety of innovative approaches, several of which are reported in this special issue. This review provides an overview of methods of heparin isolation and digestion, discusses known heparin contaminants, including OSCS, and summarizes recent publications on heparin impurity analysis using sensors, near-IR, Raman, and NMR spectroscopy, as well as electrophoretic and chromatographic separations. Figure Schematic illustrating the process for heparin impurity characterization

Keywords: Contamination; Impurity; Heparin; NMR; OSCS; Adulteration

Analysis and characterization of heparin impurities by Szabolcs Beni; John F. K. Limtiaco; Cynthia K. Larive (pp. 527-539).

Keywords: Contamination; Impurity; Heparin; NMR; OSCS; Adulteration

Hyphenated techniques for the analysis of heparin and heparan sulfate by Bo Yang; Kemal Solakyildirim; Yuqing Chang; Robert J. Linhardt (pp. 541-557).

The elucidation of the structure of glycosaminoglycan has proven to be challenging for analytical chemists. Molecules of glycosaminoglycan have a high negative charge and are polydisperse and microheterogeneous, thus requiring the application of multiple analytical techniques and methods. Heparin and heparan sulfate are the most structurally complex of the glycosaminoglycans and are widely distributed in nature. They play critical roles in physiological and pathophysiological processes through their interaction with heparin-binding proteins. Moreover, heparin and low-molecular weight heparin are currently used as pharmaceutical drugs to control blood coagulation. In 2008, the health crisis resulting from the contamination of pharmaceutical heparin led to considerable attention regarding their analysis and structural characterization. Modern analytical techniques, including high-performance liquid chromatography, capillary electrophoresis, mass spectrometry, and nuclear magnetic resonance spectroscopy, played critical roles in this effort. A successful combination of separation and spectral techniques will clearly provide a critical advantage in the future analysis of heparin and heparan sulfate. This review focuses on recent efforts to develop hyphenated techniques for the analysis of heparin and heparan sulfate.

Keywords: Heparin/heparan sulfate; High-performance liquid chromatography; Capillary electrophoresis; Tandem mass spectrometry; Nuclear magnetic resonance spectroscopy; Hyphenated techniques

Hyphenated techniques for the analysis of heparin and heparan sulfate by Bo Yang; Kemal Solakyildirim; Yuqing Chang; Robert J. Linhardt (pp. 541-557).

Keywords: Heparin/heparan sulfate; High-performance liquid chromatography; Capillary electrophoresis; Tandem mass spectrometry; Nuclear magnetic resonance spectroscopy; Hyphenated techniques

Applications of isotopes in advancing structural and functional heparanomics by Vy M. Tran; Thao K. N. Nguyen; Karthik Raman; Balagurunathan Kuberan (pp. 559-570).

Heparanomics is the study of all the biologically active oligosaccharide domain structures in the entire heparanome and the nature of the interactions among these domains and their protein ligands. Structural elucidation of heparan sulfate and heparin oligosaccharides is a major obstacle in advancing structure–function relationships and heparanomics. There are several factors that exacerbate the challenges involved in the structural elucidation of heparin and heparan sulfate; therefore, there is great interest in developing novel strategies and analytical tools to overcome the barriers in decoding the enigmatic heparanome. This review focuses on the applications of isotopes, both radioisotopes and stable isotopes, in the structural elucidation of the complex heparanome at the disaccharide or oligosaccharide level using liquid chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. This review also outlines the utility of isotopes in determining the substrate specificity of biosynthetic enzymes that eventually dictate the emergence of biologically active oligosaccharides.

Keywords: Heparin; Heparan sulfate; Proteoglycans; Glycosaminoglycans; High-performance liquid chromatography; Nuclear magnetic resonance; Mass spectrometry; Stable isotopes (¹³C; ³³S; ³⁴S); Radioisotopes (³H; ¹⁴C; ³⁵S); Sulfotransferases; Antithrombin III; Fibroblast growth factor

Applications of isotopes in advancing structural and functional heparanomics by Vy M. Tran; Thao K. N. Nguyen; Karthik Raman; Balagurunathan Kuberan (pp. 559-570).

Electrochemical heparin sensing at liquid/liquid interfaces and polymeric membranes by Shigeru Amemiya; Yushin Kim; Ryoichi Ishimatsu; Benjamin Kabagambe (pp. 571-579).

The monitoring of heparin and its derivatives in blood samples is important for the safe usage of these anticoagulants and antithrombotics in many medical procedures. Such an analytical task is, however, highly challenging due to their low therapeutic levels in the complex blood matrix, and it still relies on classical, indirect, clot-based assays. Here we review recent progress in the direct electrochemical sensing of heparin and its analogs at liquid/liquid interfaces and polymeric membranes. This progress has been made by utilizing the principle of electrochemical ion transfer at the interface between two immiscible electrolyte solutions (ITIES) to voltammetrically drive the interfacial transfer of polyanionic heparin and monitoring the resulting ionic current as a direct measure of heparin concentration. The sensitivity, selectivity, and reproducibility of the ion-transfer voltammetry of heparin are dramatically enhanced compared to those of traditional potentiometry. This voltammetric principle was successfully applied for the detection of heparin in undiluted blood samples, and was used to develop highly sensitive ion-selective electrodes based on thin polymeric membranes that are intended for analytical applications beyond heparin detection. The mechanism of heparin recognition and transfer at liquid/liquid interfaces was assessed quantitatively via sophisticated micropipet techniques, which aided the development of a powerful ionophore that can extract large heparin molecules into nonpolar organic media. Moreover, the reversible potentiometric detection of a lethal heparin-like contaminant in commercial heparin preparations was achieved through the use of a PVC membrane doped with methyltridodecylammonium chloride, which enables charge density dependent polyanion selectivity.

Keywords: Electroanalytical methods; Liquid/liquid interface; Ion-transfer voltammetry; Potentiometry; Ion selective electrodes; Molecular recognition

Electrochemical heparin sensing at liquid/liquid interfaces and polymeric membranes by Shigeru Amemiya; Yushin Kim; Ryoichi Ishimatsu; Benjamin Kabagambe (pp. 571-579).

Keywords: Electroanalytical methods; Liquid/liquid interface; Ion-transfer voltammetry; Potentiometry; Ion selective electrodes; Molecular recognition

Characterization of currently marketed heparin products: key tests for quality assurance by David A. Keire; Hongping Ye; Michael L. Trehy; Wei Ye; Richard E. Kolinski; Benjamin J. Westenberger; Lucinda F. Buhse; Moheb Nasr; Ali Al-Hakim (pp. 581-591).

During the 2007–2008 heparin crisis, it was found that the United States Pharmacopeia (USP) testing monograph for unfractionated heparin sodium (UFH) did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS) in heparin. In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to not only detect the contaminant OSCS but also to improve assurance of quality and purity of the drug product. Additional tests were also developed to monitor the heparin supply chain for other possible economically motivated additives or impurities. In 2009, a new USP monograph was put in place that includes 500 MHz ¹H NMR, SAX-HPLC, %galactosamine in total hexosamine, and anticoagulation time assays with purified factor IIa or factor Xa. These tests represent orthogonal approaches for UFH identification, measurement of bioactivity, and for detection of process impurities or contaminants in UFH. The FDA has applied these analytical approaches to the study of UFH active pharmaceutical ingredients in the marketplace. Here, we describe results from a comprehensive survey of UFH collected from seven different sources after the 2009 monograph revision and compare these data with results obtained on other heparin samples collected during the 2007–2008 crisis. Figure A plot the 1.90 to 2.30 ppm region of an overlay of the 500 MHz 1H-NMR spectra of 20 mg samples of a heparin sodium API alone or spiked with 1.0%, 5.0% or 10.0% weight percent of OSCS and the same API alone or containing 1.0%, 5.0% or 10.0% of DS. Signals associated with the presence of the spiked OSCS or DS in heparin are denoted.

Keywords: Heparin; OSCS; SAX-HPLC; NMR; CE; Factor IIa/Xa

NMR methods to monitor the enzymatic depolymerization of heparin by John F. K. Limtiaco; Szabolcs Beni; Christopher J. Jones; Derek J. Langeslay; Cynthia K. Larive (pp. 593-603).

Heparin and the related glycosaminoglycan, heparan sulfate, are polydisperse linear polysaccharides that mediate numerous biological processes due to their interaction with proteins. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, digestion to produce smaller oligosaccharides is commonly performed prior to separation and analysis. Current techniques used to monitor the extent of heparin depolymerization include UV absorption to follow product formation and size exclusion or strong anion exchange chromatography to monitor the size distribution of the components in the digest solution. In this study, we used ¹H nuclear magnetic resonance (NMR) survey spectra and NMR diffusion experiments in conjunction with UV absorption measurements to monitor heparin depolymerization using the enzyme heparinase I. Diffusion NMR does not require the physical separation of the components in the reaction mixture and instead can be used to monitor the reaction solution directly in the NMR tube. Using diffusion NMR, the enzymatic reaction can be stopped at the desired time point, maximizing the abundance of larger oligosaccharides for protein-binding studies or completion of the reaction if the goal of the study is exhaustive digestion for characterization of the disaccharide composition. In this study, porcine intestinal mucosa heparin was depolymerized using the enzyme heparinase I. The unsaturated bond formed by enzymatic cleavage serves as a UV chromophore that can be used to monitor the progress of the depolymerization and for the detection and quantification of oligosaccharides in subsequent separations. The double bond also introduces a unique multiplet with peaks at 5.973, 5.981, 5.990, and 5.998 ppm in the ¹H-NMR spectrum downfield of the anomeric region. This multiplet is produced by the proton of the C-4 double bond of the non-reducing end uronic acid at the cleavage site. Changes in this resonance were used to monitor the progression of the enzymatic digestion and compared to the profile obtained from UV absorbance measurements. In addition, in situ NMR diffusion measurements were explored for their ability to profile the different-sized components generated over the course of the digestion. Figure DOSY spectra of intact (blue) and digested (red) heparin illustrating the differences in their diffusion coefficients.

Keywords: DOSY; Diffusion NMR; Heparinase; SEC; Heparin; Heparan sulfate

NMR methods to monitor the enzymatic depolymerization of heparin by John F. K. Limtiaco; Szabolcs Beni; Christopher J. Jones; Derek J. Langeslay; Cynthia K. Larive (pp. 593-603).

Keywords: DOSY; Diffusion NMR; Heparinase; SEC; Heparin; Heparan sulfate

Comparison of established and novel purity tests for the quality control of heparin by means of a set of 177 heparin samples by Susanne Alban; Susanne Lühn; Simone Schiemann; Tanja Beyer; Jochen Norwig; Claudia Schilling; Oliver Rädler; Bernhard Wolf; Magnus Matz; Knut Baumann; Ulrike Holzgrabe (pp. 605-620).

The widespread occurrence of heparin contaminated with oversulfated chrondroitin sulfate (OSCS) in 2008 initiated a comprehensive revision process of the Pharmacopoeial heparin monographs and stimulated research in analytical techniques for the quality control of heparin. Here, a set of 177 heparin samples from the market in 2008 as well as pure heparin sodium spiked with defined amounts of OSCS and DS were used to evaluate established and novel methods for the quality control of heparin. Besides ¹H nuclear magnetic resonance spectroscopy (NMR), the assessment included two further spectroscopic methods, i.e., attenuated total reflection-infrared spectroscopy (ATR-IR) and Raman spectroscopy, three coagulation assays, i.e., activated partial thromboplastin time (aPTT) performed with both sheep and human plasma and the prothrombin time (PT), and finally two novel purity assays, each consisting of an incubation step with heparinase I followed by either a fluorescence measurement (Inc-PolyH-assay) or by a chromogenic aXa-assay (Inc-aXa-assay). NMR was shown to allow not only sensitive detection, but also quantification of OSCS by using the peak-height method and a response factor determined by calibration. Chemometric evaluation of the NMR, ATR-IR, and Raman spectra by statistical classification techniques turned out to be best with NMR spectra concerning the detection of OSCS. The validity of the aPTT, the current EP assay, could be considerably improved by replacing the sheep plasma by human plasma. In this way, most of the contaminated heparin samples did not meet the novel potency limit of 180 IU/mg. However, also more than 50% of the uncontaminated samples had <180 IU/MG. In contrast to the aPTT, the PT specifically detects OSCS and other heparin mimetics (LOD 3%). About ten times more sensitive are both the Inc-PolyH-assay and the Inc-aXa-assay, two rapid and simple quantification assays for heparin mimetics. The determined OSCS contents of the heparin samples excellently correlated with those calculated from the NMR spectra. In conclusion, NMR proved to be the current spectroscopic method of choice. The two two-step-assays represent options to supplement NMR, especially as tests for the initial screening, since they detect any heparin mimetic without requiring special expertise for interpretation of the results.

Keywords: Heparin; OSCS; ¹H NMR; Chemometric classification; Two-step-fluorescence assay; Two-step-aXa-assay; Coagulation assays; Raman spectroscopy; ATR-IR spectroscopy; Purity tests

¹H NMR signal at 2.10 ppm in the spectrum of KMnO₄-bleached heparin sodium: identification of the chemical origin using an NMR-only approach by E. Kellenbach; K. Sanders; P. J. A. Michiels; F. C. Girard (pp. 621-628).

The recently revised European Pharmacopeia and US Pharmacopeia heparin sodium monographs include nuclear magnetic resonance (NMR) tests on both identity and purity. In KMnO₄-bleached heparin, an unidentified NMR signal is present at 2.10 ppm at a level of 15–20% of the mean of signal height of the major glucosamine (GlcNAc/GlcNS,6S) anomeric proton signal at 5.42 ppm and of the major iduronic acid (IdoA2S) anomeric proton signal at 5.21 ppm. According to the new monographs, no unidentified signals greater than 4% should be detected at that position. Thus, the material did not meet the acceptance criterion. The signal at 2.10 ppm has been present at the same level in all released MSD KMnO₄-bleached heparin sodium batches analyzed over the past 10 years. The signal is a result of the KMnO₄ bleaching. No (oversulfated) chondroitin sulfate or dermatan sulfate was detected in this material. A comprehensive NMR study using long-range heteronuclear 2D techniques identifies this signal at 2.10 ppm as originating from the acetyl methyl group of (6-sulfated) 2-N-acetyl-2-deoxy-glucono-1,5-lactone. This modified monosaccharide is formed by the KMnO₄ oxidation of the reducing end of a terminal N-acetylglucosamine.

Keywords: Heparin sodium; Chondroitin sulfate; Dermatan sulfate; Glycosaminoglycans; ¹H NMR spectroscopy; Pharmacopeia

Quantification of chondroitin sulfate and dermatan sulfate in danaparoid sodium by ¹H NMR spectroscopy and PLS regression by B. Üstün; K. B. Sanders; P. Dani; E. R. Kellenbach (pp. 629-634).

Danaparoid sodium (the active pharmaceutical ingredient in Orgaran; Merck Sharp and Dohme) is a biopolymeric non-heparin drug used as anticoagulant and antithrombotic agent approved for the prophylaxis of post-operative deep-vein thrombosis, which may lead to pulmonary embolism in patients undergoing, e.g., elective hip replacement surgery. It consists of a mixture of three glycosaminoglycans (GAGs): heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate (CS). Currently, the CS and DS content are quantified by means of a time-consuming enzymatic method. In this paper the use of ¹H NMR in combination with multivariate regression (partial least-squares, PLS) is proposed as a new method. In order to evaluate the proposed method, a series of danaparoid sodium samples were analyzed and the results were compared with those obtained by the enzymatic method (reference method). The results showed that the proposed ¹H NMR method is a good alternative for analysis of CS and DS in danaparoid sodium. Accuracy of ±0.7% (w/w) and ±1.1% (w/w) for CS and DS was obtained by the ¹H NMR method and accuracy of ±1.0% (w/w) and ±1.3% (w/w) by the enzymatic method. Furthermore, the use of ¹H NMR in combination with PLS results in a fast quantification. The analysis time is reduced to 35 min per sample instead of 60 h for a maximum of 16 samples.

Keywords: Danaparoid sodium; Heparan sulfate; Chondroitin sulfate; Dermatan sulfate; Glycosaminoglycans; ¹H NMR spectroscopy; Chemometrics; Partial least-squares regression

Quantification of chondroitin sulfate and dermatan sulfate in danaparoid sodium by ¹H NMR spectroscopy and PLS regression by B. Üstün; K. B. Sanders; P. Dani; E. R. Kellenbach (pp. 629-634).

Keywords: Danaparoid sodium; Heparan sulfate; Chondroitin sulfate; Dermatan sulfate; Glycosaminoglycans; ¹H NMR spectroscopy; Chemometrics; Partial least-squares regression

Determination of galactosamine impurities in heparin samples by multivariate regression analysis of their ¹H NMR spectra by Qingda Zang; David A. Keire; Richard D. Wood; Lucinda F. Buhse; Christine M. V. Moore; Moheb Nasr; Ali Al-Hakim; Michael L. Trehy; William J. Welsh (pp. 635-649).

Heparin, a widely used anticoagulant primarily extracted from animal sources, contains varying amounts of galactosamine impurities. Currently, the United States Pharmacopeia (USP) monograph for heparin purity specifies that the weight percent of galactosamine (%Gal) may not exceed 1%. In the present study, multivariate regression (MVR) analysis of ¹H NMR spectral data obtained from heparin samples was employed to build quantitative models for the prediction of %Gal. MVR analysis was conducted using four separate methods: multiple linear regression, ridge regression, partial least squares regression, and support vector regression (SVR). Genetic algorithms and stepwise selection methods were applied for variable selection. In each case, two separate prediction models were constructed: a global model based on dataset A which contained the full range (0–10%) of galactosamine in the samples and a local model based on the subset dataset B for which the galactosamine level (0–2%) spanned the 1% USP limit. All four regression methods performed equally well for dataset A with low prediction errors under optimal conditions, whereas SVR was clearly superior among the four methods for dataset B. The results from this study show that ¹H NMR spectroscopy, already a USP requirement for the screening of contaminants in heparin, may offer utility as a rapid method for quantitative determination of %Gal in heparin samples when used in conjunction with MVR approaches.

Keywords: Heparin; Galactosamine impurities; Proton nuclear magnetic resonance (¹H NMR); Multivariate regression (MVR); Variable selection

NMR of heparin API: investigation of unidentified signals in the USP-specified range of 2.12–3.00 ppm by Sarah E. Lee; Edward K. Chess; Barrett Rabinow; G. Joseph Ray; Christina M. Szabo; Bennett Melnick; Reagan L. Miller; Lakshmy M. Nair; Edwin G. Moore (pp. 651-662).

This article addresses the identification and quantification of the chemical species resulting in resonances at 2.17 and 2.25 ppm in the ¹H nuclear magnetic resonance (NMR) spectrum of pharmaceutical-grade heparin sodium. The NMR signals in question were first confirmed to arise from chemical moieties covalently attached to the heparin molecule through NMR diffusion experiments as well as chemical treatment of heparin active pharmaceutical ingredient (API) containing the resonances. The material responsible for the extra NMR signals was then demonstrated by NMR spiking studies to be something other than oversulfated chondroitin sulfate and was finally identified as an O-acetylation product of heparin through ¹³C labeling experiments with subsequent NMR analysis. The extent of O-acetylation was quantified using three orthogonal techniques: ¹H NMR, ion chromatography, and headspace gas chromatography/mass spectrometry. The results of this work showed good agreement between the three quantitative methods developed to analyze the signals in the United States Pharmacopeia-specified region of 2.12–3.00 ppm for heparin API.

Keywords: Heparin; Acetylation; NMR; Isotopic labeling

Determination of the primary structure and carboxyl pK _As of heparin-derived oligosaccharides by band-selective homonuclear-decoupled two-dimensional ¹H NMR by Khanh Nguyen; Dallas L. Rabenstein (pp. 663-671).

Determination of the structure of heparin-derived oligosaccharides by ¹H NMR is challenging because resonances for all but the anomeric protons cover less than 2 ppm. By taking advantage of increased dispersion of resonances for the anomeric H¹ protons at low pD and the superior resolution of band-selective, homonuclear-decoupled (BASHD) two-dimensional ¹H NMR, the primary structure of the heparin-derived octasaccharide ∆UA(2S)-[(1 → 4)-GlcNS(6S)-(1 → 4)-IdoA(2S)-]₃-(1 → 4)-GlcNS(6S) has been determined, where ∆UA(2S) is 2-O-sulfated ∆^4,5-unsaturated uronic acid, GlcNS(6S) is 6-O-sulfated, N-sulfated β-d-glucosamine and IdoA(2S) is 2-O-sulfated α-l-iduronic acid. The spectrum was assigned, and the sites of N- and O-sulfation and the conformation of each uronic acid residue were established, with chemical shift data obtained from BASHD-TOCSY spectra, while the sequence of the monosaccharide residues in the octasaccharide was determined from inter-residue NOEs in BASHD-NOESY spectra. Acid dissociation constants were determined for each carboxylic acid group of the octasaccharide, as well as for related tetra- and hexasaccharides, from chemical shift–pD titration curves. Chemical shift–pD titration curves were obtained for each carboxylic acid group from sub-spectra taken from BASHD-TOCSY spectra that were measured as a function of pD. The pK _As of the carboxylic acid groups of the ∆UA(2S) residues are less than those of the IdoA(2S) residues, and the pK _As of the carboxylic acid groups of the IdoA(2S) residues for a given oligosaccharide are similar in magnitude. Relative acidities of the carboxylic acid groups of each oligosaccharide were calculated from chemical shift data by a pH-independent method.

Keywords: Heparin; Heparin octasaccharide; Oligosaccharides; Acid dissociation constants; Carboxylate pK _A ; BASHD-NMR; NMR

Simple fluorescence assay for quantification of OSCS in heparin by Susanne Lühn; Simone Schiemann; Susanne Alban (pp. 673-680).

In 2008, heparin contaminated with oversulfated chondroitin sulfate (OSCS) penetrated the worldwide market and was associated with severe adverse effects. Feasible and reliable methods to test heparin for adulteration are needed. The objective was to develop a simple approach based on a microplate assay for quantification of heparin and sulfated glycans using the fluorescent heparin sensor polymer-H (polymer-H assay). However, both heparin and OSCS concentration-dependently increase the fluorescence intensity (FI) of polymer-H, so that OSCS in heparin cannot be detected. The idea was a two-step procedure including, first, separation of heparin by degradation with heparinase I, and then measurement of the remaining OSCS. To achieve complete heparin (unfractionated heparin (UFH), enoxaparin) degradation, several conditions (e.g. incubation time and heparinase I concentration) were optimized by using the aXa assay for monitoring. Defined UFH/OSCS mixtures incubated in this way showed a concentration-dependent FI increase in the polymer-H assay (λ _(em) 330 nm, λ _(ex) 510 nm). The sensitivity was unexpectedly high with an LOD/LOQ of 0.5%/0.6% OSCS content in heparin. Further experiments testing UFH/OSCS mixtures in the aXa assay confirmed our hypothesis: OSCS inhibits heparinase I resulting in incomplete heparin degradation and thus an additional FI increase of polymer-H by intact heparin. This two-step microplate fluorescence assay is a sensitive, rapid, and simple method for quantification of OSCS in heparin. In contrast with ¹H NMR and CE, neither expensive equipment nor much experience are required. It could be applied not only in the quality control of heparin, but also in clinical practice, to check the applied heparin preparation when a patient suffers any adverse effect. Figure Assay principle of the two-step fluorescence assay for the quantification of OSCS in heparin. OSCS inhibits heparinase I resulting in incomplete heparin degradation and thus an additional FI increase of polymer-H by intact heparin. This two-step microplate fluorescence assay is a sensitive, rapid, and simple method for quantification of OSCS in heparin. FI = fluorescence intensity

Keywords: Heparin; Quantification; Counterfeit drugs; Quality assurance/control; OSCS; Fluorescence assay

Simple fluorescence assay for quantification of OSCS in heparin by Susanne Lühn; Simone Schiemann; Susanne Alban (pp. 673-680).

Keywords: Heparin; Quantification; Counterfeit drugs; Quality assurance/control; OSCS; Fluorescence assay

Combination of a two-step fluorescence assay and a two-step anti-Factor Xa assay for detection of heparin falsifications and protein in heparins by Susanne Alban; Susanne Lühn; Simone Schiemann (pp. 681-690).

There are several methods for sensitive detection of oversulfated chondroitin sulfate (OSCS) in heparin. Although contamination with OSCS is unlikely to be repeated, use of other compounds to counterfeit heparin must be considered. We have previously developed a two-step fluorescence microplate assay (two-step FI assay) for detection of OSCS. First, the heparin sample is incubated with heparinase I, then its increasing effect on the fluorescence intensity (FI) of the sensor molecule Polymer-H is measured (PolyH assay). The high sensitivity of the assay is shown to be based on heparinase I inhibition by OSCS. The objective of this study was to evaluate another assay option — indirect quantification of OSCS after heparinase I incubation by means of the anti-Factor Xa (aXa) activity of the remaining undegraded heparin (two-step aXa assay). We also examined, whether other heparin mimetics (HepM), direct Factor Xa inhibitors (DXI), and protein impurities are detectable by use of these assays. Heparin was spiked with different amounts of HepM including OSCS, pentosan polysulfate, dextran sulfate, curdlan sulfate, the natural contaminant dermatan sulfate, the DXI rivaroxaban, and BSA as a protein. These samples were compared with pure heparin in the two-step FI assay, the two-step aXa assay, and in the PolyH assay and the aXa assay without heparinase I incubation. Both two-step assays sensitively measured contamination with all the HepM (LOD ≤ 0.5%, LOQ ≤ 0.7%). The two-step aXa assay also detected rivaroxaban (LOD 0.3%, LOQ 0.4%), whereas the two-step FI assay was shown to be suited to determination of protein impurities (LOD 0.11%, LOQ 0.13%). Use of two different heparinase I inactivation procedures enabled clear differentiation between protein, HepM, and both contaminants. Finally, with the aXa assay the heparin potency can be determined in the same assay run, whereas the FI increase in the PolyH assay was shown to be useful for identification. In conclusion, both the two-step FI assay and the two-step aXa assay are sensitive, rapid, and simple tests for the detection of counterfeit heparin. Comprehensive information about heparin quality can be obtained by their combined use and the parallel measurement of non-incubated heparin samples.

Keywords: Heparin; Oversulfated contaminants; Direct factor Xa inhibitors; Protein; aXa assay; Fluorescence assay; Heparinase

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs by Vanessa Castro-López; Leanne F. Harris; James S. O’Donnell; Anthony J. Killard (pp. 691-700).

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 μM of the fluorogenic substrate Mes-D-LGR-ANSN (C₂H₅)₂ (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0–1.6 U mL⁻¹. The statistically sensitive assay range was 0–0.4 U mL⁻¹ for enoxaparin and tinzaparin and 0–0.2 U mL⁻¹ for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-d-cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.

Keywords: FXa; Fluorogenic substrate; AMC; ANSN; Low molecular weight heparins; Danaparoid

The use of circular dichroism as a simple heparin-screening strategy by F. E. Stanley; A. M. Stalcup (pp. 701-706).

Heparin, a heterogeneous polysaccharide, is used extensively as an anticoagulant. Recently, however, tainted heparin was associated with acute reactions that lead to numerous deaths. Extensive investigations ultimately showed oversulfated chondroitin sulfate, a semi-synthetic polysaccharide, to be present in the contaminated samples. These events highlighted the need for new, convenient heparin-screening methods capable of rapidly determining sample purity. In this work, we report the use of circular dichroism spectroscopy to analyze heparin samples for the presence of heparin-like adulterants (e.g., chondroitin sulfate A, dermatan sulfate, and oversulfated chondroitin sulfate) in a simple and straightforward manner. This strategy exploits the subtle differences in the optical properties of each polymer; these differences result from structural dissimilarities. To the best of our knowledge, the findings presented here are the first report of heparin purity screening using traditional spectropolarimetry techniques.

Keywords: Chondroitin sulfate A; Circular dichroism; Heparin; Glycosaminoglycan; Oversulfated chondroitin sulfate

The use of circular dichroism as a simple heparin-screening strategy by F. E. Stanley; A. M. Stalcup (pp. 701-706).

Keywords: Chondroitin sulfate A; Circular dichroism; Heparin; Glycosaminoglycan; Oversulfated chondroitin sulfate

The development of an FIA-CD strategy for screening sulfated polysaccharides using antimalarial drugs and related species as probes by F. E. Stanley; A. M. Warner; A. M. Stalcup (pp. 707-716).

Heterogeneous sulfated polysaccharides have attracted significant attention in light of their various biological activities. However, recent events involving heparin have dramatically illustrated that several analytical challenges exist in accounting for such species. In this case, tainted heparin was associated with acute reactions that lead to numerous deaths. Researchers were forced to use time-consuming, sophisticated techniques (e.g., enzymatic digestion, NMR, CE, HPLC, MS, etc.) to identify the cause of these adverse effects. Extensive investigations ultimately showed oversulfated chondroitin sulfate, a semi-synthetic sulfated polysaccharide, to be present in the contaminated samples. These events highlighted the need for a new generation of screening techniques. In this work, we report the development of a screening strategy that exploits unique circular dichroism features observed as a function of association between investigated polymers and judiciously selected probe molecules (i.e., chloroquine, N1-(7-chloro-4-quinolinyl)-N3-methyl-1,3-propanediamine, quinacrine, and N2-9-acridinyl-N1,N1-dimethyl-1,2-ethanediamine). Application of obtained spectropolarimetry results to a flow injection analysis circular dichroism platform allowed for the establishment of linear polysaccharide response curves for dextran sulfate, heparin, and oversulfated chondroitin sulfate in the low micromolar range. Lastly, through additional work with heparin, the proposed method was shown to be capable of rapidly screening sulfated polysaccharide samples for closely related impurities.

Keywords: Circular dichroism; Dextran sulfate; Flow injection analysis; Heparin; Oversulfated chondroitin sulfate

The development of an FIA-CD strategy for screening sulfated polysaccharides using antimalarial drugs and related species as probes by F. E. Stanley; A. M. Warner; A. M. Stalcup (pp. 707-716).

Keywords: Circular dichroism; Dextran sulfate; Flow injection analysis; Heparin; Oversulfated chondroitin sulfate

Development and qualification of a size exclusion chromatography coupled with multiangle light scattering method for molecular weight determination of unfractionated heparin by John Beirne; Hilary Truchan; Lin Rao (pp. 717-725).

The molecular weight of unfractionated heparin was determined by size exclusion chromatography (SEC) coupled with multiangle light scattering (MALS) detection. The SEC/MALS method determines absolute molecular weight directly from the angular dependence of scattered light intensity as a function of concentration and does not rely on molecular weight standards for column calibration. The SEC/MALS method developed at Scientific Protein Laboratories was qualified in terms of specificity, precision, robustness, and accuracy. By eliminating the requirement of well-characterized molecular weight standards derived from heparin, the present procedure represents a clear improvement over the column calibration methods used in molecular weight determination. The SEC/MALS method is suitable for routine quality control of unfractionated heparin.

Keywords: Heparin; Molecular weight and molecular weight distribution; SEC/MALS; dn/dc ; Second virial coefficient A ₂

Targeted analysis of glycomics liquid chromatography/mass spectrometry data by Jonathan M. Dreyfuss; Christopher Jacobs; Yevgeniy Gindin; Gary Benson; Gregory O. Staples; Joseph Zaia (pp. 727-735).

Hydrophilic interaction chromatography (HILIC) liquid chromatography/mass spectrometry (LC/MS) is appropriate for all native and reductively aminated glycan classes. HILIC carries the advantage that retention times vary predictably according to oligosaccharide composition. Chromatographic conditions are compatible with sensitive and reproducible glycomics analysis of large numbers of samples. The data are extremely useful for quantitative profiling of glycans expressed in biological tissues. With these analytical developments, the rate-limiting factor for widespread use of HILIC LC/MS in glycomics is the analysis of the data. In order to eliminate this problem, a Java-based open source software tool, Manatee, was developed for targeted analysis of HILIC LC/MS glycan datasets. This tool uses user-defined lists of compositions that specify the glycan chemical space in a given biological context. The program accepts high-resolution LC/MS data using the public mzXML format and is capable of processing a large data file in a few minutes on a standard desktop computer. The program allows mining of HILIC LC/MS data with an output compatible with multivariate statistical analysis. It is envisaged that the Manatee tool will complement more computationally intensive LC/MS processing tools based on deconvolution and deisotoping of LC/MS data. The capabilities of the tool were demonstrated using a set of HILIC LC/MS data on organ-specific heparan sulfates. Figure Unsupervised heat map of the 25 most abundant HS compositions among 5 bovine organs with hierarchical clustering of rows and columns

Keywords: Glycomics; Glycosaminoglycan; Heparan sulfate; Mass spectrometry; Bioinformatics; Proteoglycan

Targeted analysis of glycomics liquid chromatography/mass spectrometry data by Jonathan M. Dreyfuss; Christopher Jacobs; Yevgeniy Gindin; Gary Benson; Gregory O. Staples; Joseph Zaia (pp. 727-735).

Keywords: Glycomics; Glycosaminoglycan; Heparan sulfate; Mass spectrometry; Bioinformatics; Proteoglycan

Analysis of E. coli K5 capsular polysaccharide heparosan by Mellisa Ly; Zhenyu Wang; Tatiana N. Laremore; Fuming Zhang; Weihong Zhong; Dennis Pu; Dmitri V. Zagorevski; Jonathan S. Dordick; Robert J. Linhardt (pp. 737-745).

Heparosan is the key precursor for the preparation of bioengineered heparin, a potential replacement for porcine intestinal heparin, an important anticoagulant drug. The molecular weight (MW) distribution of heparosan produced by the fermentation of E. coli K5 was investigated. Large-slab isocratic and mini-slab gradient polyacrylamide gel electrophoresis (PAGE) were used to analyze the MW and polydispersity of heparosan. A preparative method that allowed fractionation by continuous-elution PAGE was used to obtain heparosan MW standards. The MWs of the heparosan standards were determined by electrospray ionization Fourier-transform mass spectrometry (ESI-FT-MS). A ladder of the standards was then used to determine the MW properties of polydisperse heparosan samples. Unbleached and bleached heparosan produced by fermentation of E. coli K5 had similar number-averaged MWs (M_N), weight-averaged MWs (M_W), and MW ranges of 3,000 to 150,000 Da.

Keywords: E. coli K5 capsular polysaccharide; Heparosan; Molecular weight determination; PAGE; ESI-FT-MS; Continuous-elution preparative electrophoresis

New developments in quantitative polymerase chain reaction applied to control the quality of heparins by Cécile Auguste; Stéphanie Dereux; Céline Martinez; Pascal Anger (pp. 747-755).

Heparin is a widely used intravenous anticoagulant comprised of a very complex mixture of glucosaminoglycan chains, mainly derived from porcine intestinal mucosa. Recent contamination of heparin with oversulfated (OS) chondroitin sulfate resulted in a significant number of deaths, triggering a rapid revision of product monographs and the introduction of new analytical methods to limit as far as possible the chances of another occurrence of such a phenomenon. The distribution of heparin-processing units across the globe prevents their complete fool-proof auditing. Therefore, the implementation of additional orthogonal analytical techniques for quality control (QC) of heparin batches is highly important. We perform routine quantitative polymerase chain reaction (Q-PCR) release tests to confirm the quality of all crude heparin batches received by sanofi-aventis. The routine test used provides information on the animal species of origin as requested by the US Pharmacopoeia (USP) and European Pharmacopoiea monographs. Here, we demonstrate that the Q-PCR test is inhibited by OS glycosaminoglycans at concentrations as low as 0.5% (w/w versus heparin) and can be used as an additional safeguard to monitor levels of potentially harmful contaminants without any increased workload. In response to a request from the USP, we also describe the development of a Q-PCR method for monitoring nucleotidic impurities in pure heparin, which is able to detect amplifiable DNA at concentrations lower than 0.1 ng DNA per milligram of heparin. This increased sensitivity makes this modified Q-PCR method a potential candidate for inclusion as a QC requirement in future monographs. Monitoring levels of oversulfated chondroitin sulfate (OSCS) contaminants in heparin by routine-release Q-PCR with an internal positive control

Keywords: PCR; Quality assurance/control; Heparin; Contamination

New developments in quantitative polymerase chain reaction applied to control the quality of heparins by Cécile Auguste; Stéphanie Dereux; Céline Martinez; Pascal Anger (pp. 747-755).

Keywords: PCR; Quality assurance/control; Heparin; Contamination

A quantitative PCR method to quantify ruminant DNA in porcine crude heparin by Sean P. Concannon; P. Brett Wimberley; Wesley E. Workman (pp. 757-762).

Heparin is a well-known glycosaminoglycan extracted from porcine intestines. Increased vigilance for transmissible spongiform encephalopathy in animal-derived pharmaceuticals requires methods to prevent the introduction of heparin from ruminants into the supply chain. The sensitivity, specificity, and precision of the quantitative polymerase chain reaction (PCR) make it a superior analytical platform for screening heparin raw material for bovine-, ovine-, and caprine-derived material. A quantitative PCR probe and primer set homologous to the ruminant Bov-A2 short interspersed nuclear element (SINE) locus (Mendoza-Romero et al. J. Food Prot. 67:550–554, 2004) demonstrated nearly equivalent affinities for bovine, ovine, and caprine DNA targets, while exhibiting no cross-reactivity with porcine DNA in the quantitative PCR method. A second PCR primer and probe set, specific for the porcine PRE1 SINE sequence, was also developed to quantify the background porcine DNA level. DNA extraction and purification was not necessary for analysis of the raw heparin samples, although digestion of the sample with heparinase was employed. The method exhibits a quantitation range of 0.3–3,000 ppm ruminant DNA in heparin. Validation parameters of the method included accuracy, repeatability, precision, specificity, range, quantitation limit, and linearity.

Keywords: Heparin; Ruminant; Porcine; DNA; Quantitative polymerase chain reaction; Transmissible spongiform encephalopathy

A quantitative PCR method to quantify ruminant DNA in porcine crude heparin by Sean P. Concannon; P. Brett Wimberley; Wesley E. Workman (pp. 757-762).

Keywords: Heparin; Ruminant; Porcine; DNA; Quantitative polymerase chain reaction; Transmissible spongiform encephalopathy

Protamine neutralisation of low molecular weight heparins and their oligosaccharide components by Mette Schroeder; John Hogwood; Elaine Gray; Barbara Mulloy; Anne-Marie Hackett; Kristian B. Johansen (pp. 763-771).

Protamine sulphate is an effective inhibitor of heparin and is used clinically to neutralise both low molecular weight heparins (LMWH) and unfractionated heparin (UFH). However, protamine sulphate does not fully counter the anti-Xa effect of LMWH, even in excess (>40 μg to 1 IU/ml). To investigate the molecular basis for this observation, the residual potencies in the presence and absence of plasma as well as the molecular weight profiles of commercial LMWH neutralised with increasing amounts of protamine were measured. Materials over 5000 Da are preferentially neutralised by protamine. To further investigate this molecular weight dependence, monodisperse oligosaccharides were prepared from three commercial LMWHs. The specific anti-Xa activity for the fractions increased with molecular weight, and was found to vary between the three preparations for oligosaccharides of the same molecular weight. Our results indicate that protamine sulphate neutralisation is largely dependent on molecular weight, leading to the implication that LMWHs containing a larger proportion of small oligosaccharides will not be as effectively neutralised. Protamine sulphate neutralisation of any given LMWH is also affected by the specific anticoagulant activities of its low molecular weight components, which varies between LMWH products, presumably with the method of manufacture.

Keywords: Protamine sulphate; Low molecular weight heparin; Oligosaccharides; Neutralisation; Anti-Xa activity

Heparin-immobilized microspheres for the capture of cytokines by Jia Duo; Julie A. Stenken (pp. 773-782).

The preparation and characterization of heparin-immobilized microspheres which were used to bind acidic fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), monocyte chemoattractant protein 1 (MCP-1/CCL2), and regulation upon activation normal T cell express sequence (RANTES/CCL5) is described. These beads were used as trapping agents in microdialysis sampling experiments in a separate study. Both free heparin and a synthesized heparin–albumin conjugate were immobilized onto microspheres and compared for their effectiveness. The heparin–albumin conjugate microspheres exhibited significant nonspecific adsorption which appeared to be due to the albumin content. The prepared heparin-immobilized microspheres were stable for 3 months at 4 °C. A bead-based flow cytometric assay was developed to study the binding capacity and specificity of the heparin-immobilized microspheres to cytokines. These heparin-immobilized microspheres exhibited broad dynamic ranges for binding to the four cytokines (aFGF, 1.0–1,000 ng/mL; VEGF, 0.5–1,000 ng/mL; CCL2, 1.95–1,000 ng/mL; CCL5, 1.95–500 ng/mL). Fast binding kinetics of the cytokines to the heparin-immobilized beads suggests that these beads may be useful as affinity agents in microfluidic flow systems. Figure Heparin-immobilized microspheres for cytokine binding analysis

Keywords: Heparin-immobilized microspheres; Cytokines; Flow cytometry

Heparin-immobilized microspheres for the capture of cytokines by Jia Duo; Julie A. Stenken (pp. 773-782).

Keywords: Heparin-immobilized microspheres; Cytokines; Flow cytometry

In vitro and in vivo affinity microdialysis sampling of cytokines using heparin-immobilized microspheres by Jia Duo; Julie A. Stenken (pp. 783-793).

Heparin-immobilized microspheres were included in microdialysis sampling perfusion fluids under both in vitro and in vivo conditions to improve the recovery of different cytokines, acidic fibroblast growth factor, vascular endothelial growth factor, monocyte chemoattractant protein-1 (or CCL2), and regulation upon activation normal T cell express sequence (or CCL5). Different strategies to dissociate captured CCL2 and CCL5 from the immobilized heparin were attempted, and both cytokines could be quantitatively eluted from the beads using a phosphate buffer (pH 7.4) containing 25% (v/v) acetonitrile which did not interfere with the subsequent detection of cytokine using an ELISA assay. Using these heparin-immobilized microspheres, a two to fivefold increase of microdialysis relative recovery (RR) was achieved for the four cytokines from a quiescent solution. Enhanced microdialysis RR of CCL2 using the heparin-immobilized microspheres from microdialysis probes implanted into the peritoneal cavity of a rat was performed to test the in vivo application. This work suggests that the heparin-immobilized microspheres provide an alternative affinity agent to the previously used antibody-immobilized microspheres for enhanced microdialysis sampling of cytokines. Figure Microdialysis sampling with heparin-immobilized microspheres has been used to capture cytokines aFGF, CCL2, CCL5, and VEGF

Keywords: Cytokines; Microdialysis sampling; Heparin-immobilized microspheres

In vitro and in vivo affinity microdialysis sampling of cytokines using heparin-immobilized microspheres by Jia Duo; Julie A. Stenken (pp. 783-793).

Keywords: Cytokines; Microdialysis sampling; Heparin-immobilized microspheres

Application of fully automated online solid phase extraction-liquid chromatography-electrospray-tandem mass spectrometry for the determination of sulfonamides and their acetylated metabolites in groundwater by M. Jesús García-Galán; Teresa Garrido; Josep Fraile; Antoni Ginebreda; M. Silvia Díaz-Cruz; Damià Barceló (pp. 795-806).

The present study describes an automated methodology based on a liquid chromatography-electrospray, tandem mass spectrometry method combined with online solid phase extraction (online SPE-LC-ESI-MS/MS) for the simultaneous analysis of 16 sulfonamides (SAs) and five of their acetylated metabolites in groundwater. The evaluation of the degree of SA pollution in groundwater was made through the analysis of a total of 39 samples taken in seven groundwater bodies of Catalonia (Spain). Recovery values obtained ranged from 34.3% (N ⁴-acetylsulfadiazine) to 134.4% (sulfabenzamide). The method limits of detection for all the analytes were 0.09–11 ng L⁻¹. Sulfamethoxazole was the SA detected more frequently (56.4% of the samples), with an average concentration of 2.3 ng L⁻¹, followed by sulfadimethoxine, present in 54% of the samples with an average concentration of 0.2 ng L⁻¹. It should be highlighted that the acetylated metabolites were ubiquitous in the different samples, with frequencies of detection up to 36% and maximum concentrations of 18 ng L⁻¹ (N ⁴-acetylsulfamerazine).

Keywords: Groundwater; Pharmaceuticals; Sulfonamides and metabolites; HPLC

Cleanup strategies and advantages in the determination of several therapeutic classes of pharmaceuticals in wastewater samples by SPE–LC–MS/MS by M. A. Sousa; C. Gonçalves; E. Cunha; J. Hajšlová; M. F. Alpendurada (pp. 807-822).

This work describes the development and validation of an offline solid-phase extraction with simultaneous cleanup capability, followed by liquid chromatography–(electrospray ionisation)–ion trap mass spectrometry, enabling the concurrent determination of 23 pharmaceuticals of diverse chemical nature, among the most consumed in Portugal, in wastewater samples. Several cleanup strategies, exploiting the physical and chemical properties of the analytes vs. interferences, alongside with the use of internal standards, were assayed in order to minimise the influence of matrix components in the ionisation efficiency of target analytes. After testing all combinations of adsorbents (normal-phase, ion exchange and mixed composition) and elution solvents, the best results were achieved with the mixed-anion exchange Oasis MAX cartridges. They provided recovery rates generally higher than 60%. The precision of the method ranged from 2% to 18% and 4% to 19% (except for diclofenac (22%) and simvastatin (26%)) for intra- and inter-day analysis, respectively. Method detection limits varied between 1 and 20 ng L⁻¹, while method quantification limits were <85 ng L⁻¹ (both excluding ibuprofen). This analytical method was applied to gather preliminary results on influents and effluents of two wastewater treatment plants (WWTPs) located in the urban region of Porto (Portugal). Typically, paracetamol, hydrochlorothiazide, furosemide, naproxen, ibuprofen, diclofenac and bezafibrate were detected in concentrations ranging from 1 to 20 μg L⁻¹, while gemfibrozil, simvastatin, ketoprofen, azithromycin, bisoprolol, lorazepam and paroxetine were quantified in levels below 1 μg L⁻¹. These WWTPs were given particular attention since they discharge their effluents into the Douro river, where water is extracted for the production of drinking water. Some sampling spots in this river were also analysed.

Keywords: Pharmaceuticals; Wastewater; Cleanup; MAX cartridges; Liquid chromatography–tandem mass spectrometry (LC–MS/MS); Surface water

Shotgun lipidomics for candidate biomarkers of urinary phospholipids in prostate cancer by Hye Kyeong Min; Sangsoo Lim; Bong Chul Chung; Myeong Hee Moon (pp. 823-830).

Qualitative and quantitative profiling of six different categories of urinary phospholipids (PLs) from patients with prostate cancer was performed to develop an analytical method for the discovery of candidate biomarkers by shotgun lipidomics method. Using nanoflow liquid chromatography–electrospray ionization–tandem mass spectrometry, we identified the molecular structures of a total of 70 PL molecules (21 phosphatidylcholines (PCs), 11 phosphatidylethanolamines (PEs), 17 phosphatidylserines (PSs), 11 phosphatidylinositols (PIs), seven phosphatidic acids, and three phosphatidylglycerols) from urine samples of healthy controls and prostate cancer patients by data-dependent collision-induced dissociation. Identified molecules were quantitatively examined by comparing the MS peak areas. From statistical analyses, one PC, one PE, six PSs, and two PIs among the PL species showed significant differences between controls and cancer patients (p < 0.05, Student’s t test), with concentration changes of more than threefold. Cluster analysis of both control and patient groups showed that 18:0/18:1-PS and 16:0/22:6-PS were 99% similar in upregulation and that the two PSs (18:1/18:0, 18:0/20:5) with two PIs (18:0/18:1 and 16:1/20:2) showed similar (>95%) downregulation. The total amount of each PL group was compared among prostate cancer patients according to the Gleason scale as larger or smaller than 6. It proposes that the current study can be utilized to sort out possible diagnostic biomarkers of prostate cancer. Figure Dendrogram of urinary phospholipids from prostate cancer

Keywords: Phospholipids; Quantitative analysis; nLC–ESI–MS/MS; Urine; Prostate cancer; Biomarker

Shotgun lipidomics for candidate biomarkers of urinary phospholipids in prostate cancer by Hye Kyeong Min; Sangsoo Lim; Bong Chul Chung; Myeong Hee Moon (pp. 823-830).

Keywords: Phospholipids; Quantitative analysis; nLC–ESI–MS/MS; Urine; Prostate cancer; Biomarker

Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli by P. Noguera; G. A. Posthuma-Trumpie; M. van Tuil; F. J. van der Wal; A. de Boer; A. P. H. A. Moers; A. van Amerongen (pp. 831-838).

The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay, NALFIA). Prior to the detection with NALFIA, a rapid amplification method with tagged primers was applied. In the evaluation of the optimised NALFIA strips, no cross-reactivity was found for any of the antibodies used. The limit of detection was higher than for quantitative PCR (q-PCR), in most cases between 10⁴ and 10⁵ colony forming units/mL or 0.1–0.9 ng/μL DNA. NALFIA strips were applied to 48 isolates from cattle faeces, and results were compared to those achieved by q-PCR. E. coli virulence factors identified by NALFIA were in very good agreement with those observed in q-PCR, showing in most cases sensitivity and specificity values of 1.0 and an almost perfect agreement between both methods (kappa coefficient larger than 0.9). The results demonstrate that the screening method developed is reliable, cost-effective and user-friendly, and that the procedure is fast as the total time required is <1 h, which includes amplification. Figure Results achieved with multi-analyte NALFIA for E. coli virulence factors. First strip: blank; second to sixth strip: each of the STEC factors; seventh strip: all factors

Keywords: E. coli ; Verotoxin; Shiga toxin; Screening method; Nucleic acid lateral flow immunoassay; Carbon nanoparticles

Simultaneous quantitative profiling of N-acyl-l-homoserine lactone and 2-alkyl-4(1H)-quinolone families of quorum-sensing signaling molecules using LC-MS/MS by Catharine A. Ortori; Jean-Frédéric Dubern; Siri Ram Chhabra; Miguel Cámara; Kim Hardie; Paul Williams; David A. Barrett (pp. 839-850).

An LC-MS/MS method, using positive mode electrospray ionization, for the simultaneous, quantitative and targeted profiling of the N-acyl-l-homoserine lactone (AHL) and 2-alkyl 4-(1H)-quinolone (AQ) families of bacterial quorum-sensing signaling molecules (QSSMs) is presented. This LC-MS/MS technique was applied to determine the relative molar ratios of AHLs and AQs produced by Pseudomonas aeruginosa and the consequences of mutating individual or multiple QSSM synthase genes (lasI, rhlI, pqsA) on AHL and AQ profiles and concentrations. The AHL profile of P. aeruginosa was dominated by N-butanoyl-l-homoserine lactone (C4-HSL) with lesser concentrations of N-hexanoyl-l-homoserine lactone (C6-HSL) and 3-oxo-substituted longer chain AHLs including N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL). The AQ profile of P. aeruginosa comprised the C7 and C9 long alkyl chain AQs including 2-heptyl-4-hydroxyquinoline (HHQ), 2-nonyl-4-hydroxyquinoline, the “pseudomonas quinolone signal” (2-heptyl-3-hydroxy-4-quinolone) and the N-oxides, 2-heptyl-4-hydroxyquinoline N-oxide and 2-nonyl-4-hydroxyquinoline N-oxide. Application of the method showed significant effects of growth medium type on the ratio and the nature of the QSSMs synthesized and the dramatic effect of single, double and triple mutations in the P. aeruginosa QS synthase genes. The LC-MS/MS methodology is applicable in organisms where either or both AHL and AQ QSSMs are produced and can provide comprehensive profiles and concentrations from a single sample.

Keywords: Bioanalytical methods; Mass spectrometry; HPLC; Pseudomonas aeruginosa ; N-acyl-l-homoserine lactones; 2-alkyl-4-(1H)-quinolones

Retinol fluorescence in lecithin/n-butanol/water aggregates: a new improvement for its analysis in cosmetics without pretreatment by M. P. San Andrés; S. Vera; M. Torre; M. Valiente (pp. 851-859).

The possibilities of different media formed by lecithin/n-butanol (n-BuOH)/water ternary mixtures for the analysis of all-trans-retinol by fluorescence have been studied. Fluorescence intensity of retinol increases in the presence of different types of aggregates formed in these media. Analytical features are good, the detection limit and quantification limit have micrograms per liter levels, and the linear range and sensitivity are appropriate to determine retinol in cosmetic samples. The analysis of retinol in anti-wrinkle creams can be achieved directly without any pretreatment of the sample. The vesicles built up from a biocompatible surfactant (lecithin) in aqueous solution with a low amount of n-BuOH permit an appropriated media for a simple, rapid, and sensitive analytical method. This method has a linear range between 64.1 and 800 μg L⁻¹, a sensitivity of 202.3 L mg⁻¹, and a low detection and quantification limit at 19.2 and 64.1 μg L⁻¹, respectively. Figure Fluorescence contour graph of (a) 0.1% lecithin/4.9% n-BuOH/95% water and (b) cosmetic sample 1 in this medium

Keywords: All-trans-retinol; Lecithin; Cosmetics; Fluorescence

Retinol fluorescence in lecithin/n-butanol/water aggregates: a new improvement for its analysis in cosmetics without pretreatment by M. P. San Andrés; S. Vera; M. Torre; M. Valiente (pp. 851-859).

Keywords: All-trans-retinol; Lecithin; Cosmetics; Fluorescence

Accelerated sample treatment for screening of banned doping substances by GC–MS: ultrasonication versus microwave energy by M. Galesio; M. Mazzarino; X. de la Torre; F. Botrè; J. L. Capelo (pp. 861-875).

A comparison between ultrasonication and microwave irradiation as tools to achieve a rapid sample treatment for the analysis of banned doping substances in human urine by means of gas chromatography–mass spectrometry (GC–MS) was performed. The following variables were studied and optimised: (i) time of treatment, (ii) temperature, (iii) microwave power and (iv) ultrasonic amplitude. The results were evaluated and compared with those achieved by the routine method used in the World Anti-Doping Agency (WADA) accredited Antidoping Laboratory of Rome. Only under the effect of the ultrasonic field was it possible to enhance the enzymatic hydrolysis reaction rate of conjugated compounds. Similar reaction yield to the routine method was achieved after 10 min for most compounds. Under microwave irradiation, denaturation of the enzyme occurs for high microwave power. The use of both ultrasonic or microwave energy to improve the reaction rate of the derivatisation of the target compounds with trimethyliodosilane/methyl-N-trimethylsilyltrifluoroacetamide (TMSI/MSTFA/NH₄I/2-mercaptoethanol) was also evaluated. To test the use of the two systems in the acceleration of the reaction with TMSI, a pool of 55 banned substances and/or their metabolites were used. After 3 min of ultrasonication, 34 of the 55 compounds had recoveries similar to those obtained with the classic procedure that lasts for 30 min (Student’s t test, n = 5), 18 increased to higher silylation yields, and for the compounds 13β,17α-diethyl-3α,17β-dihydroxy-5α-gonane (norboletone metabolite 1), metoprolol and metipranolol the same results were obtained increasing the ultrasonication time to 5 min. Similar results were obtained after 3 min of microwave irradiation at 1,200 W. In this case, 30 of the 55 compounds had recoveries similar to the classic procedure (Student’s t test, n = 5) whilst 18 had higher silylation yields. For the compounds 3α-hydroxy-1α-methyl-5α-androstan-17-one (mesterolone metabolite 1), 17α-ethyl-5β-estrane-3α,17β,21-triol (norethandrolone metabolite 1), epioxandrolone, 4-chloro-6β,17β-dihydroxy-17α-methyl-1,4-androstadien-3-one (chlormetandienone metabolite 1), carphedon, esmolol and bambuterol the same results were obtained after 5 min under microwave irradiation.

Keywords: Doping substances; GC–MS; β-Glucuronidase hydrolysis; MSTFA derivatisation; Ultrasonic energy; Microwave energy; Sample treatment; Anti-doping

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of phenolic polycyclic aromatic hydrocarbons (OH-PAH) in urine of non-smokers and smokers by Bernhard Ramsauer; Katharina Sterz; Heinz-Werner Hagedorn; Johannes Engl; Gerhard Scherer; Mike McEwan; Graham Errington; Jim Shepperd; Francis Cheung (pp. 877-889).

Polycyclic aromatic hydrocarbons (PAH) are products of the incomplete combustion of organic materials and, therefore, occur ubiquitously in the environment and also in tobacco smoke. Since some PAH have been classified as carcinogens, it is important to have access to suitable analytical methods for biomarkers of exposure to this class of compounds. Past experience has shown that measuring a profile of PAH metabolites is more informative than metabolites of a single PAH. Assessment of environmental and smoking-related exposure levels requires analytical methods with high sensitivity and specificity. In addition, these methods should be fast enough to allow high throughput. With these pre-conditions in mind, we developed and validated a high-performance liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of phenolic metabolites of naphthalene, fluorene, phenanthrene and pyrene in urine of smokers and non-smokers. Sample work-up comprised enzymatic hydrolysis of urinary conjugates and solid-phase extraction on C18 cartridges. The method showed good specificity, sensitivity, and accuracy for the intended purpose and was also sufficiently rapid with a sample throughput of about 350 per week. Application to urine samples of 100 smokers and 50 non-smokers showed significant differences between both groups for all measured PAH metabolites, and strong correlations with markers of daily smoke exposure in smoker urine. Urinary levels were in good agreement with previously reported data using different methodologies. In conclusion, the developed LC-MS/MS method is suitable for the quantification of phenolic PAH metabolites of naphthalene, fluorene, phenanthrene, and pyrene in smoker and non-smoker urine. Figure Cumulative frequencies of urinary 4-OH-Phe (left, not significantly influenced by smoking) and 1-OH-Pyr (right, significantly elevated in smokers)

Keywords: Polycyclic aromatic hydrocarbons; Urine; LC-MS/MS; Smokers; Non-smokers

Determination of pharmaceuticals, steroid hormones, and endocrine-disrupting personal care products in sewage sludge by ultra-high-performance liquid chromatography–tandem mass spectrometry by Yiyi Yu; Qiuxin Huang; Jianlan Cui; Kun Zhang; Caiming Tang; Xianzhi Peng (pp. 891-902).

A sensitive method has been developed and validated for the determination of diverse groups of pharmaceuticals, steroid hormones, and hormone-like personal care products in sewage sludge. Samples were extracted by ultrasonic-assisted extraction followed by solid-phase extraction cleanup. For determination of estrogens and hormone-like phenolic compounds, sample extracts were further derivatized with dansyl chloride and purified with silica gel column chromatography to improve the analytical sensitivity. The chemicals were determined by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) in multiple-reaction monitoring mode. Recoveries ranged mostly from 63% to 119% with relative standard deviations within 15%. Method quantification limits were 0.1–3 ng g⁻¹ dry weight (dw) for sewage sludge. The method was applied to a preliminary investigation of pharmaceuticals and personal care products (PPCPs) in sewage sludge and sediment in the Pearl River Delta, South China. Triclosan, triclocarban, 2-phenylphenol, bisphenol A, and parabens were ubiquitously detected at 3.6–5088.2 ng g⁻¹ dw in sludge and 0.29–113.1 ng g⁻¹ dw in sediment samples, respectively. Estrone, carbamazepine, metoprolol, and propranolol were also frequently quantified in the sludge and sediment samples. The dewatering process caused no significant losses of these PPCPs in sewage sludge. Figure MRM chromatograms of the analytes in a standard solution at 5 ng mL⁻¹ (A), sewage sludge (B), river sediment (C), and estuary sediment (D) samples. ATL, atenolol; MPL, metoprolol; CBZ, carbamazepine; E1, estrone; E2,17β-estradiol, E3, estriol; EE2,17α-ethynylestradiol; MedP, medroxyprogesterone; MP, methylparaben; EP, ethylparaben; PP, propylparaben, PHP, 2-phenylphenol; TCS, triclosan; TCC, triclocarban; BPA, bisphenol A.

Keywords: Pharmaceuticals and personal care products (PPCPs); Sewage sludge; Derivatization; UHPLC-MS/MS; China

Retention modelling of polychlorinated biphenyls in comprehensive two-dimensional gas chromatography by Angelo Antonio D’Archivio; Angela Incani; Fabrizio Ruggieri (pp. 903-913).

In this paper, we use a quantitative structure–retention relationship (QSRR) method to predict the retention times of polychlorinated biphenyls (PCBs) in comprehensive two-dimensional gas chromatography (GC×GC). We analyse the GC×GC retention data taken from the literature by comparing predictive capability of different regression methods. The various models are generated using 70 out of 209 PCB congeners in the calibration stage, while their predictive performance is evaluated on the remaining 139 compounds. The two-dimensional chromatogram is initially estimated by separately modelling retention times of PCBs in the first and in the second column (¹ t _R and ² t _R, respectively). In particular, multilinear regression (MLR) combined with genetic algorithm (GA) variable selection is performed to extract two small subsets of predictors for ¹ t _R and ² t _R from a large set of theoretical molecular descriptors provided by the popular software Dragon, which after removal of highly correlated or almost constant variables consists of 237 structure-related quantities. Based on GA-MLR analysis, a four-dimensional and a five-dimensional relationship modelling ¹ t _R and ² t _R, respectively, are identified. Single-response partial least square (PLS-1) regression is alternatively applied to independently model ¹ t _R and ² t _R without the need for preliminary GA variable selection. Further, we explore the possibility of predicting the two-dimensional chromatogram of PCBs in a single calibration procedure by using a two-response PLS (PLS-2) model or a feed-forward artificial neural network (ANN) with two output neurons. In the first case, regression is carried out on the full set of 237 descriptors, while the variables previously selected by GA-MLR are initially considered as ANN inputs and subjected to a sensitivity analysis to remove the redundant ones. Results show PLS-1 regression exhibits a noticeably better descriptive and predictive performance than the other investigated approaches. The observed values of determination coefficients for ¹ t _R and ² t _R in calibration (0.9999 and 0.9993, respectively) and prediction (0.9987 and 0.9793, respectively) provided by PLS-1 demonstrate that GC×GC behaviour of PCBs is properly modelled. In particular, the predicted two-dimensional GC×GC chromatogram of 139 PCBs not involved in the calibration stage closely resembles the experimental one. Based on the above lines of evidence, the proposed approach ensures accurate simulation of the whole GC×GC chromatogram of PCBs using experimental determination of only 1/3 retention data of representative congeners. Figure Agreement between experimental two-dimensional GCxGC chromatogram of 139 polychlorinated biphenyls and the one predicted by PLS-1 regression based on 237 molecular descriptors

Keywords: Polychlorinated biphenyls; Two-dimensional gas chromatography; QSRR modelling; Molecular descriptors

Retention modelling of polychlorinated biphenyls in comprehensive two-dimensional gas chromatography by Angelo Antonio D’Archivio; Angela Incani; Fabrizio Ruggieri (pp. 903-913).

Keywords: Polychlorinated biphenyls; Two-dimensional gas chromatography; QSRR modelling; Molecular descriptors

LC–MS–MS determination of ibuprofen, 2-hydroxyibuprofen enantiomers, and carboxyibuprofen stereoisomers for application in biotransformation studies employing endophytic fungi by Keyller Bastos Borges; Anderson Rodrigo Moraes de Oliveira; Thiago Barth; Valquíria Aparecida Polizel Jabor; Mônica Tallarico Pupo; Pierina Sueli Bonato (pp. 915-925).

The purpose of this study was the development and validation of an LC–MS–MS method for simultaneous analysis of ibuprofen (IBP), 2-hydroxyibuprofen (2-OH-IBP) enantiomers, and carboxyibuprofen (COOH-IBP) stereoisomers in fungi culture medium, to investigate the ability of some endophytic fungi to biotransform the chiral drug IBP into its metabolites. Resolution of IBP and the stereoisomers of its main metabolites was achieved by use of a Chiralpak AS-H column (150 × 4.6 mm, 5 μm particle size), column temperature 8 °C, and the mobile phase hexane–isopropanol–trifluoroacetic acid (95: 5: 0.1, v/v) at a flow rate of 1.2 mL min⁻¹. Post-column infusion with 10 mmol L⁻¹ ammonium acetate in methanol at a flow rate of 0.3 mL min⁻¹ was performed to enhance MS detection (positive electrospray ionization). Liquid–liquid extraction was used for sample preparation with hexane–ethyl acetate (1:1, v/v) as extraction solvent. Linearity was obtained in the range 0.1–20 μg mL⁻¹ for IBP, 0.05–7.5 μg mL⁻¹ for each 2-OH-IBP enantiomer, and 0.025–5.0 μg mL⁻¹ for each COOH-IBP stereoisomer (r ≥ 0.99). The coefficients of variation and relative errors obtained in precision and accuracy studies (within-day and between-day) were below 15%. The stability studies showed that the samples were stable (p > 0.05) during freeze and thaw cycles, short-term exposure to room temperature, storage at −20 °C, and biotransformation conditions. Among the six fungi studied, only the strains Nigrospora sphaerica (SS67) and Chaetomium globosum (VR10) biotransformed IBP enantioselectively, with greater formation of the metabolite (+)-(S)-2-OH-IBP. Formation of the COOH-IBP stereoisomers, which involves hydroxylation at C3 and further oxidation to form the carboxyl group, was not observed. Figure Enantioselective biotransformation of ibuprofen to 2-hydroxyibuprofen by endophytic fungi

Keywords: LC–MS–MS; Ibuprofen; 2-hydroxyibuprofen; Carboxyibuprofen; Biotransformation; Endophytic fungi

A novel total reflection X-ray fluorescence procedure for the direct determination of trace elements in petrochemical products by Amedeo Cinosi; Nunzio Andriollo; Giancarlo Pepponi; Damiano Monticelli (pp. 927-933).

A total reflection X-ray fluorescence (TXRF) procedure was developed for the determination of metal traces in petrochemical end products or intermediates for surfactant synthesis. The method combines a fast and straightforward sample preparation, i.e. deposition on the sample holder and evaporation of the sample matrix, with an efficient quantification method based on internal standardization (organic gallium standard). The method developed showed detection limits below 0.05 μg g^-1 and in most cases below 0.005 μg g^-1. Fifteen elements (Ca, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Rh, Sn, Sr, V and Zn) were determined in matrices such as paraffins, n-olefins, linear alkylbenzenes, long-chain alkyl alcohols and esters: typical metal contents were below 1 μg g^-1. The results were compared with the reference method ASTM D5708 (test method B) based on inductively coupled plasma optical emission spectroscopy: advantages and drawbacks of the two procedures were critically evaluated. The TXRF method developed showed comparable precision and absence of bias with respect to the reference method. A comparison of the performances of the two methods is presented.

Keywords: Total reflection X-ray fluorescence; Inductively coupled plasma optical emission spectroscopy; Trace elements; Petrochemical products; Direct analysis

Development and in-house validation of allergen-specific ELISA tests for the quantification of Dau c 1.01, Dau c 1.02 and Dau c 4 in carrot extracts (Daucus carota) by Kay Foetisch; Lotte Dahl; Baerbel Jansen; Wolf-Meinhard Becker; Jonas Lidholm; Ronald van Ree; Hermann Broll; Susanne Kaul; Stefan Vieths; Thomas Holzhauser (pp. 935-943).

Even though carrot allergy is common in Europe, the amount of different allergens in carrots is still unknown due to a lack of methods for quantitative allergen measurements. The current study aimed at the development of quantitative ELISA tests for the known carrot allergens, namely Dau c 1.01, Dau c 1.02, and Dau c 4 in pure carrot extracts. Monoclonal antibodies targeting the major carrot allergen isoforms Dau c 1.01 and Dau c 1.02 were generated and combined in sandwich ELISA with rabbit antisera against Api g 1, the celery homologue of Dau c 1. A competitive ELISA for the carrot profilin Dau c 4 was based on a polyclonal rabbit antiserum. The three ELISA tests were allergen-specific and displayed detection limits between 0.4 and 6 ng allergen/ml of carrot extract. The mean coefficient of variation (CV) as a means of intraassay variability of the Dau c 1.01, Dau c 1.02 and Dau c 4 ELISA tests was 8.1%, 6.9%, and 11.9%, and the mean interassay CV 13.3%, 37.1% and 15.6%, respectively. Target recovery ranged between 93 and 113%. In conclusion, the specific, accurate and reproducible quantification of three important carrot allergens may help to identify less allergenic carrot varieties, as well as to standardize the amount of allergens in extracts used for carrot allergy diagnosis.

Keywords: Allergen; Daucus carota ; ELISA; Dau c 1.01; Dau c 1.02; Dau c 4

GC-MS determination of parabens, triclosan and methyl triclosan in water by in situ derivatisation and stir-bar sorptive extraction by Ana María Casas Ferreira; Monika Möder; María Esther Fernández Laespada (pp. 945-953).

Stir-bar sorptive extraction in combination with an in situ derivatisation reaction and thermal desorption–gas chromatography–mass spectrometry was successfully applied to determine parabens (methylparaben, isopropylparaben, n-propylparaben, butylparaben and benzylparaben), triclosan and methyltriclosan in water samples. This approach improves both the extraction efficiency and the sensitivity in the GC in a simple way since the derivatisation reaction occurs at the same time as the extraction procedure. The in situ derivatisation reaction was carried out with acetic anhydride under alkaline conditions. Thermal desorption parameters (cryofocusing temperature, desorption flow, desorption time, desorption temperature) were optimised using a Box–Behnken experimental design. All the analytes gave recoveries higher than 79%, except methylparaben (22%). The method afforded detection limits between 0.64 and 4.12 ng/L, with good reproducibility and accuracy values. The feasibility of the method for the determination of analytes in water samples was checked in tap water and untreated and treated wastewater.

Keywords: Stir-bar sorptive extraction; In situ derivatisation; Parabens; Water; Wastewater

Dispersive solid-phase extraction for in-sorbent Fourier-transform infrared detection and identification of nerve agent simulants in analysis for verification of chemical weapon convention by Prabhat Garg; Deepak Pardasani; Avik Mazumder; Ajay Purohit; D. K. Dubey (pp. 955-963).

The combination of dispersive solid-phase extraction (DSPE) and Fourier-transform infrared (FTIR) spectroscopy is presented for detection and quantification of markers and simulants of nerve agents. Hydrophilic–lipophilic balance (HLB) sorbent was used for extraction and enrichment of organophosphonates from water. When the extraction efficiency of DSPE was compared with that of conventional solid-phase extraction (SPE), DSPE was more efficient. Extraction conditions such as extraction time, and type and quantity of sorbent material were optimized. In DSPE, extracted analytes are detected and quantified on the sorbent using FTIR as analytical technique. Absorbance in FTIR due to P–O–C stretching was used for detection and quantification. Infrared absorbance of different analytes were compared by determining their molar absorptivities (ε _max). Quantitative analyses were performed employing modified Beer’s law, and relative standard deviations (RSDs) for intraday repeatability and interday reproducibility were found to be in the range 0.30–0.90% and 0.10–0.80% respectively. The limit of detection (LOD) was 5–10 μg mL⁻¹. The applicability of the method was tested with an unknown sample prepared by mimicking the sample obtained in an international official proficiency test. Figure Dispersive solid-phase extraction of nerve agents simulants coupled with Fourier transform infrared detection

Keywords: Chemical warfare agents; Molar extinction coefficient; Dispersive solid-phase extraction; FTIR; Organophosphonates

Automatic optosensing device based on photo-induced fluorescence for determination of piceid in cocoa-containing products by Lucía Molina-García; Antonio Ruiz-Medina; Maria Luisa Fernández-de Córdova (pp. 965-972).

Piceid (3,4′,5-trihydroxystilbene-3-β-d-glucoside) is a stilbene which occurs naturally in various families of plants and has been shown to protect lipoproteins from oxidative damage and to have cancer chemopreventive activity. This paper deals with the determination of piceid in cocoa-containing products by using photo-induced fluorescence and the aid of a multicommutated continuous-flow assembly which was provided with an on-line photoreactor. A strongly fluorescent photoproduct is generated from piceid when it is irradiated under UV light for 30 s, which is retained on Sephadex QAE A-25 and directly monitored on this active solid support at 257/382 nm (λ _exc/λ _em, respectively). The pre-concentration of the photoproduct of piceid on the solid support greatly improves both sensitivity and selectivity. The influence of different experimental parameters, both chemical (pH, ionic strength) and hydrodynamic (irradiation time, flow rate, photoreactor length, sampling time), was tested. The sample pre-treatment included delipidation with toluene and cyclohexane, stilbene extraction with ethanol/water (80:20, v/v) and clean-up by solid-phase extraction on C₁₈ cartridges and methanol/water (40:20, v/v) as eluting solution. This procedure allowed the elimination of the aglycon of piceid, resveratrol and other potential interfering species and a recovery of about a 90% piceid. The method was applied to the analysis of piceid in cocoa powder, dark chocolate and milk chocolate. The quantification limits were 1.4, 1.1 and 0.09 mg kg⁻¹, respectively. Relative standard deviations ranged from 1.8% to 3.1%. This is the first reported non-chromatographic method for determination of piceid in these foods.

Keywords: Piceid; Multicommutation; Optosensor; Photo-induced fluorescence; Cocoa powder; Chocolate

Particle size characterization of titanium dioxide in sunscreen products using sedimentation field-flow fractionation–inductively coupled plasma–mass spectrometry by Atitaya Samontha; Juwadee Shiowatana; Atitaya Siripinyanond (pp. 973-978).

Sedimentation field-flow fractionation–inductively coupled plasma–mass spectrometry (SdFFF-ICP-MS) was successfully applied to investigate particle size distribution of titanium dioxide (TiO₂) in sunscreen samples after hexane extraction to remove organic components from the samples. Three brands of sunscreen products of various sun protection factor (SPF) value were used as samples. Different particle size distribution profiles were observed for sunscreen samples of various brands and SPF values; however, the particle size distributions of titanium dioxide in most sunscreen samples investigated in this work were larger than 100 nm. The titanium dioxide concentrations were higher for the products of higher SPF values. By comparing the results obtained from online SdFFF-ICP-MS and those from the off-line ICP-MS determination of titanium after acid digestion, ICP-MS was found to effectively atomize and ionize the titanium dioxide particle without the need for acid digestion of the samples. Therefore, the online coupling between SdFFF and ICP-MS could be effectively used to provide quantitative information of titanium dioxide concentrations across particle size distribution profiles.

Keywords: Titanium dioxide; Sunscreen; Particle size; Sedimentation field-flow fractionation; Inductively coupled plasma–mass spectrometry

Production and analytical characterization of neopterin immunoreagents for biosensor developments by Ivo Cernoch; Erwin Schleicher; Milan Franek (pp. 979-986).

Neopterin is a valuable biomarker of cellular immunity associated with various pathological situations such as viral and bacterial infections, autoimmune, cardiovascular, neurodegenerative and malignant disorders. To produce specific antibodies against neopterin for a rapid multi-biomarker-based diagnosis, a novel hapten derivative was synthesized and attached to carrier proteins. The thoroughly characterized conjugates were used for immunization of BALB/c mice and rabbits. The produced monoclonal antibody reached in both direct and indirect enzyme-linked immunosorbent assay (ELISA) format LoD of 0.18 and 0.45 μg L⁻¹, respectively, and was a superior immunoreagent for further biosensor developments with regard to assay sensitivity and material availability. The best polyclonal antibody was somewhat more sensitive in direct ELISA with LoD of 0.05 μg L⁻¹. The optimized ELISA method was evaluated with blood samples collected from patients with renal insufficiency, patients with sepsis, patients without confirmed clinical diagnosis, and healthy volunteers. In plasma samples, neopterin concentrations ranging from 3.2 to 103 μg L⁻¹ could be determined with the monoclonal ELISA whereas twofold lower results were obtained with the polyclonal ELISA. A satisfactory correlation of results was found between the polyclonal ELISA and IBL Neopterin ELISA kit within the concentration range 0.5–16 μg L⁻¹ (R = 0.874; n = 40), and slightly lower correlation was found for monoclonal-based ELISA (R = 0.819; n = 40). These data show that the generated antibodies may be used as functional analytical reagents for the integration into multianalyte biochip detection systems.

Keywords: Neopterin; Hapten density; Monoclonal and polyclonal antibody; Real blood samples; ELISA evaluation; Multianalyte chip

Production and analytical characterization of neopterin immunoreagents for biosensor developments by Ivo Cernoch; Erwin Schleicher; Milan Franek (pp. 979-986).

Keywords: Neopterin; Hapten density; Monoclonal and polyclonal antibody; Real blood samples; ELISA evaluation; Multianalyte chip

pH optimization for a reliable quantification of brain tumor cell and tissue extracts with ¹H NMR: focus on choline-containing compounds and taurine by O. Robert; J. Sabatier; D. Desoubzdanne; J. Lalande; S. Balayssac; V. Gilard; R. Martino; M. Malet-Martino (pp. 987-999).

The aim of this study was to define the optimal pH for ¹H nuclear magnetic resonance (NMR) spectroscopy analysis of perchloric acid or methanol–chloroform–water extracts from brain tumor cells and tissues. The systematic study of the proton chemical shift variations as a function of pH of 13 brain metabolites in model solutions demonstrated that recording ¹H NMR spectra at pH 10 allowed resolving resonances that are overlapped at pH 7, especially in the 3.2–3.3 ppm choline-containing-compounds region. ¹H NMR analysis of extracts at pH 7 or 10 showed that quantitative measurements of lactate, alanine, glutamate, glutamine (Gln), creatine + phosphocreatine and myo-inositol (m-Ino) can be readily performed at both pHs. The concentrations of glycerophosphocholine, phosphocholine and choline that are crucial metabolites for tumor brain malignancy grading were accurately measured at pH 10 only. Indeed, the resonances of their trimethylammonium moieties are cleared of any overlapping signal, especially those of taurine (Tau) and phosphoethanolamine. The four non-ionizable Tau protons resonating as a singlet in a non-congested spectral region permits an easier and more accurate quantitation of this apoptosis marker at pH 10 than at pH 7 where the triplet at 3.43 ppm can be overlapped with the signals of glucose or have an intensity too low to be measured. Glycine concentration was determined indirectly at both pHs after subtracting the contribution of the overlapped signals of m-Ino at pH 7 or Gln at pH 10. Figure Partial ¹H NMR spectra (2.9-3.5 ppm region) of a D₂O solution containing glutamine (Gln), taurine (Tau), hypotaurine (HTau), myo-inoitol (m-Ino), betaine (Bet), glycerophosphocholine (GPC), phosphocholine (PC), choline (Cho), phosphoethanolamine (PE), creatine (Cr) standards and MeOH at pH 7.0 and 10.0. The figure clearly shows the superimposition of choline-containing-compounds (GPC, PC, Cho) resonances with those of Tau and PE.

Keywords: In vitro ¹H NMR; Cell and tissue extracts; Brain tumor; Optimal pH; Choline-containing compounds; Taurine

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Analytical and Bioanalytical Chemistry (v.399, #2)