l-α-Phosphatidylglycerol Chlorohydrins as Potential Biomarkers for Chlorine Gas Exposure.

Chlorine is a widely available toxic chemical that has been repeatedly used in armed conflict globally. The Organization for the Prohibition of Chemical Weapons (OPCW) have on numerous occasions found "compelling confirmation" that chlorine gas has been used against civilians in northern Syria. However, currently, there are no analytical methods available to unambiguously prove chlorine gas exposure. In this study, we describe the screening for chlorinated biomolecules by the use of mass isotope ratio filters followed by the identification of two biomarkers present in bronchoalveolar lavage fluid (BALF) from chlorine gas exposed mice. The relevance of these markers for human exposure was verified by their presence in in vitro chlorinated human BALF. The biomarkers were detectable for 72 h after exposure and were absent in nonexposed control animals. Furthermore, the biomarkers were not detected in humans diagnosed with chronic respiratory diseases. The potential chlorine specific markers were all chlorohydrins of unsaturated pulmonary surfactant phospholipids; phosphatidylglycerols, and phosphatidylcholines. Mass spectrometry fragmentation characteristics were favorable for the phosphatidylglycerol chlorohydrins, and they were therefore proposed as the best biomarker candidates.

Characterization of intact neo-glycoproteins by hydrophilic interaction liquid chromatography.

In this study, an HPLC HILIC-UV method was developed for the analysis of intact neo-glycoproteins. During method development the experimental conditions evaluated involved different HILIC columns (TSKgel Amide-80 and ZIC-pHILIC), and water-acetonitrile mixtures containing various types of acids and salts. The final selected method was based on a TSKgel Amide-80 column and a mobile phase composed of acetonitrile and water both containing 10 mM HClO4. The influence of temperature and sample preparation on the chromatographic performances of the HILIC method was also investigated. The method was applied to the separation of neo-glycoproteins prepared starting from the model protein RNase A by chemical conjugation of different glycans. Using the method here reported it was possible to monitor by UV detection the glycosylation reaction and assess the distribution of neo-glycoprotein isoforms without laborious sample workup prior to analysis.

Phospholipid chlorohydrins as chlorine exposure biomarkers in a large animal model.

Chlorine is a toxic industrial chemical that has been used as a chemical weapon in recent armed conflicts. Confirming human exposure to chlorine has proven challenging, and there is currently no established method for analyzing human biomedical samples to unambiguously verify chlorine exposure. In this study, two chlorine-specific biomarkers: palmitoyl-oleoyl phosphatidylglycerol chlorohydrin (POPG-HOCl) and the lipid derivative oleoyl ethanolamide chlorohydrin (OEA-HOCl) are shown in bronchoalveolar lavage fluid (BALF) samples from spontaneously breathing pigs after chlorine exposure. These biomarkers are formed by the chemical reaction of chlorine with unsaturated phospholipids found in the pulmonary surfactant, which is present at the gas-liquid interface within the lung alveoli. Our results strongly suggest that lipid chlorohydrins are promising candidate biomarkers in the development of a verification method for chlorine exposure. The establishment of verified methods capable of confirming the illicit use of toxic industrial chemicals is crucial for upholding the principles of the Chemical Weapons Convention (CWC) and enforcing the ban on chemical weapons. This study represents the first published dataset in BALF revealing chlorine biomarkers detected in a large animal. Furthermore, these biomarkers are distinct in that they originate from molecular chlorine rather than hypochlorous acid.

Column comparison and method development for the analysis of short-chain carboxylic acids by zwitterionic hydrophilic interaction liquid chromatography with UV detection.

Short-chain carboxylic acids are relevant in pharmaceutical, food quality control, and biomedical analysis. In this study, 11 acids commonly found in drugs and in food products were selected. Wine was chosen as matrix for testing the method. The test compounds were used for comparing the selectivity of four 150 × 2.1 mm zwitterionic hydrophilic interaction LC (HILIC) columns (ZIC-HILIC 5 µm, 200 Å, and 3.5 µm, 100 Å, ZIC-pHILIC 5 µm, ZIC-cHILIC 3 µm, 100 Å) while varying the conditions to optimize for low UV wavelength detection and achieve high sensitivity. Retention using potassium phosphate and ammonium carbonate as mobile-phase components at pH 6.0, 7.5, and 8.5-8.9 was studied considering recent hypotheses on HILIC mechanism-related with the Hofmeister series effect and ion hydration. An isocratic method with UV detection at 200 nm and mobile phase consisting of 75% acetonitrile and 10 mM potassium phosphate at pH 6.0 applied to a ZIC-cHILIC column was found provisionally optimal and partially validated for the 11 analytes. Satisfactory results (R(2) from 0.9940 to >0.9999), and recoveries from 93-106% for all analytes evidenced the method as suitable for wine analysis. To the best of our knowledge, no previous study has reported on the direct ZIC-HILIC separation and UV detection of the acids considered here in wine.

Nasal Lavage Fluid as a Biomedical Sample for Verification of Chlorine Exposure.

Chlorine is a toxic chemical that has been used as a chemical warfare agent in recent armed conflicts. There is an urgent need for methods to verify alleged uses of chlorine, and phospholipid chlorohydrins (PL-HOCl) derived from the pulmonary surfactant of exposed victims have previously been proposed as biomarkers of chlorine exposure. Here, we describe an improved protocol for the chemical analysis of these biomarkers and its applicability to biomedical samples from chlorine-exposed animals. By the use of a polymeric solid-phase-supported transesterification of PL-HOCl using ethanolamine, a common biomarker, oleoyl ethanolamide chlorohydrin (OEA-HOCl), was derived from all the diverse oleoyl PL-HOCl that may be formed by chlorine exposure. Compared to native lipid biomarkers, OEA-HOCl represents a larger biomarker pool and is better suited for nano-liquid chromatography--tandem mass spectrometry (nLC-MS-MS analysis), generating 3 amol Limit of Detection (LOD) and a reduced sample carry-over. With the improved protocol, significantly elevated levels of OEA-HOCl were identified in bronchoalveolar lavage fluid (BALF) of chlorine-exposed rats, 2-48 hours after exposure. The difficulty of BALF sampling from humans limits the methods usefulness as a verification tool of chlorine exposure. Conversely, nasal lavage fluid (NLF) is readily collected without advanced equipment. In NLF from chlorine-exposed rats, PL-HOCl were identified and significantly elevated levels of the OEA-HOCl biomarker were detected 2-24 hours after exposure. In order to test the potential of NLF as a biomedical sample for verification of human exposure to chlorine, in-vitro chlorination of human NLF samples was performed. All human in-vitro chlorinated NLF samples exhibited elevated OEA-HOCl biomarker levels, following sample derivatization. These data indicate the potential of human NLF as a biomedical sample for the verification of chlorine exposure, but further work is required to develop and validate the method for the use on real-world samples.

Preparation of a sorbitol methacrylate grafted silica as stationary phase for hydrophilic interaction chromatography.

A new highly hydrophilic stationary phase based on graft polymerization of sorbitol methacrylate from the surface of Kromasil silica particles is described. Polymerization was initiated by thermal cleavage of tert-butyl hydroperoxide covalently attached to the silica particle surface. Due to the highly amphiphilic properties of the monomer, an extensive search was needed to find solvent conditions that enabled surface-initiated polymerization. This was finally solved by using a mixture of solvents that only partially dissolved the monomer. The graft polymerization was confirmed by Fourier transform infrared spectroscopy and elemental analysis. The resulting stationary phase was evaluated by HPLC and exhibited a selectivity markedly different from that of commercially available columns and of neat silica.

Alternative organic solvents for HILIC separation of cisplatin species with on-line ICP-MS detection.

Several low volatile organic solvents were evaluated as organic modifiers in eluents for HILIC separations of cisplatin species to optimize the on-line coupling of HILIC to inductively coupled plasma MS (ICP-MS). The aim was to identify a solvent giving low solvent vapor loading of the ICP, to maximize analyte sensitivity and minimize carbon depositions on instrumental parts, while retaining chromatographic performance. The best overall performance of the HILIC-ICP-MS system for the analysis of cisplatin was achieved using 1,4-dioxane as eluent, yielding high retention and an HILIC type retention mechanism, at the expense of a 50% drop in column efficiency due to the higher viscosity of 1,4-dioxane compared to the more commonly used HILIC solvent ACN. Using 1,4-dioxane as solvent in HILIC provides the best compromise between carbon deposition and separation efficiency among a series of high-boiling water-miscible solvents tested.

Hydrophilic interaction chromatography.

Separation of polar compounds on polar stationary phases with partly aqueous eluents is by no means a new separation mode in LC. The first HPLC applications were published more than 30 years ago, and were for a long time mostly confined to carbohydrate analysis. In the early 1990s new phases started to emerge, and the practice was given a name, hydrophilic interaction chromatography (HILIC). Although the use of this separation mode has been relatively limited, we have seen a sudden increase in popularity over the last few years, promoted by the need to analyze polar compounds in increasingly complex mixtures. Another reason for the increase in popularity is the widespread use of MS coupled to LC. The partly aqueous eluents high in ACN with a limited need of adding salt is almost ideal for ESI. The applications now encompass most categories of polar compounds, charged as well as uncharged, although HILIC is particularly well suited for solutes lacking charge where coulombic interactions cannot be used to mediate retention. The review attempts to summarize the ongoing discussion on the separation mechanism and gives an overview of the stationary phases used and the applications addressed with this separation mode in LC.

Atom-transfer radical graft polymerization initiated directly from silica applied to functionalization of stationary phases for high-performance liquid chromatography in the hydrophilic interaction chromatography mode.

Initiation of atom-transfer radical polymerization of a number of monomers (styrene, methyl acrylate, 3-[N,N-dimethyl-N-(methacryloyloxyethyl)ammonium] propanesulfonate, butyl methacrylate, 2,3-epoxypropyl methacrylate) directly from chlorinated porous silica particles has been performed. The grafting has been confirmed and evaluated by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. This initiation technique results in a hydrolytically stable initial Si-C bond, tethering the polymer to the silica substrate. The resulting grafted particles have been used as separation materials for both reversed-phase and hydrophilic interaction chromatography.

Polymer-based monolithic microcolumns for hydrophobic interaction chromatography of proteins.

Monolithic capillary columns for hydrophobic interaction chromatography (HIC) have been prepared by thermally initiated, single-step in situ polymerization of mixtures of monovinyl monomers including butyl methacrylate and/or 2-hydroxyethyl methacrylate, with a divinyl crosslinker glycerol dimethacrylate or 1,4-butanediol dimethacrylate using two different porogen systems. Two porogenic solvent mixtures were used; one "hydrophilic", consisting of water, butanediol, and propanol, and one "hydrophobic," comprising dodecanol and cyclohexanol. The porous structures of the monoliths were characterized and their performance was demonstrated with a separation of a mixture of myoglobin, ribonuclease A, and lysozyme under conditions typical of HIC.