A new approach towards highly sensitive detection of endogenous N-acetylaspartic acid, N-acetylglutamic acid, and N-acetylaspartylglutamic acid in brain tissues based on strong anion exchange monolith microextraction coupled with UHPLC-MS/MS.

A novel in-tube solid-phase microextraction coupled with an ultra-high performance liquid chromatography-mass spectrometry method has been established for simultaneous quantification of three crucial brain biomarkers N-acetylaspartic acid (NAA), N-acetylglutamic acid (NAG), and N-acetylaspartylglutamic acid (NAAG). A polymer monolith with quaternary ammonium as the functional group was designed and exhibited efficient enrichment of target analytes through strong anion exchange interaction. Under the optimized conditions, the proposed method displayed wide linear ranges (0.1-80 nM for NAA and NAG, 0.2-160 nM for NAAG) with good precision (RSDs were lower than 15%) and low limits of detection (0.019-0.052 nM), which is by far the most sensitive approach for NAA, NAG, and NAAG determination. Furthermore, this approach has been applied to measure the target analytes in mouse brain samples, and endogenous NAA, NAG, and NAAG were successfully detected and quantified from only around 5 mg of cerebral cortex, cerebellum, and hippocampus. Compared with existing methods, the newly developed method in the current study provides highest sensitivity and lowest sample consumption for NAA, NAG, and NAAG measurements, which would potentially be utilized in determining and tracking these meaningful brain biomarkers in diseases or treatment processes, benefiting the investigations of pathophysiology and treatment of brain disorders.

Imidazole-octyl mixed-mode stationary phase based on macroporous silica for the purification of ovomucoid and ovotransferrin.

A process-simplified hard template approach was established to synthesize the monodisperse macroporous silica microspheres with homogeneous structures by twice alkali-thermal treatment and calcination routes. Porous vinyl-functionalized polysesquioxane microspheres (V-PMSQ) were synthesized through a hydrolyzation-polycondensation method and used as templates. The template particles with large aperture and high pore volume were obtained by adjusting the pH value and reaction time of the twice alkali-thermal reaction. After calcination, monodisperse silica microspheres with an average pore size of 30 nm, homogeneous pore structures, and narrow particle size distribution were fabricated, which can be directly used as chromatographic matrices without classification. After that, a new reversed-phase/strong anion-exchange (RP/SAX) mixed-mode stationary phase Sil-S-VOIM was prepared by bonding the 1-vinyl-3-octyl-imidazole ligands to the above silica microspheres through a "thiol-ene" click reaction. The performance of the Sil-S-VOIM column was evaluated by one acidic protein (transferrin) and two basic proteins (lysozyme, α-chymotrypsin) and compared to a single imidazole-modified Sil-S-VIM column and an octyl-modified Sil-C8 column, respectively. Due to the synergistic effect of electrostatic repulsion and hydrophobic interactions, baseline separations of the above proteins were observed only on the Sil-S-VOIM column, with resolutions of 2.55 and 2.01 between lysozyme and transferrin, and between transferrin and α-chymotrypsin, respectively, indicating good selectivity and separation ability compared with single-mode stationary phases. It was applied to the isolation of egg white samples with peaks identified by SDS-PAGE and MALDI-TOF-MS. The results showed that the selective retention and isolation of ovomucoid and ovotransferrin were successfully achieved, with yields of 78.8% and 67.2%, respectively. The protocol described in this work is simpler, faster, and has higher protein recovery. Overall, this new mixed-mode stationary phase provided a promising potential for the separation and determination of intact proteins.

Purification and characterisation of heparin-like sulfated polysaccharides with potent anti-SARS-CoV-2 activity from snail mucus of Achatina fulica.

Heparin-like sulfated polysaccharide, acharan sulfate, was purified from the mucus of an African giant snail with unique sulfated glycosaminoglycans (GAGs). This study reported on finding novel and safe heparin resources from Achatina fulica for further use as well as easy isolation and purification of the active fraction from the initial raw material. Its structure was characterised by a strong-anion exchange combined with high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy. The results indicated that the potential acharan sulfate fraction is a glycosaminoglycan composed of several repeating disaccharide units, namely, of →4)-α-IdoA(2S)(1→4)-α-GlcNAc/GlcNAc(6S)/GlcNSO3(6S)(1→, and hence, presents heterogeneity regarding negative net charge density. Furthermore, the heparinase digests inhibit the binding of SARS-CoV-2 spike protein to the ACE2 receptor. In summary, the acharan sulfate presented in this work has shown its great potential for application in the preparation of sulfated polysaccharides as an alternative to heparin with important biological activity.

Development of a safer and improved analytical method for polycyclic aromatic hydrocarbons in creosote products.

Polycyclic aromatic hydrocarbons (PAHs) in creosote products used for wood preservation are regulated in Japan. Although the analytical method for this regulation has been stipulated by law, two main problems have been highlighted, namely the use of dichloromethane, a potential carcinogen, as a solvent and inadequate purification. Therefore, an analytical method to solve these problems was developed in this study. Actual creosote-treated wood samples were examined, and it was found that acetone could be used as an alternative solvent. Purification methods using centrifugation, silica gel cartridges, and strong anion exchange (SAX) cartridges were also developed. It was found that the SAX cartridges strongly retained PAHs, and an effective purification method was developed using this phenomenon, in which contaminants were removed by washing with diethyl ether/hexane (1/9 v/v), which could not be achieved with a silica gel cartridge. This strong retention was attributed to cation-π interactions. The analytical method developed in this study yielded good recoveries (81.4-113.0%) with low relative standard deviations (<6.8%), and the limit of quantification (0.02-0.29 µg/g) was significantly lower than the current creosote product regulation. Therefore, this method can safely and effectively extract and purify PAHs from creosote products.

Enhanced selectivity for acidic contaminants in drinking water: From suspect screening to toxicity prediction.

A novel analytical workflow for suspect screening of organic acidic contaminants in drinking water is presented, featuring selective extraction by silica-based strong anion-exchange solid-phase extraction, mixed-mode liquid chromatography-high resolution accurate mass spectrometry (LC-HRMS), peak detection, feature reduction and compound identification. The novel use of an ammonium bicarbonate-based elution solvent extended strong anion-exchange solid-phase extraction applicability to LC-HRMS of strong acids. This approach performed with consistently higher recovery and repeatability (88 ± 7 % at 500 ng L-1), improved selectivity and lower matrix interference (mean = 12 %) over a generic mixed-mode weak anion exchange SPE method. In addition, a novel filter for reducing full-scan features from fulvic and humic acids was successfully introduced, reducing workload and potential for false positives. The workflow was then applied to 10 London municipal drinking water samples, revealing the presence of 22 confirmed and 37 tentatively identified substances. Several poorly investigated and potentially harmful compounds were found which included halogenated hydroxy-cyclopentene-diones and dibromomethanesulfonic acid. Some of these compounds have been reported as mutagenic in test systems and thus their presence here requires further investigation. Overall, this approach demonstrated that employing selective extraction improved detection and helped shortlist suspects and potentially toxic chemical contaminants with higher confidence.

Evaluating the "wrong-way-round" electrospray ionization of antiretroviral drugs for improved detection sensitivity.

The presence of antiretroviral drugs (ARVDs) in the aquatic environment poses a significant health risk to the ecosystem. The dilution of these compounds during wastewater treatment processes, followed by discharge into the environment, results in extremely low concentrations in the range of ng/L. Therefore, to enable detection of these low concentrations, it is important to determine the most efficient electrospray ionization (ESI) mode using the right mobile phase modifier and to establish a selective extraction procedure. In this study, we compared the ESI intensity in the positive and negative mode using both formic acid (FA) and ammonium hydroxide (NH4OH) as mobile phase modifiers. The results revealed a phenomenon known as the "wrong-way-round" (WWR) ESI in which high intensity [M + H]+ ions were detected under basic conditions using NH4OH as modifier and, similarly, high intensity [M-H]- ions were detected under acidic conditions using FA as modifier. Furthermore, mixed-mode strong cation (MCX) and mixed-mode strong anion (MAX) exchange sorbents were evaluated for extraction recoveries, which yielded extraction recoveries between 60 and 100%. Finally, the recoveries obtained using mixed-mode ion exchange sorbents compared to ion production during the ESI process provide evidence that ions produced in solution do not necessarily reflect the ions that are produced during the ESI process. Based on the results of this study, it is recommended to evaluate the optimal ionization mode under basic and acidic conditions, instead of defaulting to the use of acidic modifiers with positive ion detection.

Isolation and Compositional Analysis of Glycosaminoglycans.

The compositional and structural analysis of GAGs is challenging due to their heterogenous structures. Strong anion exchange (SAX) HPLC can aid in the compositional analysis of GAGs and can separate complex mixtures based on charge and degree of sulfation. Herein we describe the digestion and release of GAGs from tissue, and the compositional analysis using SAX-HPLC.

Arsenosugar extracted from algae: Isolation by anionic exchange solid-phase extraction.

Obtaining reliable speciation data for evaluating dietary exposure, and increasing understanding of arsenic biochemistry in algae, are hindered by the availability of suitable standards of arsenosugars, the major species in these types of samples. Moreover, chemical syntheses of such compounds have been reported to be complex and tedious. The aim of this work was to investigate the feasibility of the anionic exchange SPE cartridges (SAX and WAX) as an easy and quick alternative for the isolation and preconcentration of arsenosugars. Two commercial silica-based SPE cartridges strong anion exchange sorbent (DSC-SAX) and weak anion exchange sorbent (DSC-NH2) were compared for the SPE of three arsenosugars (PO4-Sug, SO3-Sug and SO4-Sug). The effect of pH, ionic strength, type of salt and elution solvent on the elution protocols of these arsenosugars are studied. Eluted solutions from SPE were analyzed by ICP-MS for total arsenic content and IC-ICP-MS for the study of arsenic speciation. The developed SPE procedure allows to obtain a solution containing the three arsenosugars isolated from other arsenic species with recoveries over 75% for SO3-Sug and SO4-Sug, whereas for PO4-Sug were around 45%.

Ratiometric fluorescent nanosystem based on upconversion nanoparticles for histamine determination in seafood.

Histamine is the prime culprit of toxicity resulting from seafood consumption, whereas conventional detection methods are not convenient to meet the needs of rapid histamine analysis nowadays. Based on upconversion nanoparticles (UCNPs) and inner filter effects (IFE), a novel ratiometric fluorescence nanosystem was developed for the efficient detection of histamine. Through pre-treatment with solid-phase extraction (SPE) and colorific azo coupling reaction of histamine, the fluorescence of UCNPs at 548 nm was quenched, while fluorescence at 664 nm was unchanged. Thus, ratiometric fluorescence I548/I664 was inversely proportional to histamine concentration at a wide range of 10-200 mg/L. The detection limit was 7.34 mg/L, one order of magnitude lower than that of the traditional colorimetric method (25 mg/L). In addition, such a convenient and environment-friendly detection system was further employed to quantify the histamine in fish, shrimp, and shellfish samples, showing excellent application potential in seafood safety.

Determination of Background Concentrations of Ag, Pd, Pt and Au in Highly Mineralized Ground Waters at Sub-ng L-1 Concentrations by Online Matrix Separation/Pre-Concentration Coupled to ICP-SFMS.

Though not regulated in directives such as the Water Framework Directive of the European Union, the investigation of geogenic background concentrations of certain elements such as precious metals is of increasing interest, in particular for the early detection of a potential environmental pollution due to the increased use in various industrial and technological applications and in medicine. However, the precise and accurate quantification of precious metals in natural waters is challenging due to the complex matrices and the ultra-low concentrations in the (sub-) ng L-1 range. A methodological approach, based on matrix separation and pre-concentration on the strong anion exchange resin TEVA® Resin in an online mode directly coupled to ICP-SFMS, has been developed for the determination of Ag, Pt, Pd and Au in ground water. Membrane desolvation sample introduction was used to reduce oxide-based spectral interferences, which complicate the quantification of these metals with high accuracy. To overcome errors arising from matrix effects-in particular, the highly varying major ion composition of the investigated ground water samples-an isotope dilution analysis and quantification based on standard additions, respectively, were performed. The method allowed to process four samples per hour in a fully automated mode. With a sample volume of only 8 mL, enrichment factors of 6-9 could be achieved, yielding detection limits <1 ng L-1. Validation of the trueness was performed based on the reference samples. This method has been used for the analysis of the total concentrations of Ag, Pt, Pd and Au in highly mineralized ground waters collected from springs located in important geological fault zones of Austria's territory. Concentrations ranges of 0.21-64.2 ng L-1 for Ag, 0.65-6.26 ng L-1 for Pd, 0.07-1.55 ng L-1 for Pt and 0.26-1.95 ng L-1 for Au were found.

Analyses of Inositol Phosphates and Phosphoinositides by Strong Anion Exchange (SAX)-HPLC.

The phosphate esters of myo-inositol (Ins) occur ubiquitously in biology. These molecules exist as soluble or membrane-resident derivatives and regulate a plethora of cellular functions including phosphate homeostasis, DNA repair, vesicle trafficking, metabolism, cell polarity, tip-directed growth, and membrane morphogenesis. Phosphorylation of all inositol hydroxyl groups generates phytic acid (InsP6), the most abundant inositol phosphate present in eukaryotic cells. However, phytic acid is not the most highly phosphorylated naturally occurring inositol phosphate. Specialized small molecule kinases catalyze the formation of the so-called myo-inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8. These molecules are characterized by one or several "high-energy" diphosphate moieties and are ubiquitous in eukaryotic cells. In plants, PP-InsPs play critical roles in immune responses and nutrient sensing. The detection of inositol derivatives in plants is challenging. This is particularly the case for inositol pyrophosphates because diphospho bonds are labile in plant cell extracts due to high amounts of acid phosphatase activity. We present two steady-state inositol labeling-based techniques coupled with strong anion exchange (SAX)-HPLC analyses that allow robust detection and quantification of soluble and membrane-resident inositol polyphosphates in plant extracts. These techniques will be instrumental to uncover the cellular and physiological processes controlled by these intriguing regulatory molecules in plants.

Underwater suspended bifunctionalized polyethyleneimine-based sponge for selective removal of anionic pollutants from aqueous solution.

Highly selective and efficient removal of ionic pollutants, including ionic organic compounds and heavy metal ions from water, is still a huge challenge due to the complex nature of polluted water. To meet this challenge, we presented the synthesis of bifunctionalized polyethyleneimine-based sponges through cryo-polymerization via BDDE as the crosslinker followed by bifunctional modification with glycidyl trimethylammonium chloride (GTAC) and phenyl glycidyl ether (PGE), which simultaneously afford quaternary ammonium cation (strongly basic and hydrophilic) and phenyl (hydrophobic) functionalities, respectively. As a result, a hybrid hydrophilic-hydrophobic sponge is generated that could stably be suspended underwater due to the co-operative effect of the water-absorbing hydrophilic domain and the hydrophobic domain generating buoyancy. The quaternized and phenyl-functionalized PEI-based sponge (SQP-PEI) demonstrated highly selective and efficient removal of anionic pollutants from water, including diclofenac sodium (DIC), methyl orange (MO) and chromium (Cr(VI)) with co-existing interferences. The Langmuir isotherms revealed the maximum adsorption capacities of 342.7 mg/g, 491.9 mg/g, and 242.7 mg/g for DIC, MO, and Cr(VI), respectively. The studies of adsorption mechanism suggested that the bifunctional SQP-PEI sponge indeed afford both strong anion-exchange interaction and π-π interaction toward organic pollutants DIC and MO, and the strong anion-exchange interaction can be the dominated adsorption mechanism for anionic DIC, MO and Cr(VI) species. The suspended SQP-PEI also demonstrated excellent reusability, which shows the potential of SQP-PEI for real applications.

Antibody-free enrichment method for proteome-wide analysis of endogenous SUMOylation sites.

SUMOylation is a reversible post-translational modification that plays crucial roles in numerous cellular processes. Although anti-SUMO antibodies have been applied to analyze exogenous and endogenous SUMOylation, such immunoprecipitation enrichment strategy is applicable only for the enrichment of one specific SUMO type in mammalian cells, unable to map the global landscape of all endogenous SUMOylation simultaneously. To address this issue, we proposed an antibody-free strategy to enrich and profile endogenous SUMO1/2/3-modified peptides simultaneously. Upon trypsin digestion, the SUMO1- and SUMO2/3-modified peptides contained SUMO remnants with 7 and 9 acidic amino acids respectively, which carried more negative charges at high pH and could interact with strong anion exchange (SAX) materials more strongly than non-SUMOylated peptides, thus enabling the specific enrichment of endogenous SUMOylated peptides. Followed by the secondary digestion with Asp-N/Glu-C to generate smaller SUMOylated peptides with proper length for MS identification, off-line high-pH C18 pre-fractionation and low pH nanoRPLC-ESI-MS/MS analysis, 177 SUMO1-modified sites and 74 SUMO2/3-modified sites were unbiasedly identified in HeLa cell lysate. To the best of our knowledge, this was the first antibody-free strategy to comprehensively profile various endogenous SUMOylation sites, demonstrating the great potential in the comprehensive analysis of endogenous SUMOylation across various species and organs, which might further facilitate the understanding of SUMO's function in physiology and pathology.

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry-Based Glycoproteomics.

Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography and its derivatives, porous graphitic carbon, reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as mass spectrometry instrumentation and software improve, so this review aims to help equip researchers with the necessary information to choose appropriate enrichment strategies that best complement these efforts.

Biomedical copper speciation in relation to Wilson's disease using strong anion exchange chromatography coupled to triple quadrupole inductively coupled plasma mass spectrometry.

Biomedical analytical methods often rely on indirect measurements, such as immunoassays, which can lack effective metrological traceability. In the nephelometric determination of ceruloplasmin (Cp), an important protein whose circulating level is altered in Wilson's disease (WD), the anti-Cp antibody used is not specific for the biologically active holoprotein so the assay can overestimate the concentration of Cp due to the presence of the apoprotein. By providing quantitation using elemental standards, the use of strong anion exchange chromatography (SAX) coupled to triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS-MS) can overcome the drawbacks of methods for the measurement of metalloproteins reliant on immunoassays. In the current study, a SAX-ICP-MS-MS method for Cp quantification was designed and tested in samples of blood serum of WD patients and healthy controls. Using standards based on a copper-EDTA complex for calibration, the method provides relatively simple quantification of Cp with the limit of detection of 0.1 µg L-1 (limit of quantification 0.4 µg L-1). The method was also used to investigate the copper species separated by using a 30 kDa cut-off ultrafiltration device. The so-called "exchangeable" copper fraction is considered as an alternative clinical biomarker of WD. Using the designed speciation approach, it was shown that the ultrafiltration method can overestimate the "exchangeable" copper fraction due to a removal of copper from Cp. This was confirmed by comparing the enzymatic activity of the fractions. Thus, the specificity of the "exchangeable" copper test can be ensured only under strict maintenance of ultrafiltration conditions.

High-Throughput Characterization of Histidine Phosphorylation Sites Using UPAX and Tandem Mass Spectrometry.

Liquid chromatography (LC)-tandem mass spectrometry (MS/MS) is key for the characterization of phosphorylation sites in a high-throughput manner, and its application has proven essential to elucidate the phosphoproteome of many biological systems. Following proteolytic digestion of proteins extracted from tissues or cells, phosphopeptides are typically enriched by affinity chromatography using TiO2 or metal-ions (e.g., Fe3+) coupled to solid-phase materials, prior to LC-MS/MS analysis. Separation of relatively low abundance phosphopeptides from nonphosphorylated peptides in these types of extremely complex mixtures is essential to maximize coverage of the phosphoproteome. Maintaining acidic conditions during these IMAC or TiO2-based enrichment minimizes the concurrent unwanted binding of highly acidic peptides. However, while peptides containing phosphomonoesters, namely, phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr), are stable under these acidic binding conditions, phosphopeptides containing acid-labile phosphate group such as phosphohistidine (pHis), are not. Consequently, hydrolysis of these types of phosphopeptides occurs during standard phosphopeptide enrichment, and subsequent phosphosite identification by LC-MS/MS is severely compromised. Here we describe UPAX, unbiased phosphopeptide enrichment using strong anion exchange, for the separation of both acid-stable (pSer, pThr, pTyr) and acid-labile phosphopeptides (including those containing pHis) from nonphosphorylated peptides. We outline how implementation of UPAX prior to a minimally modified standard proteomics workflow can be used to identify sites of pHis as well as other acid-labile, as well as acid-stable phosphosites.

Strong anion exchange-mediated phosphoproteomics reveals extensive human non-canonical phosphorylation.

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site-specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other "non-canonical" amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non-canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry-based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non-canonical phosphosites is approximately one-third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high-throughput exploration of non-canonical phosphorylation in all organisms.

Finding the Sweet Spot in ERLIC Mobile Phase for Simultaneous Enrichment of N-Glyco and Phosphopeptides.

Simultaneous enrichment of glyco- and phosphopeptides will benefit the studies of biological processes regulated by these posttranslational modifications (PTMs). It will also reveal potential crosstalk between these two ubiquitous PTMs. Unlike custom-designed multifunctional solid phase extraction (SPE) materials, operating strong anion exchange (SAX) resin in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) mode provides a readily available strategy to analytical labs for enrichment of these PTMs for subsequent mass spectrometry (MS)-based characterization. However, the choice of mobile phase has largely relied on empirical rules from hydrophilic interaction chromatography (HILIC) or ion-exchange chromatography (IEX) without further optimization and adjustments. In this study, ten mobile phase compositions of ERLIC were systematically compared; the impact of multiple factors including organic phase proportion, ion pairing reagent, pH, and salt on the retention of glycosylated and phosphorylated peptides was evaluated. This study demonstrated good enrichment of glyco- and phosphopeptides from the nonmodified peptides in a complex tryptic digest. Moreover, the enriched glyco- and phosphopeptides elute in different fractions by orthogonal retention mechanisms of hydrophilic interaction and electrostatic interaction in ERLIC, maximizing the LC-MS identification of each PTM. The optimized mobile phase can be adapted to the ERLIC HPLC system, where the high resolution in separating multiple PTMs will benefit large-scale MS-based PTM profiling and in-depth characterization.

Elimination of ghost peaks by optimization of anion exchange chromatography method for determination of gamma-carboxyglutamic acid (Gla)-domainless impurity in recombinant activated clotting factor VII drug products.

Secreted recombinant activated clotting factor VII activated (rFVIIa) in cell culture media missing gamma-carboxyglutamic acid (Gla) domain as a result of failure in gamma-carboxylation or cell lysis is called Gla-domainless impurity which has less negative charge compared to native rFVIIa. Based on risk assessment, this type of impurity is considered as critical drug product quality attribute of rFVIIa and its quantitative analysis in product batches is a critical issue in quality control laboratories. Analysis of Gla-domainless impurity is accomplished by Strong Anion Exchange Chromatography (SAX) in recombinant factor VIIa using Tris and Bis-Tris propane salt buffers as equilibrating buffers and high concentration ammonium acetate as an eluent. Appearance of ghost peaks with notable intensity during elution time of Gla-domainless impurity caused distortion of the related peak and interference with robust and accurate quantification of this impurity. Subsequently, the ghost peak was analyzed by LC-ESI-MS to determine the structure which showed the m/z values at 905.27, 623.53 and 341.60 and 563.73. To find the source of these ghost peaks, quality of water, buffer salts and Chelex-100 together with ionic strength of mobile phase A (addition of 25 mM NaCl) were considered as affecting parameters and several experiments designed with DOE software to optimize the best condition of highest quality the method with lowest signal of ghost peak noises. By interpretation of DOE result, it is concluded that high grade water and buffer salt along with high quality Chelex-100 resins are important factors to achieve a method with lowest ghost peaks. However, addition of 25 mM NaCl to mobile phase A with either lower quality buffer salts or lower water grade yields high quality chromatogram peak with acceptable ghost peaks. LC/MS analysis indicates that macrostructures of Bis-Tris propane made up as a result of hydrogen bonds with each other or Tris molecules can be the source of ghost peaks.

Hypercrosslinked strong anion-exchange polymers for selective extraction of fluoroquinolones in milk samples.

In this work, hypercrosslinked strong anion-exchange polymer resins (HXLPP-SAX) were synthesized and evaluated as solid-phase extraction (SPE) sorbents for extracting fluoroquinolones (FQs) in milk samples. These particles were prepared using a protocol that was facile and cost-effective by combining hypercrosslinking reactions and quaternisation reactions in one step. After the synthesis, the morphological properties and anion-exchange, adsorption, and selectivity performances of the HXLPP-SAX resins were characterized and evaluated. It was shown that the polymers were monodisperse microspheres, with the mean diameters of 2-4 µm, ion-exchange capacity (IEC) of 0.311 meq/g, and specific surface area more than 1000 m2/g. The resins applied as SPE sorbents possessed high selectivity in the extraction of amphoteric compounds (enoxacin and enrofloxacin as the representatives) followed by HPLC-UV analysis, as compared to commercially available strong anion-exchange sorbents (Oasis MAX). The SPE-HPLC-UV method was fully validated and exhibited good linearity (10-2000 ng/g, R2 ≥ 0.997), low limits of detection (2.8-5.1 ng/g), good recoveries (85.8%-117.9%, RSDs ≤ 7.1%) and precisions (intra-day RSDs ≤ 7.7% and inter-day RSDs ≤ 9.4%). Then the method was performed the selective enrichment of six FQs (enoxacin, norfloxacin, ciprofloxacin, lomefloxacin, enrofloxacin, and sparfloxacin) in milk samples. The present results demonstrated that the proposed resins exhibited a promising potential for the enrichment and determination of FQ residues in milk samples as well as in other samples with complex matrices.