Strong Cation-exchange Material Research Articles

Analysis of acrylamide in vegetable chips after derivatization with 2-mercaptobenzoic acid by liquid chromatography\u2013mass spectrometry

Since many years, acrylamide (AA) is a well-known toxicologically relevant processing contaminant (“food-borne toxicant”). However, only during the recent years, high levels of acrylamide have been reported in vegetable chips. In the present study, AA was quantitated via a modified derivatization procedure with 2-mercaptobenzoic acid based on stable isotope dilution analysis and liquid chromatography–mass spectrometry. Extraction with a modified QuEChERS (quick, easy, cheap, efficient, rugged, safe) method, defatting with n-hexane, and a solid phase extraction clean-up with strong cation-exchange material were performed prior to the derivatization step. Limits of detection and quantitation (LoD and LoQ) were 12 and 41 µg of AA/kg of vegetable chips (estimated via signal-to-noise ratios of 3:1 and 10:1, respectively), and thus below the LoQ of 50 µg/kg requested by the European Food Safety Authority. Recovery rates between 92 and 101% at four spiking levels with a good precision expressed as a relative standard deviation < 7% were determined. With this method at hand, a survey of the current AA amounts in 38 vegetable chips from the worldwide market was performed, showing a remarkable variability between the different vegetables, but also between different products of the same vegetable. Thereby, the AA amounts ranged between 77.3 and 3090 µg/kg, with an average of 954 µg/kg which was distinctly higher in comparison to commercially available potato chips also analyzed in the present study (12 samples, range: 117–832 µg/kg, average: 449 µg/kg). While for sweet potato and parsnip relatively low AA amounts were found, beetroot and carrot showed rather high contents.

Column Classification/Characterisation of Strong Cation Exchange Phases for the Liquid Chromatographic Analysis of Small Molecular Weight Bases

A simple, rapid and robust protocol for the characterisation of strong cation exchange columns for the analysis of small molecular weight bases is described. A range of ten different phases were characterised, and the resultant selectivity and retention factors analysed using Principal Component Analysis. The score plots for the first and second principal components described 83% of the variability within the dataset. Score plots highlighted the large chromatographic differences observed between the phases, the validity of which was established using a larger range of bases. All the strong cation exchange materials demonstrated a synergistic mixed mode (i.e. ion exchange and hydrophobic) retention mechanism. Principal Component Analysis also highlighted the potential difficulty in locating suitable strong cation exchange “back-up” columns for the analysis of small molecular weight bases in that the characterised columns all displayed very different selectivities. The robustness of the protocol was confirmed by a factorial design experiment.

Trimodal Mixed Mode Chromatography That Enables Efficient Offline Two-Dimensional Peptide Fractionation for Proteome Analysis.

Offline two-dimensional chromatography is a common means to achieve deep proteome coverage. To reduce sample complexity and dynamic range and to utilize mass spectrometer (MS) time efficiently, high chromatographic resolution of and good orthogonality between the two dimensions are needed. Ion exchange and high pH reversed phase chromatography are often used for this purpose. However, the former requires desalting to be MS-compatible, and the latter requires fraction pooling to create orthogonality. Here, we report an alternative first-dimension separation technique using a commercial trimodal phase incorporating polar embedded reversed phase, weak anion exchange, and strong cation exchange material. The column is capable of retaining polar and nonpolar peptides alike without noticeable breakthrough. It allows separating ordinary and TMT-labeled peptides under mild acidic conditions using an acetonitrile gradient. The direct MS compatibility of solvents and good orthogonality to online coupled C18 columns enable a straightforward workflow without fraction pooling and desalting while showing comparable performance to the other techniques. The method scales from low to high microgram sample quantity and is amenable to full automation. To demonstrate practical utility, we analyzed the proteomes of 10 human pancreatic cancer cell lines to a depth of >8,700 quantified proteins.

A simple and rapid screening method for glyphosate in water using flow-injection with electrochemiluminescence detection

A flow-injection (FI) system coupled with the electrochemiluminescence (ECL) detection method for rapid screening of glyphosate (GLY) in water has been developed. A wall-jet type ECL detector coupled with an indium/tin oxide (ITO)-coated glass working electrode was modified from a thin-layer electrochemical cell, and tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+)-based ECL reaction was used for the sensitive detection of GLY. Under optimized conditions, the linear range of the method was 0.03–2.81 mg L−1 GLY with R2 = 0.9998 (n = 7). Repeatability was 5.3% (n = 27) and the detection limit (3σ) was 0.03 mg L−1 GLY. Sample throughput was about 100 injections h−1. Possible interference from some multivalent cations can be readily eliminated by passing the acidified water sample through a solid-phase extraction column packed with strong cation-exchange materials before analysis. The method was examined by application to surface waters including tap water and irrigation water.

Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex

Here we describe the function of a previously uncharacterized protein, named family with sequence similarity 60 member A (FAM60A) that maps to chromosome 12p11 in humans. We use quantitative proteomics to determine that the main biochemical partners of FAM60A are subunits of the Sin3 deacetylase complex and show that FAM60A resides in active HDAC complexes. In addition, we conduct gene expression pathway analysis and find that FAM60A regulates expression of genes that encode components of the TGF-beta signaling pathway. Moreover, our studies reveal that loss of FAM60A or another component of the Sin3 complex, SDS3, leads to a change in cell morphology and an increase in cell migration. These studies reveal the function of a previously uncharacterized protein and implicate the Sin3 complex in suppressing cell migration.

A novel solid phase extraction method for pre-concentration of gadolinium and gadolinium based MRI contrast agents from the environment

An efficient solid phase extraction (SPE) method using bis-(2-ethylhexyl)-phosphate (HDEHP) coated reverse phase C18 support has been developed for the pre-concentration of Gadolinium (Gd) and Gd contrast agents widely used in magnetic resonance imaging (MRI). Enrichment of Gd in the ionic form has been compared with strong cation exchange material Chromabond SA (SCX), weak ion exchange material Chelex-100 and also with lanthanide specific HDEHP modified reverse phase C18. The determination of Gd and its complexes after enrichment were performed using inductively coupled plasma mass spectrometry (ICP-MS) and on the basis of 158Gd. Among the three SPE materials, HDEHP coated reverse phase C18 SPE has been found to be most efficient, yielding a hundredfold Gd enrichment with > 95% recovery for linear and cyclic contrast agents like Gd-DTPA (Magnevist), Gd-DOTA (Dotarem), Gd-BOPTA (Multihance), and Gd-BT-DO3A (Gadovist). The developed SPE method has been successfully applied to the surface water and waste water samples originated from different places in Berlin, Germany. The results were in good agreement with the results obtained with direct measurement with ICP-MS. The developed pre-concentration method can be efficiently used for the determination of trace levels of gadolinium in the environment even with less sensitive analytical techniques.

Determination of the interparticle void volume in packed beds via intraparticle Donnan exclusion

Interparticle void volumes and porosities of packed capillaries have been determined using intraparticle Donnan exclusion of a small, unretained, co-ionic tracer (nitrate ions). The operational domain of this approach has been characterized for bare silica, reversed-phase, and strong cation-exchange materials (with different particle sizes and intraparticle pore sizes) in dependence of the mobile phase ionic strength. Interparticle porosities agree well with those analyzed by inverse size-exclusion chromatography (ISEC). Limitations to the use of Donnan exclusion (electrostatic exclusion) and ISEC (mechanical exclusion) arise as either type of exclusion becomes noticeable also in the cusp regions between the particles, or as the intraparticle pores are so large that complete electrostatic and size-exclusion are difficult to realize. Our data confirm that intraparticle Donnan exclusion presents a most simple, fast, and reliable approach for the analysis of packing densities.

HPLC of basic drugs using non‐aqueous ionic eluents: evaluation of a 3 μm strong cation‐exchange material

HPLC columns packed with 3 microm particle size HPLC Technology Techsphere SCX (propylsulfonic acid-modified) silica offer considerable advantages over 5 microm SCX packings in the analysis of basic drugs using 100% methanol eluents containing an ionic modifier such as ammonium perchlorate. The basic drugs studied included clozapine and norclozapine, olanzapine, quinine and quinidine, and amitriptyline, nortriptyline, imipramine and desipramine. The 3 microm column was not only more efficient for a given column length compared with 5 microm materials, but also elution times were less, a phenomenon observed in reversed-phase systems. The high efficiencies and excellent peak shapes obtained with the 3 microm SCX-modified packing together with the relatively low back-pressures attained show that such materials deserve serious consideration by laboratories involved in the analysis of basic drugs. Manufacturers should offer such packings as a matter of routine. Alternative ionic modifiers such as ammonium acetate are available for use with mass spectrometric detection if required.

Phosphorylation by Casein Kinase 1 Regulates Tonicity-induced Osmotic Response Element-binding Protein/Tonicity Enhancer-binding Protein Nucleocytoplasmic Trafficking

The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, is the only known osmo-sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. Although it is well documented that the subcellular localization and transactivation activity of OREBP/TonEBP are tightly regulated by extracellular tonicity, the molecular mechanisms involved remain elusive. Here we show that nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by the dual phosphorylation of Ser-155 and Ser-158. Alanine scanning mutagenesis revealed that Ser-155 is an essential residue that regulates OREBP/TonEBP nucleocytoplasmic trafficking. Tandem mass spectrometry revealed that Ser-155 and Ser-158 of OREBP/TonEBP are both phosphorylated in living cells under hypotonic conditions. In vitro phosphorylation assays further suggest that phosphorylation of the two serine residues proceeds in a hierarchical manner with phosphorylation of Ser-155 priming the phosphorylation of Ser-158 and that these phosphorylations are essential for nucleocytoplasmic trafficking of the transcription factor. Finally, we have shown that the pharmacological inhibition of casein kinase 1 (CK1) abolishes the phosphorylation of Ser-158 and impedes OREBP/TonEBP nuclear export and that recombinant CK1 phosphorylates Ser-158. Knockdown of CK1alpha1L, a novel isoform of CK1, inhibits hypotonicity-induced OREBP/TonEBP nuclear export. Together these data highlight the importance of Ser-155 and Ser-158 in the nucleocytoplasmic trafficking of OREBP/TonEBP and indicate that CK1 plays a major role in regulating this process.

Modular Stop and Go Extraction Tips with Stacked Disks for Parallel and Multidimensional Peptide Fractionation in Proteomics

Proteome complexity necessitates protein or peptide separation prior to analysis. We previously described a pipet-tip based peptide micropurification system named StageTips (STop and Go Extraction Tips), which consists of a very small disk of membrane-embedded separation material. Here, we extend this approach in several dimensions by stacking disks containing reversed phase (C(18)) and strong cation exchange (SCX) materials. Multidimensional fractionation as well as desalting, filtration, and concentration prior to mass spectrometry in single or tandem columns is described. C(18)-SCX-C(18) stacked disks significantly improved protein identification by LC-MS/MS for an E. coli protein digest and by MALDI-MS for a 12 standard protein digest. Sequential fractionation based on C(18)- followed by SCX material was also developed. This multidimensional fractionation approach was expanded to parallel sample preparation by incorporating C(18)-SCX-StageTips into a 96-well plate (StagePlate). Fractions were collected into other C(18)-StagePlates and desalted and eluted in parallel to sample well plates or MALDI targets. This approach is suitable for high throughput protein identification for moderately complex, low abundance samples using automated nanoelectrospray-MS/MS or MALDI-MS.

An automated multidimensional protein identification technology for shotgun proteomics.

We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10000 to 1 between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated. By improving sample preparation along with separations, the method improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.

Strategy for the development of automated methods involving dialysis and trace enrichment as on-line sample preparation for the determination of basic drugs in plasma by liquid chromatography

Among the sample preparation techniques, dialysis followed by clean-up and enrichment of the dialysate on a pre-column has proved to be a useful approach for the LC determination of drugs in plasma. By use of sample processors, like the ASTED system, such bioanalytical methods can be fully automated, the dialysis and trace enrichment steps being directly coupled to LC. In order to facilitate the development of such automated methods, a strategy based on a decision tree has been elaborated. After the selection of appropriate conditions for the LC analysis, the decision tree provides information about suggested starting conditions and guidelines for the optimisation of the most important parameters likely to influence analyte recovery and method selectivity. The plasma samples are dialysed on a cellulose acetate membrane in the static-pulsed mode and the dialysate is enriched on a trace enrichment pre-column packed with octadecyl silica or with a strong cation-exchange material. This decision tree is until now restricted to the analysis of basic drugs in plasma. In order to demonstrate the applicability of this method development strategy, an automated procedure based on the coupling of dialysis with trace enrichment has been developed for the LC determination of antifungal agents (clotrimazole, econazole and miconazole) in plasma.

HPLC of basic drugs on microparticulate strong cation-exchange materials — a review

Propylsulphonic acid (SCX)-modified silica HPLC columns used with methanol or aqueous methanol eluents of appropriate pH and ionic strength can give good retention and peak shape for basic drugs. In the system studied, eluent pH influenced retention via protonation of basic analytes, the p K a of the analyte indicating the pH where retention begins to decrease at constant ionic strength. At constant pH, retention is inversely proportional to ionic strength for protonated bases and quaternary ammonium compounds. The underlying retention mechanism appears to be ion-exchange with the SCX moieties, although ionized surface silanols may also contribute to retention at higher eluent pH values. In capillary electrochromatography (CEC) unprecedented efficiencies, but similar selectivity, to that observed in conventional HPLC have been obtained for a standard range of basic drugs using Waters Spherisorb S3SCX. SCX-modified silica columns can be used in the HPLC of many basic drugs, including some compounds that are poorly retained on unmodified silica using methanol-rich eluents. N-Desalkyl and sulphoxide metabolites are often resolved at an appropriate eluent pH. Even analogues differing by a methylene unit in a side-chain remote from a basic centre are often resolved. Applications of Waters Spherisorb S5SCX columns include HPLC of antimalarials such as chloroquine and quinine, cardioactive drugs, for example amiodarone and flecainide, antipsychotics (clozapine, olanzapine), and antidepressants (amitriptyline, clomipramine, dothiepin, fluoxetine) and their N-desalkyl metabolites. Major practical features of these systems are that (i) acidic and neutral compounds are not retained, (ii) solvent extracts can be injected directly, and (iii) eluent recycling can be performed routinely.

Automated liquid chromatographic determination of atenolol in plasma using dialysis and trace enrichment on a cation-exchange precolumn for sample handling

A fully automated method involving dialysis combined with trace enrichment was developed for the liquid chromatographic (LC) determination of atenolol, a hydrophilic β-blocking agent, in human plasma. The plasma samples were dialysed on a cellulose acetate membrane and the dialysate was reconcentrated on a short trace enrichment column (TEC) packed with a strong cation-exchange material. All sample handling operations can be executed automatically by a sample processor (ASTED system). After TEC conditioning, the plasma sample, to which the internal standard (sotalol, another hydrophilic β-blocker) was automatically added, was introduced in the donor channel and dialysed in the static/pulsed mode. The dialysis liquid consisted of 4.3 m M phosphoric acid. When the dialysis process was discontinued, the analytes were eluted from the TEC in the back-flush mode by the LC mobile phase and transferred to the analytical column, packed with octyl silica. The LC mobile phase consisted of phosphate buffer, pH 7.0–methanol (81:19; v/v) with 1-octanesulfonate. Atenolol and the internal standard were monitored photometrically at 225 nm. The different parameters influencing the dialysis and trace enrichment processes were optimised with respect to analyte recovery. The influence of two different kinds of cation-exchange material on analyte recovery and peak efficiency was also studied. The method was then validated in the concentration range 25–1000 ng/ml. The mean recovery for atenolol was 65% and the limit of quantitation was 25 ng/ml.

HPLC of basic drugs and quaternary ammonium compounds on microparticulate strong cation-exchange materials using methanolic or aqueous methanol eluents containing an ionic modifier

Sulphopropyl (SCX)-method silica HPLC columns used with methanolic or aqueous methanol eluents of appropriate pH and ionic strength can give good retention and peak shape for quaternary ammonium compounds and basic drugs. In the system studied, eluent pH influenced retention via protonation of basic analytes, the p Ka indicating the pH where retention began to decrease at constant ionic strength. At constant eluent pH retention was inversely proportional to ionic strength for protonated bases and quaternary ammonium compounds. However, this effect was less marked at pH 8.3 as compared to results obtained under acidic conditions. Except for codeine, morphine and lignocaine, the addition of water had no major effects on retention or selectivity at concentrations up to 30% (v/v) at pH 6.7. However, and in contrast to behaviour on unmodified silica, the addition of up to 5% (v/v) water under strongly acidic conditions caused a doubling of retention for most analytes studied. SCX-modified silica columns can be used in the HPLC of a range of basic drugs, including many compounds which are poorly retained on unmodified silica using methanol-rich eluents. The underlying retention mechanism appears to be ion exchange with the SCX moieties, although ionized surface silanols may also contribute to retention at higher eluent pH values. Applications of SCX columns in the HPLC of basic drugs include the analysis of antimalarials such as chloroquine, desethylchloroquine, hydroxychloroquine and quinine, benzodiazepines such as clonazepam, bronchodilators such as salbutamol and terbutaline, cardioactive drugs such as flecainide and lignocaine, and tricyclic antidepressants such as amitriptyline, dothiepin, and imipramine, and their N-demethyl, hydroxyl and sulphoxide metabolites.

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene-divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.

Determination of arsenic in a nickel based alloy using a microwave-digestion procedure and a continuous flow hydride generation atomic absorption system incorporating on-line matrix removal

A continuous flow hydride generation system incorporating a micro-column of strong cation-exchange material (SCX) was used in conjunction with flame heated quartz tube atomic absorption spectrometry for the determination of arsenic in a nickel based alloy. The system is capable of on-line matrix removal of nickel, which would cause severe depression of the arsenic signal. The resulting matrix stripped solution allowed hydride generation of arsenic to be achieved interference free. The columns were regenerated when necessary by using 1 M HCl. The procedure was successfully applied to a standard reference nickel based alloy (BCS 346) and the accuracy assessed through a recovery experiment for a 10 p.p.b. sample. The system was found to have a limit of detection of 1.3 ng and an RSD of 3.0%.

Cation-exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica

A strong cation-exchange material, poly(2-sulfoethyl aspartamide)-silica (PolySULFOETHYL Aspartamide) was developed for purification and analysis of peptides by high-performance liquid chromatography. All peptides examined were retained at pH 3, even when the amino terminus was the only basic group. Peptides were eluted in order of increasing number of basic residues with a salt gradient. Capacity was high, as was selectivity and column efficiency. This new column material displays modest mixed-mode effects, allowing the resolution of peptides having identical charges at a given pH. The selectivity can be manipulated by the addition of organic solvent to the mobile phases; this increases the retention of some peptides and decreases the retention of others. The retention in any given case may reflect a combination of steric factors and non-electrostatic interactions. Selectivity was complementary to that of reversed-phase chromatography (RPC) materials. Excellent purifications were obtained by sequential use of Poly-SUFLOETHYL Aspartamide and RPC columns for purification of peptides from crude tissue extracts. The new cation exchanger is quite promising as a supplement to RPC for general peptide chromatography.

Measurement of Cardioactive Drugs in Biological Samples by HPLC

Abstract Unmodified silica columns used with non-aqueous, usually methanolic, eluents containing an ionic modifier provide simple, flexible systems for the HPLC analysis of a wide range of basic cardioactive drugs and some quaternary ammonium compounds. Retention and peak shape are influenced primarily by eluent pH and ionic strength although changes in eluent composition and in the stationary phase may also prove useful. Recently it has been found that chemically-bonded strong cation exchange materials provide enhanced retention for analytes which are poorly retained on silica alone. Solvent extracts and elution fractions from bonded-phase extraction tubes may be analysed directly and, in addition to low-wavelength UV and fluorescence, electrochemical oxidation detection may be used routinely for a wide range of analytes including secondary and tertiary aliphatic amines.

Separation of plant betaines and their sulphur analogues by cation-exchange high-performance liquid chromatography

Recent interest in stress-induced accumulation of glycinebetaine in plants has stimulated the search for improved methods of assaying betaines. A method based on high-performance liquid chromatographic separation on a strong cation-exchange material combined with low-wavelength ultraviolet absorbance detection is described. This forms the basis of a rapid assay procedure for glycinebetaine using plant saps as the starting material and avoiding the need for derivatization.