Strong Cation-exchange High-performance Liquid Research Articles

Rapid and Efficient Enrichment of Snake Venoms from Human Plasma Using a Strong Cation Exchange Tip Column to Improve Snakebite Diagnosis.

Snake envenomation is a serious public health issue in many tropical and subtropical countries. Accurate diagnosis and immediate antivenom treatment are critical for effective management. However, the venom concentration in the victims’ plasma is usually low, representing one of the bottlenecks in developing clinically applicable assays for venom detection and snakebite diagnosis. In this study, we attempted to develop a simple method for rapid enrichment of venom proteins from human plasma to facilitate detection. Our experiments showed that several major protein components of both Naja atra (N. atra) and Bungarus multicinctus (B. multicinctus) venoms have higher isoelectric point (pI) values relative to high-abundance human plasma proteins and could be separated via strong cation exchange–high-performance liquid chromatography (SCX-HPLC). Based on this principle, we developed an SCX tip column-based protocol for rapid enrichment of N. atra and B. multicinctus venom proteins from human plasma. Application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) led to the identification of cytotoxin and beta-bungarotoxin as the major proteins enriched by the SCX tip column in each venom sample. The entire process of venom enrichment could be completed within 10–15 min. Combination of this method with our previously developed lateral flow strip assays (rapid test) significantly enhanced the sensitivity of the rapid test, mainly via depletion of the plasma protein background, as well as increase in venom protein concentration. Notably, the SCX tip column-based enrichment method has the potential to efficiently enrich other Elapidae snake venoms containing proteins with higher pI values, thereby facilitating venom detection with other assays. This simple and rapid sample preparation method should aid in improving the clinical utility of diagnostic assays for snakebite.

Simultaneous Determination of Six Protoalkaloids in Chinese Local Varieties of Loose-Skin Mandarins and Sweet Oranges by Strong Cation Exchange-High Performance Liquid Chromatography

Six protoalkaloids in the fruits of 34 Chinese local loose-skin mandarins and 25 sweet oranges were detected simultaneously by strong cation exchange-high performance liquid chromatography with an ultraviolet detector (SCX-HPLC-UV). Linear regression analysis showed satisfactory linearity for all the analytes with the correlation coefficients (R2) higher than 0.9998. The satisfactory recoveries of the six protoalkaloids in pulps at two spiked levels ranged from 82.8 to 93.4% (RSDs, 1.5–6.1%) and those of synephrine in peels ranged from 89.3 to 91.9% (RSDs, 4.8–5.3%). The limits of quantification (LOQs) were 0.2 mg kg−1. The proposed method was used to determinate the protoalkaloids in loose-skin mandarins and sweet oranges to evaluate its feasibility and accuracy. The major alkaloid in the tested samples was synephrine, and the predominant alkaloid was N-methyltyramine, followed by octopamine. The variations in alkaloid components and contents were both largely the same for the tested varieties, verifying that alkaloids in plants are genetically controlled.

Comparison of Aurantii Fructus Immaturus and Aurantii Fructus based on multiple chromatographic analysis and chemometrics methods

To get a better understanding of the bioactive constituents in Aurantii Fructus Immaturus (AFI) and Aurantii Fructus (AF), in the present study, a comprehensive strategy integrating multiple chromatographic analysis and chemometrics methods was firstly proposed. Based on segmental monitoring, a high-performance liquid chromatography (HPLC)-variable wavelength detection method was established for simultaneous quantification of ten major flavonoids, and the quantitative data were further analyzed by hierarchical cluster analysis (HCA) and principal component analysis (PCA). A strong cation exchange-high performance liquid chromatography (SCX-HPLC) method combined with t-test and one-way analysis of variance (ANOVA) was developed to determine synephrine, the major alkaloid in AFI and AF. The essential oils were analyzed by gas chromatography-mass spectrometry (GC–MS) and further processed by partial least squares discrimination analysis (PLS-DA). The results indicated that the contents of ten flavonoids and synephrine in AFI were significantly higher than those in AF, and significant difference existed in samples from different geographical origins. Also, 9 differential volatile constituents detected could be used as chemical markers for discrimination of AFI and AF. Collectively, the proposed comprehensive analysis might be a well-acceptable strategy to evaluate the quality of traditional citrus herbs.

Sample Preparation Approaches for iTRAQ Labeling and Quantitative Proteomic Analyses in Systems Biology.

Among a variety of global quantification strategies utilized in mass spectrometry (MS)-based proteomics, isobaric tags for relative and absolute quantitation (iTRAQ) are an attractive option for examining the relative amounts of proteins in different samples. The inherent complexity of mammalian proteomes and the diversity of protein physicochemical properties mean that complete proteome coverage is still unlikely from a single analytical method. Numerous options exist for reducing protein sample complexity and resolving digested peptides prior to MS analysis. Indeed, the reliability and efficiency of protein identification and quantitation from an iTRAQ workflow strongly depend on sample preparation upstream of MS. Here we describe our methods for: (1) total protein extraction from immortalized cells; (2) subcellular fractionation of murine tissue; (3) protein sample desalting, digestion, and iTRAQ labeling; (4) peptide separation by strong cation-exchange high-performance liquid chromatography; and (5) peptide separation by isoelectric focusing.

The physiological and mononuclear cell activation response to cryotherapy following a mixed martial arts contest: a pilot study

Abstract Cold water immersion is thought to reduce the inflammatory response to injury. Using cultured mononuclear cells and human subjects in a mixed martial arts (MMA) contest, we examined the effect of cryotherapy on 7,8-dihydroneopterin and neopterin generation. Urine was collected from 10 elite male mixed martial artists before, immediately post and 1, 2, 24 and 48 h following a contest. Myoglobin was analysed by reverse-phase high performance liquid chromatography, and urinary neopterin and total neopterin (neopterin+7,8-dihydroneopterin) were measured by strong cation exchange high-performance liquid chromatography. Cold water immersion and passive recovery were compared using changes in these markers, while cryotherapy tested total neopterin production in γ-interferon and phorbol myristate acetate (PMA)-stimulated blood-derived mononuclear cells (monocytes/T cells). Myoglobin significantly increased (p<0.05) at 1 h post-contest, neopterin significantly increased at 1 and 24 h (p<0.05), total neopterin significantly increased (p<0.05) at 1 h post for the passive group only, and significant individual variation was observed for all markers (p<0.01). Cold water immersion attenuated total neopterin production (p<0.05), while cryotherapy significantly reduced total neopterin production in PMA-stimulated mononuclear cells (p<0.01). Cryotherapy attenuates the post-exercise inflammatory response following an MMA contest. The evidence also suggests that the mechanisms responsible for this may be related to direct immune cell suppression.

Impact‐induced muscle damage and urinary pterins in professional rugby: 7,8‐dihydroneopterin oxidation by myoglobin

Muscle damage caused through impacts in rugby union is known to increase oxidative stress and inflammation. Pterins have been used clinically as markers of oxidative stress, inflammation, and neurotransmitter synthesis. This study investigates the release of myoglobin from muscle tissue due to force-related impacts and how it is related to the subsequent oxidation of 7,8-dihydroneopterin to specific pterins. Effects of iron and myoglobin on 7,8-dihydroneopterin oxidation were examined in vitro via strong cation-exchange high-performance liquid chromatography (SCX-HPLC) analysis of neopterin, xanthopterin, and 7,8-dihydroxanthopterin. Urine samples were collected from 25 professional rugby players pre and post four games and analyzed for myoglobin by enzyme-linked immunosorbent assay, and 7,8-dihydroneopterin oxidation products by HPLC. Iron and myoglobin oxidized 7,8-dihydroneopterin to neopterin, xanthopterin, and 7,8-dihydroxanthopterin at concentrations at or above 10 μM and 50 μg/mL, respectively. All four games showed significant increases in myoglobin, neopterin, total neopterin, biopterin, and total biopterin, which correlated between each variable (P < 0.05). Myoglobin and iron facilitate 7,8-dihydroneopterin oxidation to neopterin and xanthopterin. In vivo delocalization of myoglobin due to muscle damage may contribute to oxidative stress and inflammation after rugby. Increased concentrations of biopterin and total biopterin may indicate production of nitric oxide and monoamine neurotransmitters in response to the physical stress.

Simultaneous production of multi-functional peptides by pancreatic hydrolysis of bovine casein in an enzymatic membrane reactor via combinational chromatography

Three bioactive peptides, angiotensin-converting enzyme-inhibitory peptides (ACEIPs), casein phosphopeptides (CPPs) and antimicrobial peptides (AMPs), were simultaneously prepared from casein by pancreatic hydrolysis via an enzymatic membrane reactor (EMR) and combinational chromatography. The reaction was performed at 37°C and pH 8.0 for 3h followed by ultrafiltration. ACEIPs were purified by size exclusion chromatography (SEC) from permeate fractions with molecular weight (MW) below 1kDa. Concurrently, strong cation exchange high-performance liquid chromatography (SCE-HPLC) was used to isolate CPPs and AMPs from retentate fractions ranging from 1kDa and 5kDa. Following reverse-phase high performance liquid chromatography–electrospray ionization tandem mass spectrometry (RP-HPLC–ESI-MS/MS) analysis, potential ACEIPs and a total of 34 CPPs were identified (18 corresponded to αs1-casein, 2 to αs2-casein, 10 to β-casein and 4 to κ-casein). Additionally, the ACEIPs in fraction B from SEC had the highest ACE-inhibiting-activity (73.5% in 1.0mg/ml), while total concentrations of AMPs were directly proportional to overall antibacterial activity. Uniquely, fraction 5 from SCE exhibited the highest activity against Staphylococcus aureus (97.49% in 1.0mg/ml).

An approach to speed up the isolation of hydrophilic metabolites from natural sources at semipreparative level by using a hydrophilic–lipophilic balance/mixed-mode strong cation exchange–high-performance liquid chromatography/mass spectrometry system

An approach to speed up the isolation of hydrophilic metabolites in complex natural matrixes by using a HLB/MCX–HPLC/MS system based on the retention properties of hydrophilic–lipophilic and cation exchange polymeric cartridges was developed. This methodology was successfully applied to the re-isolation of small water soluble compounds with completely different structures from two different natural extracts such as a dipeptide (vanchrobactin) from a bacterium culture broth and a pyrrolidine bearing a carboxylic acid moiety (clionapyrrolidine A) from a sponge. This method improved not only the efficiency of the isolation methodology but also the isolation time in relation to the existing methods.

Pharmacokinetics of LJP 993, a tetrameric conjugate of domain 1 of β2-glycoprotein I for antiphospholipid syndrome

beta2-glycoprotein I is the best-characterized antigenic target for antiphospholipid autoantibodies. We synthesized a tetrameric conjugate of the domain 1 of beta2-glycoprotein I (LJP 993) aimed at developing the conjugate as a Toleragen to suppress antiphospholipid syndrome. The present studies focused on determining the stability, tissue distribution, plasma concentration-time profile and excretion of the LJP 993 in mice. The stability of LJP 993 in mouse plasma was quantitatively evaluated using strong cation-exchange high performance liquid chromatography. ( 125)I-labeled LJP 993 was intravenously injected to mice, and levels of (125)I-labeled LJP 993 in plasma, tissues, urine and feces were determined at known intervals. Incubation of LJP 993 with mouse serum at 37 degrees C for 8 h resulted in a decrease by 34% of LJP 993 concentration. No degradation fragment was observed during the incubation. After a single intravenous administration of (125)I-LJP 993 (0.5 and 5 mg/kg) to mice, both C(max) and area-under-curve values increased in a dose-proportional manner, and blood radioactivity disappeared in a bi-exponential manner with the distribution half-lives equal to 1.7 min, and the elimination half-lives 188 and 281 min, respectively. The (125)I-LJP 993 was moderately distributed into organs and tissues with the exception that brain level of ( 125)I-LJP 993 was negligible. The major sites of (125)I-LJP 993 uptake were the kidney (at 30 min post dosing), and kidney, lung, liver, heart, spleen, skin, muscle and fat tissues (at 4 h post dosing). Cumulative urinary and fecal radioactivity for 0-48 h post dosing accounted for 44.7% and 4.2% of the administered dose, respectively, with the fast rate of urinal excretion occurring within the first 8 h. In summary, LJP 993 was fairly stable in mouse plasma. After administration to mice, (125)I-LJP 993 was taken up mainly by kidney and then distributed extensively to tissues except brain. Both C(max) and area-under-curve values increased in a dose-proportional manner. It was predominantly excreted in the urine with an elimination half-life longer than 3 h. Kidney is a major route to excrete the tetrameric conjugate.

Development of a strong cation-exchange high-performance liquid chromatography method for analysis of isoaspartate in proteins

Development of a strong cation-exchange high-performance liquid chromatography method for analysis of isoaspartate in proteins

The application of strong cation exchange high-performance liquid chromatography to drug analysis

The application of strong cation exchange (SCX) HPLC to the analysis of drugs in plasma, tissue and rodent feed is presented. This approach is shown to give useful separations allowing the separation of physiochemically diverse compounds in a simple isocratic system. The SCX columns are not as robust as standard reversed-phase materials and to ensure maximum column lifetimes the use of a guard column is recommended.

Strong cation-exchange high-performance liquid chromatography as a versatile tool for the characterization and purification of peptides

Synthetic peptides play a major role in all areas of biomedical research. The ease with which virtually any sequence of both natural and unnatural amino acids can be synthesized is a primary reason for their popularity. Often, the final product not only contains the target peptide sequence but contaminating species that differ in subtle ways such as minor deletions, incomplete deprotected side chains, fragmentation at unexpected residues, and residue adducts as a consequence of improper resin cleavage. The investigator must then purify the desired target molecule from the crude mixture in order to conduct meaningful experiments. A widely practiced and highly successful method of analysis and purification utilizes reversed-phase high-performance liquid chromatography (RP-HPLC). An alternative Chromatographic procedure uses strong-cation exchange (SCX)-HPLC. Here the primary mode of separation is the interaction of the peptides' positive charges with the negatively charged functional moiety of the Chromatographic support. At a mobile phase of pH 3, peptides are expected to possess a net positive charge as a result of protonation of carboxyl groups and C-terminus, leaving arginine, lysine, histidine and the N-terminus to contribute to the net positive charge. Thus, as a first approximation separation will be a monotonic function of differences in net positive charge. Considerable success has been realized with a sulfoethyl aspartamide SCX-HPLC support for the analysis and purification of well over 500 distinct synthetic and proteolytically derived peptide fragments. This short practical review will present examples of these separations with emphasis on synthetic peptides. For completeness, a section on SCX-HPLC of proteolytically derived peptides will also be presented.

Re-evaluation of strong cation-exchange high-performance liquid chromatography for the analysis of basic drugs

Strong cation-exchange (SCX) HPLC has been systematically evaluated for the chromatography of basic compounds using an eluent of 80% methanol containing ammonium formate (0.02 M) with the pH adjusted to 2.45 with trifluroacetic acid. Contrary to common perception SCX-HPLC is capable of high efficiencies (> 60 000 plates/m) and good peak symmetries ( A s typically <1.2). Retention shows the expected dependency on the reciprocal of the eluent ammonium concentration and eluent pH. Other mechanisms of retention have also been demonstrated including partitioning and hydrogen bond interactions. Control of the separation selectivity is possible through systematic adjustment of the eluent parameters such as ionic strength, pH and methanol concentration. SCX-HPLC is shown to be a powerful separation procedure for the analysis of basic compounds.

Hydralazine and Other Hydrazine Derivatives and the Formation of DNA Adducts

Previous work has demonstrated that hydrazine after formylation to its corresponding hydrazone may be activated both in vivo and in vitro to a methylating intermediate resulting in the formation of O6-methyl- and N7-methylguanines in DNA. Incubation of calf thymus DNA with the hydrazine derivative, hydralazine, and formaldehyde resulted in the production of N7-methylguanine and two aberrant bases in DNA. These bases were separated by strong cation-exchange high-performance liquid chromatographic fractionation of neutral thermal hydrolysates. Administration of hydralazine to rats resulted in the formation of N7-methylguanine in liver DNA, but the two unknown bases observed in the in vitro experiment could not be demonstrated in vivo. In contrast to hydrazine, administration of hydralazine resulted in the methylation of DNA only at doses approaching the LD50, suggesting that formylation does not represent a significant mechanism for hydralazine toxicity in the system described. Hydralazine in combination with formaldehyde resulted in the formation of triazolophthalazine, a metabolite which has been characterized in man. The ability of 17 other hydrazine derivatives to alkylate liver DNA was determined after single administration to young adult male Sprague-Dawley rats or C57BL6 mice. Quantifiable amounts of N7-methylguanine were measured in liver DNA from animals treated with 10 of the 17 compounds. In 3 of the 10 cases quantifiable amounts of O6-methylguanine were also measured. Methylation of liver DNA guanine was obtained with hydrazine, hydralazine, procarbazine, isoniazid, phenylhydrazine, nialamide, nitrofurazone, maleic hydrazide, sulfomethoxypyridazine, and sulfamethiazole and two hydrazine-formaldehyde polymerization products, formalazine and tetraformyltrisazine.

Optimization of high-performance liquid chromatographic peptide separations with alternative mobile and stationary phases

Peptides are routinely separated with reversed-phase high-performance liquid chromatography using increasing concentrations of acetonitrile in the presence of trifluoroacetic acid. While these separations may be improved by adjustments of gradient slope or substitutions of different solid-phase chemistries, many mixtures would benefit from systematic optimization of mobile phase components. Tryptic digests of cytochrome c from various species were separated on Waters Delta-Pak TM C 18. The effects of varying pH as well as the concentration and type of ion-pair reagent were examined. In addition, low pH, ion-suppression/ion-pairing chromatography was inverted using a polymeric reversed-phase column at high pH with alkyl amine ion pairing. Finally, a tryptic digest of cytochrome c was resolved by ion-exchange chromatography with a strong cation-exchange high-performance liquid chromatography column. These data suggest a framework for dramatically changing the selectivity of peptide separations, leading to more satisfactory peptide mapping.

Strong cation-exchange high-performance liquid chromatography of peptides : Effect of non-specific hydrophobic interactions and linearization of peptide retention behaviour

Strong cation-exchange chromatography (strong CEX) is probably the most useful mode of high-performance ion-exchange chromatography (IEC) for peptide separations. Although the hydrophobic character of high-performance ion-exchange packings, often giving rise to mixed-mode contributions to solute separations, has long been recognized, a systematic approach to examining the effect and magnitude of the hydrophobicity of these packings during IEC of peptides has so far been lacking. In the present study, we report the synthesis of three series of positively charged peptide polymers which vary significantly in overall hydrophobicity and polypeptide chain length (5–50 amino acid residues): Ac-(Gly-Lys-Gly-Leu-Gly) n -amide, Ac(Leu-Gly-Leu-Lys-Ala) n -amide ( n = 1, 2, 4, 6, 8, 10). We have examined non-specific hydrophobic interactions of these peptides with both silica- and polymer-based ion-exchange packings, demonstrating how these interactions are overcome by the addition of acetonitrile to the mobile phase. It was also shown that removal of non-specific hydrophobic interactions may be necessary just to elute peptides from the ion-exchange matrix. In addition, from the observed retention times of these three peptide polymer series and other peptides which vary substantially in charge density, net charge, polypeptide chain length and hydrophobicity, we have established a simple approach to linearization and, thus, prediction of peptide retention behaviour in CEX.

Separation of peptides by strong cation-exchange high-performance liquid chromatography

The effects of pH and gradient conditions on the separation of a series of ten peptides (9–36 residues) and carboxamidomethylated troponin I (CM-TnI, 178 residues) on a new commercially available strong cation-exchange silica based 300-Å column (Synchropak S300) were examined. The elution times of the peptides were linear with respect to their net charge at pH 3.0 and pH 6.5. The basic protein CM-TnI (p I ≈ 9.5) and peptides with net charges from + 2 to + 10 were separated with linear AB salt gradients varying from 5 m M to 10 m M B per min (A  5 m M KH 2PO 4 buffer, pH 6.5 or 3.0; B  5 m M KH 2PO 4 buffer, pH 6.5 or 3.0, containing 1 M KCl). All peptides and CM-TnI were eluted with KCl concentrations below ca. 0.6 M. The advantage of performing cation-exchange chromatography over anion-exchange chromatography was demonstrated for the separation of peptides which, while acidic or weakly basic at neutral pH, through protonation of the acidic functions results in positively charged peptides at pH 3.0.