Ion-exchange Liquid Chromatography Research Articles

A 9-Month-Old Boy with Seizures and Discrepant Urine Tryptophan Concentrations

A 9-month-old boy with a history of seizures underwent a neurologic and biochemical-genetic evaluation. The brain MRI results were compatible with a diagnosis of Leigh disease, also known as subacute necrotizing encephalomyelopathy, a rare neurometabolic disorder that affects the central nervous system. The patient had been prescribed several antiepileptic medications, including levetiracetam, lamotrigine, phenobarbital, vigabatrin, and topiramate. Metabolic screening for free amino acids was performed on the child's urine, with concentrations quantified with an automated amino acid analyzer (Hitachi L-8800). This commercially available system couples ion-exchange liquid chromatography with postcolumn ninhydrin derivatization before UV detection. This analysis revealed a very large peak with a retention time consistent with the elution of tryptophan (Fig. 1). The calculated urinary excretion was 125 204 μmol/g creatinine. In addition, the urinary concentrations of γ-aminobutyric acid (GABA),5 β-alanine, β-aminoisobutyric acid, and glutamine were also increased substantially. The concentrations of the remaining amino acids were within their respective reference intervals. For confirmation, we submitted a urine aliquot to an external reference laboratory for analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS). This analysis revealed a tryptophan excretion of 71 μmol/g creatinine (reference interval, 15–302 μmol/g creatinine). Fig. 1. Chromatogram of the patient's urinary amino acid concentrations as generated by the Hitachi L-8800 automated amino acid analyzer. The increased analyte assigned to tryptophan eluted at 77.22 min. The gross discrepancy in tryptophan concentration between the 2 assays, combined with the increased urinary excretion of GABA, β-alanine, β-aminoisobutyric acid, and glutamine, triggered an inquiry into the potential causes. The patient had a neonatal-onset seizure disorder, hypotonia, a developmental delay, and MRI findings compatible with Leigh disease, but isolated hypertryptophanuria is not a finding for this disorder. In addition, the overall prognosis of patients with Leigh disease is poor. The disease represents the clinical and radiologic expression of a group of inherited disorders of energy metabolism, specifically disorders of oxidative phosphorylation or pyruvate oxidation. The ultimate cause of Leigh …

Quantitative UPLC-MS/MS analysis of underivatised amino acids in body fluids is a reliable tool for the diagnosis and follow-up of patients with inborn errors of metabolism

Background An electro-spray ionisation ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) application for the quantitative analysis of amino acids was developed. The suitability for the detection and follow-up of patients suffering from inborn errors of metabolism (IEM) was assessed by extensive cross-validation with ion-exchange liquid chromatography (IEX-LC) with post-column ninhydrin derivatisation, participation in external quality control (ERNDIM) and analysis of samples of patients with confirmed IEM. Methods Prior to analysis plasma and urine samples were merely diluted 150-fold in mobile phase. Amino acids were detected in the multiple reaction monitoring mode (MRM) in the ESI-positive mode. The analytical results were compared with IEX-LC. External quality control scheme performance is presented. Results Comprehensive analysis of amino acids in plasma and urine was achieved with a run-to-run time of 30 min. Validation results were satisfactory and there was a very good correlation between UPLC-MS/MS and IEX-LC. Analytical results obtained in the external quality control scheme were essentially the same as those of the other participants. Patients suffering from IEM were readily identified. Conclusion UPLC-MS/MS analysis of amino acids in body fluids is rapid, reliable and suitable for the diagnosis and follow-up patients with IEM.

Liquid Chromatography/Tandem Mass Spectrometry of Glycolytic Intermediates: Deconvolution of Coeluting Structural Isomers Based on Unique Product Ion Ratios

A method has been developed for rapid quantification of nine glycolytic intermediates using ultraperformance liquid chromatography/electrospray-tandem mass spectrometry (UPLC/ESI-MS/MS) to monitor the metabolism of glucose during microbial fermentation. Because comprehensive chromatographic separation is not required, analysis time is significantly less than traditional ion exchange liquid chromatography assays or enzymatic assays. Complete glycolytic intermediate analysis by LC/MS/MS can be achieved in less than 7 min per sample. Quantification is accomplished using isotopically labeled glucose, glucose-6-phosphate, and pyruvate as internal standards. In addition, a method to deconvolute peak areas of coeluting structural isomers based on unique product ion ratios has been developed to allow accurate quantification of the individual isomers 2-phosphoglycerate and 3-phosphoglycerate, as well as glucose-6-phosphate and fructose-6-phosphate. Intrasample precisions for glycolytic intermediate measurements in cell-free extracts using this method vary between 0.9% and 11.8%, averaging 3.5% (RSD). Calibration curves are linear over the range 0.1-100 microg/mL, and detection limits are estimated at 2-49 ng/mL. Spike recoveries in cell extracts vary from 53% to 127% averaging 91%. This method has the potential to demonstrate correlation of glycolytic intermediate flux to microbial production profiles toward acceleration of the bioprocess development cycle.

Analysis of charge heterogeneities in mAbs using imaged CE

An analytical method using the imaged CE (iCE) technology has been developed and validated for measuring contents of charge variants for mAb molecules. The method could generate similar information that require both conventional IEF electrophoresis and ion exchange liquid chromatography to generate as an iCE analysis would produce both the pI of the molecules and quantitative contents of charge variants. Thus, it offers unique advantages over the IEF and ion exchange methods in terms of identification, separation and quantitation of charge variants. The data presented in this study demonstrated that the iCE method is suitable not only for research purposes but also for quality control purpose of mAb clinical or commercial manufacture as the technique can be validated and possesses sufficient robustness. The developed generic iCE method has been tested for a wide range of therapeutic mAbs and proved its suitability for multiple mAb molecules. The tested mAbs are a group of molecules with a wide range of charge compositions (acidic species ranging from 10 to 70%) and a diverse range of pI of 6.9-9.6. Developing platform analytical technologies is an efficient way to meet the demand of rapid growth of therapeutic mAb candidates under clinical and preclinical development. The iCE technology is a good candidate to become a platform method as it could cover a broad range of pH gradient. The experiences learned during the developmental process would provide important and valuable information to the biotech industry for the evaluation of charge heterogeneity of mAbs in terms of release testing, characterization, stability study, process development support and comparability study.

Determination of four arsenic species in soil by sequential extraction and high performance liquid chromatography with post-column hydride generation and inductively coupled plasma optical emission spectrometry detection

Several procedures, involving various solvents and ultrasound, were evaluated for the extraction of four arsenic species, arsenite (As(III)), arsenate (As(V)), monomethylarsonate (MMA) and dimethylarsinate (DMA), from a silt loam soil to which species had been added at a concentration of 20 mg kg−1. The best extraction was by a two-stage procedure: shaking for 24 h in the presence of 0.1 mol l−1 phosphoric acid followed by shaking for 24 h in 1.0 mol l−1 sodium hydroxide solution. The arsenic species in the extracts were separated by high performance ion-exchange liquid chromatography, derivatized to hydrides by reaction with tetrahydroborate(III) in a multi-mode sample introduction system (MSIS) and quantified by ICP-OES. Detection limits in solution ranged from 0.4 (As(III) and DMA) to 1 (MMA and As(V)) µg l−1, corresponding to 10 and 25 µg kg−1 in a 0.2 g soil sample and 5 ml of extractant. The most significant change over time was that As(III) was converted to As(V). When each species was added individually, arsenic was 100% recovered over a period of several months. When all four species were added together, the recovery was 89%. As the precipitation of humic acids was slow, the sodium hydroxide extract could be acidified and analyzed without loss of analyte species.

Determination of glyphosate and its biodegradation products by chromatographic methods

Conditions were selected for the separation of the herbicide glyphosate (N-(phosphonomethyl)glycine) and products of its microbiological utilization as N-acylated derivatives by ion-exchange liquid chromatography. The order of the elution of compounds on a Repro-Gel H column with UV detection correlates with their structures. The detection limits of the derivatives (wavelength 210 nm) are as follows (ng): glyphosate, 30; glycine and sarcosine, 20 and 43, respectively; aminomethylphosphonic acid, 45. The detection limit of methylphosphonic acid is 14 μg. Glyphosate and its biodegradation products were separated by thin-layer chromatography on plates with silica gel in the system isopropanol-5% aqueous ammonia solution (1: 1).

Determination of Nucleotides in Infant Formula by Ion-Exchange Liquid Chromatography

Nucleotide-supplemented infant formula has been shown to positively modify the composition of intestinal microflora, emulating the attribute of human milk. Quantification of nucleotides in infant formula is of interest because of its applicability in quality and safety assessments. There is no standard method for the analysis of nucleotides in infant formula. In the present study, ion-exchange liquid chromatography (IELC)- and centrifugal ultrafiltration (CUF)-based protocols were developed for routine determination of additive nucleotides in infant formula. Five target nucleotides, guanosine 5'-monophosphate (GMP), inosine 5'-monophosphate (IMP), uridine 5'-monophosphate (UMP), cytidine 5'-monophosphate (CMP), and adenosine 5'-monophosphate (AMP) were measured by IELC with a mobile phase of 50 mM diammonium hydrogen phosphate buffer, pH 4.0, with UV detection at 254 nm. The calibration was linear over the range 0.5-50 microg/mL; R(2) = 0.999. The calculated LOD and LOQ were 0.01-0.05 microg/mL and 0.05-0.5 microg/mL, respectively. Recovery values (spiked concentration levels: 0.5, 5, and 10 microg/mL) ranged from 85.0 +/- 1.4% to 92.3 +/- 2.1% using only CUF preparation. This was applied to measure the concentration of five nucleotides in common infant formulas.

Metabolite Profiling of Human Amniotic Fluid by Hyphenated Nuclear Magnetic Resonance Spectroscopy

The metabolic profiling of human amniotic fluid (HAF) is of potential interest for the diagnosis of disorders in the mother or the fetus. In order to build a comprehensive metabolite database for HAF, hyphenated NMR has been used, for the first time, for systematic HAF profiling. Experiments were carried out using reverse-phase (RP) and ion-exchange liquid chromatography (LC), in order to detect less and more polar compounds, respectively. RP-LC conditions achieved good separation of amino acids, some sugars, and xanthines. Subsequent NMR and MS analysis enabled the rapid identification of 30 compounds, including 3-methyl-2-oxovalerate and 4-aminohippurate identified in HAF for the first time, to our knowledge. Under ion-exchange LC conditions, a different set of 30 compounds was detected, including sugars, organic acids, several derivatives of organic acids, and amino acids. In this experiment, five compounds were identified for the first time in HAF: D-xylitol, amino acid derivatives (N-acetylalanine, N-acetylglycine, 2-oxoleucine), and isovalerate. The nonendogenous nature of some metabolites (caffeine, paraxanthine, D-xylitol, sorbitol) is discussed. Hyphenated NMR has allowed the rapid detection of approximately 60 metabolites in HAF, some of which are not detectable by standard NMR due to low abundance (microM) and signal overlap thus enabling an extended metabolite database to be built for HAF.

Strategy combining separation of isotope-labeled unfolded proteins and matrix-assisted laser desorption/ionization mass spectrometry analysis enables quantification of a wide range of serum proteins

A novel strategy for the quantitative profiling of serum proteome is described. It includes an ammonium sulfate depletion of the serum, an affordable stable isotope labeling chemistry for samples with a large amount of protein, separation of the unfolded proteins, and relative quantification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Labeling of unfolded proteins was performed using normal (D0) acrylamide and deuterated (D3) acrylamide. The workflow for separating the unfolded proteins includes whole gel elution and ion exchange liquid chromatography, and it combines electrophoretic separation based on the protein molecular weight followed by chromatographic separation in the presence of 8M urea based on protein charge. This was followed by trypsinolysis and MALDI MS analysis, leading to the quantification of a large number of serum proteins, including those with an abundance of 10–5 less than albumin. This robust and inexpensive workflow is suitable for the quantitative profiling of protein changes in serum associated with preanalytical variables.

Direct analysis of un-derivatized asymmetric dimethylarginine (ADMA) and l-arginine from plasma using mixed-mode ion-exchange liquid chromatography–tandem mass spectrometry

A high-throughput analytical method was developed for the measurement of asymmetric dimethylarginine (ADMA) and l-arginine (ARG) from plasma using LC/MS/MS. The sample preparation was simple and only required microfiltration prior to analysis. ADMA and ARG were assayed using mixed-mode ion-exchange chromatography which allowed for the retention of the un-derivatized compounds. The need for chromatographic separation of ADMA from symmetric dimethylarginine (SDMA) was avoided by using an ADMA specific product ion. As a result, the analytical method only required a total run time of 2 min. The method was validated by linearity, with r 2 ≥ 0.995 for both compounds, and accuracy, with no more than 7% deviation from the theoretical value. The estimated limit of detection and limit of quantification were suitable for clinical evaluations. The mean values of plasma ADMA and ARG taken from healthy volunteers ( n = 15) were 0.66 ± 0.12 and 87 ± 35 μM, respectively; the mean molar ratio of ARG to ADMA was 142 ± 81.

210Po and major ions in drainage water from soil treated with various types of fertilizers

The levels of 210Po, nutrients (NH4 +, NO3 −, PO4 3 −) and major ions (Na+, K+, Mg2 +, Ca2 +, F−, NO2 −, Br−, Cl−, SO4 2 −) were determined, by means of lysimeter experiences, in drainage waters for agricultural soils untreated and treated with different types of fertilizers (animal manure, sewage sludge and NPK synthetic fertilizer) applied at several rates. Analytical determinations were performed by using α -spectrometry in the case of 210Po, or Ion Exchange liquid chromatography for the other ionic species. Statistical uni and multivariate analysis of the results shown significant differences among lixiviates according to the different fertilizer treatments. Sewage sludge and manure applications resulted in similar compositions of lixiviates with low 210Po levels, whereas synthetic fertilizers produced higher 210Po concentrations and different concentration patterns of ionic species when applied at or above the recommended rates. All 210Po levels were well below the limits proposed by the 2001/928/ Euratom Recommendation. The concentrations of the rest of the ionic species, exception made from NH4 + and NO3 −, were also below the limits proposed by Spanish regulations.

Quantitative In Silico Analysis of Ion Exchange from Chromatography to Protein

Molecular interaction forces are based on solubility factors. Coulombic force is the strongest, followed by the Lewis acid‐base interaction, including hydrogen bonding and charge‐transfer effects, and van der Waals forces. Steric hindrance affects the molecular interaction. The individual interaction force can be studied using chromatography. The main interaction force in reversed‐phase liquid chromatography is the van der Waals force. The main interaction force in normal phase liquid chromatography is the Lewis acid-base interaction, including hydrogen bonding, and that in ion exchange liquid chromatography is the Coulombic force. Enantiomer separation is achieved by the combination of these molecular interaction forces with steric hindrance as the molecular recognition of proteins. Previously, the retention mechanism in reversed‐phase liquid chromatography was demonstrated using quantitative in silico analysis of chromatographic data of various compounds, such as phenolic compounds, aromatic acids, and acidic and basic drugs.1 The retention mechanism of ion exchange liquid chromatography was then quantitatively analyzed in silico. Carboxyl and guanidino phases were selected for studying the basic molecular recognition mechanism of proteins, and the selective molecular recognition of d‐amino acid oxidase was quantitatively analyzed in silico. The main molecular interaction was the Coulombic force, and the enantioselectivity was affected by the steric hindrance of the surrounding amino acid residues.

Improved proteomic discovery by sample pre-fractionation using dual-column ion-exchange high performance liquid chromatography

Clinically relevant biomarkers are urgently needed for improving patient diagnosis, risk stratification, prognosis and therapeutic treatments. There is a particularly compelling motivation for identifying protein-based indicators of early-stage disease for more effective interventions. Despite recent progress, the proteomic discovery process remains a daunting challenge due to the sheer heterogeneity and skewed protein abundances in biofluids. Even the most advanced mass spectrometry systems exhibit limiting overall dynamic ranges and sensitivities relative to the needs of modern biomedical applications. To this end, we report the development of a robust, rapid, and reproducible high performance ion-exchange liquid chromatography pre-fractionation method that allows for improved proteomic detection coverage of complex biological specimens using basic tandem mass spectrometry screening procedures. This form of sample simplification prior to global proteomic profiling, which we refer to collectively as ‘fractionomics’, increases the number and diversity of proteins that can be confidently identified in tissue and cell lysates as compared to the straight analysis of unfractionated crude extracts.

Hyperactivation and thermostabilization of Phanerochaete chrysosporium lignin peroxidase by immobilization in xerogels

This is a continuation of our previous paper on production of lignin peroxidase (LiP) by Phanerochaete chrysosporium in solid substrate fermentation (SSF) medium of corncobs. The enzyme was purified by ammonium sulphate precipitation and ion-exchange fast protein liquid chromatography. Maximum yield of LiP was 13.7 U/gds (units per gram dry substrate) after 5 days of SSF with 70% moisture and 20% (v/w) inoculum. The approximate molecular mass of purified LiP, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 38 kDa. The pH and temperature optima for the LiP were 4 and 40°C, respectively. Immobilization of LiP in hydrophobic xerogels caused hyperactivation of LiP and enhanced its thermostability properties. The KM and Vmax values for immobilized LiP were 10.56 mg/ml and 16.67 μmol/min (120.49 U/mg of protein) as compared to 13 mg/ml and 11.76 μmol/min (85 U/mg of protein), respectively, for free LiP using veratryl alcohol as substrate.

Functionalization of XAD-4 resin for the separation of lanthanides using chelation ion exchange liquid chromatography

A new chelating agent bis-2[( O-carbomethoxy)phenoxy]ethylamine has been synthesized using a facile microwave induced process. The ligand was appended on to a crosslinked polystyrene resin XAD-4 and adsorption properties of La(III), Nd(III) and Sm(III) towards this resin were studied. The selectivity sequences of the resin for these metals were in agreement with their stability constants. Pre-concentration of the metals was studied and effect of other ions on the pre-concentration has been analyzed. Lastly, the resin was used for chromatographic separation of La(III) and Nd(III), Nd(III) and Sm(III) mixtures and conditions for the same optimized.

Preliminary Study of the Oxidation of Nitrogen Compounds of Gas Oil from Brazilian Petroleum

This work describes the development of a procedure for the preferential oxidation of nitrogen compounds of a gas oil sample from Brazilian petroleum. The sample was first submitted to an extraction procedure, based on preparative liquid ion-exchange chromatography (IEC), to characterize the main nitrogen compounds. The instrumental analysis was performed by gas chromatography with a mass spectrometer detector (GC/MSD), and the compounds were identified using the GC/MSD library (Wiley). The IEC analysis shows that the nitrogen compounds were concentrated mainly in the acid and neutral fractions of the sample. The hindrance influence of the nonbonded electron pair of the N atom in the IEC process was studied with a standard mixture of three nitrogen compounds. The oxidation reaction used was efficient for the preferential oxidation of nitrogen compounds while keeping the distribution of the other class compounds of the gas oil unchanged. Experiments for the reaction scale reduction were also performed.

DIRECTION OF IN VITRO CALCIFIED CHITOSAN MEMBRANES FOR TECHNOLOGICAL APPLICATIONS

Some calcium compounds, such as hydroxyapatite (HA), are widely recognized as materials capable of separating proteins, nucleic acids, and viruses in aqueous systems using chromatography. For this reason, exploring alternative routes for forming calcified resins is of great interest. One of these routes is the biomineralization or biomimetic process. The present study analyzes the calcification deposits obtained on chitosan substrates as potential adsorbents. Adsorption experiments were performed with acids and basic proteins (cytochrome C, bovine serum albumin (BSA), and trypsin inhibitor), and the capacity results were compared to an HA ion exchange liquid chromatography model. The correlation with chemical structures of deposits was also determined. Chitosan was shown to be a very strong absorbent of acid proteins, and calcification increased this capacity by more than 10 times.

Selective separations of peptides with sequence deletions, single amino acid polymorphisms, and/or epimeric centers using macrocyclic glycopeptide liquid chromatography stationary phases

Separating closely related peptides (those differing by one or two amino acids or the chirality of a single amino acid) can be challenging using reversed-phase liquid chromatography (LC), ion-exchange LC, or using ion-pairing agents. Also, the mobile phases that give the best separations in these modes may not be electrospray ionization mass spectrometry (ESI-MS) compatible. Forty-two peptides from 11 peptide families were separated on three macrocyclic glycopeptide stationary phases in reverse-phase mode using ESI-MS-compatible mobile phases. The peptide classes studied were angiotensin, bradykinin, α-bag cell factor, β,γ-bag cell factor, β-casomorphin, dynorphin, enkephalin, leucokinin, lutinizing hormone releasing hormone, neurotinsin, substance P, and vasopressin. High selectivity was observed for single amino acid substitutions (achiral and chiral) regardless of the position of the substitution in the sequence. Mobile phase optimization, its effect on peptide elution behavior, and chromatographic efficiency is also discussed. Using LC–ESI-MS, a 2 ng limit of detection was obtained, two orders of magnitude lower than the UV detection limit.

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification.

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off-line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure-assisted flow. Unlike first-dimensional ion-exchange LC fractionation, where there is a large degree of overlap, the CZE-MS results show less than 15% overlap between neighboring RPLC fractions.

Speciation and Bioavailability of Selenium in Yeast-Based Intervention Agents Used in Cancer Chemoprevention Studies

This study investigated the speciation and bioavailability of selenium in yeast-based intervention agents from multiple manufacturers from several time points. Sources of selenized yeast included Nutrition 21 (San Diego, CA), which supplied the Nutritional Prevention of Cancer (NPC) Trial from 1981-1996; Cypress Systems (Fresno, CA; 1997-1999); and Pharma Nord (Vejle, Denmark; 1999-2000), which supplied the Prevention of Cancer by Intervention by Selenium (PRECISE) Trial pilot studies. The low-molecular-selenium species were liberated from the samples by proteolytic hydrolysis followed by separation by ion exchange liquid chromatography and detection by inductively coupled plasma-mass spectrometry. The results for the NPC tablets showed that selenomethionine, together with 3 unidentified selenium compounds, were predominant in the sample hydrolysates. The relative amounts of the 4 selenium species varied (p < 0.05) among several of the 7 tablet batches used during the course of the NPC Trial. In comparison, 5 batches of more recently produced selenized yeasts, which were used as a source of selenium in the PRECISE and other trials, contained less of the unknown compounds and more selenomethionine at 54-60% of the total selenium in the yeasts. One batch of yeast, however (from 1985), which originated from the same producer as the yeast used in the NPC tablets, contained only 27% of selenium in the sample as selenomethionine. Human subjects receiving 200 microg selenium/day in the UK PRECISE Pilot Trial showed a higher concentration (p < 0.01) and higher increase from baseline in plasma selenium than did the same dosage used in the NPC Trial. Differences in intake, speciation, or bioavailability of selenium from the yeast-based supplements in the population groups studied may explain this. Furthermore, the selenium concentration in whole blood from the Danish PRECISE Pilot Trial was higher (p < 0.001) than that obtained with synthetic L-selenomethionine in a comparable group of Danes, both groups having been treated with 300 microg selenium/day.