Anion-exchange HPLC Method Research Articles

Anion exchange-HPLC method for evaluating the encapsulation efficiency of mRNA-loaded lipid nanoparticles using analytical quality by design

Lipid nanoparticles (LNPs) are emerging nucleic acid delivery systems in the development of mRNA therapeutics such as the severe acute respiratory syndrome coronavirus 2 vaccines. However, a suitable analytical method for evaluating the encapsulation efficiency (EE) of the LNPs is required to ensure drug efficacy, as current analytical methods exhibit throughput issues and require long analysis times. Hence, we developed and validated an anion-exchange HPLC method using Analytical Quality by Design. Three critical method parameters (CMPs) were identified using risk assessment and Design of Experiments: column temperature, flow rate, and sodium perchlorate concentration. The CMPs were optimized using Face-Centered Central Composite Design. The discriminating power of the optimized HPLC method and RiboGreen assay was comparable. The main advantage of this method is that LNPs can be directly injected into the HPLC system without bursting the LNPs loaded with encapsulated poly(A). The optimized HPLC method was validated as robust, high-throughput, and sufficiently sensitive according to the ICH Q2 guidelines. We believe our findings could promote efficient LNPs-based drug development.

A mild anion-exchange HPLC method for analysis of [18F]sodium fluoride solution for injection

A mild HPLC method for the quality control of [18F]sodium fluoride solution for injection was developed. The method used anion exchange column for separation, UV and online radio-detectors for fluoride content and fluorine-18 determination. A mixture of 100 mM sodium acetate and 25 mM sodium chloride was chosen as mobile phase. The method was validated for system suitability, specificity, fluoride concentration and radiochemical purity determination with good linearity, accuracy and precision. The good robustness of the system was also verified, enabling routine analysis of fluoride content and radiochemical purity of the [18F]sodium fluoride product.

Re-assessment of the cut-off levels of Carbohydrate Deficient Transferrin (CDT) for automated immunoassay and multi-capillary electrophoresis for application in a forensic context

BackgroundThe determination of Carbohydrate Deficient Transferrin (CDT) in a forensic context should be based on the use of a screening technique followed, for the “positive samples”, by a confirmatory technique. The aim of this study was to compare the two most used automated screening methods for CDT analysis, immuno-nephelometric assay (INA) and multi-capillary electrophoresis (mCE), with a validated HPLC procedure, used as confirmation test, in order to re-evaluate the cut-off concentrations of the screening methods. Methods195 serum samples underwent CDT analysis by using the N Latex CDT direct immuno-nephelometric assay, the multicapillary system Capillarys™ and an anion exchange HPLC method with UV-visible detection at 460nm developed and validated at our laboratories. Statistical analyses were performed by using Bland–Altman plots and ROC curves. Results and discussionThe 95% limits of agreement were ±0.94% when comparing INA and HPLC and ±0.60% when comparing mCE and HPLC. The ROC analysis of both INA and mCE, using HPLC as the reference method, showed that no false negative results were found when the cut-off was fixed to 1.2% for mCE and to 2.3% for INA. ConclusionsThe study showed a good agreement among CDT determinations carried out either with mCE or INA or HPLC. However, the usual cut-offs of both mCE (1.3%) and INA (2.5%) should be lowered to minimize false negatives at the screening analysis.

Development and validation of an anion-exchange HPLC method for the determination of fluoride content and radiochemical purity in [18F]NaF

(18)F-labeled sodium fluoride ([(18)F]NaF) is a useful bone imaging agent that has been demonstrated to be significantly more accurate than (99m)Tc-labeled methylene diphosphonate for the detection of both sclerotic and lytic lesions in various malignancies. A reliable anion-exchange HPLC method equipped with suppressed conductivity and radioactive detectors has been developed in order to analyze the content of NaF and radiochemical purity in [(18)F]NaF radiopharmaceuticals. The method described for fluoride analysis uses an isocratic elution of NaF in a Hamilton anion-exchange column using a mobile phase that consists of 7.5 mM sodium carbonate and 0.018 mM potassium thiocyanate. The flow rate was 1.0 ml/min. The method was validated in accordance with several parameters, including system suitability, specificity, precision, accuracy, linearity, robustness, limit of detection and limit of quantification. The results are described as follows: (1) The system suitability includes the tailing factor, theoretical plate number and resolution, which are 1.192534, 2729.6594 and 16.7415, respectively. (2) For specificity, the solvent peak and chloride ion did not interfere with the retention time of the fluoride. (3) The percentage coefficient of variation for analysis of precision, including repeatability and intermediate precision, is less than 2.0%. (4) Accuracy of method is within the range of 98%-102%. (5) The range of linearity is from 10 to 400 μg/ml, with the correlation coefficient (R(2)) always being above 0.9985. (6) The data of method robustness are within acceptance criteria. (7) The limit of detection and limit of quantification are 0.0678 and 0.20 μg/ml, respectively. All of the analysis results demonstrate that this method is highly sensitive, convenient, specific and suitable for quantification of NaF over a wide linear range. Therefore, the method can be successfully performed for routine analysis of fluoride content in [(18)F]NaF radiopharmaceuticals and reduce the time required for analysis.

Pre-Clinical Characterization and Analysis of Long Lasting Recombinant Factor IXFc

AbstractAbstract 4415A recombinant Factor IXFc fusion molecule has been demonstrated to prolong half-life in several species (normal mice, rats, monkeys and FIX-deficient mice and dogs). The recombinant fusion molecule consists of a single rFIX moiety fused to the Fc region of Immunoglobulin G. A number of post-translational modifications exist on the rFIX portion of the fusion protein. These PTMs consist of γ-carboxylation, in the Gla domain, of the first 12 glutamic acids; the majority of which occupy glutamic acids 1–10 and partial occupation of glutamic acids 11–12. Other possible modifications include: hydroxylation of an aspartic acid residue, sulfation of a tyrosine residue and phosphorylation of a serine residue. In addition, there are several N- and O- linked glycosylation sites. One of the more important PTMs that may impact biological activity is γ-carboxy-glutamic acid (Gla) content thus several analytical methods were developed to monitor Gla content. Analytical methods include: amino acid analysis, AAA, (total Gla content), peptide mapping by HPLC-MS (Gla occupation), FIX coagulation activity by aPTT and analytical anion-exchange HPLC (iso-form separation). Material from the polishing ion-exchange step; purified peak and strip peak fractions, were analyzed. The average Gla content, measured by AAA and quantified on a molar basis relative to plasma derived FIX, of the purified peak fraction was 11.0 (3.9 % RSD) and, relative to plasma derived FIX which was fixed at 12.0 Gla. The average Gla content and biological activity of the strip peak fraction was reduced relative to the purified peak fraction. Gla occupancy was determined by digesting FIXFc with Lys-C and separating the peptides by HPLC with detection by a Mass Spectrometer (HPLC-MS). The primary peptides monitored were K1K2 and K3, each peptide has a maximum occupancy of 6 Gla's. The K1K2 peptide with 6 Gla was detected in the purified peak fraction, and 5 Gla was below detection whereas the strip peak fraction exhibited a reduced level of 6 Gla's and consequently K1K2 peptide with 5 Gla's was detected. The K3 peptide exhibits a distribution of Gla's ranging from 4 to 6, for both purified and strip peak fractions. An anion-exchange HPLC method was used to separate intact rFIXFc iso-forms. Purified peak fraction was injected onto an anion-exchange column and was separated into three species. Each species was collected and analyzed by aPTT, AAA and peptide mapping. Each species retained biological activity as well as Gla content ≥9.5. Peptide mapping indicated 6 Gla's in the K1K2 peptide (5 Gla's if present were below detection), while 4, 5 and 6 Gla's were detected in the K3 peptide. Strip peak fraction was also injected on to the anion-exchange column and two primary species were separated, each species was collected and analyzed as the purified peak fraction. No biological activity was measured for species 1, and species 2 had equivalent activity as the strip peak fraction (starting material) at 0.40 IU/nmol. Gla content by AAA indicated the strip peak fraction (starting material) to be reduced relative to the purified peak fraction; species 1 and 2 were determined to have a Gla content of 8.5 and 9.9, respectively. Peptide mapping of the strip peak fraction (starting material and species 1 and 2, derived from analytical anion-exchange HPLC), indicated peptides corresponding to K1K2 with 5 Gla's as well as 6 Gla's, and the K3 peptide exhibited 4, 5 and 6 Gla's. In summary, a number of analytical methods were developed and utilized to assess the Gla content of rFIXFc in purified peak fractions as well as the strip peak fraction. The data suggests the purified peak fraction is highly γ-carboxylated. Further fractionation of the rFIXFc iso-forms derived from the purified peak fraction was performed by analytical anion-exchange into several species. The separated species (species 1 and 2) retained biological activity with a reduction in Gla content, relative to the un-fractionated material and species 3. Analysis of the strip peak fraction exhibited a reduction of biological activity and Gla content, as well as an increased population of K1K2 5 Gla's. These analytical techniques were used to provide analytical as well characterization methodologies to support process development for designing a manufacturing process for production of clinical material. Disclosures:No relevant conflicts of interest to declare.

Oxidative Stress and Atherosclerosis

Oxidative stress is a continuous level of oxidative damage in animal cells, which is caused by an overabundance of reactive oxygen species or a decline in antioxidant ability against them. Oxidative stress increases with individual risk factors of atherosclerosis such as obesity, hypertension, hyperlipidemia, diabetes and smoking. Thus, oxidative stress is considered to play a key role in the pathogenesis of atherosclerosis. This review discusses the relationship between oxidative stress and atherosclerosis based on findings from our research group. We have found that atherosclerotic lesions are formed in the aorta of mice fed a high-cholesterol and high-linoleic diet, in parallel with elevated serum lipid peroxide levels. This model is useful for primary screening of antiatherosclerotic agents with antioxidative activity. One notable factor in the development of atherosclerosis is oxidized low-density lipoprotein (OxLDL). In order to examine OxLDL levels in blood, we have developed anion-exchange HPLC methods using stepwise elution. Using these methods, we have found that OxLDL markedly increases in a rat model of metabolic syndrome, in animals exposed to cigarette smoke and in smokers in parallel with other oxidative stress markers. These oxidative stress markers have been attenuated by administration of several antioxidants. In addition, we have found that smoking accelerates atherogenesis in the aorta of apoE-deficient mice and this acceleration can be ameliorated by administration of vitamin E. These observations suggest that antioxidant supplementation may be an effective therapeutic strategy for metabolic syndrome and smoking-induced diseases in which elevated oxidative stress plays a pivotal role.

Comparative study between anion-exchange HPLC and homogeneous assay methods in regard to the accuracy of high- and low-density lipoprotein cholesterol measurement

Objectives: A convenient method based on anion-exchange HPLC was recently developed to determine cholesterol levels of lipoproteins (HDL, LDL, IDL, VLDL, and chylomicron). The present study was performed to compare this HPLC method to homogenous assay in regard to measurement accuracy of HDL and LDL cholesterol. Design and methods: Serum samples ( n = 105), including three samples from cholestasis patients, were measured by homogenous assay with Cholestest-LDL and CholestestN-HDL (Daiichi Chemicals, Tokyo) and by HPLC as reported previously ( J Lipid Res 2003; 44: 1404–12). Results: The homogenous assay for HDL cholesterol correlated strongly with the HPLC method for HDL cholesterol ( r = 0.976). Two samples from cholestasis patients could not be measured by homogenous assay but were measured by HPLC. The homogenous assay for LDL cholesterol correlated modestly with the HPLC method for LDL cholesterol ( r = 0.823). Three outlier samples, from cholestasis patients with serum cholesterol levels > 17 mmol/L, were observed in this correlation analysis. Homogenous assay data showed that these LDL cholesterol levels were 15.2–34.7 mmol/L. However, HPLC data showed that these LDL cholesterol levels were 3.6–8.2 mmol/L, and that the major lipoprotein fractions were VLDL and IDL. The difference in LDL cholesterol levels (homogenous assay data minus HPLC data) was positively correlated with VLDL cholesterol levels. Conclusions: When measuring samples from cholestasis patients, homogenous assay may give inaccurate results. In contrast, the HPLC method is likely to be capable of accurately measuring HDL and LDL cholesterol levels without the involving VLDL.

Separation of topological forms of plasmid DNA by anion-exchange HPLC: Shifts in elution order of linear DNA

We sought to establish a single anion-exchange HPLC method for the separation of linear, open circular and supercoiled plasmid topoisomers using purified topoisomeric forms of three plasmids (3.0, 5.5 and 7.6 kb). However, finding one condition proved elusive as the topoisomer elution order was determined to depend on salt gradient slope. The observed change in selectivity increased with plasmid size and was most pronounced for the linear form. Indeed, the elution order of the linear 7.6 kb plasmid was reversed relative to the supercoiled form. This observation may have implications for methods used in quality control of plasmid DNA.

Quantification of O-acetyl, N-acetyl and phosphate groups and determination of the extent of O-acetylation in bacterial vaccine polysaccharides by high-performance anion-exchange chromatography with conductivity detection (HPAEC-CD)

The O-acetyl groups in meningococcal A and typhoid Vi polysaccharides (PSs) are functional immunogenic epitopes in humans. To quantify and determine the extent of O-acetylation in these and other bacterial vaccine PSs, anion-exchange HPLC methods have been developed for quantification of O-acetyl, N-acetyl, and phosphate groups in the PSs after these groups were hydrolyzed into anions. The O-acetylation in meningococcal A, C, Y and W-135, pneumococcal 9V and 18C and typhoid Vi PSs were analyzed. The O-acetyl group was selectively released from a PS as acetate by mild alkaline hydrolysis in 10 or 20 mM NaOH at 37 °C until maximum release. The acetate in the hydrolysate was then quantified by high-performance anion-exchange chromatography with conductivity detection (HPAEC-CD) after removal of the PS by filtration with a 10,000 molecular-weight-cut-off membrane. Since the extent of O-acetylation on the PSs depends on bacterial species, strains and growth conditions, the N-acetyl group of amino-sugars, phosphate or monosaccharide components of the PSs were also quantified using HPAEC with conductivity or amperometry detection to determine the molar ratios of the O-acetyl group to these components. The average numbers of O-acetyl molecules in one PS repeating unit of the PSs were obtained from the molar ratios. Besides the O-acetyl determination, the pyruvate component in non- O-acetylated pneumococcal type 4 PS was analyzed by the HPAEC method. The HPAEC method can quantify the O-acetyl content in 0.2 μg of the meningococcal C PS and has a sensitivity at least 10 times higher than that of the colorimetric Hestrin assay. The method can be used for routine analysis of O-acetylation of PSs for quality control of vaccine PSs.

Is There an Analytical or Diagnostic Advantage from Including Trisialo Transferrin into the Fraction of Carbohydrate-Deficient Transferrin? Lessons from a Comparison of Two Commercial Turbidimetric Immunoassays with the Carbohydrate-Deficient Transferrin Determination by High-Performance Liquid Chromatography

Objectives: Carbohydrate-deficient transferrin CDT has originally been defined as the sum of isotransferrins exhibiting isoelectric point values ≥ 5.7 asialo, monosialo, and disialo transferrin but may also include at least in part trisialo transferrin when measured by modern commercial immunoassays. To examine the effects of divergently defining the analyte CDT, we compared two commercial assays yielding differently composed CDT fractions with a high-performance liquid chromatography HPLC assay commonly regarded as a reference method of CDT determination. Methods: Relative CDT levels (CDT concentrations expressed as percent of total transferrin) were determined in 142 sera by (i) a turbidimetric immunoassay (ChronAlco I.D.) reportedly detecting asialo to disialo transferrin as CDT, (ii) an analogous assay (CDT Turbidimetric ImmunoAssay [TIA]) said to additionally include part of trisialo transferrin into the CDT fraction measured, and (iii) an anion-exchange HPLC method. Isotransferrins separated by the two commercial assays were also investigated by isoelectric focusing. Results: Data from HPLC and isoelectric focusing indicate that the ChronAlco assay detects major parts of asialo, monosialo, and disialo transferrin as CDT while the CDT TIA yields CDT as the total of asialo, monosialo, disialo, and trisialo transferrin. When relative CDT concentrations obtained by both assays were classified as either normal or elevated according to reference ranges cited by the manufacturer and then were compared to analogously classified HPLC data, there were clearly more discrepancies between corresponding results from CDT TIA and HPLC (22%) than between ChronAlco and HPLC results (9%). Conclusion: Including trisialo transferrin into the CDT fraction enlarges the analytical signal and therefore slightly improves assay precision but also results in a significant number of pathologic results in samples exhibiting physiologic levels of the classical CDT components asialo to disialo transferrin. As long as the diagnostic information of the trisialo transferrin concentration is largely unknown, we strongly recommend not to include this isotransferrin into the determination of CDT.

High-performance liquid chromatographic assay of hydroperoxide levels in oxidatively modified lipoproteins

An anion-exchange HPLC method has been developed for the chemiluminescence (CL) assay of hydroperoxide (HPO) levels in native and oxidized low density lipoproteins (N- and Ox-LDLs, respectively) of Watanabe heritable hyperlipidemic (WHHL) rabbits. The method involves anion-exchange HPLC separation in N- and Ox-LDLs using a DEAE–glucomannan gel, and direct CL detection of HPOs in them without extraction of the lipids following postcolumn reaction with isoluminol, microperoxidase and Triton X-100. Addition of Triton X-100, which could solubilize lipids, was essential for the detection of HPOs in N- and Ox-LDLs. With an increase in the degree of oxidation, Ox-LDL was more retained on the DEAE–glucomannan gel with a concomitant increase in the CL intensity. The proposed method could analyze the HPO levels in N- and Ox-LDLs of WHHL rabbits without extraction of the lipids.

Expression in Escherichia coli, purification and characterization of heparinase I from Flavobacterium heparinum

The use of heparin for extracorporeal therapies has been problematical due to haemorrhagic complications; as a consequence, heparinase I from Flavobacterium heparinum is used for the determination of plasma heparin and for elimination of heparin from circulation. Here we report the expression of recombinant heparinase I in Escherichia coli, purification to homogeneity and characterization of the purified enzyme. Heparinase I was expressed with an N-terminal histidine tag. The enzyme was insoluble and inactive, but could be refolded, and was purified to homogeneity by nickel-chelate chromatography. The cumulative yield was 43%, and the recovery of purified heparinase I was 14.4 mg/l of culture. The N-terminal sequence and the molecular mass as analysed by matrix-assisted laser desorption MS were consistent with predictions from the heparinase I gene structure. The reverse-phase HPLC profile of the tryptic digest, the Michaelis-Menten constant Km (47 micrograms/ml) and the specific activity (117 units/mg) of purified recombinant heparinase I were similar to those of the native enzyme. Degradation of heparin by heparinase I results in a characteristic product distribution, which is different from those obtained by degradation with heparinase II or III from F. heparinum. We developed a rapid anion-exchange HPLC method to separate the products of enzymic heparin degradation, using POROS perfusion chromatography media. Separation of characteristic di-, tetra- and hexa-saccharide products is performed in 10 min. These methods for the expression, purification and analysis of recombinant heparinase I may facilitate further development of heparinase I-based medical therapies as well as further investigation of the structures of heparin and heparan sulphate and their role in the extracellular matrix.

HPLC characterization of Ag+ and Cu+ metal exchange reactions with Zn- and Cd-metallothioneins.

Anion exchange HPLC methods were developed to study the metal exchange reactions of ZnMT and CdMT with Ag+ and Cu+. The kinetics of these reactions revealed the existence of a slow interprotein metal redistribution process that follows initial rapid displacement of metal from Zn7MT or Cd7MT by Ag+ or Cu+. Kinetically-favored products of the reaction of Ag+ with Zn7MT in the presence of Chelex resin include Zn4Ag6MT and Ag12MT. Subsequent slow reaction between Zn7MT and Ag12MT leads to the thermodynamically-favored product, Zn4Ag6MT. Analogous behavior was observed for Cd7MT titrated with Ag+ and Zn7MT with Cu+. The use of Chelex resin to remove unbound metal ions was found to influence the reactions of Zn7MT and Cd7MT with Ag+. Upon addition of Ag+ to Zn7MT in the absence of Chelex, a Ag18MT species forms as the kinetically-favored product in addition to Zn4Ag6MT and Ag12MT. Subsequently, Ag18MT was found to donate Ag+ to Zn7MT and Zn4Ag6MT via slow interprotein metal exchange reactions. Chelex resin influences the titration reaction either by limiting formation of Ag18MT and/or by binding the positively charged Ag18MT species and removing it from solution. The formation of Ag18MT in the absence of Chelex explains why more than 12 equiv of Ag+ is required to displace all of the Zn2+ or Cd2+ from Zn7MT and Cd7MT. The HPLC methods developed in this study allow homogeneous preparations to be made of metal-hybrid MT species containing Ag+ in the beta-domain and Cd2+ or Zn2+ in the alpha-domain.

1206 The role of neurotrophin for the neurite extension and cell migration of cerebellar granule cells in vitro

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.

An Efficient Method for the Isolation and Purification of Oligoribonucleotides

Abstract Problems associated with the use of tetrabutylammonium fluoride like incomplete desilylation and removal of the tetrabutylammonium salts during large scale syntheses of oligoribonucleotides (RNA) have been eliminated by the use of triethylamine trihydrofluoride and precipitation of the RNA with 1-butanol. An efficient anion-exchange HPLC method has been developed for the purification of chemically synthesized RNA and the resulting product precipitated directly by the addition of 1-propanol. A new activator, 5-ethylthio-1H-tetrazole significantly enhances the synthesis quality and yield of oligoribonucleotides. RNA synthesized using these improvements has been shown to be biologically active by a comparative ribozyme-substrate assay.

A novel oligodeoxynucleotide inhibitor of thrombin. I. In vitro metabolic stability in plasma and serum.

To determine the degradation rates and pathways of GS-522, a potent oligodeoxynucleotide (GGTTGGTGTGGTTGG) inhibitor of thrombin, in serum and plasma. A stability-indicating, anion-exchange HPLC method was developed and used to determine concentrations of GS-522 and metabolites. In monkey plasma at 2 microM or below, the degradation of GS-522 can be fit to a first-order exponential with a kpobs approximately 0.01 min-1. At 3 microM and above the degradation process deviates from a monoexponential decay profile. An initial fast degradation process is followed by a slower phase with an observed rate constant equal to that observed at 2 microM and below. In monkey serum, the KM and Vmax are 8.4 microM and 0.87 microM min-1, respectively. The kinetics are consistent with an equilibrium binding of GS-522 to prothrombin in plasma (Kd = 50 nM) which saturates at GS-522 concentrations > 2 microM. Compared to a scrambled sequence (GGTGGTGGTTGTGGT), with no defined tertiary structure, GS-522 is 4-fold more stable in serum. The metabolic profile in plasma is consistent with a 3'-exonuclease catalyzed hydrolysis of GS-522.

Separation and Analysis of 4′-Epimeric UDP-Sugars, Nucleotides, and Sugar Phosphates by Anion-Exchange High-Performance Liquid Chromatography with Conductimetric Detection

An anion-exchange HPLC method coupled with conductimetric detection was developed for the analysis of UDP-sugars, nucleotides, and sugar phosphates, each in a single chromatographic run. The analysis was applicable to concentrations over 50 pmol. The utility of this technique was demonstrated by the measurement of UDP-sugar 4′-epimerase activity of cell-free extracts from wild-type and mutant Neisseria meningitidis serogroup B strains, Additionally, the method has been applied to the analysis of the intermediates of glycolysis in human erythrocytes.

Improved separation of isomeric gangliosides by anion-exchange high-performance liquid chromatography.

A new anion-exchange HPLC method for separation of gangliosides has been developed using TMAE (trimethylaminoethyl)-Fractogel as the stationary phase and a gradient system of ammonium acetate in methanol for elution. Chromatography of mouse brain gangliosides resulted in separation according not only to their degree of sialylation into mono-, di-, tri- and tetrasialogangliosides, but also in separation of positional isomers within the four elution domains, e.g. GD1a (IV3Neu5Ac,II3Neu5Ac-GgOse4Cer) and GD1b (II3(Neu5Ac)2-GgOse4Cer) of the disialoganglioside fraction. Furthermore, base-line separation of alpha 2-3 and alpha 2-6 sialylated neolacto-series gangliosides e.g. IV3Neu5Ac-nLcOse4Cer and IV6Neu5Ac-nLcOse4Cer, isolated from human granulocytes, was achieved and pure fractions of each ganglioside were obtained. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the isolation of pure gangliosides on a preparative scale by one-step column chromatography.

High-performance liquid chromatographic analysis of radiolabeled inositol phosphates

Separation of inositol phosphates by low-pressure anion-exchange chromatography yields unsatisfactory results, while previously described anion-exchange HPLC methods require such extensive processing times that they preelude efficient sample analysis. Using a low-capacity Vydac nucleotide anion-exchange column, we have developed a method which allows complete separation of myo-inositol, inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate in approximately 10 min followed by a 5-min column regeneration time. This method provided exceptional reproducibility and quantitative recovery of each inositol phosphate. One column was used for over 300 separations with no loss in performance or alteration in elution pattern. A modifled procedure with a 14-min gradient was developed to separate the 1,3,4- and 1,4,5-isomers of inositol trisphosphate. These separation procedures were used to characterize the kinetics of degradation of inositol phosphates by lysates of erythrocytes and neutrophils. We conclude that these procedures are applicable for rapid and quantitative analysis of radiolabeled inositol phosphates in cellular extracts.

Lectin affinity high-performance liquid chromatography: Interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia villosa agglutinin

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH 4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.