Analysis of bile acids and their conjugates using high-pH anion-exchange chromatography with pulsed amperometric detection

Bile acids and their conjugated forms may be separated by anion-exchange chromatography in alkaline media (0.9 M sodium acetate, 0.1 M sodium hydroxide, 15% v/v acetonitrile) on a CarboPac PA-100 column. The efflient was monitored at high sensitivity, with detection limits of less than 10 μM, using a pulsed amperometric detector. Free bile acids and their glyco- and tauro-conjugated forms were separated and detected within 40 min under isocratic conditions.     

A strategy for the mapping of N-glycans by high-pH anion-exchange chromatography with pulsed amperometric detection.

We have evaluated the high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with respect to its suitability to establish a carbohydrate mapping database that would enable carbohydrate structural analysis by mere comparison of retention times. The suitability of HPAE-PAD for carbohydrate structural analysis was ascertained by validation experiments. The retention times of distinct N-glycans, prepared and measured on different days, were shown to be highly reproducible, with a coefficient of variation (CV) of less than 0.5%, requiring less than 100 pmol of N-glycan per injection for reliable measurements. Including appropriate internal chromatographic standards, such as (Neu5Ac)1, (Neu5Ac)2, (Neu5Ac)3, and Neu5Gc, the HPAE-PAD method fulfills the analytical requirements with respect to accuracy, precision, reproducibility, and sensitivity. The N-glycan mapping database was established, using two optimized linear gradients "S" and "A" for sialylated and asialo N-glycans, respectively. Approximately 100 different N-glycans of known structure, which have thus far been measured and characterized, have entered our Lotus 1-2-3 mapping database. The efficiency of the database for structural determinations was tested, using the N-linked carbohydrates isolated from rhuEPO, expressed in BHK cells. Nine different sialylated N-glycans of rhuEPO (BHK) could be assigned with a deviation of less than +/- 0.5%, using gradient S, and six of the eight asialo N-glycans of rhuEPO (BHK) detected with gradient A could be assigned with an accuracy of less than +/- 1%, three of them even with an accuracy of less than 0.1%, providing the reliability of the established HPAE-PAD mapping database.     

High-performance anion-exchange chromatography with pulsed amperometric detection for carbohydrate analysis of glycoproteins

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an established technique for the carbohydrate analysis of glycoproteins. HPAE-PAD is routinely used for determinations of monosaccharide, sialic acid, mannose-6-phosphate (M-6-P), and oligosaccharide contents of a glycoprotein. This is true for both the initial investigation of a glycoprotein and routine assays of recombinant therapeutic glycoproteins. This contribution reviews the fundamentals of HPAE-PAD, recent technological improvements, and advances in the last ten years in its application to carbohydrate analysis of glycoproteins. The application areas reviewed include monosaccharide determinations, sialic acid determinations, M-6-P determinations, sugar alcohol determinations, analysis of polysialic acids, neutral and charged oligosaccharide analysis, following glycosidase and glycosyltransferase reactions, and coupling HPAE-PAD to mass spectrometry (MS).     

Sialyl-Tn-KLH, glycoconjugate analysis and stability by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

The quantitation of sialyl-Tn (STn) conjugated to keyhole limpethaemocyanin (KLH) can be determined by quantitating the amountof ^N-acetylneuraminic acid (NANA) released by acid or enzymaticdigestion. An optimal 0.1 N H₂SO₄ acid hydrolysis at 80°Cresults in quantitative release of NANA with minimal loss. Arapid isocratic method for the quantitation and separation ofNANA is described using high-pH anion-exchange chromatographyand pulsed amperometric detection (PAD). Multiple injectionof NANA standard and/or samples containing protein led to adecrease in the PAD response which was corrected by additionof internal standard, -2-keto-3-deoxyoctonate (KDO). The ratioof NANA/KDO peak area or peak height gives a linear responsewith increasing amount of NANA in the range 2.5–20 µg/ml(r² = 0.99). The limit of quantitation (LOQ) for NANA usingthis isocratic method is 1.9 µg/ml ({small tilde}160 pmol/25µl injection). Based on the multiple determination theglycoconjugate, STn-KLH, showed a NANA content of 2.9% (w/w).Acid hydrolysis and the sialidase treatment of STn-KLH bothyielded a similar NANA content. The carrier protein, KLH, showedthe absence of NANA. The stability of glycoconjugate STn-KLHwas monitored by a gradient method which separated possibledegradation products STn-crotyl, NANA and GalNAc. Subjectingthe glycoconjugate STn-KLH to various stress conditions of temperature,pH and oxidation does not result in any release of sialic acid,GalNac and STn-crotyl group. high-pH anion-exchange chromatography mono-saccharide analysis pulsed amperometric detection sialyl-Tn stability of glycoconjugate     

Exoglycosidase purity and linkage specificity: assessment using oligosaccharide substrates and high-pH anion-exchange chromatography with pulsed amperometric detection

Simplified HPLC protocols to determine the activity and linkagespecificity and to detect the most commonly-encountered contaminantsin available exoglycosidase preparations (Jacob and Scudder,Methods Enzymol., 230, 280–300, 1994) were developed.Monosaccharides and oligosaccharides were analyzed in a singlechromatographic step using high-pH anion-exchange chromatographywith pulsed amperometric detection. All analyses were performedwith underivatized oligosaccharide substrates and by directinjection of unprocessed, diluted enzyme digests into the chromatograph.The sialidase from Newcastle disease virus was found to releaseboth a(2     

Monosaccharide and oligosaccharide analysis of isoelectric focusing-separated and blotted granulocyte colony-stimulating factor glycoforms using high-pH anion-exchange chromatography with pulsed amperometric detection

In this study, a sensitive, straightforward technique is developedfor the analysis of glycoprotein O-linked oligosaccharides.Specifically, O-linked oligosaccharides of granulocyte colony-stimulatingfactor (G-CSF) are analysed by separating charged glycoformsusing isoelectric focusing, electroblotting to polyvinylidenedifluoride, releasing monosaccharides and oligosaccharide alditolsfrom the blotted glycoprotein bands, and producing chromatographsusing high-pH anion-exchange chromatography with pulsed amperometricdetection. Using this technique, the O-linked structures ofG-CSF produced by recombinant Chinese hamster ovary (CHO) cellsare deduced by comparison with monosaccharide and oligosaccharidestandards. Lectin blotting and peptide sequencing support theidentities of the presumed G-CSF glycoforms. The two major glycoformsdetermined using this methodology correspond to those determinedpreviously for CHO-produced G-CSF using NMR. Additional glycoformsare also identified in this study, presumably resulting fromthe presence of N-glycolyl-neuraminic acid in place of N-acetylneuraminicacid. The utility of this analytical approach is then demonstratedin an analysis of the effect of the extracellular environmenton the O-linked glycosylation of G-CSF by recombinant CHO cells.Increasing the level of ammonium ion in the culture medium isshown to reduce the percentage of G-CSF produced with sialicacid linked (2,6) to N-acetylgalactosamne. ammonium ion granulocyte colony-stimulating factor high-pH anion-exchange chromatography isoelectric focusing O-linked glycosylation     

The mapping by high-pH anion-exchange chromatography with pulsed amperometric detection and capillary electrophoresis of the carbohydrate moieties of human plasma alpha 1-acid glycoprotein.

The reducing oligosaccharides released from alpha 1-acid glycoprotein (AGP) by conventional hydrazinolysis have been analyzed by two different mapping techniques, using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and capillary electrophoresis (CE) with uv detection at 190 nm. The CE measurements proved about 4000 times more sensitive than the measurements by HPAE-PAD. The N-glycan pool was fractionated by Mono Q anion-exchange chromatography, and individual fractions so obtained were desialylated using Vibrio cholerae neuraminidase. The resulting asialo-N-glycans were further analyzed by HPAE-PAD, revealing 2 major, 4 intermediate, and 4 small peaks and at least 3 spikes, which counted for at least 13 different asialo-N-glycans. The carbohydrate structures were tentatively assigned by comparison of the Mono Q-separated N-glycans with the known AGP carbohydrate structures and known structures contained in a mapping database that allows structural assignment of N-glycans by mere comparison of retention times. In addition to the hitherto known AGP carbohydrate structures, we have tentatively identified a number of sulfated N-glycans that are currently being analyzed in more detail. We have also compared the glycan pools recovered from AGP using hydrazinolysis and glycopeptidase F (PNGase F). Approximately 40 distinct peaks could be detected in the hydrazinolysis-derived N-glycan pool by either technique (HPAE-PAD and CE), while about 30 distinct peaks were detected in the N-glycan pool derived by PNGase F digestion of the tryptic AGP digest of the same batch of AGP. These differences were attributed to an increased desialylation (approximately 3 mol%) during hydrazinolysis, based on the detection by HPAE-PAD and CE of free sialic acid and monosialylated oligosaccharides in the glycan pool derived by conventional hydrazinolysis. The integrity of the N-glycans' chitobiose core was examined by 500-MHz 1H NMR spectoscopy. The hydrazinolysis procedure could be optimized such that the hydrazinolysis-derived N-glycan pool was chromatographically essentially identical to the PNGase F-derived N-glycan pool. Hydrazinolysis proved best, with practically no loss of N-acetlylneuraminic acid and the closest resemblance to the PNGase F-derived N-glycan pool, using an automated apparatus. Notably, it was recognized that, in our hands, PNGase F digestion in the presence of sodium dodecyl sulfate resulted in partial desialylation of the liberated N-glycans.     

Analysis of oligogalacturonic acids with 50 or fewer residues by high-performance anion-exchange chromatography and pulsed amperometric detection

Underivatized oligogalacturonic acids with a degree of polymerization (DP) ranging from 2 to 50 have been separated for the first time on a high-performance CarboPac PA1 pellicular anion-exchange stationary phase column. Baseline separation of these pectic fragments was accomplished using a nonlinear gradient of pH 6 potassium oxalate buffer as the mobile phase. Acetate buffer linear gradients were also useful as mobile phases, but only for separations of oligogalacturonic acids that were soluble in this solvent (DP less than 20). Additionally, oligogalacturonic acid separations were accomplished on a lower capacity AS4A stationary phase column. Triple pulse amperometric detection was selective, sensitive, and reproducible, nevertheless, oligogalacturonic acid response factors were affected by DP and compositional changes in the mobile phase.     

High-performance anion-exchange chromatography of oligosaccharides using pellicular resins and pulsed amperometric detection

High-performance liquid chromatography using pellicular quaternary amine-bonded resins was used to separate a variety of neutral, sialylated, and phosphorylated oligosaccharides. At pH 4.6, sialylated compounds were separated according to number of negative charges, sialic acid linkage [alpha(2,3) compared to alpha(2,6)], and position of sialic acid linkage along a linear saccharide chain. At pH 13, the neutral sugar portion of the sialylated chain had a significant effect on the separation, due to oxyanion formation. Specifically, sialylated tetrasaccharides containing the Gal beta(1,3)GlcNAc sequence were retained much more than their Gal beta(1,4)GlcNAc- or Gal-beta(1,4)GalNAc-sialylated counterparts. Linear phosphorylated oligosaccharides could be completely separated according to number of charges and net carbohydrate content. Partial separation of linear-chain positional isomers, differing in either location of Man-6-PO4 in the chain or linkage position of Man or Man-6-PO4, was accomplished. Branched-chain phosphorylated compounds could be completely separated according to which antennae contained the Man-6-PO4. The electrochemical current generated by oxidation of sialylated, phosphorylated, and neutral oligosaccharides was compared to that of a glucose. The relative molar response factors for neutral, sialylated, and phosphorylated oligosaccharides ranged from 0.2 to 3.2. Neutral oligosaccharides gave the following molar responses for each group of structurally related compounds: (1) mono- and disaccharide, 1-1.3; (2) linear tri- and tetrasaccharides, 1.5-2.0; and (3) branched pentasaccharide-nonasaccharides, 2.4-3.1. Response factors for the sialyated compounds were not as consistent and were affected by linkage position of sialic acid. For oligosaccharides of the same size, increasing phosphorylation resulted in a twofold decrease in response factor for each added phosphate group. Therefore, conversion of sialylated and phosphorylated oligosaccharides to their neutral counterparts, using alkaline phosphatase or neuraminidase, respectively, was required for quantitative analysis of oligosaccharide mixtures using electrochemical response. Using this approach, complete separation of the parent neutral structures was obtained, the relative proportions of the neutral species were quantified, and the amount of sialic acid released was easily determined in a neuraminidase digest.     

Direct determination of tryptophan using high-performance anion-exchange chromatography with integrated pulsed amperometric detection

Here we present a new method to rapidly quantify tryptophan (Trp) in proteins, animal feed (Mehaden fishmeal), cell cultures, and fermentation broths. Trp is separated from common amino acids by anion-exchange chromatography in 12min and directly detected by integrated pulsed amperometry. The estimated lower detection limit for this method is 1pmol. Alkaline (4M NaOH) hydrolysates can be directly injected, and therefore we used this method to determine the optimum alkaline hydrolysis conditions for the release of Trp from a model protein, bovine serum albumin (BSA). This method accurately determined the Trp content of BSA and fishmeal. High levels of glucose (2%, w/w) do not interfere with the chromatography or decrease recovery of Trp. We used this method to monitor free Trp during an Escherichia coli fermentation.     

Direct determination of polyglucose metabolites in plasma using anion-exchange chromatography with pulsed amperometric detection

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE–PAD) was evaluated for the quantitation of polyglucose metabolites (DP2–DP7) in human plasma. The method was investigated for accuracy, precision, specificity, linearity, range and analyte stability. Samples were prepared by dilution into the standard range (0.1–10 μg/ml) followed by deproteinization using a 30?000 molecular mass cut-off filtration device. The limit of detection was 0.05 μg/ml for all metabolites. Method precision for DP2–DP7 varied from approximately 2% R.S.D. in the upper range to approximately 15% R.S.D. at the limit of quantitation. Samples were stable following one or two freeze–thaw cycles and, after preparation, they could be refrigerated for up to 72 h. Application of this method to clinical plasma samples from continuous ambulatory peritoneal dialysis (CAPD) patients administered one daily night-time intraperitoneal exchange of 2 l of 7.5% polyglucose solution for four weeks indicated that plasma levels of DP2, DP3 and DP4 increased from baseline levels of <0.01 g/l to steady-state levels of 1.2±0.3, 1.2±0.3 and 0.4±0.1 g/l (mean±S.D.), respectively. These steady state plasma levels for DP2 and DP3 are comparable to previously reported levels in patients administered daily overnight 7.5% polyglucose dialysis solution.     

High-pH anion-exchange chromatography with pulsed amperometric detection and molar response factors of human milk oligosaccharides

A method is described to separate and characterize neutral and acidic lactose-derived oligosaccharides without prior derivatization or reduction by high-pH anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD). This method has been applied to human milk oligosaccharides from donors with different blood group specificity (A, Le^a and A, Le^b). Neutral and acidic components were separated from each other by anion-ecchange chromatography. A distinct separation of individual components was obtained by size-exclusion chromatography on Fractogel TSK HW 50S (acidic oligosaccharides) or Fractogel TSK HW 40S (neutral oligosaccharides containing up to 6 monomers) and Bio-Gel P-4 size exclusion (neutral oligosaccharides containing more than 6 monomers). Furthermore, the molar response factors after HPAEC-PAD have been determined for 8 components.     

Analysis of nonstructural carbohydrates in storage organs of 30 ornamental geophytes by high-performance anion-exchange chromatography with pulsed amperometric detection

A comprehensive analysis of nonstructural carbohydrates in storage organs (bulbs and corms) of 30 ornamental geophytes was conducted by employing a variety of extraction techniques followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Among species, starch, fructan, glucomannan and soluble sugars accounted for 50-80% of storage organ dry weight (DW). Starch ranged from 24 to 760 mg g(-1) DW, fructan (commonly occurring with starch) from 25 to 500 mg g(-1) DW, and glucomannan from 15 to 145 mg g(-1) DW. An acid hydrolysis protocol for concurrent determination of fructan and glucomannan was developed. The average degree of polymerization (DP) of ethanol and water-soluble fructan and the man : glu ratio of glucomannan also varied between species. The 80% ethanol fraction contained soluble sugars and short-chain fructans (< 25 DP), whereas water extracts contained soluble sugars, fructans (both short- and long-chain, 相似文献   

Separation and identification of neisserial lipooligosaccharide oligosaccharides using high-performance anion-exchange chromatography with pulsed amperometric detection

We determined the optimal conditions for high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) of oligosaccharides (OS) released from neisserial lipooligosaccharides (LOS) by mild acid hydrolysis. We efficiently obtained detailed composition, sequence, and linkage information about high Mr LOS. We found that HPAE-PAD can discriminate isobaric (same Mr) molecules of different structure, for example, nLc4 and Gb4, distinguish alpha from beta chain extensions, and determine the number of phosphoethanolamine (PEA) substituents. HPAE-PAD provided quantitative information that could be used to compare the relative abundances of OS. We used HPAE-PAD to identify all of the known LOS alpha chain antennae. When used with antibody-binding profiles and exoglycosidase digestion results, HPAE-PAD can provide nearly complete structures rapidly.     

Sugar analysis of glycoproteins and glycolipids after methanolysis by high-performance liquid chromatography with pulsed amperometric detection

A procedure for the analysis of the monosaccharide composition of glycoproteins and glycolipids by methanolysis and high-performance liquid chromatography with pulsed amperometric detection is described. The advantage over previous methods is the analysis of underivatized methyl glycosides of all glycoconjugate monosaccharides including sialic acid and uronic acid in a single chromatographic step at the subnanomolar level.     

Method for monitoring alginate released in biological fluids by high-performance anion-exchange chromatography with pulsed amperometric detection

The use of alginate-entrapped cells in cell therapy requires a method for monitoring possible released compound within biological fluids following either their implantation or inoculation in artificial organs. Oligomannuronic and oligoguluronic acids were prepared by enzymatic depolymerization with alginate lyase from Pseudomonas alginovora, characterized by high-performance anion-exchange chromatography with pulsed amperometric detection and quantitated in human, pig and rabbit blood, urine and tissue samples. The method was tested for linearity and detection limit, accuracy, intra- and inter-day precision. The limit of detection was 3 microgram/ml in both urine and plasma and 5 mg/g of tissues. The relative standard deviations (RSDs) of intra-day precision were 6.0-16.6% and 4.8-8.7% in plasma and urine, respectively; the RSDs of inter-day precision were 5.1-14.4% and 5.0-11.6% in plasma and urine, respectively. Thus, this method appears suitable for the measurement of released alginate from entrapped cells used in cell therapy.     

Isolation of oligogalacturonic acids up to DP 20 by preparative high-performance anion-exchange chromatography and pulsed amperometric detection

Milligram quantities of oligogalacturonic acids up to a degree of polymerization (DP) of 20 were purified by high-performance anion-exchange chromatography utilizing a preparative-scale (21-mm i.d.) CarboPac PA1 column and a nonlinear potassium acetate (pH 7.5) gradient. Detection was accomplished by pulsed amperometry without post-column addition of hydroxide. Pulsed amperometry at near-neutral pH is an excellent detection method for preparative separations of carbohydrates because it avoids base-catalyzed degradation reactions that can occur at high pH. This method was simpler, faster, had higher sample loading capacity and allowed for the isolation of higher DP oligogalacturonic acids than other methods reported previously. With this improved method, multi-milligram quantities of valuable oligogalacturonic acids (up to DP 20) can be readily isolated.     

Preparation of high purity monodisperse oligosaccharides derived from mannuronan by size-exclusion chromatography followed by semi-preparative high-performance anion-exchange chromatography with pulsed amperometric detection

Oligosaccharides of ([4)-β-d-ManpA-(→]_n) with a degree of polymerisation (DP) of 5, 10 and 15 were generated by partial acid hydrolysis of alginate mannuronan. These were subsequently purified by a combination of size-exclusion chromatography and semi-preparative high-performance anion-exchange chromatography with pulsed amperometric detection. The purity of the isolated oligosaccharides was greater than 96%. With automated operation of the chromatography system, milligram quantities can be generated over a period of a few days. Thus, our methodology now offers some significant advantages over earlier, including our own, protocols focused on uronic acid oligomers, where the final products are either not as pure or more starting material is needed to generate an equivalent yield of product. Removal of ammonium ions in collected fractions after size-exclusion chromatography and prior to freeze-drying was found to be essential to prevent the formation of imines and subsequent Maillard browning products.