Weak Anion-exchange High-performance Liquid Research Articles

Determination of Inosine Triphosphate Pyrophosphatase Phenotype in Human Red Blood Cells Using HPLC

Thiopurine drugs, widely used in cancer chemotherapy, inflammatory bowel disease, and autoimmune hepatitis, are responsible for common adverse events. Only some of these may be explained by genetic polymorphism of thiopurine S-methyltransferase. Recent articles have reported that inosine triphosphate pyrophosphatase (ITPase) deficiency was associated with adverse drug reactions toward thiopurine drug therapy. Here, we report a weak anion exchange high-performance liquid chromatography method to determine ITPase activity in red blood cells and to investigate the relationship with the occurrence of adverse events during azathioprine therapy. ITPase activity was assessed by the enzymatic conversion of inosine triphosphate (ITP) to inosine monophosphate (IMP). The reaction was stopped by heating for 3 minutes at 120°C. IMP, inosine diphosphate, and ITP were analyzed on a Hypersil APS-2 column, a weak anion exchange phase that exhibits both ionic and hydrophobic properties. The chromatographic method reported allows the analysis of IMP, inosine diphosphate, and ITP in a single run in <12.5 minutes. The method was linear in the range 5-1500 μmole/L of IMP. Intraassay and interassay precisions were <5% for red blood cell lysates supplemented with 50, 500, and 1000 μmole/L IMP. Km and Vmax evaluated by Lineweaver-Burk plot were 677.4 μmole/L and 19.6 μmole·L·min, respectively. The frequency distribution of ITPase from 73 patients was investigated. The method described is useful to determine the ITPase phenotype from patients on thiopurine therapy and to investigate the potential relation between ITPase deficiency and the occurrence of adverse events.

Heparin identification test and purity test for OSCS in heparin sodium and heparin calcium by weak anion-exchange high-performance liquid chromatography

Heparin sodium and heparin calcium, which are widely used as anti-coagulants, are known to potentially contain the natural impurity dermatan sulfate (DS). Recently serious adverse events occurred in patients receiving heparin sodium in the US, and a contaminant oversulfated chondroitin sulfate (OSCS) was found to be a cause of the events. To ensure the quality and safety of pharmaceutical heparins, there is need of a physicochemical identification test that can discriminate heparin from the heparin-related substances as well as a sensitive purity test for OSCS. Recently, HPLC with a strong-anion exchange column was proposed as the methods for identifying heparin and determination of OSCS in heparin sodium. Although this method is convenient and easy to perform, the only column suitable for this purpose is the Dionex IonPac AS11-HC column. In this study, we developed alternative identification test and test for OSCS in both heparin sodium and heparin calcium using a weak anion-exchange column. The identification test allowed for separation of heparin from the impurity DS and contaminant OSCS in a shorter time. The purity test provided enough sensitivity, specificity, linearity, recovery and repeatability for OSCS. We believe that our methods will be useful for quality control of pharmaceutical heparins.

Common aberrations from the normal human plasma N-glycan profile

After performing hydrophilic interaction and weak anion exchange high-performance liquid chromatography to analyze N-glycans in the plasma of 1991 people, we identified several individuals that differed significantly from the "normal" profile of N-glycans. By performing consensus scoring of pairwise distances between vectors containing measured glycan values, we formed six groups of individuals with specific glyco-phenotypes. Some aberrations from the normal plasma protein patterns were found to be associated with clinical conditions (such as renal problems in people with increased monosialylated biantennary glycans, A2G2S1), while other substantial changes in N-glycan structure, such as the near complete absence of neutral glycans or antennary fucosylated tri- and tetraantennary glycans, were not associated with any observed adverse health outcomes. These results demonstrate the existence of specific altered glyco-phenotypes in some individuals and indicate that in some cases they might represent risk factors for the development of specific diseases.

Changes of Serum Glycans During Sepsis and Acute Pancreatitis

Acute inflammatory response is a complex process associated with the production of both pro- and anti-inflammatory mediators. Although it is generally considered to be a single homeostatic mechanism, there are differences associated with the nature and the site of inflammation. We examined the changes of N-linked glycans released from the serum of a patient with sepsis and a patient with acute pancreatitis during the first eight days of the disease. Sera were taken from patients at the time of reporting to hospital and then three more times. The blood from a healthy individual was drawn on one occasion only. Glycans were released using N-glycosidase F and were subjected to normal phase and weak anion exchange high-performance liquid chromatography, exoglycosidase digestions, and mass spectrometry. The levels of identified structures have been followed through the course of disease and compared to the control levels. Changes in serum glycans were found to occur very early in acute inflammation. The most prominent differences include the increase in ratio of outer arm to core fucose, increase in the amount of tetrasialylated structures, changes in the levels of mannose structures, and in the degree of branching. The relative proportions of different glycans changed daily and some differences were also observed between sepsis and pancreatitis, probably reflecting that in these two conditions, the acute phase response is triggered by a different stimulus that is associated with different patterns of production of cytokines.

Direct analysis of mannitol, lactulose and glucose in urine samples by high-performance anion-exchange chromatography with pulse amperometric detection. Clinical evaluation of intestinal permeability in human immunodeficiency virus infection.

Clinically, the ratio of lactulose/mannitol excretion in urine after administration of these non-metabolized sugars has been used to evaluate the extent of malabsorption and intestinal permeability disruption in several infections and nutritional diseases, including human immunodeficiency virus (HIV) infection. A range of methodologies have been reported to determine the lactulose/mannitol ratio, including enzymatic assay, gas-liquid chromatography (GC), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Most published methods involve tedious sample preparations, rendering them unsuitable for routine or automated clinical laboratory testing. We describe in this paper a method in which weak anion-exchange high-performance liquid chromatography in conjunction with a pulsed amperometric detector was used. It requires very simple sample preparation and avoids interference by other components present in the urine. The linear range of determination for mannitol, lactulose and glucose are up to 10 nmol, in a single injection. The limits of detection are 8, 12, 47 and 52 pmol, respectively, for mannitol, glucose, lactose and lactulose. The separation and quantification using this method are highly reproducible, yielding standard errors of less than 2.5% for retention times and less than 3.5% for quantitation. The ratios of lactulose/mannitol recovery in controls and in HIV-infected subjects with and without diarrhea showed striking differences, which are in close agreement with the published results derived with similar HPLC methods.

Analytical and Preparative Separation of Anionic Oligosaccharides by Weak Anion-Exchange High-Performance Liquid Chromatography on an Inert Polymer Column

The use of ammonium formate buffers for analytical and preparative weak anion-exchange high-performance chromatography of anionic sugars is described. The method can be used for structures containing sulfate, phosphate, sialic acid, and uronic acid moieties. Excellent separation of the anionic sugars was achieved from pH 5.5 to 9. In contrast to silica matrix columns the polymer matrix column used can tolerate a wide range of pH values for the eluting buffer and it does not shed material into the eluent. The use of a single volatile buffer avoids additional purification steps to recover separated sugars suitable for further structural and functional analyses.

Determination of alkaline phosphatase isoenzymes in serum by high-performance liquid chromatography with post-column reaction detection

A weak anion-exchange high-performance liquid chromatographic procedure with post-column reaction detection for simultaneous determination of alkaline phosphatase (EC 3.1.3.1, ALP) isoenzymes is described. We identified six peaks with ALP isoenzyme activity in normal serum. The peaks were, in order of elution, one intestinal/bone, two bone and three liver ALP isoenzymes. This new assay with automatic injection, on-line post-column reaction detection and powerful integration data system could be of significant value in the routine clinical biochemistry laboratory. The advantages include improved sensitivity and selectivity over previous methods for the determination of ALP isoenzymes.

Weak anion-exchange high-performance liquid chromatography of peptides

In this survey, the principles and applications of a method recently developed for peptide separations are given. This method uses a bonded weak anion-exchange column and mixtures of volatile triethylammonium acetate buffer and acetonitrile as eluent. Its applications to the separation of a large number of peptides including diastereomeric and other closely related peptides are discussed. Separation of the enzymatic digests of some proteins is also presented. The complementary use of this method to the reversed-phase methods is outlined and their combined use for separation of enzymatic digests of proteins and assessment of purity of synthesized peptides is demonstrated. The results reviewed show that the weak anion-exchange method is an excellent approach for peptide separations and could be an important partner of reversed-phase methods for achieving optimal results.

Separation and purification of diastereomers of angiotensin I by weak anion-exchange high-performance liquid chromatography

Several diasteroemers of angiotensin I were resolved by weak anion-exchange high-performance liquid chromatography (HPLC). All of the diastereomers which were examined contained significant amounts of peptides whose amino acid composition differed from the designated diastereomer of angiotensin I. The des-Asp 1 forms of angiotensin I and the impurities were weakly retained on the anion-exchange column and were well separated from the rest of the peptides. Many of the impurities contained fractional amounts of amino acids suggesting that peptides which contained variable amounts of histidine and arginine are not resolved by this method. The results are compared with the results of separations of the same peptides by reversed-phase HPLC. This comparison strongly suggests that the two HPLC methods, utilizing different separation principles, are complementary; hence their combined use leads to a more confident assessment of the purity of a given peptide preparation.

Comparison of reversed-phase and weak anion-exchange high-performance liquid chromatographic methods for peptide separations

Weak anion-exchange and reversed-phase high-performance liquid chromtographic methods for peptide separations were compared using a tryptic digest of “rat small myelin basic protein”. In these experiments, a number of tryptic peptides that were not resolved on the reversed-phase column could be separated on the weak anion-exchange column, and in other instances, as might be expected, reversed-phase chromatography provided better resolution of certain peptides than did the weak anion-exchange method. The results obtained strongly suggest that the combined use of these two methods of separation, which utilize different selectivities, can provide an excellent improvement in resolving power for a number of peptide separations.