Glycan Mapping of Low-Molecular-Weight Heparin Using Mass Spectral Correction Based on Chromatography Fitting with "Glycomapping" Software.

In this work, the first version of "Glycomapping" software is developed for the analysis of the most common low-molecular-weight heparin (LMWH), enoxaparin. Using ultrahigh-performance liquid chromatography-mass spectrometry, size exclusion chromatography is applied, and a virtual database of glycans in enoxaparin is established for the initial searching. With "Glycomapping", a complex chromatogram can be fitted, significantly improving resolution and confirming an accurate distribution range for each size of glycan within enoxaparin. In addition, randomly matched MS data can be corrected, with the constraint of the corresponding chromatographic retention time range, to remove most false positive data. The analytical stability of "Glycomapping" software was confirmed. Enoxaparin, prepared by different manufacturers and from different animal sources, was analyzed using "Glycomapping." Compared to raw data, data processed with "Glycomapping" are more robust and accurate. Another two LMWHs, nadroparin and dalteparin could also be analyzed with this software. This work lays a solid foundation for the automated analysis of heterogeneous mixtures of natural glycans, such as LMWHs and other complex oligosaccharides and polysaccharides.

Analysis of heparinase derived LMWH products using a MHC 2D LC system linked to Q-TOF MS.

Low molecular weight heparins (LMWHs), depolymerized from unfractionated heparin (UFH), are widely used as anticoagulant drugs in clinic. The variable degradation methods result the different types of LMWHs, such as enoxaparin prepared by alkaline degradation following benzylation and nadroparin degraded by nitrous acid and subsequent reduction. They have different anticoagulant activities, molecular weight and special oligosaccharide sequences. Oligosaccharide analysis of the heparinase-catalyzed digestion products of heparin and LMWHs is an important way to explore the fine structural composition. In this work, a MHC-2D-LC-MS system using SAX followed by SEC and tandem to MS was applied to analyze the heparinase-products of LMWHs. 15 components of enoxaparin and 20 components of nadroparin were separated and unambiguously characterized with mass spectrum, including eight common disaccharides, and the special structural domains resistant to enzyme digestion which have the 3-O sulfated residue and/or characteristic terminal residues and the linkage region tetrasaccharides.

[Determination of sulfation degree of heparin and low molecular weight heparins by capillary electrophoresis].

Heparin is composed of a highly sulfated linear saccharide and is widely used as an anticoagulant. Low molecular weight heparins (LMWHs) are derived from the unfractionated heparin (UFH) by enzymatic or chemical degradation. LMWHs have largely replaced heparin as an anticoagulant for treatment and prevention of thrombosis because of the advantages of less bleeding, greater bioavailability, and more predictable anticoagulant effects in comparison to heparin. Enoxaparin, produced by the alkaline degradation of UFH through ß-eliminative cleavage, represents the most commonly used LMWH. The structural characteristics of LMWHs differ from their parent heparin not only in terms of molecular weight but also in the sulfation degree as a result of losing the sulfate ester groups during the manufacturing process. The resulting compositional variation directly leads to a fluctuation in anticoagulant activity. In vitro functional assays showed that there is a wide variation in anticoagulant activity among the various LMWHs from different manufacturers owing to slight differences in the manufacturing process. This will directly affect heparin drug safety. In order to ensure the stability of product quality, it is necessary to develop a method for detecting the degree of heparin sulfation to monitor the stability of UFH and processing conditions. During the last two decades, various analytical methods based on chromatography or NMR have been developed for structural characterization of UFH and LMWHs. However, the reported methods require expensive equipment and professional data processing. These limitations make it difficult to apply the current methods to quality control via sulfation degree determination. Herein, we report a simple and robust method for the detection of the sulfation degree of UFH and LMWHs. The determination is based on the separation of building blocks of heparin obtained by exhaustive digestion of UFH and LMWHs in a mixture of heparinases. A mixed solution of heparinase Ⅰ, Ⅱ, and Ⅲ was prepared to give a final content of 0.13 IU/mL for each enzyme. The digestion of enoxaparin and heparin samples was performed at 25 ℃ for 48 h. By using a capillary electrophoresis (CE) method, a total of 18 oligosaccharides building blocks of heparin, including ten disaccharides, one trisaccharide, three tetrasaccharides, and four 1,6-anhydro derivatives, can be baseline separated. Then, the compositions of enoxaparin and UFH can be precisely determined. Based on the assumption that the molar extinction coefficient of each oligosaccharide at UV 232 nm is the same, the concentration of each oligosaccharide can be conveniently replaced by their peak area, and the accurate number of sulfate ester groups in each disaccharide unit can be determined, hence the average sulfation degree (SD). The developed method allows us to compare the sulfation degree data between the enoxaparin batches from the different manufacturers to evaluate the composition similarity. Herein, eight batches of commercially available enoxaparin from two manufacturers and four batches of UFH source materials were measured. Each sample was measured in triplicate, and the average values as well as the relative standard deviations (RSD) were calculated. The total sulfation degree (T-SD), the individual degree of N-sulfation (N-SD) and O-sulfation (O-SD) data were also determined and compared. A significant difference was observed in the SD of the products from the different manufacturers, which indicated that our method can be used as one of the quantitative compositional analysis methods for quality control of LMWHs and UFH. The variation in terms of the sulfation degree of enoxaparin products from different manufacturers can be precisely identified using this method. This allows us to determine the detailed compositional differences between products from the different manufacturers. The obtained satisfactory data show that high fluctuation in the sulfation degree of UFH could transmit to the final enoxaparin products. The consistency of the products can also be evaluated by using these methods. The CE method has several advantages for quantitative compositional analysis of LMWHs, such as high separation efficiency, high sensitivity, automation, short analysis time and low consumption of both sample and reagents. It has a good application potential in the quality control heparin production.

[Determination of the anticoagulant activity of low molecular weight heparins by micellar electrokinetic chromatography combined with on-column enzymatic reaction].

Low molecular weight heparins (LMWHs) have largely replaced heparin for the treatment and prevention of thrombosis because of their various advantages over unfractionated heparins (UFHs) such as less bleeding, greater bioavailability, and more predictable anticoagulant effects. For special groups of patients, such as pregnant women, children, and patients with renal failure, it is necessary to monitor the anticoagulant activity of LMWHs in the blood. The traditional method used to determine the anticoagulant activity of heparin is the coagulation test. However, the results are various from different laboratories and different reagents. In contrast, the chromogenic substrate method is more accurate, sensitive and is easy to automate. Here, a method for the determination of the anticoagulant activity of LMWHs was developed by using a capillary-electrophoresis-based substrate chromogenic method. In this method, micellar electrokinetic chromatography (MEKC) was used in combination with electrophoretically mediated microanalysis to determine the anti-factor Xa (FXa) activity of LMWHs. The inhibition was measured by employing a chromogenic peptide substrate (CPS) with a p-nitroaniline (p-NA) moiety as the chromophore. The injection end of the capillary was used as a microreactor in which solutions of LMWHs, antithrombin Ⅲ (ATⅢ), FXa and CPS were successively injected and mixed by using diffusion, the transverse diffusion of laminar flow profiles and applied voltage. The reaction product p-NA was separated from unreacted CPS and sample matrix by using the MEKC mode with discontinuous background electrolyte system. The produced p-NA was baseline separated from the other components and detected at 380 nm to obtain maximum sensitivity. The amount of p-NA was inversely proportional to the activity of LMWHs in the sample. To improve the accuracy of quantification and the method repeatability of methods, nitrofurantoin (NF) was selected as the internal standard, which was added to the solution of CPS. The method was validated and used to measure a set of samples. The method is characterized by automation, good repeatability, high sensitivity, and cost-effectiveness. Additionally, the method does not interfere by the sample matrix, and thus can be used to monitor the anticoagulant activity of LMWHs in plasma.

Anticoagulant activity of porcine heparin: Structural-property relationship and semi-quantitative estimation by nuclear magnetic resonance (NMR) spectrometry.

Heparin is a carbohydrate polymer, which is clinically used as an anticoagulant for the treatment of thrombotic disorders. The anticoagulant process is mainly mediated by the interaction of heparin with antithrombin followed by inhibition of clotting factors IIa (FIIa) and Xa (FXa). The influence of polymer disaccharide structure, average molecular weight and impurity profiling (e.g., chloride and water content) was investigated by NMR spectrometry and principal component analysis (PCA) for a representative dataset of porcine heparin samples (n = 509). A significant linear dependence was found between anticoagulant activity and scores on the third principal component (PC3) based on the non-targeted analysis of 1H NMR fingerprints. The correlation between average molecular values and anticoagulant activity for the 24 porcine heparin samples from two manufacturers was linear (R = 0.85) over typical values for porcine heparin preparations. Chloride and water contents were identified as negatively influencing factors for the actual activity values as their presence decrease the "pharmaceutically active" organic part of heparin preparations. Some suggestions regarding manufacturing process are made according to the results.

Non-Anticoagulant Low Molecular Weight Heparins for Pharmaceutical Applications.

Heparin is a polypharmacological agent with anticoagulant activity. Periodate oxidation of the nonsulfated glucuronic acid residue results in non-anticoagulant heparin derivative (NACH) of reduced molecular weight. Similar treatment of a low molecular weight heparin, dalteparin, also removes its anticoagulant activity, affording a second heparin derivative (D-NACH). A full structural characterization of these two derivatives reveals their structural differences. SPR studies display their ability to bind to several important heparin-binding proteins, suggesting potential new therapeutic applications.

Authentication of animal origin of heparin and low molecular weight heparin including ovine, porcine and bovine species using 1D NMR spectroscopy and chemometric tools.

High resolution (600MHz) nuclear magnetic resonance (NMR) spectroscopy is used to distinguish heparin and low-molecular weight heparins (LMWHs) produced from porcine, bovine and ovine mucosal tissues as well as their blends. For multivariate analysis several statistical methods such as principal component analysis (PCA), factor discriminant analysis (FDA), partial least squares - discriminant analysis (PLS-DA), linear discriminant analysis (LDA) were utilized for the modeling of NMR data of more than 100 authentic samples. Heparin and LMWH samples from the independent test set (n=15) were 100% correctly classified according to its animal origin. Moreover, by using 1H NMR coupled with chemometrics and several batches of bovine heparins from two producers were differentiated. Thus, NMR spectroscopy combined with chemometrics is an efficient tool for simultaneous identification of animal origin and process based manufacturing difference in heparin products.

Novel method for the determination of average molecular weight of natural polymers based on 2D DOSY NMR and chemometrics: Example of heparin.

Apart from the characterization of impurities, the full characterization of heparin and low molecular weight heparin (LMWH) also requires the determination of average molecular weight, which is closely related to the pharmaceutical properties of anticoagulant drugs. To determine average molecular weight of these animal-derived polymer products, partial least squares regression (PLS) was utilized for modelling of diffused-ordered spectroscopy NMR data (DOSY) of a representative set of heparin (n=32) and LMWH (n=30) samples. The same sets of samples were measured by gel permeation chromatography (GPC) to obtain reference data. The application of PLS to the data led to calibration models with root mean square error of prediction of 498Da and 179Da for heparin and LMWH, respectively. The average coefficients of variation (CVs) did not exceed 2.1% excluding sample preparation (by successive measuring one solution, n=5) and 2.5% including sample preparation (by preparing and analyzing separate samples, n=5). An advantage of the method is that the sample after standard 1D NMR characterization can be used for the molecular weight determination without further manipulation. The accuracy of multivariate models is better than the previous results for other matrices employing internal standards. Therefore, DOSY experiment is recommended to be employed for the calculation of molecular weight of heparin products as a complementary measurement to standard 1D NMR quality control. The method can be easily transferred to other matrices as well.

Qualitative and quantitative analysis of heparin and low molecular weight heparins using size exclusion chromatography with multiple angle laser scattering/refractive index and inductively coupled plasma/mass spectrometry detectors.

Heparin, a highly sulfated glycosaminoglycan, has been used as a clinical anticoagulant over 80 years. Low molecular weight heparins (LMWHs), heparins partially depolymerized using different processes, are widely used as clinical anticoagulants. Qualitative molecular weight (MW) and quantitative mass content analysis are two important factors that contribute to LMWH quality control. Size exclusion chromatography (SEC), relying on multiple angle laser scattering (MALS)/refractive index (RI) detectors, has been developed for accurate analysis of heparin MW in the absence of standards. However, the cations, which ion-pair with the anionic polysaccharide chains of heparin and LMWHs, had not been considered in previous reports. In this study, SEC with MALS/RI and inductively coupled plasma/mass spectrometry detectors were used in a comprehensive analytical approach taking both anionic polysaccharide and ion-paired cations heparin products. This approach was also applied to quantitative analysis of heparin and LMWHs. Full profiles of MWs and mass recoveries for three commercial heparin/LMWH products, heparin sodium, enoxaparin sodium and nadroparin calcium, were obtained and all showed higher MWs than previously reported. This important improvement more precisely characterized the MW properties of heparin/LMWHs and potentially many other anionic polysaccharides.

Characterization of Low-Molecular-Weight Heparins by Strong Anion-Exchange Chromatography.

Currently, detailed structural characterization of low-molecular-weight heparin (LMWH) products is an analytical subject of great interest. In this work, we carried out a comprehensive structural analysis of LMWHs and applied a modified pharmacopeial method, as well as methods developed by other researchers, to the analysis of novel biosimilar LMWH products; and, for the first time, compared the qualitative and quantitative composition of commercially available drugs (enoxaparin, nadroparin, and dalteparin). For this purpose, we used strong anion-exchange (SAX) chromatography with spectrophotometric detection because this method is more helpful, easier, and faster than other separation techniques for the detailed disaccharide analysis of new LMWH drugs. In addition, we subjected the obtained results to statistical analysis (factor analysis, t-test, and Newman-Keuls post hoc test).

Heparinas de bajo peso molecular en pacientes con fibrilación auricular / Low-molecular-weight heparins in patients with atrial fibrillation

En la práctica clínica, las heparinas de bajo peso molecular se utilizan con relativa frecuencia en pacientes con fibrilación auricular con el objetivo de prevenir eventos embólicos. En este artículo se revisa la evidencia disponible en las siguientes situaciones clínicas: inicio rápido de anticoagulación, tratamiento «puente» (en sustitución del tratamiento anticoagulante oral crónico en relación con un procedimiento invasivo) y cardioversión guiada por ecocardiograma transesofágico (AU)

In clinical practice, low-molecular-weight heparins are used relatively frequently in patients with atrial fibrillation to prevent embolic events. In this article, it is revised the available evidence in the following clinical situations: rapid onset of anticoagulation, bridging therapy (replacing long-term oral anticoagulant therapy around an invasive procedure) and transesophageal echocardiography-guided cardioversion (AU)

Parent heparin and daughter LMW heparin correlation analysis using LC-MS and NMR.

Heparin is a structurally complex, polysaccharide anticoagulant derived from livestock, primarily porcine intestinal tissues. Low molecular weight (LMW) heparins are derived through the controlled partial depolymerization of heparin. Increased manufacturing and regulatory concerns have provided the motivation for the development of more sophisticated analytical methods for determining both their structure and pedigree. A strategy, for the comprehensive comparison of parent heparins and their LMW heparin daughters, is described that relies on the analysis of monosaccharide composition, disaccharide composition, and oligosaccharide composition. Liquid chromatography-mass spectrometry is rapid, robust, and amenable to automated processing and interpretation of both top-down and bottom-up analyses. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues and glucosamine substitution. Principal component analysis (PCA) was applied to the normalized abundance of oligosaccharides, calculated in the bottom-up analysis, to show parent and daughter correlation in oligosaccharide composition. Using these approaches, six pairs of parent heparins and their daughter generic enoxaparins from two different manufacturers were comprehensively analyzed. Enoxaparin is the most widely used LMW heparin and is prepared through controlled chemical ß-eliminative cleavage of porcine intestinal heparin. Lovenox®, the innovator version of enoxaparin marketed in the US, was analyzed as a reference for the daughter LMW heparins. The results, show similarities between LMW heparins from two different manufacturers with Lovenox®, excellent lot-to-lot consistency of products from each manufacturer, and detects a correlation between each parent heparin and daughter LMW heparin.

Qualification of HSQC methods for quantitative composition of heparin and low molecular weight heparins.

An NMR HSQC method has recently been proposed for the quantitative determination of the mono- and disaccharide subunits of heparin and low molecular weight heparins (LMWH). The focus of the current study was the validation of this procedure to make the 2D-NMR method suitable for pharmaceutical quality control applications. Pre-validation work investigated the effects of several experimental parameters to assess robustness and to optimize critical factors. Important experimental parameters were pulse sequence selection, equilibration interval between pulse trains and temperature. These observations were needed so that the NMR method was sufficiently understood to enable continuous improvement. A standard validation study on heparin then examined linearity, repeatability, intermediate precision and limits of detection and quantitation; selected validation parameters were also determined for LMWH.

Top-down and bottom-up analysis of commercial enoxaparins.

A strategy for the comprehensive analysis of low molecular weight (LMW) heparins is described that relies on using an integrated top-down and bottom-up approach. Liquid chromatography-mass spectrometry, an essential component of this approach, is rapid, robust, and amenable to automated processing and interpretation. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues comprising a low molecular weight heparin. Using our integrated approach four different low molecular weight heparins prepared from porcine heparin through chemical ß-eliminative cleavage were comprehensively analyzed. Lovenox™ and Clexane™, the innovator versions of enoxaparin marketed in the US and Europe, respectively, and two generic enoxaparins, from Sandoz and Teva, were analyzed. The results which were supported by analysis of variation (ANOVA), while showing remarkable similarities between different versions of the product and good lot-to-lot consistency of each product, also detects subtle differences that may result from differences in their manufacturing processes or differences in the source (or parent) porcine heparin from which each product is prepared.

Collaborative study for the calibration of replacement batches for the heparin low-molecular-mass for assay biological reference preparation.

The European Pharmacopoeia (Ph. Eur.) prescribes the control of the activity of low molecular mass heparins by assays for anti-Xa and anti-IIa activities (monograph 0828), using a reference standard calibrated in International Units (IU). An international collaborative study coded BSP133 was launched in the framework of the Biological Standardisation Programme (BSP) run under the aegis of the Council of Europe and the European Commission to calibrate replacement batches for the dwindling stocks of the Heparin low-molecular-mass for assay Biological Reference Preparation (BRP) batch 8. Thirteen official medicines control and manufacturers laboratories from European and non-European countries took part in this study to calibrate two freeze-dried candidate batches against the 3rd International Standard (IS) for heparin, low molecular weight (11/176; 3rd IS). The Heparin low-molecular-mass for assay BRP (batch 8) was also included in the test panel to check the continuity between subsequent BRP batches. Taking into account the stability data, the results of this collaborative study and on the basis of the central statistical analysis performed at the European Directorate for the Quality of Medicines & HealthCare (EDQM), the 2 candidate batches were officially adopted by the Commission of the European Pharmacopoeia as Heparin low-molecular-mass for assay BRP batches 9 and 10 with assigned anti-Xa activities of 102 and 100 IU/vial and anti-IIa activities of 34 and 33 IU/vial respectively.

Comprehensive Identification and Quantitation of Basic Building Blocks for Low-Molecular Weight Heparin.

Low-molecular weight heparins (LMWHs) are widely used anticoagulant drugs. They inherit the heterogeneous backbone sequences of the parent heparin, while the chemical depolymerization process modifies the nonreducing end (NRE) and reducing end (RE) of their sugar chains. Some side reactions may also occur and increase the structural complexity of LMWHs. It is important to precisely characterize the structures of LMWHs, especially their chemical modifications, to ensure drug quality and safety. Compositional analysis provides a powerful approach to reveal the building blocks that make up the LMWHs, which are the mutual consequence of the heparin starting materials and the manufacturing process. Here, we introduce a comprehensive analytical method to recover the most basic building blocks of LMWHs. A strategy of combining both enzymatic digestion and oxidative degradation of LMWH was used to make the NRE, RE, and backbone structures differentiable from one another. Satisfactory separation, identification, and quantitation were achieved by coupling hydrophilic interaction chromatography with a triple quadrupole mass spectrometer operating under the multiple reaction monitoring mode. After enzymatic digestion, over 30 species were detected, with both natural and chemically modified heparin basic building blocks. Two novel structures, including a trisaccharide containing two glucosamine residues and a tetrasaccharide containing a 3-O-sulfated uronic acid residue, were discovered. Reduced and oxidatively degraded samples were analyzed to provide the complementary information on both termini of LMWHs. The reproducibility of this method was evaluated, and enoxaparin injections were analyzed to demonstrate the application of this method for evaluating the sameness of LMWH products.

Comparison of Low-Molecular-Weight Heparins Prepared From Bovine Lung Heparin and Porcine Intestine Heparin.

Currently porcine intestine is the only approved source for producing pharmaceutical heparin in most countries. Enoxaparin, prepared by benzylation and alkaline depolymerization from porcine intestine heparin, is prevalent in the anticoagulant drug market. It is predicted that porcine intestine heparin-derived enoxaparin (PIE) will encounter shortage, and expanding its production from heparins obtained from other animal tissues may, therefore, be inevitable. Bovine lung heparin is a potential alternative source for producing enoxaparin. Critical processing parameters for producing bovine lung heparin-derived enoxaparin (BLE) are discussed. Three batches of BLEs were prepared and their detailed structures were compared with PIEs using modern analytical techniques, including disaccharide composition, intact chain mapping by liquid chromatography-mass spectrometry and 2-dimensional nuclear magnetic resonance spectroscopy. The results suggested that the differences between PIEs and BLEs mainly result from N-acetylation differences derived from the parent heparins. In addition, bioactivities of BLEs were about 70% of PIEs based on anti-factor IIa and Xa chromogenic assays. We conclude that BLE has the potential to be developed as an analogue of PIE, although some challenges still remain.

Development of hydrophilic interaction chromatography with quadruple time-of-flight mass spectrometry for heparin and low molecular weight heparin disaccharide analysis.

RATIONALE: Heparin and low molecular weight heparin (LMWH) are widely used as clinical anticoagulants. The determination of their composition and structural heterogeneity still challenges analysts. METHODS: Disaccharide compositional analysis, utilizing heparinase-catalyzed depolymerization, is one of the most important ways to evaluate the sequence, structural composition and quality of heparin and LMWH. Hydrophilic interaction chromatography coupled with quadruple time-of-flight mass spectrometry (HILIC/QTOFMS) has been developed to analyze the resulting digestion products. RESULTS: HILIC shows good resolution and excellent MS compatibility. Digestion products of heparin and LMWHs afforded up to 16 compounds that were separated using HILIC and analyzed semi-quantitatively. These included eight common disaccharides, two disaccharides derived from chain termini, three 3-O-sulfo-group-containing tetrasaccharides, along with three linkage region tetrasaccharides and their derivatives. Structures of these digestion products were confirmed by mass spectral analysis. The disaccharide compositions of a heparin, two batches of the LMWH, enoxaparin, and two batches of the LMWH, nadroparin, were compared. In addition to identifying disaccharides, 3-O-sulfo-group-containing tetrasaccharides, linkage region tetrasaccharides were observed having slightly different compositions and contents in these heparin products suggesting that they had been prepared using different starting materials or production processes. CONCLUSIONS: Thus, compositional analysis using HILIC/QTOFMS offers a unique insight into different heparin products.

Capillary Electrophoresis-Mass Spectrometry for the Analysis of Heparin Oligosaccharides and Low Molecular Weight Heparin.

Heparins, highly sulfated, linear polysaccharides also known as glycosaminoglycans, are among the most challenging biopolymers to analyze. Hyphenated techniques in conjunction with mass spectrometry (MS) offer rapid analysis of complex glycosaminoglycan mixtures, providing detailed structural and quantitative data. Previous analytical approaches have often relied on liquid chromatography (LC)-MS, and some have limitations including long separation times, low resolution of oligosaccharide mixtures, incompatibility of eluents, and often require oligosaccharide derivatization. This study examines the analysis of glycosaminoglycan oligosaccharides using a novel electrokinetic pump-based capillary electrophoresis (CE)-MS interface. CE separation and electrospray were optimized using a volatile ammonium bicarbonate electrolyte and a methanol-formic acid sheath fluid. The online analyses of highly sulfated heparin oligosaccharides, ranging from disaccharides to low molecular weight heparins, were performed within a 10 min time frame, offering an opportunity for higher-throughput analysis. Disaccharide compositional analysis as well as top-down analysis of low molecular weight heparin was demonstrated. Using normal polarity CE separation and positive-ion electrospray ionization MS, excellent run-to-run reproducibility (relative standard deviation of 3.6-5.1% for peak area and 0.2-0.4% for peak migration time) and sensitivity (limit of quantification of 2.0-5.9 ng/mL and limit of detection of 0.6-1.8 ng/mL) could be achieved.

Analysis of 3-O-sulfo group-containing heparin tetrasaccharides in heparin by liquid chromatography-mass spectrometry.

Complete heparin digestion with heparin lyase 2 affords a mixture of disaccharides and resistant tetrasaccharides with 3-O-sulfo group-containing glucosamine residues at their reducing ends. Quantitative online liquid chromatography-mass spectrometric analysis of these resistant tetrasaccharides is described in this article. The disaccharide and tetrasaccharide compositions of seven porcine intestinal heparins and five low-molecular-weight heparins were analyzed by this method. These resistant tetrasaccharides account for from 5.3 to 7.3wt% of heparin and from 6.2 to 8.3wt% of low-molecular-weight heparin. Because these tetrasaccharides are derived from heparin's antithrombin III-binding sites, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. The content of 3-O-sulfo group-containing tetrasaccharides in a heparin correlated positively (r=0.8294) to heparin's anticoagulant activity.