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.

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.

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.

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.

Liquid chromatography-diode array detection-mass spectrometry for compositional analysis of low molecular weight heparins.

Low molecular weight heparins (LMWHs) are important artificial preparations from heparin polysaccharide and are widely used as anticoagulant drugs. To analyze the structure and composition of LMWHs, identification and quantitation of their natural and modified building blocks are indispensable. We have established a novel reversed-phase high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry approach for compositional analysis of LMWHs. After being exhaustively digested and labeled with 2-aminoacridone, the structural motifs constructing LMWHs, including 17 components from dalteparin and 15 components from enoxaparin, were well separated, identified, and quantified. Besides the eight natural heparin disaccharides, many characteristic structures from dalteparin and enoxaparin, such as modified structures from the reducing end and nonreducing end, 3-O-sulfated tetrasaccharides, and trisaccharides, have been unambiguously identified based on their retention time and mass spectra. Compared with the traditional heparin compositional analysis methods, the approach described here is not only robust but also comprehensive because it is capable of identifying and quantifying nearly all components from lyase digests of LMWHs.

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.

Structural features of glycol-split low-molecular-weight heparins and their heparin lyase generated fragments.

Periodate oxidation followed by borohydride reduction converts the well-known antithrombotics heparin and low-molecular-weight heparins (LMWHs) into their "glycol-split" (gs) derivatives of the "reduced oxyheparin" (RO) type, some of which are currently being developed as potential anti-cancer and anti-inflammatory drugs. Whereas the structure of gs-heparins has been recently studied, details of the more complex and more bioavailable gs-LMWHs have not been yet reported. We obtained RO derivatives of the three most common LMWHs (tinzaparin, enoxaparin, and dalteparin) and studied their structures by two-dimensional nuclear magnetic resonance spectroscopy and ion-pair reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. The liquid chromatography-mass spectrometry (LC-MS) analysis was extended to their heparinase-generated oligosaccharides. The combined NMR/LC-MS analysis of RO-LMWHs provided evidence for glycol-splitting-induced transformations mainly involving internal nonsulfated glucuronic and iduronic acid residues (including partial hydrolysis with formation of "remnants") and for the hydrolysis of the gs uronic acid residues when formed at the non-reducing ends (mainly, in RO-dalteparin). Evidence for minor modifications, such as ring contraction of some dalteparin internal aminosugar residues, was also obtained. Unexpectedly, the N-sulfated 1,6-anhydromannosamine residues at the enoxaparin reducing end were found to be susceptible to the periodate oxidation. In addition, in tinzaparin and enoxaparin, the borohydride reduction converts the hemiacetalic aminosugars at the reducing end to alditols. Typical LC-MS signatures of RO-derivatives of individual LMWH both before and after digestion with heparinases included oligosaccharides generated from the original antithrombin-binding and "linkage" regions.

Structural analysis of low molecular weight heparin by ultraperformance size exclusion chromatography/time of flight mass spectrometry and capillary zone electrophoresis.

Although low molecular weight heparins (LMWHs) have been used as anticoagulant agents for over 2 decades, their structures have not been fully characterized. In this work, we propose a new strategy for the comprehensive structural analysis of LMWHs based on the combination of ultraperformance size exclusion chromatography/electrospray quadruple time-of-flight-mass spectrometry (UPSEC/Q-TOF-MS) and capillary zone electrophoresis (CZE). More than 70 components, including oligosaccharides with special structures such as 1,6-anhydro rings, saturated uronic acid at the nonreducing end and odd-numbered saccharides units were identified with UPSEC/Q-TOF-MS. Furthermore, a more detailed compositional analysis was accomplished by CZE analysis. PEG10000 and MgCl(2) were added to the background electrolyte to separate those saccharides with the nearly same charge-to-mass ratio. Baseline separation and quantification of all the building blocks of the most complex LMWH, namely, enoxaparin, which include 10 disaccharides, 1 trisaccharide, 2 tetrasaccharides, and, of particular importance, 4 1,6-anhyro derivatives, was achieved using CZE for the first time. Additionally, the peaks of oligosaccharides, in the absence of commercially available standards, were assigned on the basis of the linear correlation between the electrophoretic mobilities of oligosaccharides and their charge-to-mass ratios. These two approaches are simple and robust for structural analysis of LMWHs.

Low molecular weight heparin dosing and monitoring in solid organ transplant recipients.

Anti-Xa monitoring for low molecular weight heparin (LMWH) is currently recommended in obese, renally impaired, and pregnant patients. Substantial evidence indicates that solid organ transplant (SOT) patients are at an increased risk of renal impairment, thus representing a population at risk of LMWH accumulation. The purpose of this study was to review our experience with LMWH dosing and monitoring in a cohort of transplant recipients. This was a retrospective, single-center review of 96 SOT patients receiving enoxaparin treatment and anti-Xa monitoring. The percent of patients with supratherapeutic anti-Xas (>1 IU/mL) was determined, as was the relationship between enoxaparin dosages and anti-Xa levels and bleeding. The cohort had a mean age of 62 yr and creatinine clearance of 59 mL/min and was primarily lung transplant recipients (73%). The mean enoxaparin dose was 0.82 mg/kg, which resulted in a mean anti-Xa level of 0.98 IU/mL. Despite the reduced enoxaparin dose, 44% of patients experienced a supratherapeutic anti-Xa level. Patients with supratherapeutic anti-Xas had higher doses than those within the therapeutic range (0.89 mg/kg vs. 0.77 mg/kg; p = 0.002). No major bleeds occurred. Supratherapeutic anti-Xa levels are common in transplant patients receiving enoxaparin therapy. Empirically reduced dosing of enoxaparin and monitoring may warrant consideration in this population.

Top-down approach for the direct characterization of low molecular weight heparins using LC-FT-MS.

Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics, and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography, and chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency, and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycReSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy.

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.

Ultra-performance ion-pairing liquid chromatography with on-line electrospray ion trap mass spectrometry for heparin disaccharide analysis.

A high-resolution method for the separation and analysis of disaccharides prepared from heparin and heparan sulfate (HS) using heparin lyases is described. Ultra-performance liquid chromatography in a reverse-phase ion-pairing mode efficiently separates eight heparin/HS disaccharides. The disaccharides can then be detected and quantified using electrospray ionization mass spectrometry. This method is particularly useful in the analysis of small amounts of biological samples, including cells, tissues, and biological fluids, because it provides high sensitivity without being subject to interference from proteins, peptides, and other sample impurities.

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs.

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 µM of the fluorogenic substrate Mes-D-LGR-ANSN (C(2)H(5))(2) (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0-1.6 U mL(-1). The statistically sensitive assay range was 0-0.4 U mL(-1) for enoxaparin and tinzaparin and 0-0.2 U mL(-1) for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-D: -cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.

Chitosan-genipin microspheres for the controlled release of drugs: clarithromycin, tramadol and heparin.

The aim of this study was to first evaluate whether the chitosan hydrochloride-genipin crosslinking reaction is influenced by factors such as time, and polymer/genipin concentration, and second, to develop crosslinked drug loaded microspheres to improve the control over drug release. Once the crosslinking process was characterized as a function of the factors mentioned above, drug loaded hydrochloride chitosan microspheres with different degrees of crosslinking were obtained. Microspheres were characterized in terms of size, morphology, drug content, surface charge and capacity to control in vitro drug release. Clarithromycin, tramadol hydrochloride, and low molecular weight heparin (LMWH) were used as model drugs. The obtained particles were spherical, positively charged, with a diameter of 1-10 microm. X-Ray diffraction showed that there was an interaction of genipin and each drug with chitosan in the microspheres. In relation to the release profiles, a higher degree of crosslinking led to more control of drug release in the case of clarithromycin and tramadol. For these drugs, optimal release profiles were obtained for microspheres crosslinked with 1 mM genipin at 50 °C for 5 h and with 5 mM genipin at 50 °C for 5 h, respectively. In LMWH microspheres, the best release profile corresponded to 0.5 mM genipin, 50 °C, 5 h. In conclusion, genipin showed to be eligible as a chemical-crosslinking agent delaying the outflow of drugs from the microspheres. However, more studies in vitro and in vivo must be carried out to determine adequate crosslinking conditions for different drugs.

[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.

[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.

Isolation and characterization of contaminants in recalled unfractionated heparin and low-molecular-weight heparin.

Recently, a contaminant was found in some clinically used unfractionated heparin (UFH) preparations. Administration of this UFH was associated with an increased risk of developing a wide range of adverse effects including death. To further investigate the chemical profile of the contaminant, contaminated batches of UFH were treated by exhaustive nitrous acid depolymerization followed by methanol precipitation to remove heparin oligosaccharides. Because contaminated heparins may have been used as starting material in the production of low-molecular-weight heparins (LMWHs), a similar procedure was carried out using an experimental batch of enoxaparin prepared from contaminated heparin. While high-pressure liquid chromatography (HPLC) analysis of contaminated heparin did not distinguish the presence of the contaminant, it could readily be observed as a high-molecular weight shoulder in the elution profile of contaminated enoxaparin. Digesting contaminated heparin with heparinase-I prior to HPLC analysis showed the presence of a nondigestible component (15%-30% of the mixture). This contaminant was also resistant to degradation by chondroitinases A, B, and C. Proton nuclear magnetic resonance (NMR) indicated that the contaminant was oversulfated chondroitin sulfate (OSCS). Size-exclusion chromatography indicated that the mean molecular weight of the OSCS was 16.8 kD, comparable to that of a synthetic porcine cartilage OSCS preparation that was used as a reference material (17.2 kD). While varying degrees of high-molecular weight dermatan sulfate and other minor impurities were detected, OSCS appeared to be the major contaminant in these preparations. The process involved in the production of enoxaparin does not significantly degrade OSCS.

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.

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.

A capillary electrophoretic method for fingerprinting low molecular weight heparins.

Clinically used low molecular weight heparins (LMWH) are anticoagulants of choice and are phenomenally complex mixtures of millions of distinct natural and unnatural polymeric sequences. The FDA recommends that each LMWH be considered as an independent drug with its own activity profile, placing significant importance on the biophysical characterization of each intact LMWH. We report a robust protocol for fingerprinting these pharmaceutical agents. Capillary electrophoresis of three LMWHs, enoxaparin, tinzaparin, and a Sigma preparation, under reverse polarity conditions in the presence of selected linear alkyl polyamines gives an electrophoretic pattern that is characteristic of the nature of the starting material. The buffers that best provided optimal resolution without compromising sensitivity and speed of analysis were 50 mM sodium phosphate, pH 2.3, and 100 mM ammonium formate, pH 3.5. Resolution was strongly dependent on the structure of polyamine with pentaethylenehexamine being most effective for enoxaparin and Sigma LMWH. In contrast, tinzaparin could be best resolved with tetraethylenepentamine. Cyclic polyamines were ineffective. Resolution was also dependent on the concentration of resolving agents and displayed a narrow window that provides optimal resolution. These features suggest a strong structural origin of the fingerprint pattern. Overall, the simple protocol will find special use in assessing LMWH quality and batch-to-batch variability.