Quantification Of Heparin Research Articles

New Fluorescent Assay for the Direct Quantification of Heparin and Low-Molecular-Weight Heparins In Clinical Samples

AbstractAbstract 3325Heparin, a linear polydisperse polysaccharide, is used to prevent blood clotting in various diseases. It consists predominantly of a trisulfated disaccharide repeating unit and has a high negative charge density. Currently accepted methods to control blood levels of heparin or of its more commonly used low-molecular-weight (LMW) fractions include chromogenic substrate assays that analyze the biological inhibition of blood coagulation factor Xa (FXa). There are no established methods to directly determine the concentration of heparins in clinical samples.A modified positively charged perylene diimide (Heparin Red) was developed that binds specifically to heparin and LMW-heparin (Szelke H et al., Chem. Commun. 2010;46:1667). A red fluorescence signal is dose dependently quenched in the presence of heparin and LMW-heparins. LMW-Heparin was added to human serum, plasma or urine. The chromogenic S2222 substrate assay was used to determine the anti-FXa activity of the anticoagulants. Fluorescence was recorded with a portable fluorimeter, equipped with a 550 nm excitation filter and a 610 nm emission filter.For plasma samples spiked with 0.6–1.0 IU/mL LMW-heparin, the intraday (n=5) and interday (n=5) coefficient of variations (CVs) of the fluorimetric Heparin Red assay were in the range of 2.9–6.1% and 4.3–13.8%, respectively. The CVs of the chromogenic S2222 assay for same plasma samples were the range of 3.3–18.8% (intraday) and 6.2–13.7 (interday).In serum and plasma samples of patients treated with LMW-heparin, correlation between the Heparin Red assay and the chromogenic anti-FXa test (S-2222) was r = 0.66. The correlation between the Heparin Red assay and the anti-FXa test in urine samples was r = 0.83. This is probably due to its lower protein concentration in urine compared to plasma and serum samples.The fluorescent perylene diimide Heparin Red allows sensitive quantification of heparin and LMWHs in plasma, serum and urine samples. Ease of application combined with matrix-independent response make this heparin probe attractive for both routine laboratory and fast point-of-care testing as well as high-throughput pharmacokinetic studies, ideally without the need for patient-specific calibration. Disclosures:No relevant conflicts of interest to declare.

Simple fluorescence assay for quantification of OSCS in heparin

In 2008, heparin contaminated with oversulfated chondroitin sulfate (OSCS) penetrated the worldwide market and was associated with severe adverse effects. Feasible and reliable methods to test heparin for adulteration are needed. The objective was to develop a simple approach based on a microplate assay for quantification of heparin and sulfated glycans using the fluorescent heparin sensor polymer-H (polymer-H assay). However, both heparin and OSCS concentration-dependently increase the fluorescence intensity (FI) of polymer-H, so that OSCS in heparin cannot be detected. The idea was a two-step procedure including, first, separation of heparin by degradation with heparinase I, and then measurement of the remaining OSCS. To achieve complete heparin (unfractionated heparin (UFH), enoxaparin) degradation, several conditions (e.g. incubation time and heparinase I concentration) were optimized by using the aXa assay for monitoring. Defined UFH/OSCS mixtures incubated in this way showed a concentration-dependent FI increase in the polymer-H assay (λ ((em)) 330nm, λ ((ex)) 510nm). The sensitivity was unexpectedly high with an LOD/LOQ of 0.5%/0.6% OSCS content in heparin. Further experiments testing UFH/OSCS mixtures in the aXa assay confirmed our hypothesis: OSCS inhibits heparinase I resulting in incomplete heparin degradation and thus an additional FI increase of polymer-H by intact heparin. This two-step microplate fluorescence assay is a sensitive, rapid, and simple method for quantification of OSCS in heparin. In contrast with (1)H NMR and CE, neither expensive equipment nor much experience are required. It could be applied not only in the quality control of heparin, but also in clinical practice, to check the applied heparin preparation when a patient suffers any adverse effect.

A fluorescent probe for the quantification of heparin in clinical samples with minimal matrix interference

A red-fluorescent perylene diimide probe allows sensitive quantification of heparin, a widely used antithrombotic drug, in plasma and serum samples with minimal interference from matrix components.

Naked-Eye Detection and Quantification of Heparin in Serum with a Cationic Polythiophene

A strategy for naked-eye detection and quantification of heparin in biological media, such as fetal bovine serum (FBS) is demonstrated by monitoring the absorbance change of a water-soluble cationic polythiophene. The negatively charged heparin interacts with positively charged polythiophene through electrostatic interaction, which leads to polymer conformation and color change from yellow to orange in solution. Under optimized conditions, addition of heparin derivatives, such as hyaluronic acid or chondroitin 4-sulfate to the same polymer solution leads to less change in polymer conformation and solution color due to their lower charge density as compared to that of heparin. Increasing the detection temperature or simply adding some organic solvent to the aqueous media reduces the polymer-polymer interchain pi stacking, and the polymer color change can be used to clearly differentiate heparin from its analogues in homogeneous solutions. Quantification of heparin is also demonstrated by correlating the changes in polymer absorbance to the heparin concentration. A linear calibration curve is observed in the 0-6.7 U/mL and 0-2.2 U/mL ranges for heparin quantification in pure water and in FBS, respectively.

Cationic Conjugated Polymer/Heparin Interpolyelectrolyte Complexes for Heparin Quantification

AbstractA cationic conjugated polymer (P1) containing 20 mol‐% 2,1,3‐benzothiadiazole (BT) content is synthesized through a post‐polymerization method. With a good water solubility (≈20 mg · mL−1), this polymer has shown a low inherent BT emission in aqueous solution. Formation of interpolyelectrolyte complexes between oppositely charged P1 and heparin facilitates energy transfer from the fluorene segments to the BT units within P1, leading to the intensity increase for the orange emission peak at 585 nm and the intensity decrease for the blue emission at 412 nm. A practical calibration curve ranging from 0.2 to 76 × 10−6 M is thus obtained by correlating the changes in BT emission with the heparin concentration in buffer. The significant advantage of this heparin macromolecular probe is that the calibration curve could cover the whole range of the therapeutic dosing level of heparin.magnified image

Conjugated Polyelectrolytes as Light‐Up Macromolecular Probes for Heparin Sensing

AbstractTwo cationic poly(fluorene‐alt‐benzothiadiazole)s with different side chains are designed and synthesized. Both polymers show low fluorescence in aqueous solution due to the charge‐transfer character of the polymer's excited states. Fluorescence turn‐on biosensors for heparin detection and quantification are developed, taking advantage of complexation‐induced aggregation, which increases the polymer fluorescence in aqueous solution. It is found that good polymer water‐solubility is beneficial to the sensitivity and fluorescence contrast of the heparin turn‐on sensor as a result of the low fluorescence background. Moreover, stronger complexation between the polymer/heparin leads to a substantially larger fluorescence increase in the presence of heparin relative to that in the presence of its analog, hyaluronic acid (HA), allowing discrimination of heparin from HA. Heparin quantification with a practical calibration range covering the whole therapeutic dosing levels (0.2–8 U mL−1) is realized based on the polymer with good water‐solubility. This investigation provides a new insight for designing conjugated polymers with a light‐up signature for biomolecular sensing.

Detection and neutralisation of heparin by a fluorescent ruthenium compound

Heparin and low-molecular-weight heparin (LMWH) are commonly monitored by determination of activated clotting times or chromogenic assays. Despite their wide use, these assays determine the biological activity and not the concentration of the anticoagulants. They may be inaccurate in some circumstances such as certain disease states. In addition, there is a significant interest in alternative tests for the point-of-care detection of heparin and LMWH. Their binding to small molecules for the detection in biological matrices is poorly explored. We describe here a new optical molecular probe for the detection of LMWH in serum samples. The polycationic ruthenium compound 1 is applicable to the quantification of heparin by monitoring 630 nm fluorescence. In addition, compound 1 is a rare example of a non-polymeric low molecular weight compound which neutralises the anticoagulant activity of heparin and LMWH in plasma samples. Limitation of the method is its low sensitivity currently being improved by structural modification of compound 1.

A Multicolor Cationic Conjugated Polymer for Naked-Eye Detection and Quantification of Heparin

A new cationic polyfluorene derivative with 20 mol % 2,1,3-benzothiadiazole (BT) content was synthesized via Suzuki cross-coupling polymerization. The high charge density and cationic oligo(ethylene oxide) side chains endue the polymer with a good water solubility (∼12 mg/mL), leading to a low inherent BT emission background in buffer. Addition of negatively charged heparin into the polymer solution induces polymer aggregation, giving rise to enhanced energy transfer from the fluorene segments to the BT units. With increasing heparin concentrations, the orange BT emission intensity progressively increases at the expense of the blue fluorene emission. In contrast, addition of hyaluronic acid, an analogue of heparin, results in an insignificant enhancement in BT emission. This selective optical signature not only allows distinguishing heparin from hyaluronic acid but also highlights the importance of electrostatic attraction between the polymer and the analyte in an energy transfer process. Heparin quantifi...

New method for low molecular weight heparin quantification in tablets by suppressed conductivity detection and cryptand column

Unfractioned Heparins (UFH) and Low Molecular Weight Heparins (LMWHs) are non-chromophoric, high charged sulphated molecules which are difficult to elute and detect with conventional liquid chromatography methods. Moreover, the detection of LMWHs at low concentration level, like in samples coming from dissolution test of tablets formulated with Heparins is problematic due to the weak response by UV or refractive index detection. According to the European Pharmacopoeia requirements, the assay of LMWHs is based on the anti-Xa activity and anti-IIa activity factors, which are biological parameters related to the antithrombotic activity of this compound family. The same assay method could also be applied to determine the dissolution rate of LMWHs contained in tablets, but it is time consuming and expensive test. The challenge was to develop a simpler and faster alternative method based on Ion Chromatography coupled with Suppressed Conductivity detection, but the particular polycharge interaction of LMWHs with anions exchanger makes the analyte with standard eluents and columns difficult to elute . The analytical problem could be solved by using a particular chromatographic phase with 2,2,2 cryptand sites which gives variable capacity in anion exchange depending on the used eluent. Such phase was very effective for the elution of strongly retained polyanions using simple inorganic eluent phase suitable for suppressed conductivity detection as well. The developed method allows the elution of a unique peak, which represents the whole polymeric distribution, and therefore it makes easy to quantify quickly and selectively the active ingredient in case of multiple analysis like when a dissolution test is required to characterize and discriminate between different tablets formulations. The method was tested and developed in Cosmo Pharmaceuticals QC labs and the validation results are reported in the present paper. Besides, a comparison between biological and chromatographic methods was carried out as well.

Ultraperformance Ion-Pair Liquid Chromatography Coupled to Electrospray Time-of-Flight Mass Spectrometry for Compositional Profiling and Quantification of Heparin and Heparan Sulfate

Heparin and heparan sulfate (HS) are important pharmaceutical targets because they bind a large number of proteins, including growth factors and cytokines, mediating many biological processes. Because of their biological significance and complexity, there is a need for development of rapid and sensitive analytical techniques for the characterization and compositional analysis of heparin and HS at the disaccharide level, as well as for the structure elucidation of larger glycosaminoglycan (GAG) sequences important for protein binding. In this work, we present a rapid method for analysis of disaccharide composition using reversed-phase ion-pairing ultraperformance liquid chromatography coupled with electrospray time-of-flight mass spectrometry ((RPIP)-UPLC-MS). Heparin disaccharide standards were eluted in less than 5 min. The method was used to determine the constituents of GAGs from unfractionated heparin/HS from various bovine and porcine tissues, and the results were compared with literature values.

Cyclic voltammetry of biopolymer heparin at PVC plasticized liquid membrane

Cyclic voltammetry is used to study the charge transfer reaction of the biopolymer heparin at the PVC plasticized 1,6-dichlorohexane membrane–solution interface. Several unique feature of the reaction are demonstrated including the apparent diffusion control and the specific interaction of heparin with the cation of the membrane phase, which probably acts as the heparin carrier (ionophore). Voltammetric behaviour corresponds to the interfacial transfer of a mixture of polyanions with a relatively high content of polyanions carrying a low charge. Amperometry appears to be a suitable method for the quantification of heparin in various preparations with the sensitivity of ca. 1 U mL −1 ( S/ N=3).

Compositional analysis and quantification of heparin and heparan sulfate by electrospray ionization ion trap mass spectrometry.

A new method using a combination of electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MSn) was developed for the identification and quantitative analysis of eight heparan sulfate (HS)- and heparin-derived delta-disaccharides obtained by enzymatic depolymerization. The compositional analysis of nonisomeric disaccharide constituents of heparin/HS was achieved from full-scan MS1 spectra using an internal standard and a calculated response factor for each disaccharide. Diagnostic product ions from MSn spectra of isomeric disaccharides were used for the quantitative analysis of the relative amounts of each of the isomers in mixtures. The protocol was validated using several quality control samples and showed satisfactory accuracy and precision. The analysis is rapid, accurate, and uses no purification or separation steps prior to analysis by MS, thus reducing sample consumption and analysis time of traditional methods. Using this quantitative analysis procedure, percentages of disaccharide compositions for heparins from porcine and bovine intestinal mucosa and heparan sulfate from bovine kidney were determined.