Analysis of crude heparin by 1H NMR, capillary electrophoresis, and strong-anion-exchange-HPLC for contamination by over sulfated chondroitin sulfate

Keire, David A.; Trehy, Michael L.; Reepmeyer, John C.; Kolinski, Richard E.; Ye, Wei; Dunn, Jamie D.; Westenberger, Benjamin J.; Buhse, Lucinda F.

doi:10.1016/j.jpba.2009.10.017

Cited by 60 publications

(68 citation statements)

References 15 publications

(24 reference statements)

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“…There are literature methods 15,16 for Crude Heparin recommended by the recent FDA guidance -one is a colorimetric microplate assay method and the other is a SAX HPLC method which uses a high salt concentration of up to 2.5 M sodium chloride in the mobile phase, a polyvinylbenzyl ammonium (divinyl benzene) resin based SAX column with medium-low hydrophobicity and the LOD of OSCS by this method is less than 0.1% 7 . There is another method in the literature for determination of OSCS in Heparin API using CD (Circular dichroism) detection and the LOD of OSCS by this method is stated as 0.22 mg/ml 17 .…”

Section: Heparin Sodiummentioning

confidence: 99%

Untitled

2014

IJPSR

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ABSTRACT:A rapid and sensitive pH-gradient SAX HPLC method for the determination of Dermatan sulphate (DS) and trace levels of Oversulphated chondroitin sulphate (OSCS) in Heparin sodium was developed and validated. A pH gradient was opted to strike a balance between resolution and sensitivity because the response of OSCS was found to be better at neutral pH and the resolution between DS and Heparin was good at acidic pH. The use of commercially available lithium perchlorate was avoided and an in situ preparation of the same was opted to decrease the baseline drift which is a major concern for gradient methods using perchlorate mobile phases for compounds being analyzed at lower wavelengths. Chromatographic separation was achieved using a hydrophilic polymer resin-based SAX column, a linear gradientelution from 15% to 70% of solution-B which is 1.0M lithium perchlorate containing 5mM sodium dihydrogen phosphate at pH 6.0 and solution-A being 5mM sodium dihydrogen phosphate at pH 3.0 and a UV detection at 202 nm. The method has been validated and the limit of detection for DS and OSCS were found to be 15µg/ml and 20µg/ml respectively which is 0.03% &0.04% respectively with respect to the method concentration of 50 mg/mL. The method was found to be linear over the range of 50-150% of the specifications of the impurities with respect to the working sample concentration. The precision, accuracy, specificity robustness and solution stability were determined.

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Section: Heparin Sodiummentioning

confidence: 99%

Untitled

2014

IJPSR

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“…Compared with other techniques such as nuclear magnetic resonance (Guerrini et al, 2008), high performance liquid chromatography (Keire et al, 2010), colorimetry (Guarise et al, 2006;Fu et al, 2012) and fluorometry (Pu and Liu., 2008;Li et al, 2011;Hu et al, 2012;Fan et al, 2012) for protease or heparin assay, the potentiometric method has advantages of portability, ease of use, simple and reliable instrument, and resistance to color and turbid interference. However, unlike classical ion-selective electrodes working under fully equilibrium conditions, the response of a polyion-sensitive electrode is a quasi-steady-state one highly dependent on the polyion flux from the sample bulk into the sample-membrane interface (Fu et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

A moving-part-free protamine-sensitive polymeric membrane electrode for sensitive biomedical analyses

Wang

Qin

2012

Biosensors and Bioelectronics

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“…The analytical method reported for the analysis of contaminants like OSCS and the impurities of Hep is NMR [5,6], CE [1,7,8], and HPLC [6,9]. The USP official method uses a polymer-based strong anion exchange column and a 1% limit is set for the galactosamine impurities expressed as dermatan sulfate and other galactosamines along with the absence of OSCS.…”

Section: Introductionmentioning

confidence: 99%

Novel and highly sensitive mixed‐polymeric electrokinetic chromatography system for determination of contaminants and impurities of heparin samples

et al. 2010

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A mixed-polymeric electrokinetic chromatography system has been developed for the simultaneous determination of a contaminant like oversulfated condroitin sulfate (OSCS) and impurities expressed as dermatan (Der) in heparin (Hep) samples. The EKC system consisted of 0.5% w/v polymeric β-CD, 0.4% w/v tetronic(®) 1107 and 400 mM tris-phosphate buffer at pH 3.5. The optimized electrophoretic conditions included the use of an uncoated-silica capillary of 50 cm of total length and 75 μm id, an applied voltage of -7 kV, a temperature of 30°C and 200 nm UV-detection. The highly sensitive method developed showed low values of LOD, 0.07% w/w (0.07 μg/mL) (OSCS) and 0.1% w/w (0.1 μg/mL) (Der), and values of LOQ 0.2% w/w (0.2 μg/mL) (OSCS) and 0.3% w/w (0.3 μg/mL) (Der) with a concentration level of Hep sample as low as 0.1 mg/mL. Additional parameters of validation such as specificity, linearity, accuracy, and robustness were evaluated according to international guidelines. Owing to its simplicity, high sensitivity, and reliability, the proposed method can be an advantageous alternative to the traditional methodologies for the analysis of Hep in raw material and specially in finished products because of the low amounts of Hep sample required.

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Analysis of crude heparin by 1H NMR, capillary electrophoresis, and strong-anion-exchange-HPLC for contamination by over sulfated chondroitin sulfate

Cited by 60 publications

References 15 publications

Untitled

Untitled

A moving-part-free protamine-sensitive polymeric membrane electrode for sensitive biomedical analyses

Novel and highly sensitive mixed‐polymeric electrokinetic chromatography system for determination of contaminants and impurities of heparin samples

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