Author(s)Ansunman Mahato & Alan Brookes

Agilent Technologies India & EMEA

ACCURATE MOLECULAR WEIGHT DETERMINATION OF PENTOSAN POLYSULPHATE BY AGILENT 1260 INFINITY II MULTI DETECTOR GPC SYSTEM

PHARMA MARKET

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Pentosan Polysuphate is a low molecular weight heparin-like compound. It has anticoagulant and fibrinolytic effects. Orally administered Pentosan Polysulphate is used primarily in the treatment of interstitial cystitis. It is one of only two products approved for treatment of this condition. With its small, heparin-like molecular structure, Pentosan has anticoagulant and fibrinolytic properties.

ABSTRACT

INTRODUCTIONProprietary names: Elmiron, Cartrophen, Fibrase, Fibrenzym, Hemoclar, SP-54, Thrombocid

The molecular weight of Pentosan Polysulphate is in the range of 4000 to 6000 Da. Conventional GPC with RI detection produces molecular weights relative to the standards used to generate the column calibration, however to obtain true or absolute molecular weights the GPC system can be enhanced with the addition of light scattering.

Figure 2: PEG standards

EXPERIMENTAL

InstrumentationAgilent 1260 Infinity II Isocratic pump (G7110B)Agilent 1260 Infinity II Vial sampler (G7129A)Agilent 1260 Infinity II MCT (G7116A)Agilent 1260 Infinity GPC/SEC Multi-Detector Suite (G7800A)MDS Light Scattering Detector MDS Refractive Index Detector

Method for AnalysisDetectors: MDS LS, DRI,Mobile Phase: 25mM potassium phosphateColumns: 1 X PL aquagel - OH 30Standards: PEGTemperature: 40˚C (column and detector)Injection volume 100μLFlow rate: 1ml/minSoftware: Agilent GPC/SEC software

Instrument/Detector/Column CalibrationPEG standards were used to generate the column calibration and to perform the detector calibration. The PEG standards chromatograms are overlaid in Figure 2 and the PEG calibration is shown in Figure 3

In this study the Light Scattering detector is used together with RI to generate accurate Mw information of Pentosan Polysulphate. Molecular weight analysis is performed by conventional calibration and by Light Scattering for comparison.

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Figure 3: PEG calibration

Figure 5: Overlay of Pentosan Polysulphate samples

Figure 6: Overlay of molecular weight distribution of eight Pentosan Polysulphate injections

RT PEG Mp8.18 123208.48 78309.00 38709.68 14809.95 101010.28 615

SAMPLE ANALYSISThe Pentosan Polysulphate sample was supplied in the form of a 100mg capsule. This was dissolved at a concentration of approximately 8mg/ml for analysis. To ensure complete dissolution the sample was dissolved for up to 1 hour, and then followed by filtration through a 0.4μm PTFE syringe filter. A typical chromatogram of the Pentosan Polysulphate sample is shown in Figure 4. Replicate injections were made to show reproducibility, and are shown in Figures 5 - 6

Figure 4: Pentosan Polysulphate sample, RI and LS signal

# Peak RT_RIPSS_replicate 1 8.23PSS_replicate 2 8.25PSS_replicate 3 8.23PSS_replicate 4 8.25PSS_replicate 5 8.26PSS_replicate 6 8.23PSS_replicate 7 8.25PSS_replicate 8 8.25

AVG 8.24SD 0.01

RSD % 0.14

Sample Name Mp Mn Mw PDPentosan Polysul-phate_Replicate 1 3122 3563 5179 1.4

Pentosan Polysul-phate_Replicate 2 3191 3647 5316 1.4

Pentosan Polysul-phate_Replicate 3 3017 3440 5136 1.4

Average 3110 3550 5210 1.4

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Molecular weight averages calculated through the conventional calibration (PEG) and those calculated using the Light Scattering detector are shown below. In this example, the molecular weights by conventional GPC with PEG calibration are overestimated, whilst the molecular weights obtained using Light Scattering are close to the literature values expected for this polymer.

Molecular Weight Analysis

Conventional GPC LS GPC

Literature value of

PPS, MwMn 12825 3563

4000 to 6000Mw 18515 5179PD 1.4 1.4

CONCLUSIONThe Agilent Infinity 1260 Infinity II GPC system equipped with light scattering detection is ideal for the analysis of Pentosan Polysulphate samples. The system provided accurate and reliable analysis of this polymer and by using the Light Scattering detection we can calculate molecular weights that are within the expected range for this polymer.

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This information is subject to change without notice.© Agilent Technologies, Inc. 2017

Printed in EU, 2017-05-85991-8196EN