U.S. Department of Energy

Pacific Northwest National Laboratory

Mass spectrometry profiling of pentosan polysulfate sodium (PPS)

Introduction 

Pentosan polysulfate (PPS) is a semisynthetic heterogenous sulfated polysaccharide derived from xylan, the β-1,4-linked polymer of xylose. PPS sold by the brand name Elmiron in United States is taken orally to alleviate pain associated with interstitial cystitis. PPS is a mixture of hundreds or more discrete molecules built from a range of oligoxylose lengths modified with different combinations of functional group modifications, including sulfation, 4-O-methyl-glucuronidylation, acetylation, and others. The overall goal of our research is to develop an approach using MS together with other methods such as NMR to profile PPS at the molecular level. Profiling PPS according to its molecular composition would be invaluable for understanding biological activity, bioavailability, and pharmacokinetics, as well as for quality control.

Method 

One Elmiron (100 mg PPS) capsule was extracted with 1 ml of HPLC-grade water, and further dilutions were made with this stock solution. Diluted PPS at a concentration of 0.5mg/ml was treated with an ion exchange resin for few hours, centrifuged and the supernatant collected. To this supernatant butylamine (15mM) and hexafluoroisopropanol (60mM) were added as an ion-pair reagent (final pH ~8.5). The treated sample was fractionated on C18 SPE cartridge using acetonitrile (ACN) starting from concentration of 10% up to 100% ACN. Each fraction was individually analyzed by FTICR and IMS-MS both in positive and negative mode. Agilent drift tube-IMS-QTOF MS and home-built drift tube IMS-MS were used to characterize PPS from different lots and locations of production.

Preliminary Results 

The mass spectrum obtained from PPS directly dissolved in water is complex and difficult to interpret due in-source fragmentation of sulfated oligosaccharides and presence of multiple metal ion adducts [M+Na]. We have explored the potential of ion-pair reversed phase chromatography to extract and analyze PPS using C18-SPE followed by MS detection using FTICR and IMS. When each eluate was injected directly in FTICR without any chromatographic separation, most of the PPS eluted in fraction containing 10% and 20% ACN. Analysis of mass spectra revealed presence of multiply charged state species, mostly +2, +3 and +4 for data collected in positive mode. Analysis of deconvulated peaks in positive mode displayed abundant neutral loss of 171.03 across the entire MS1 spectrum. This neutral loss of 171.03 units is most likely coming from the group –OSO3NH2(CH2)3CH3 from PPS backbone. IMS-MS is capable of separating molecules that have the same mass-to-charge (m/z) ratio but different sizes, shapes or conformations. Therefore it is appealing for separating PPS with different polymerized sizes and different charge states and for reducing the complexity of mass spectra. Low-molecular-weight heparin, another sulfated oligosaccharide, was used as a standard to develop IMS-MS method. Heparin DP10 which has molecular weight around 3000 Da has shown a 2D IMS-MS spectrum with trend lines for charge +2 and +3 and m/z range from 1000 to 2000. Preliminary data of PPS showed 2D IMS-MS profiles with charge states from +1 to +5 and m/z range from 300 to 2500. These results show that IMS-MS can reduce the complexity of sulfated polysaccharide spectra by additional separation of different charge states and polysaccharide sizes. However the spectra are still complex for peak assignment without any pre-treatment. The uses of ion exchange resin and ion-pairs have shown improved sensitivity and separation in IMS-MS.

Novel Aspect 

We have demonstrated a novel approach for molecular profiling and characterization of the highly sulfated oligosaccharide mixture pentosan polysulfate.

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