Table 3. Detection of oversulfated heparan sulfate in contaminated heparin by liquid chromatography/mass spectrometry.
A. Heparin lyase digestion and liquid chromatography/mass spectrometry analysis. LSCH4 and OSCH2 were digested by a mixture of heparin lyase I, II, and III. OSCH2 was tagged with H-aniline and LSCH4 was tagged with D-aniline. Both tagged samples were reduced with NaBH4. An equal amount of samples was mixed and analyzed simultaneously by liquid chromatography/mass spectrometry. The elution time and total ion current for the disaccharides, tetrasaccharides, and novel tetrasaccharides corresponding to the proposed structures were listed. The ratio of each structure in the two differentially tagged samples was determined.
| Proposed structures | A. Total Ion Current |
|||
|---|---|---|---|---|
| Elution time (min) | OSCH2 | LSCH4 | Ratio | |
| DA+0S | 15.6 | 7559 | 13000 | 0.58 |
| DH+1S | 17.0 | 453 | 846 | 0.54 |
| DH+3S | 20.1 | 0 | 37000 | |
| DAUH+4S | 23.3 | 0 | 1092 | |
| DHUH+6S | 24.7 | 0 | 1675 | |
| DAUA+1S+103 | 25.8 | 0 | 4885 | |
| DAUH+3S+103 | 28.0 | 0 | 1003 | |
DA refers to a disaccharide containing an Δ4,5-unsaturated uronic acid (U) linked to N-acetylglucosamine (A). UAUH refers to a tetrasaccharide composed of uronic acid (U, GlcA or IdoA)-N-acetylglucosamine (A)-uronic acid (U)-glucosamine (H). S refers to sulfate.