Advertisement

Chromatographia

, Volume 72, Issue 3–4, pp 215–223 | Cite as

An Improved Ion-Pair Reversed Phase LC Method for Analysis of Major Impurities of Phosphorothioate Oligonucleotide Cantide

Original

Abstract

An improved ion-pair reversed phase LC (IP-RP-LC) method was developed using triethylammonium/hexafluoroisopropanol (TEA/HFIP) buffer as the mobile phase as an alternative to capillary gel electrophoresis (CGE) and ion-exchange chromatography (IEC) for routine quality control of the synthetic 20-mer phosphorothioate oligonucleotide (PS-ODN) Cantide. The most commonly observed impurities included an oligomer with a deletion at the 5′-end (5′n-1) and a monophosphodiester (P=S/O). The method described here was sufficiently selective to simultaneously separate and identify these impurities. To the best of our knowledge, this is the first report on the separation of P=S/O analogs from PS-ODN using IP-RP-LC. Relatively high TEA concentrations and higher pH values were recommended. The separation performance was maintained even in the presence of multiple P=S/O fragments containing different PO residues. The method was successfully applied to the analysis of failure sequences of authentic crude Cantide as well as the purified product. In addition to 5′n-1 and P=S/O analogs, some minor impurities such as 3′n-1 oligomers were also partially resolved.

Keywords

Ion-pair chromatography Ion-exchange chromatography Capillary gel electrophoresis Phosphorothioate oligonucleotides Cantide 

Notes

Acknowledgments

The authors thank the Beijing Proteome Research Center for their kindly support in the MALDI-TOF-MS analysis. This research was funded by National 863 High-Tech project from the Ministry of Science and Technology of the People’s Republic of China (No. 2007AA021602) and Important National Science and Technology Specific Projects (No. 2009ZX09503-021 and 2009ZX09102-012).

References

  1. 1.
    Srivatsa GS, Klopchin P, Batt M, Feldman M, Carlson RH, Cole DL (1997) J Pharm Biomed Anal 16:619–630. doi: 10.1016/S0731-7085(97)00180-5 CrossRefGoogle Scholar
  2. 2.
    Leeds JM, Graham MJ, Truong L, Cummins LL (1996) Anal Biochem 235:36–43. doi: 10.1006/abio.1996.0088 CrossRefGoogle Scholar
  3. 3.
    Palm AK, Marko-Varga G (2004) J Pharm Biomed Anal 35:415–423. doi: 10.1016/j.jpba.2004.01.008 CrossRefGoogle Scholar
  4. 4.
    Chen R, Luo XF, Di X, Li Y, Sun YQ, Hu YZ (2006) J Chromatogr B 843:334–338. doi: 10.1016/j.jchromb.2006.05.034 CrossRefGoogle Scholar
  5. 5.
    Bergot BJ (1992) J Chromatogr 599:35–42. doi: 10.1016/0021-9673(92)85456-4 CrossRefGoogle Scholar
  6. 6.
    Bourque AJ, Cohen AS (1993) J Chromatogr 617:43–49. doi: 10.1016/0378-4347(93)80419-5 CrossRefGoogle Scholar
  7. 7.
    Bergot BJ, Zon G (1992) Ann N Y Acad Sci 660:310–312. doi: 10.1111/j.1749-6632.1992.tb21096.x CrossRefGoogle Scholar
  8. 8.
    Bourque AJ, Cohen AS (1994) J Chromatogr B Biomed Appl 662:343–349. doi: 10.1016/0378-4347(94)00207-X CrossRefGoogle Scholar
  9. 9.
    Zhang HY, Lu DD, Wu LX, Zhou Z, Wang SQ (2008) Chin J Chromatogr 26:540–543. doi: 10.1016/S1872-2059(08)60028-0 CrossRefGoogle Scholar
  10. 10.
    Chen SH, Qian MX, Brennan JM, Gallo JM (1997) J Chromatogr B Biomed Sci Appl 692:43–51. doi: 10.1016/S0378-4347(96)00499-9 CrossRefGoogle Scholar
  11. 11.
    Huber CG, Oefner PJ, Bonn GK (1992) J Chromatogr 599:113–118. doi: 10.1016/0021-9673(92)85463-4 CrossRefGoogle Scholar
  12. 12.
    Apffer A, Chakel JA, Fischer S, Lichtenwalter K, Hancock WS (1997) Anal Chem 69:1320–1325. doi: 10.1021/ac960916h CrossRefGoogle Scholar
  13. 13.
    Huber CG, Krajete A (2000) J Chromatogr A 870:413–424. doi: 10.1016/S0021-9673(99)00953-X CrossRefGoogle Scholar
  14. 14.
    Premstaller A, Oberacher H, Huber CG (2000) Anal Chem 72:4386–4393. doi: 10.1021/ac000283d CrossRefGoogle Scholar
  15. 15.
    Oberacher H, Parson W, Muhlmann R, Huber CG (2001) Anal Chem 73:5109–5115. doi: 10.1021/ac010587f CrossRefGoogle Scholar
  16. 16.
    Gilar M, Belenky A, Cohen AS (2000) Electrophoresis 21:2999–3009. doi: 10.1002/1522-2683(20000801)21:14<2999:AID-ELPS2999>3.0.CO;2-1 CrossRefGoogle Scholar
  17. 17.
    Mayr B, Holzl G, Eder K, Buchmeiser MR, Huber CG (2002) Anal Chem 74:6080–6087. doi: 10.1021/ac025919a CrossRefGoogle Scholar
  18. 18.
    Fountain KJ, Gilar M, Gebler JC (2003) Rapid Commun Mass Spectrom 17:646–653. doi: 10.1002/rcm.959 CrossRefGoogle Scholar
  19. 19.
    Gilar M, Fountain KJ, Budman Y, Holyoke JL, Davoudi H, Gebler JC (2003) Oligonucleotides 13:229–243. doi: 10.1089/154545703322460612 CrossRefGoogle Scholar
  20. 20.
    Gilar M (2001) Anal Biochem 298:196–206. doi: 10.1006/abio.2001.5386 CrossRefGoogle Scholar
  21. 21.
    Dai GW, Wei XH, Liu ZF, Liu SJ, Marcucci G, Chan KK (2005) J Chromatogr B 825:201–213. doi: 10.1016/j.jchromb.2005.05.049 CrossRefGoogle Scholar
  22. 22.
    Wei XH, Dai GW, Liu ZF, Cheng H, Xie ZL, Marcucci G, Chan KK (2006) The AAPS Journal 8, Article 84 (http://www.aapsj.org) E743-755
  23. 23.
    Lin ZJ, Li WK, Dai GW (2007) J Pharm Biomed Anal 44:330–341. doi: 10.1016/j.jpba.2007.01.042 CrossRefGoogle Scholar
  24. 24.
    Wang SQ, Lin L, Chen ZB, Lin RX, Chen SH, Guan W, Wang XH (2002) Chin Sci Bull 47:993–997Google Scholar
  25. 25.
    Lin RX, Tuo CW, Lv QJ, Zhang W, Wang SQ (2005) Acta Pharmacol Sin 26:762–768. doi: 10.1111/j.1745-7254.2005.00762.x CrossRefGoogle Scholar
  26. 26.
    Du QY, Wang XB, Chen XJ, Zheng W, Wang SQ (2003) World J Gastroenterol 9:2030–2035Google Scholar
  27. 27.
    Webster DR, Boston GD, Patno DM (1985) J Liq Chromatogr 8:603–618. doi: 10.1080/01483918508067107 CrossRefGoogle Scholar
  28. 28.
    Knox JH, Laird GR (1976) J Chromatogr 122:17–34. doi: 10.1016/S0021-9673(00)82234-7 CrossRefGoogle Scholar
  29. 29.
    Tilly-Melin A, Askemark Y, Wahlund KG, Schill G (1979) Anal Chem 51:976–983. doi: 10.1021/ac50043a045 CrossRefGoogle Scholar
  30. 30.
    Uesugi T, Sano K, Uesawa Y, Ikegami Y, Mohri K (1997) J Chromatogr B Biomed Sci Appl 703:63–74. doi: 10.1016/S0378-4347(97)00430-1 CrossRefGoogle Scholar
  31. 31.
    Zhang W, Yang BH, Liang QD, Lu DD, Lin RX, Wang SQ (2005) Chin J Chromatogr 23:374–377Google Scholar
  32. 32.
    Wilk A, Grajkowski A, Phillips RL, Beaucage SL (1999) J Org Chem 64:7515–7522. doi: 10.1021/jo990835w CrossRefGoogle Scholar

Copyright information

© Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH 2010

Authors and Affiliations

  • Shu Li
    • 1
  • Dan-Dan Lu
    • 1
  • Yu-Lin Zhang
    • 1
  • Sheng-Qi Wang
    • 1
  1. 1.Beijing Institute of Radiation MedicineBeijingChina

Personalised recommendations