Skip to main content
SpringerLink
Log in

Simultaneous Determination of Polyethylene Glycol-Conjugated Liposome Components by Using Reversed-Phase High-Performance Liquid Chromatography with UV and Evaporative Light Scattering Detection

  • Research Article
  • Published:
AAPS PharmSciTech Aims and scope Submit manuscript

Abstract

Liposomes incorporating polyethylene glycol (PEG)-conjugated lipids (PEGylated liposomes) have attracted attention as drug delivery carriers because they show good in vivo stability. The lipid component of PEGylated liposomal formulations needs to be quantified for quality control. In this study, a simple reversed-phase high-performance liquid chromatography (HPLC) method with an evaporative light-scattering detector (ELSD) was established for simultaneous determination of hydrogenated soy phosphatidylcholine, cholesterol, PEG-conjugated lipid, and hydrolysis products of phospholipid in PEGylated liposomal formulations. These lipids were separated using a C18 column with a gradient mobile phase consisting of ammonium acetate buffer and ammonium acetate in methanol at a flow rate of 1.0 ml/min. This method provided sufficient repeatability, linearity, and recovery rate for all lipids. However, the linearity and recovery rates of cholesterol achieved using a ultraviolet (UV) detector were better than those achieved using an ELSD. This validated method can be applied to assess the composition change during the preparation process of liposomes and to quantify lipid components and hydrolysis products contained in a commercially available liposomal formulation DOXIL®. Taken together, this reversed-phase HPLC-UV/ELSD method may be useful for the rapid or routine analysis of liposomal lipid components in process development and quality control.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

REFERENCES

  1. Allen TM, Hansen C, Martin F, Redemann C, Yau-Young A. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim Biophys Acta. 1991;1066(1):29–36.

    Article  CAS  PubMed  Google Scholar 

  2. Papahadjopoulos D, Allen TM, Gabizon A, Mayhew E, Matthay K, Huang SK, et al. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc Natl Acad Sci U S A. 1991;88(24):11460–4.

    Article  CAS  PubMed  Google Scholar 

  3. Adler-Moore J, Proffitt RT. Effect of tissue penetration on AmBisome efficacy. Curr Opin Investig Drugs. 2003;4(2):179–85.

    CAS  PubMed  Google Scholar 

  4. Zhao Y, Zhang S, Cui S, Wang B. Peptide-based cationic liposome-mediated gene delivery. Expert Opin Drug Deliv. 2012;9(1):127–39. doi:10.1517/17425247.2011.630387.

    Article  CAS  PubMed  Google Scholar 

  5. Inoue K, Kitagawa T. Effect of exogenous lysolecithin on liposomal membranes. Its relation to membrane fluidity. Biochim Biophys Acta. 1974;363(3):361–72.

    Article  CAS  PubMed  Google Scholar 

  6. Zuidam NJ, Gouw HK, Barenholz Y, Crommelin DJ. Physical (in) stability of liposomes upon chemical hydrolysis: the role of lysophospholipids and fatty acids. Biochim Biophys Acta. 1995;1240(1):101–10.

    Article  PubMed  Google Scholar 

  7. Zuidam NJ, Crommelin DJ. Chemical hydrolysis of phospholipids. J Pharm Sci. 1995;84(9):1113–9.

    Article  CAS  PubMed  Google Scholar 

  8. Grit M, Underberg WJ, Crommelin DJ. Hydrolysis of saturated soybean phosphatidylcholine in aqueous liposome dispersions. J Pharm Sci. 1993;82(4):362–6.

    Article  CAS  PubMed  Google Scholar 

  9. Zhang JA, Pawelchak J. Effect of pH, ionic strength and oxygen burden on the chemical stability of EPC/cholesterol liposomes under accelerated conditions. Part 1: lipid hydrolysis. Eur J Pharm Biopharm. 2000;50(3):357–64.

    Article  CAS  PubMed  Google Scholar 

  10. FDA US (2002). Draft Guidance for Industry “Liposome Drug Products: Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation”. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070570.pdf.

  11. Mazzella N, Molinet J, Syakti AD, Dodi A, Doumenq P, Artaud J, et al. Bacterial phospholipid molecular species analysis by ion-pair reversed-phase HPLC/ESI/MS. J Lipid Res. 2004;45(7):1355–63. doi:10.1194/jlr.D300040-JLR200.

    Article  CAS  PubMed  Google Scholar 

  12. Rodriguez-Alcala LM, Fontecha J. Major lipid classes separation of buttermilk, and cows, goats and ewes milk by high performance liquid chromatography with an evaporative light scattering detector focused on the phospholipid fraction. J Chromatogr A. 2010;1217(18):3063–6. doi:10.1016/j.chroma.2010.02.073.

    Article  CAS  PubMed  Google Scholar 

  13. Schonherr C, Touchene S, Wilser G, Peschka-Suss R, Francese G. Simple and precise detection of lipid compounds present within liposomal formulations using a charged aerosol detector. J Chromatogr A. 2009;1216(5):781–6. doi:10.1016/j.chroma.2008.11.080.

    Article  CAS  PubMed  Google Scholar 

  14. Mengesha AE, Bummer PM. Simple chromatographic method for simultaneous analyses of phosphatidylcholine, lysophosphatidylcholine, and free fatty acids. AAPS PharmSciTech. 2010;11(3):1084–91. doi:10.1208/s12249-010-9470-4.

    Article  CAS  PubMed  Google Scholar 

  15. Holland WL, Stauter EC, Stith BJ. Quantification of phosphatidic acid and lysophosphatidic acid by HPLC with evaporative light-scattering detection. J Lipid Res. 2003;44(4):854–8. doi:10.1194/jlr.D200040-JLR200.

    Article  CAS  PubMed  Google Scholar 

  16. Simonzadeh N. An isocratic HPLC method for the simultaneous determination of cholesterol, cardiolipin, and DOPC in lyophilized lipids and liposomal formulations. J Chromatogr Sci. 2009;47(4):304–8.

    Article  CAS  PubMed  Google Scholar 

  17. Jaaskelainen I, Urtti A. Liquid chromatography determination of liposome components using a light-scattering evaporative detector. J Pharm Biomed Anal. 1994;12(8):977–82.

    Article  CAS  PubMed  Google Scholar 

  18. Zhong Z, Ji Q, Zhang JA. Analysis of cationic liposomes by reversed-phase HPLC with evaporative light-scattering detection. J Pharm Biomed Anal. 2010;51(4):947–51. doi:10.1016/j.jpba.2009.10.001.

    Article  CAS  PubMed  Google Scholar 

  19. Maitani Y, Soeda H, Junping W, Takayama K. Modified ethanol injection method for liposomes containing beta-sitosterol beta-D-glucoside. J Liposome Res. 2001;11(1):115–25. doi:10.1081/LPR-100103174.

    Article  CAS  PubMed  Google Scholar 

  20. Shimizu Y, Nakata M, Matsunuma J, Mizuochi T. Simultaneous quantification of components of neoglycolipid-coated liposomes using high-performance liquid chromatography with evaporative light scattering detection. J Chromatogr B Biomed Sci Appl. 2001;754(1):127–33.

    Article  CAS  PubMed  Google Scholar 

Download references

ACKNOWLEDGMENTS

We thank Professor Maitani, Hoshi University, and Professor Mruyama and Dr. Suzuki, Teikyo University, for their advice regarding the preparation of PEGylated liposomes. This study was supported by the Research on Publicly Essential Drugs and Medical Devices from the Japan Health Sciences Foundation (KHB1005 and KHB1206).

Author information

Authors and Affiliations

  1. National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-8501, Japan

    Hiroko Shibata, Chikako Yomota & Haruhiro Okuda

Authors
  1. Hiroko Shibata
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chikako Yomota
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Haruhiro Okuda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hiroko Shibata.

Electronic Supplementary Materials

Below is the link to the electronic supplementary material.

Fig. 1

Effects of ammonium acetate amount (percent volume) (a) and concentration (b) in the mobile phase on the retention times of Chol, HSPC, and DSPE-PEG. (PDF 19 kb)

Fig. 2

HPLC-ELSD detection of a standard mixture (a) and DOXIL® (b) in the mobile phase with methanol–200 mM ammonium acetate buffer (99:1, v/v). A standard mixture (20 μl) containing 300 μg/ml of Chol, and 750 μg/ml each of HSPC and DSPE-PEG was injected. DOXIL® was diluted 10-fold with methanol, and 20-μl aliquots of the diluted solution were injected. (PDF 34 kb)

Fig. 3

Effect of the pH of ammonium acetate in the mobile phase with methanol–200 mM ammonium acetate buffer (98:2, v/v) on the retention times of SA, S-LysoPC, Chol, HSPC, and DSPE-PEG. (PDF 15 kb)

Fig. 4

HPLC-ELSD detection of a standard mixture (a) and DOXIL® (b) in the mobile phase with methanol–200 mM ammonium acetate buffer (pH 4.0) (98:2, v/v). A standard mixture (20 μl) containing 150 μg/ml (each) of P-LysoPC, S-LysoPC, PA, and SA; 300 μg/ml of Chol; and 750 μg/ml each of HSPC and DSPE-PEG was injected. (PDF 114 kb)

Fig. 5

Standard curves of Chol, HSPC-2, HSPC-2, DSPE-PEG, S-LysoPC, and SA. Log (concentration) was plotted against log (area) for each lipid. (PDF 45 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shibata, H., Yomota, C. & Okuda, H. Simultaneous Determination of Polyethylene Glycol-Conjugated Liposome Components by Using Reversed-Phase High-Performance Liquid Chromatography with UV and Evaporative Light Scattering Detection. AAPS PharmSciTech 14, 811–817 (2013). https://doi.org/10.1208/s12249-013-9967-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1208/s12249-013-9967-8

KEYWORDS

Search

Navigation