Abstract
Background: Liposomes continue to play an important role in drug delivery research due to their ability to improve transport and targeting of a wide range of active molecules. Analysis of liposomal components is a key point in the characterization and evaluation of formulation stability. The aim of this work was to develop and validate an HPLC-ELSD method for the characterization and quality control of liposomes.
Methods: HPLC-ELSD method was validated by assessing selectivity, linearity, precision, accuracy, limit of detection and quantitation. The mobile phase consisted of a 0.1% (v/v) of trifluoroacetic acid (TFA) and methanol in gradient elution. Initial rate was 20:80 (0.1% TFA: methanol), with a ramp reaching 100% methanol. HPLC-MS/MS was used to confirm the presence of the fatty acid mixture in the analyzed lipids, as well as sub-products generated under pre-determined conditions in the stability study.
Results: A HPLC-ELSD method has been developed to detect and measure cholesterol, phosphatidylcholine and lysophosphatidylcholine. High specificity, sensitivity and linearity within the predetermined range for all the compounds analyzed (R2>0.99) were obtained. Accuracy and precision results for all the compounds were within the acceptance limit of ≤5% and 90-110%, respectively. Mass spectrometry results showed complementary information about the phospholipid composition to evaluate the degree of degradation of liposomes over different storage conditions.
Conclusion: The method was successfully applied as a quality control tool for the analysis of a wide range of lipids, present in liposomal formulations. HPLC-MS/MS was used to ensure complete elucidation of the lipid components and the detected lyso-forms.
Keywords: HPLC-ELSD, LC-MS/MS, phospholipid, degradation, drug delivery systems, lipid-based formulation.
Graphical Abstract
[1]
Adachi, J.; Asano, M.; Yoshioka, N.; Nushida, H.; Ueno, Y. Analysis of phosphatidylcholine oxidation products in human plasma using quadrupole time-of-flight mass spectrometry. Kobe J. Med. Sci., 2006, 52(5), 127-140.
[PMID: 17006053]
[PMID: 17006053]
[2]
Roces, C.B.; Kastner, E.; Stone, P.; Lowry, D.; Perrie, Y. Rapid quantification and validation of lipid concentrations within liposomes. Pharmaceutics, 2016, 8(3)E29
[http://dx.doi.org/10.3390/pharmaceutics8030029] [PMID: 27649231]
[http://dx.doi.org/10.3390/pharmaceutics8030029] [PMID: 27649231]
[3]
Ioffe, V.; Kalendarev, T.; Rubinstein, I.; Zupkovitz, G. Reverse phase HPLC for polar lipids. Simple and selective HPLC procedures for analysis of phospholipid-based derivatives of valproic acid and various non-steroidal anti-inflammatory drugs. J. Pharm. Biomed. Anal., 2002, 30(3), 391-403.
[http://dx.doi.org/10.1016/S0731-7085(02)00220-0] [PMID: 12367664]
[http://dx.doi.org/10.1016/S0731-7085(02)00220-0] [PMID: 12367664]
[4]
Lin, J.T.; McKeon, T.A. separation of intact phosphatidylcholine molecular species by high performance liquid chromatography. J. Liq. Chromatogr. Relat. Technol., 2000, 23(6), 813-829.
[http://dx.doi.org/10.1081/JLC-100101491]
[http://dx.doi.org/10.1081/JLC-100101491]
[5]
Patton, G.M.; Fasulo, J.M.; Robins, S.J. Separation of phospholipids and individual molecular species of phospholipids by high-performance liquid chromatography. J. Lipid Res., 1982, 23(1), 190-196.
[PMID: 7057106]
[PMID: 7057106]
[6]
Mazzella, N.; Molinet, J.; Syakti, A.D.; Dodi, A.; Doumenq, P.; Artaud, J.; Bertrand, J-C. Bacterial phospholipid molecular species analysis by ion-pair reversed-phase HPLC/ESI/MS. J. Lipid Res., 2004, 45(7), 1355-1363.
[http://dx.doi.org/10.1194/jlr.D300040-JLR200] [PMID: 15102893]
[http://dx.doi.org/10.1194/jlr.D300040-JLR200] [PMID: 15102893]
[7]
Nichols, P.D.; Shaw, P.M.; Mancuso, C.A.; Franzmann, P.D. Analysis of archaeal phospholipid-derived di- and tetraether lipids by high temperature capillary gas chromatography. J. Microbiol. Methods, 1993, 18(1), 1-9.
[http://dx.doi.org/10.1016/0167-7012(93)90066-Q]
[http://dx.doi.org/10.1016/0167-7012(93)90066-Q]
[8]
Zhong, Z.; Ji, Q.; Zhang, J.A. Analysis of cationic liposomes by reversed-phase HPLC with evaporative light-scattering detection. J. Pharm. Biomed. Anal., 2010, 51(4), 947-951.
[http://dx.doi.org/10.1016/j.jpba.2009.10.001] [PMID: 19896790]
[http://dx.doi.org/10.1016/j.jpba.2009.10.001] [PMID: 19896790]
[9]
Avalli, A.; Contarini, G. Determination of phospholipids in dairy products by SPE/HPLC/ELSD. J. Chromatogr. A, 2005, 1071(1-2), 185-190.
[http://dx.doi.org/10.1016/j.chroma.2005.01.072] [PMID: 15865192]
[http://dx.doi.org/10.1016/j.chroma.2005.01.072] [PMID: 15865192]
[10]
Bird, S.S.; Marur, V.R.; Stavrovskaya, I.G.; Kristal, B.S. Qualitative Characterization of the Rat Liver Mitochondrial Lipidome using LC-MS Profiling and High Energy Collisional Dissociation (HCD) All Ion Fragmentation, Metabolomics : Official journal of the Metabolomic Society.. 2013, 9(1 Suppl), 67-83.
[11]
Fagan, P.; Wijesundera, C. Liquid chromatographic analysis of milk phospholipids with on-line pre-concentration. J. Chromatogr. A, 2004, 1054(1-2), 241-249.
[http://dx.doi.org/10.1016/j.chroma.2004.04.051] [PMID: 15553150]
[http://dx.doi.org/10.1016/j.chroma.2004.04.051] [PMID: 15553150]
[12]
Lutzke, B.S.; Braughler, J.M. An improved method for the identification and quantitation of biological lipids by HPLC using laser light-scattering detection. J. Lipid Res., 1990, 31(11), 2127-2130.
[PMID: 2086710]
[PMID: 2086710]
[13]
Simonzadeh, N.; Ronsen, B. An Isocratic HPLC Method for the Determination of Sorbitol and Glycerol in Pharmaceutical Formulations., 2012, 50, 644-647.
[14]
Becart, I.; Chevalier, C.; Biesse, J.P. Quantitative analysis of phospholipids by HPLC with a light scattering evaporating detector – application to raw materials for cosmetic use. J. High Resolut. Chromatogr., 1990, 13(2), 126-129.
[http://dx.doi.org/10.1002/jhrc.1240130210]
[http://dx.doi.org/10.1002/jhrc.1240130210]
[15]
Olsson, N.U.; Harding, A.J.; Harper, C.; Salem, N., Jr High-performance liquid chromatography method with light-scattering detection for measurements of lipid class composition: analysis of brains from alcoholics. J. Chromatogr. B Biomed. Appl., 1996, 681(2), 213-218.
[http://dx.doi.org/10.1016/0378-4347(95)00576-5] [PMID: 8811429]
[http://dx.doi.org/10.1016/0378-4347(95)00576-5] [PMID: 8811429]
[16]
Pimentel, L. #xed, gia, A. Gomes, M. Pintado, Rodr, #xed, guez-Alcal, #xe1, and L.M., Isolation and Analysis of Phospholipids in Dairy Foods. J. Anal. Methods Chem., 2016, 2016, 12.
[17]
Holland, W.L.; Stauter, E.C.; Stith, B.J. Quantification of phosphatidic acid and lysophosphatidic acid by HPLC with evaporative light-scattering detection. J. Lipid Res., 2003, 44(4), 854-858.
[http://dx.doi.org/10.1194/jlr.D200040-JLR200] [PMID: 12562857]
[http://dx.doi.org/10.1194/jlr.D200040-JLR200] [PMID: 12562857]
[18]
Yon, C.; Han, J-S. Analysis of trimethylsilyl derivatization products of phosphatidylethanol by gas chromatography-mass spectrometry. Exp. Mol. Med., 2000, 32(4), 243-245.
[http://dx.doi.org/10.1038/emm.2000.40] [PMID: 11190278]
[http://dx.doi.org/10.1038/emm.2000.40] [PMID: 11190278]
[19]
Coene, J.; Van den Eeckhout, E.; Herdewijn, P.; Sandra, P. Gas chromatographic determination of alkyl lysophospholipids after solid-phase extraction from cell culture media. J. Chromatogr. A, 1993, 612(1), 21-26.
[http://dx.doi.org/10.1016/0378-4347(93)80362-8] [PMID: 8454698]
[http://dx.doi.org/10.1016/0378-4347(93)80362-8] [PMID: 8454698]
[20]
Hu, S.; Zhang, L.; Dovichi, N.J. Characterization of the interaction between phospholipid and protein by capillary electrophoresis with laser-induced fluorescence detection. J. Chromatogr. A, 2001, 924(1-2), 369-375.
[http://dx.doi.org/10.1016/S0021-9673(01)00988-8] [PMID: 11521886]
[http://dx.doi.org/10.1016/S0021-9673(01)00988-8] [PMID: 11521886]
[21]
Olsson, N.U.; Kaufmann, P. Optimized method for the determination of 1,2-diacyl-sn-glycero-3-phosphocholine and 1,2-diacyl-sn-glycero-3-phosphoethanolamine molecular species by enzymatic hydrolysis and gas chromatography. J. Chromatogr. A, 1992, 600(2), 257-266.
[http://dx.doi.org/10.1016/0021-9673(92)85556-9]
[http://dx.doi.org/10.1016/0021-9673(92)85556-9]
[22]
Mourey, T.H.; Oppenheimer, L.E. Principles of operation of an evaporative light-scattering detector for liquid chromatography. Anal. Chem., 1984, 56(13), 2427-2434. [http://dx.doi.org/10.1021/ac00277a039].
[23]
Zhang, L.; Krylov, S.; Hu, S.; Dovichi, N.J. Methyl-β-cyclodextrin modified micellar electrokinetic capillary chromatography with laser-induced fluorescence for separation and detection of phospholipids, 2000, Vol. 894, 129-134.
[24]
Simonzadeh, N. An Isocratic HPLC Method for the Simultaneous Determination of Cholesterol, Cardiolipin, and DOPC in Lyophilized Lipids and Liposomal Formulations., 2009, 47, 304-308.
[25]
Veloso, D.F.M.C.; Benedetti, N.I.G.M.; Ávila, R.I.; Bastos, T.S.A.; Silva, T.C.; Silva, M.R.R.; Batista, A.C.; Valadares, M.C.; Lima, E.M. Intravenous delivery of a liposomal formulation of voriconazole improves drug pharmacokinetics, tissue distribution, and enhances antifungal activity. Drug Deliv., 2018, 25(1), 1585-1594.
[http://dx.doi.org/10.1080/10717544.2018.1492046] [PMID: 30044149]
[http://dx.doi.org/10.1080/10717544.2018.1492046] [PMID: 30044149]
[26]
Gregoriadis, G. Liposome research in drug delivery: the early days. J. Drug Target., 2008, 16(7), 520-524.
[http://dx.doi.org/10.1080/10611860802228350] [PMID: 18686120]
[http://dx.doi.org/10.1080/10611860802228350] [PMID: 18686120]
[27]
Brasil.. Agência Nacional de Vigilância Sanitária, RDC Nº 166, DE
24 DE JULHO DE 2017; , 2017. Brasília.
[28]
FDA. Analytical Procedures and Methods Validation for Drugs and Biologics; New Hampshire, 2015, p. 15.
[29]
Oak, J-H.; Nakagawa, K.; Miyazawa, T. UV analysis of Amadori-glycated phosphatidylethanolamine in foods and biological samples. J. Lipid Res., 2002, 43(3), 523-529.
[PMID: 11893788]
[PMID: 11893788]
[30]
Sala Vila, A.; Castellote-Bargalló, A.I.; Rodríguez-Palmero-Seuma, M.; López-Sabater, M.C. High-performance liquid chromatography with evaporative light-scattering detection for the determination of phospholipid classes in human milk, infant formulas and phospholipid sources of long-chain polyunsaturated fatty acids. J. Chromatogr. A, 2003, 1008(1), 73-80.
[http://dx.doi.org/10.1016/S0021-9673(03)00989-0] [PMID: 12943251]
[http://dx.doi.org/10.1016/S0021-9673(03)00989-0] [PMID: 12943251]
[31]
Shin, H-D.; Suh, J-H.; Kim, J-H.; Lee, H-Y.; Eom, H.Y.; Kim, U-Y.; Yang, D-H.; Han, S.B.; Youm, J-R. Determination of Betaine in Fructus Lycii Using Hydrophilic Interaction Liquid Chromatography with Evaporative Light Scattering Detection; , , Vol. 33, . 2012.
[32]
Stolyhwo, A.; Colin, H.; Guiochon, G. Use of light scattering as a detector principle in liquid chromatography. J. Chromatogr. A, 1983, 265, 1-18.
[http://dx.doi.org/10.1016/S0021-9673(01)96693-2]
[http://dx.doi.org/10.1016/S0021-9673(01)96693-2]
[33]
Rabinovich-Guilatt, L.; Dubernet, C.; Gaudin, K.; Lambert, G.; Couvreur, P.; Chaminade, P. Phospholipid hydrolysis in a pharmaceutical emulsion assessed by physicochemical parameters and a new analytical method. Eur. J. Pharm. Biopharm., 2005, 61(1-2), 69-76.
[http://dx.doi.org/10.1016/j.ejpb.2005.03.001] [PMID: 15913973]
[http://dx.doi.org/10.1016/j.ejpb.2005.03.001] [PMID: 15913973]
[34]
Zhou, Q.; Liu, L.; Zhang, D.; Fan, X. Analysis of gemcitabine liposome injection by HPLC with evaporative light scattering detection. J. Liposome Res., 2012, 22(4), 263-269.
[http://dx.doi.org/10.3109/08982104.2012.668553] [PMID: 22428966]
[http://dx.doi.org/10.3109/08982104.2012.668553] [PMID: 22428966]
[35]
Restuccia, D.; Spizzirri, U.G.; Puoci, F.; Cirillo, G.; Vinci, G.; Picci, N. Determination of Phospholipids in Food Samples. Food Rev. Int., 2012, 28(1), 1-46.
[http://dx.doi.org/10.1080/87559129.2011.563398]
[http://dx.doi.org/10.1080/87559129.2011.563398]
[36]
Olsson, P.; Holmbäck, J.; Herslöf, B. A single step reversed-phase high performance liquid chromatography separation of polar and non-polar lipids. J. Chromatogr. A, 2014, 1369, 105-115.
[http://dx.doi.org/10.1016/j.chroma.2014.10.010] [PMID: 25441077]
[http://dx.doi.org/10.1016/j.chroma.2014.10.010] [PMID: 25441077]
[37]
Lang, J.K.; Vigo-Pelfrey, C. Quality control of liposomal lipids with special emphasis on peroxidation of phospholipids and cholesterol. Chem. Phys. Lipids, 1993, 64(1-3), 19-29.
[http://dx.doi.org/10.1016/0009-3084(93)90054-7] [PMID: 8242833]
[http://dx.doi.org/10.1016/0009-3084(93)90054-7] [PMID: 8242833]
[38]
Duan, Y.; Wei, L.; Petryk, J.; Ruddy, T.D. Formulation, characterization and tissue distribution of a novel pH-sensitive long-circulating liposome-based theranostic suitable for molecular imaging and drug delivery. Int. J. Nanomedicine, 2016, 11, 5697-5708.
[http://dx.doi.org/10.2147/IJN.S111274] [PMID: 27843312]
[http://dx.doi.org/10.2147/IJN.S111274] [PMID: 27843312]
[39]
Grit, M.; Underberg, W.J.M.; Crommelin, D. Hydrolysis of saturated soybean phosphatidylcholine in aqueous liposome dispersion, 1993, Vol. 82, 362-366.
[40]
Grit, M.; Underberg, W.J.; Crommelin, D.J. Hydrolysis of saturated soybean phosphatidylcholine in aqueous liposome dispersions. J. Pharm. Sci., 1993, 82(4), 362-366.
[http://dx.doi.org/10.1002/jps.2600820405] [PMID: 8468678]
[http://dx.doi.org/10.1002/jps.2600820405] [PMID: 8468678]
[41]
Daraee, H.; Etemadi, A.; Kouhi, M.; Alimirzalu, S.; Akbarzadeh, A. Application of liposomes in medicine and drug delivery. Artif. Cells Nanomed. Biotechnol., 2016, 44(1), 381-391.
[http://dx.doi.org/10.3109/21691401.2014.953633] [PMID: 25222036]
[http://dx.doi.org/10.3109/21691401.2014.953633] [PMID: 25222036]
[42]
Grit, M.; Crommelin, D.J.A. Chemical stability of liposomes: implications for their physical stability. Chem. Phys. Lipids, 1993, 64(1-3), 3-18.
[http://dx.doi.org/10.1016/0009-3084(93)90053-6] [PMID: 8242840]
[http://dx.doi.org/10.1016/0009-3084(93)90053-6] [PMID: 8242840]
[43]
Domingues, M.R.M.; Reis, A.; Domingues, P. Mass spectrometry analysis of oxidized phospholipids. Chem. Phys. Lipids, 2008, 156(1-2), 1-12.
[http://dx.doi.org/10.1016/j.chemphyslip.2008.07.003] [PMID: 18671956]
[http://dx.doi.org/10.1016/j.chemphyslip.2008.07.003] [PMID: 18671956]
[44]
Food_and_Drug_Administration, Liposome Drug Products Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation Guidance for Industry; Pharmaceutical Quality, 2018, p. 15.
[45]
Reis, A.; Spickett, C.M. Chemistry of phospholipid oxidation. Biochimica et Biophysica Acta (BBA) -. Biomembranes, 2012, 1818(10), 2374-2387.
[http://dx.doi.org/10.1016/j.bbamem.2012.02.002]
[http://dx.doi.org/10.1016/j.bbamem.2012.02.002]
[46]
Crowe, J.H.; McKersie, B.D.; Crowe, L.M. Effects of free fatty acids and transition temperature on the stability of dry liposomes. Biochim. Biophys. Acta, 1989, 979(1), 7-10.
[http://dx.doi.org/10.1016/0005-2736(89)90516-6] [PMID: 2917168]
[http://dx.doi.org/10.1016/0005-2736(89)90516-6] [PMID: 2917168]
[47]
Toh, M-R.; Chiu, G.N.C. Liposomes as sterile preparations and limitations of sterilisation techniques in liposomal manufacturing. Asian Journal of Pharmaceutical Sciences, 2013, 8(2), 88-95.
[http://dx.doi.org/10.1016/j.ajps.2013.07.011]
[http://dx.doi.org/10.1016/j.ajps.2013.07.011]
[48]
Mills, J.K.; Needham, D. Lysolipid incorporation in dipalmitoylphosphatidylcholine bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition. Biochim. Biophys. Acta, 2005, 1716(2), 77-96.
[http://dx.doi.org/10.1016/j.bbamem.2005.08.007] [PMID: 16216216]
[http://dx.doi.org/10.1016/j.bbamem.2005.08.007] [PMID: 16216216]
[49]
Samuni, A.M.; Lipman, A.; Barenholz, Y. Damage to liposomal lipids: protection by antioxidants and cholesterol-mediated dehydration. Chem. Phys. Lipids, 2000, 105(2), 121-134.
[http://dx.doi.org/10.1016/S0009-3084(99)00136-X] [PMID: 10823461]
[http://dx.doi.org/10.1016/S0009-3084(99)00136-X] [PMID: 10823461]
[50]
Gabrielska, J.; Sarapuk, J.; Przestalski, S. Antioxidant protection of egg lecithin liposomes during sonication. Z. Natforsch. C J. Biosci., 1995, 50(7-8), 561-564.
[http://dx.doi.org/10.1515/znc-1995-7-814] [PMID: 7546044]
[http://dx.doi.org/10.1515/znc-1995-7-814] [PMID: 7546044]
Article Metrics
33
6