Thin layer chromatography as a simple and quick inprocess tool for qualitative and semi-quantitative determination of unentrapped drugs in liposomal formulations

Control of free or unentrapped drug is one of the key quality attributes for liposomal drug deliverysystems. Conventional methodologies for estimating unentrapped drug in liposomal formulations require priorseparation, which are time-consuming and complex. There is a need for a quick in-process analytical technique, whichcan enable monitoring of drug loading during various stages of product development and manufacturing. A novel simple and rapid TLC procedure was developed for evaluating entrapment efficiency in liposomal formulations. The method is based on differential radial migration of free and entrapped drug on silica gel TLC plates. The method can be employed either as a simple visual qualitative tool in the early screening studies or in a semi-quantitative mode for detection of free and unentrapped drugs. In case of doxorubicin hydrochloride, a deep-red coloured molecule, the method enabled free drug detection up to the levels as low as 2.5%. This method precluded the need of developing the plate further with the mobile phase. The method was used successfully in product development or as an in-process monitoring tool. The method’s potential for characterizing entrapment efficiency was evaluated for less intensely coloured or UV absorbing drug candidates such as amphotericin B liposome and curcumin liposome. The preliminary results post derivatization with ninhydrin reagent or visualization under UV light indicated applicability of technique in general for liposomal delivery systems of various other drugs. According to the best of the author’s knowledge, therehas been no reports on the evaluation of drug entrapment using such TLC based technique so far.

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[1] Bulbake U, Doppalapudi S, Kommineni N, Khan W. Liposomal formulations in clinical use: an updated review. Pharmaceutics. 2017; 9(2): 12. [CrossRef]
[2] Chang HI, Yeh MK. Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy. Int J Nanomedicine. 2012; 7: 49-60. [CrossRef]
[3] Kapoor M, Lee SL, Tyner KM. Liposomal drug product development and quality: current US experience and perspective. The AAPS Journal. 2017; 19(3): 632-641. [CrossRef]
[4] Allen TM. Liposomal drug formulations. Drugs. 1998; 56(5): 747-756. [CrossRef]
[5] Panwar P, Pandey B, Lakhera PC, Singh KP. Preparation, characterization, and in vitro release study of albendazoleencapsulated nanosize liposomes. Int J Nanomedicine. 2010; 5: 101-108. [CrossRef]
[6] Gabizon AA. Pegylated liposomal doxorubicin: metamorphosis of an old drug into a new form of chemotherapy. Can. Invest. 2001; 19(4): 424-436. [CrossRef]
[7] Ali MZ, Foad AS, Zarrabi A, Reza TM. Liposomal doxorubicin delivery systems: effects of formulation and processing parameters on drug loading and release behavior. Current Drug Deliv. 2016; 13(7): 1065-1070.
[8] Szebeni J, Fülöp T, Dézsi L, Metselaar B, Storm G. Liposomal doxorubicin: the good, the bad and the not-so-ugly. J Drug Target. 2016; 24(9): 765-767. [CrossRef]
[9] Chen D, Cole DL, Srivatsa GS. Determination of free and encapsulated oligonucleotides in liposome formulated drug product. J Pharmaceut Biomed. 2000; 22(5): 791-801. [CrossRef]
[10] Mayer LD, Onge GS. Determination of free and liposome-associated doxorubicin and vincristine levels in plasma under equilibrium conditions employing ultrafiltration techniques. Anal Biochem. 1995; 232(2): 149-157. [CrossRef]
[11] Thies RL, Cowens DW, Cullis PR, Bally MB, Mayer LD. Method for rapid separation of liposome-associated doxorubicin from free doxorubicin in plasma. Anal Biochem. 1990; 188(1): 65-71. [CrossRef]
[12] Griese N, Blaschke G, Boos J, Hempel G. Determination of free and liposome-associated daunorubicin and daunorubicinol in plasma by capillary electrophoresis. J Chromatogr A. 2002; 979(1-2): 379-388. [CrossRef]
[13] Druckmann S, Gabizon A, Barenholz Y. Separation of liposome-associated doxorubicin from non-liposomeassociated doxorubicin in human plasma: implications for pharmacokinetic studies. Biochim Biophys Acta. 1989; 980(3): 381-384. [CrossRef]
[14] Edwards KA, Baeumner AJ. Analysis of liposomes. Talanta. 2006; 68(5): 1432-1441. [CrossRef]
[15] Gomez-Hens A, Fernandez-Romero JM. Analytical methods for the control of liposomal delivery systems. Trend Anal Chem. 2006; 25(2): 167-178. [CrossRef]
[16] Dipali SR, Kulkarni SB, Betagiri GV. Comparative study of separation of non‐ encapsulated drug from unilamellar liposomes by various methods. J Pharm Pharmacol. 1996; 48(11): 1112-1115. [CrossRef]
[17] Yamamoto E, Miyazaki S, Aoyama C, Kato M. A simple and rapid measurement method of encapsulation efficiency of doxorubicin loaded liposomes by direct injection of the liposomal suspension to liquid chromatography. Int JPharm. 2018; 536(1): 21-28. [CrossRef]
[18] Dhanesar SC, Peeler TR, Engel BS. Evaluation of phospholipids in liposomes by HPTLC. J Planar Chromat. 1992; 5(1): 45-49.
[19] Holzer M, Burghardt A, Schubert R. Quantitative high-performance thin-layer chromatography determination of common liposome components and critical parameters influencing the analysis results. J Liposome Res. 2010; 20(2): 124-133. [CrossRef]
[20] Hatziantoniou S, Demetzos C. Method of simultaneous analysis of liposome components using HPTLC/FID. In: D’Souza (Eds). Liposomes. Humana Press, New York, NY, USA, 2010, pp. 363-368. [CrossRef]
[21] Hatziantoniou S, Demetzos C. Method of simultaneous analysis of liposome components using HPTLC/FID. In: D’Souza (Eds). Liposomes. Humana Press, New York, NY, USA, 2017, pp. 49-54 [CrossRef]
[22] Brailoiu E, Saila L, Huhurez G, Costuleanu M, Filipeanu CM, Slatineanu S, Cotrutz C, Branisteanu DD. TLC—A rapid method for liposome characterization. BMC. 1994; 8(4): 193-195. [CrossRef]
[23] Brailoiu E, Huhurez G, Slatineanu S, Filipeanu CM, Costuleanu M, Branisteanu DD. TLC characterization of liposomes containing D‐ myo‐ inositol derivatives. BMC. 1995; 9(4): 175-178. [CrossRef]
[24] Brailoiu E, Beschea C, Brailoiu C, Costuleanu A, Filipeanu CM, Costuleanu M, Fallgrens B, Branisteanu DD. TLC Characterization of Small Unilamellar Liposomes Containing D‐ myo‐ Inositol Derivatives. BMC. 1996; 10(5): 233-236. [CrossRef]
[25] Van Renswoude AJ, Blumenthal R, Weinstein JN. Thin-layer chromatography with agarose gels: A quick, simple method for evaluating liposome size. Biochim Biophys Acta. 1980; 595(1): 151-156. [CrossRef]
[26] Nicoletti M. HPTLC fingerprint: a modern approach for the analytical determination of botanicals. Rev Bras Farmacogn. 2011; 21(5): 818-823. [CrossRef]
[27] Ansari MJ, Ahmad S, Kohli K, Ali J, Khar RK. Stability-indicating HPTLC determination of curcumin in bulk drug and pharmaceutical formulations. J PharmBiomed. 2005; 39(1-2): 132-138. [CrossRef]
[28] Fritze A, Hens F, Kimpfler A, Schubert R, Peschka-Süss R. Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient. Biochim Biophys Acta. 2006; 1758(10): 1633-1640. [CrossRef]
[29] Sarfraz M, Afzal A, Yang T, Gai Y, Raza SM, Khan MW, Cheng Y, Ma X, Xiang G. Development of dual drug loaded nanosized liposomal formulation by a reengineered ethanolic injection method and its pre-clinical pharmacokinetic studies. Pharmaceutics. 2018; 10(3): 151. [CrossRef]
[30] IB times (2017). India suspends licences of 10 drug firms over product quality, safety issues. Accessed on 25th January, 2020. http://www.ibtimes.co.in/india-suspends-licences-10-drug-firms-over-product-quality-safety-issues-678258 (Accessed on 25th January 2020).
[31] Sainz V, Conniot J, Matos AI, Peres C, Zupanǒiǒ E, Moura L, Silva LC, Florindo HF, Gaspar RS. Regulatory aspects on nanomedicines. Biochem Biophys Res. 2015; 468(3): 504-510. [CrossRef]
[32] Wang JP, Maitani Y, Takayama K, Nagai T. Pharmacokinetics and antitumor effect of doxorubicin carried by stealth and remote loading proliposome. Pharm Res. 2000; 17(7): 782-787. [CrossRef]
[33] Patel NA, Patel NJ, Patel RP. Formulation and evaluation of curcumin gel for topical application. Pharm Dev Technol. 2009; 14(1): 83-92. [CrossRef]