Development of quality Indicators of liposomal Preparations "EFIAL"

 

Borshchevskiy G.I.¹, Yarnykh T.G.², Konovalenko V.A.¹, Chabaniy V.N.¹

¹PJSC "Pharmak", Kiev National University of Pharmacy, Kharkov

²Professor, Head of the Department of Drug Technology, National University of Pharmacy, Kharkov- 61168

 

ABSTRACT:

Phospholipids recently considered only as an object of chemical, biophysical, physiological studies. However, some properties, such as the ability to form isolated volumes in an aqueous medium, lowering the surface energy of the phase, but also technology development phospholipids from natural raw materials have allowed their use in the preparation of various drugs. One promising area of application is the creation of phospholipid liposomal formulations that imitate natural ekzosomes mechanism of intercellular communication. "Efial" spray is an emulsion of a yellowish color, which includes: concentrate deproteinization dermal layer of the skin of pigs, phosphatidylcholine from soybean and auxiliaries. As preservative is used the sodium salt of propyl parahydroxybenzoate and metilgidrobenzoata. It is known that phospholipid liposomes significantly affect the properties of the liposomes and therefore requires a thorough physical and chemical analysis , which is particularly important at the stage of intra- stage control. The aim of this work is to develop techniques basic quality spray "Efial". In accordance with the requirements of SPhU held standardization Efial preparation, spray for external use. Methods have been developed to identify and quantify the active ingredients of the drug "Efial", using high performance liquid chromatography. Quality indicators can characterize the efficacy and safety "Efial" within the expiry date.

 

 

INTRODUCTION:

Phospholipids recently considered only as an object of chemical, biophysical, physiological studies. However, some properties, such as the ability to form isolated volumes in an aqueous medium, lowering the surface energy of the phase, but also technology development phospholipids from natural raw materials have allowed their use in the preparation of various drugs [2,8,11].

 

One promising area of application is the creation of phospholipid liposomal formulations that imitate natural ekzosomes mechanism of intercellular communication [6, 7,12,13].

 

At PJSC "Farmak" designed liposomal spray with phosphatidylcholine from soybean , which is intended for the treatment of wounds of various etiologies [1, 5].

 

"Efial" spray is an emulsion of a yellowish color , which includes : concentrate deproteinization dermal layer of the skin of pigs, phosphatidylcholine from soybean and auxiliaries. As preservative is used the sodium salt of propyl parahydroxybenzoate and metilgidrobenzoata [3].

 

It is known that phospholipid liposomes significantly affect the properties of the liposomes and therefore requires a thorough physical and chemical analysis, which is particularly important at the stage of intra- stage control [4, 9, 10]. The aim of this work is to develop techniques basic quality spray "Efial."

 

MATERIALS AND METHODS :

Drug "Efial" spray s.10810; standard samples: phosphatidylcholine (Cat. «Sigma», Cat. № R7443), lysophosphatidylcholine (Cat. «Sigma», Cat. № L 0906), BSA (Cat. «Sigma», Cat. № 7906 A), organic solvents for HPLC : propanol , acetonitrile , hexane .

 

Set chromatographic equipment with high-performance liquid chromatography , manufactured by «Agilent 1260 " cprogramnym software for processing the results of the analysis OpenLAB, laboratory centrifuge Eppendorf 5810R. Software for digital image processing TotallabQuant version 13.01.

 

Quantitative determination of the peptides in liposomal fractions spray for topical use "Efial " was conducted by a comparative adsorption chromatography on a nitrocellulose membrane for protein blotting followed by densitometry digital fingerprint [1]. Previously precipitated liposomes (centrifugation for 4 hours , 21,000 g) at room temperature and then homogenized in an ultrasonic bath in the original volume of phosphate buffer solution pH 7.2 . Aliquots of a suspension of the samples and comparison of the calibration solutions of bovine serum albumin were transferred onto nitrocellulose membrane sheets which are stained KumassiG -250 according to the standard protocol. Washed unbound protein and dye image zatemtsifrovye membranes with colored spots adsorbed proteins were scanned using TotallabQuant version 13.01 (Totallab Ltd, England). On the basis of comparative densitometry , the concentration of peptides in a spray for external use " Efial ." Peptide content of the lipid fraction is at least 0.12 mg / ml preparation.

 

Quantification of phosphatidylcholine and lysophosphatidylcholine was assessed using a validated methodology developed and high-performance liquid chromatography (2.2.29 SPU). Test solution: 1.0 ml of the preparation was transferred into a volumetric flask and diluted with 100 ml of methanol P till mark.

 

Reference solution: 100.0 mg of phosphatidylcholine (Cat. «Sigma», Cat. № R7443) and 10.0 mg of lysophosphatidylcholine ( Cat. «Sigma», Cat . № L 0906 ) was transferred to a volumetric flask of 100 ml and adjusted to metanol till mark at a temperature (20 ± 0,5) ° C. Filtered through a PVDF filter with pore size no greater than 0.45 microns.

 

Chromatography was carried out on a liquid chromatograph with a UV detector, under the following conditions: a steel column used 250h4 size, 6 mm, filled with sorbent KromasilSi particle size of 5 microns, which maintains system suitability requirements . Used the following mobile phase: isopropanol - n-hexane - water P (67:16, 5:16). Mobile phase velocity of 1.0 ml / min detecting at 205 nm wavelength, column temperature 40 0C; injection volume 50 microliters.

 

The chromatographic system is considered suitable if : the number of theoretical plates calculated by pikufosfatidilholina shall be at least 2000; relative standard deviation calculated for 5 pikovfosfatidilholina areas in the chromatogram obtained with reference solution does not exceed 2.0 %, the separation factor between the peaks and lisophosphatidyl phosphatidylcholine - choline is not less than 4.0.

 

Contents of Na- methyl parahydroxybenzoate and propyl parahydroxybenzoate Na- evaluated and designed by means of a validated HPLC methodology (2.2.29 SPU) for hromatograficheskoykolonke size 250 × 4,6 mm oktadetsilsililnym silica with particle diameter of 5 microns and a pore size of 100 Aº, to UV- spectrophotometer of wavelength 255 nm, room temperature thermostat . Attribute Validation methodology presented in Table 1 (for the quantitative determination of Na- propyl parahydroxybenzoate and for the quantitative determination of Na- methyl parahydroxybenzoate)

 

Table 1: Validation characteristics of the quantitative determination of Na-methyl parahydroxybenzoate and Na-propyl parahydroxybenzoate

 

Fig.1. Photo nitrocellulose membrane deposited sample solution: 1. Upper Row 6 BSA solution concentrations comparisons (Sigma kat.nom. A7906) 500 ug / ml, 250 pg / ml, 150 pg / ml, 100 pg / ml, 80 micrograms / ml, 50 micrograms / ml, respectively, 2. Samples of the test drug "Efial spray" 3. Liposome fraction of the drug "Efial spray" (bottom row, 6 parallels).

 

RESULTS AND DISCUSSION.

Photo nitrocellulose membrane deposited sample solution are presented in Fig. 1.

Typical prints spots were transferred to a nitrocellulose membrane protein solutions by passive dot blot and presented in Figure 2.

 

A B C

Fig.2. Typical densitograms fingerprint stains, transferred to nitrocellulose membrane protein solutions by the passive dot blot.  A. Solutions comparison (upper row: 500 ug / mL, 250 micrograms / mL, 150 micrograms / ml, 100  ug / ml 80 ug / ml 50 ug / mlrozvedeniyrastvorov BSAsootvestvenno);  B. typical sample test drug "Efial spray" (6 parallels);  C. liposomal drug faction "Efial spray" (bottom row, parallels

 

 

In typical densitograms submitted test solution and the comparison solution stained protein bands peaks surely detected and allow to calculate the relative concentration of proteins in the composition liposomnoyfraktsii (peptide content is not less than 0.12 mg / ml).

 

In the chromatogram obtained with reference solution impurity lysophosphatidylcholine (Fig. 3) eluted at 22.17 minutes peak lysophosphatidylcholine. Peak area at the limit of quantitation.

 

 

Figure 3. Chromatogram obtained with reference solution impurity lysophosphatidylcholine.

 

Figure 4. Chromatogram obtained with reference solution of phosphatidylcholine.

 

Figure 5. The chromatogram of the test solution. Control of the content of phosphatidylcholine and lysophosphatidylcholine

 

Fig. 6. The test solution. Determination of Na-methylparahydroxybenzoate and Na-propyl parahydroxybenzoate

 

Fig. 7. Reference solution. Determination of Na-methylparahydroxybenzoate and Na-propyl parahydroxybenzoate.

 

Table 2. Indicators of quality of the finished product

Value	Requirement
Description	The emulsion is a white to yellowish
Identification peptides -Na-methyl parahydroxybenzoate -Na-Propyl parahydroxybenzoate -phosphatidylcholine	Must withstand requirements
рН	For 5.2 till 5.6
The volume of content  container	Not less 15 ml
Homogeneity of dose mass	Must withstand requirements
Microbiological purity	In 1 ml of preparation may be no more than 100 microorganisms (aerobic bacteria, and fungi in total). Absence of enterobacteria and some other gram-negative bacteria in 1 ml. Absence in 1 ml Pseudomonasaeruginosa. Absence in 1 ml Staphylococcusaureus
Content of peptide	Not less than 0.12 mg/ml
Content of  phosphatidylcholine  and lysophosphatidylcholine	For 90 till 110 mg/ml Not more 12 mg/ml
Content of Na-methyl parahydroxybenzoate Na-propyl parahydroxybenzoate	For 1.62 till 1.98 mg/ml For 0.18 till 0.22 mg/ml

 

 

In the chromatogram obtained with reference solution of phosphatidylcholine (Fig. 4), the peak of phosphatidylcholine out 10-11 minutes, symmetry and efficiency of chromatographic systems meet pharmacopoeial requirements.

 

Represented on the chromatogram of the test solution Fig. 5 peaks phosphatidylcholine and lysophosphatidylcholine were well separated and there was no interference with other peaks. Total analysis time of the two components does not exceed 25 minutes.

 

 

Submitted on the chromatograms of the test solution (Fig. 6) and reference solution (Fig. 7), the peaks of the sodium salts preservatives well detected and not overlap adjacent peaks. Total analysis time of the two components does not exceed 20 minutes.

 

Indicators of quality of the final product are shown in Table. 2.

 

CONCLUSIONS:

1. In accordance with the requirements of SPhU held standardization Efial preparation, spray for external use.

2. Methods have been developed to identify and quantify the active ingredients of the drug "Efial", using high performance liquid chromatography.

3. Quality indicators can characterize the efficacy and safety "Efial" within the expiry date.

 

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Received on 18.06.2014                Modified on 05.07.2014

Accepted on 10.07.2014                © RJPT All right reserved