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81BIOPHARMACEUTICAL JUSTIFICATION FOR THE CHOICE OF EMULSION BASE IN VITRO
Zuikina Ye.,
PhD of Pharmacy, Department of Pharmaceutical Technology of Drugs, NUPh
Polovko N.
Doctor of Pharmacy, Professor, Head of the Department of Pharmaceutical
Technology of Drugs, NUPh
Abstract
The aim of the study was to investigate the influence of the nature of the base on the release of salicylic and boric acid from soft dosage forms by in vitro method followed by quantification of salicylic acid by HPLC and boric acid by alkalimetry. It was found that the lowest amount of drugs is released by the vaseline base, more completely salicylic acid is released from the oil / water emulsion base and the maximum - from the water / oil, while the maximum release of boric acid occurs from the oil / water emulsion base.
Keywords: release, HPLC, alkalimetry, semi-solid dosage forms.
Introduction.The urgent task of modernization of extemporaneous production is to improve the composition of medicines produced in stock. First of all due to replacement the bases in structure of soft dosage forms (SDF) on emulsion. An important stage in the development and improvement of the composition of drugs, including SDF is to determine the factors that affect the bioavailability and, above all, the rate and completeness of drug release. This is due to the fact that the necessary release of active substances from the dosage form is a prerequisite for the therapeutic effectiveness of treatment [14, 19, 20].
There are several methods for determining bioa-vailability, the most widely used is the method of dialysis through a semipermeable membrane into the acceptor (dialysis) fluid. Nowadays, as a rule, complex multiphase compositions are prefered when creating a dosage form, these formulations usually contain two phases (water and oil), one - the outer phase, and the other - the inner [1, 16]. One example of such systems is emulsion bases, which contribute to better release of active substances compared to the fat bases [7]. The active compound is usually dissolved in one of the above phases. However, the active compound is not always completely soluble [23]. When developing and selecting the optimal formulation, it is necessary to control the size of the dispersed particles, the surface tension
between the phases and the rheological properties of the base, among other things, it is necessary to determine the degree of release of the drug from the system [3]. The rate of release of active substances in vitro is influenced by various physical and chemical parameters: solubility, number and particle size of active substance, type and nature of the base, rheological properties of semi-solid dosage form and it's solubility in the acceptor medium in which the active compound is released [21]. Therefore, we investigated the influence of the nature of the base on the release of salicylic and boric acids from soft dosage forms by in vitro method followed by quantification of salicylic acid by HPLC and boric acid by alkalimetry.
Materials and methods. Emulsifiers which were used for preparation of emulsion bases: Span 80, (Sor-bitan oleate), Span 60, (Sorbitan monostearate), cetyl-stearyl alcohol, Olevem 1000 (Cetearyl Olivate / Sorbi-tan Olivat), Emulpharma 1000 (Cetearyl alcohol, Glyceryl stearate, Cebitan stearate, Sorbitan stearate ). Salicylic acid at a concentration of 20% and boric acid at a concentration of 5% were used as the active substances. Studies of active pharmaceutical ingredients (API) release were performed in comparison with salicylic ointment with fat vaseline base and boric ointment with fat vaseline base. The composition of the samples is shown in table 1.
Table 1
Name of components Quantitative content,% / № samples
1 2 3 4 5 6 7 8
Salicylic acid 20
Boric acid 5
Corn oil 40 10 15 40 10 15
Span 80, (Sorbitan oleate) 2 2
Span 60, (Sorbitan monostearate) 3 3
Cetylstearyl alcohol 5.5 5.5
Olevem 1000 (Cetearyl Olivate / Sorbitan Olivat) 4 4
Emulpharma 1000 (Cetearyl alcohol, Glyceryl stearate, Sorbitan stearate, Cetearyl glucoside) 6 6
Vaseline 80 80
Purified water up to 100 up to 100
The composition of experimental samples
Samples № 1 - 3 were obtained by the following technology: the required number of emulsifiers was weighed on electronic scales and melted in a water bath to a temperature of about 60 - 80 ° C. Salicylic acid was pre-dissolved in weighed corn oil and added to the molten emulsifier mixture. After melting the oil phase, the calculated amount of purified water heated to 80 ° C was gradually added to it, homogenized using a laboratory homogenizer (Homogenizer HG - 15A) for 25 minutes 3000 rpm to a homogeneous consistency, cooled to room temperature and was studied the next day. Sample № 4 was obtained by the following technology: the required amount of salicylic acid was weighed on electronic scales and grinded with the required amount of ethanol. Weighed vaseline was melted in a water bath. Vaseline was mixed with crushed salicylic acid in a pounder, cooled to room temperature and a day later the study was performed.
In vitro studies of the release of salicylic acid from the ointments with emulsion and vaseline bases were performed using modified Franz-type diffuse cells with vertical diffusion cells and regenerated dialysis cellulose membranes Cuprophan (Medicell International Ltd., London, UK) using the HDT 1000 test system for vertical diffusion cell (VDC) tests. Filling of the chambers for samples of the tested SDF was from 250 mg to 425 mg. In vitro release experiments were performed (n= 2). The area of membrane diffusion was 1.13 cm2. The solution of tween-80 20% 30 ml was used as an acceptor medium. This is due to the fact that salicylic acid was indefinitely soluble in this acceptor medium. The temperature of the acceptor medium was 32 ± 1 ° C. Samples of the acceptor medium (1 ml) were taken after 15 min, 1, 1.5; 2 and 3 hours, the same amount of fresh medium was added to maintain a constant volume. The samples were filtered through a membrane filter (0.22 ^m) and analyzed by HPLC.
UPLC-ESI-MS conditions
Separation of salicylic acid in samples was carried out with Acquity H-class UPLC system (Waters, USA) equipped with triple quadrupole tandem mass spectrometer (Xevo, Waters, USA) with an electrospay ion-ization source (ESI) was used to obtain MS / MS data. BEH C18 (50 x 2.1 mm 1.7 ^m) column was used for analysis. The column temperature was maintained at 40 ° C. Gradient elution was performed with a mobile phase consisting of 0.1% formic acid water solution (solvent A) and acetonitrile (solvent B) with the flow rate set to 0.5 ml / min. Linear gradient profile was applied with the following proportions of solvent A: 0 to 0.5 min - 95%, 3.5 min. - 5%, 3.55 min back to 95%. Negative electrospray ionization was applied for analysis with the following settings: capillary voltage - 2.5 kV, source temperature - 150 ° C, desolvation temperature - 500 ° C, desolvation gas flow - 800 l / h, cone gas flow - 20 l / h. Collision energy was set to 12 eV and cone voltage was maintained at 30 V. Precursor and product ions were selected at 137 m / z and at 93 m / z respectively.
Production of standard solutions of salicylic acid. A solution of salicylic acid in methanol with a drug concentration of 4 mg / ml was prepared from a standard sample. By diluting this solution with methanol, a series of solutions were prepared; 0.04; 0.40; 4.0; 40.0; 400.0 and 4000.0 ^g / ml. The solutions were used to construct a calibration graph.
Samples № 5 - 7 were obtained by the following technology: pre-weighed emulsifier (or mixture of emulsifiers) was melted in a water bath to a temperature of 60 - 80 ° C, weighed corn oil was added. The purified water was measured separately and heated in a water bath to a temperature of 80 ± 5 ° C. The weighed boric acid was dissolved in purified water with stirring. After fusion, the oil phase was transferred to a heat-resistant container, an aqueous solution of boric (80 ° C) acid was gradually added, homogenized using a laboratory homogenizer (Homogenizer HG - 15A) for 25 minutes 3000 rpm. Cooled for 15 minutes with stirring.
Sample № 8 was obtained by the following technology: the required amount of boric acid was weighed on electronic scales. Weighed vaseline was melted on a water bath. Vaseline was mixed with crushed acid in a pounder, cooled to room temperature and the study was performed a day later.
Method of dialysis through a semipermeable membrane
A dialysis chamber consisting of two chambers was used for the experiment. The chambers were numbered; to one of the chambers with a diameter of 50 mm, and the area of 1963 mm2, a portion of the prototype ointment (20.0 g) was placed and evenly distributed on the surface of the membrane. Then a cellophane membrane (GSHSE 7730-89) was applied and the dia-lyzer was collected. Using a thin-tipped pipette or a syringe with a needle, dialysis fluid - phosphate buffer solution with a pH of 5.5 was places in the receptor chambers. The dialyzer was placed in a thermostat TC-20 at a temperature of 32 ± 1 ° C. The drug substance was released from the ointment samples into the buffer solution due to passive diffusion. Dialysate sampling (1 ml) was performed at regular intervals (1, 4, 6, 8 and 24 hours). This was followed by quantification of boric acid in the selected samples.
Method of quantitative determination of boric acid (Alkalimetry).
To 5.0 ml of the drug is added 20.0 ml of glycerol, pre-neutralized with a solution of phenolphthalein P1, mixed and titrated with 0.1 M sodium hydroxide solution until pink colouration. (s = 1).
1 ml of 0.1 M sodium hydroxide solution corresponds to 6.183 mg of boric acid.
The percentage of boric acid X is calculated by the formula:
_YnuOHXK XS XM.M.X100 _
X —- —
Vn X1000 YNaOH XK X0,006183 X100
V (2.5)
vn
where: Y is the volume of 0.1 M sodium hydroxide solution (ml);
K -is the correction factor for 0.1 M sodium hydroxide solution;
CM - molar concentration of sodium hydroxide;
M.m. - molecular weight of boric acid (g);
Vn is an aliquot for analysis (ml) [6].
Results. Within the concentrations of salicylic acid from 0.04 to 4000 mg / ml calibration graph (Fig. 1) is described by the dependence Y = -0.0464864 x x2 + 201.382 x x + 226.298 (correlation coefficient R2 = 0.970455), the indicator varies within the statistical error and concentrations, quantification of these substances can be performed with acceptable accuracy.
70000 60000 50000 40000 30000
c3 0)
<| 20000
10000
0
0
Y= -0.0464864 x x2 + 201.382 x x + 226.298 R2 = 0.970455
500 1000 1500 2000 2500 3000 3500 4000 4500
Concentration (x)
FIG. 1 The Graph of the linear dependence of the signal on the actual concentration of salicylic acid.
Results of the studies of salicylic acid release from model samples of ointments are shown in Figure 2.
The data obtained shows that the lowest amount of salicylic acid is released from the ointment on a fat (vaseline) basis.
Among the emulsion bases, the largest amount of salicylic acid was released from the sample № 1 - the
emulsion basis of the second type. Samples № 2 and 3 in quantitative indicators of API release, almost did not differ in the first hours. However, it should be noted that at the third hour of the experiment sample № 3 released more drug than sample № 2, with the same type of emulsion base.
S
0
1 Is
<D
o
s o
u
I
I
15 min
□ Sample 1
1 hours
I.
1,5 hours
rh
It
Sample 2 '/. Sample 3
2 hours
Sample
¿1
i
ir
++ ++
3 hours Time, hours
FIG. 2. Dynamics of salicylic acid release from model samples of ointments.
6
5
4
3
2
1
0
Summarizing the results obtained, it should be noted that the higher release from the emulsion base of the 2nd type may be associated with easier passage of salicylic acid, which is soluble in corn oil dispersion medium of the base, through the membrane.
Analysis of the data shown in Fig. 3, shows that the total amount of boric acid, which passed into the
o
U Ö o U
The greatest release of boric acid is observed from the emulsion base of the 1st kind using a complex emul-sifier Emulpharma 1000 (Cetearyl alcohol, Glyceryl stearate, Sorbitan stearate, Cetearyl glucoside), this may be due to a more complete dissolution of API in water dispersion medium emulsion base in emulsions of the first kind in much larger quantities compared to emulsions of the second kind.
Results. The dependence of the degree of release of salicylic and boric acids on the nature and composition of the ointment base was studied. By the method of modified diffuse Franz-type cells with vertical diffusion cells using HPLC to identify API in the acceptor fluid, it was found that salicylic acid is more intensely and completely released from the water / oil emulsion base. By dialysis through a semipermeable membrane followed by quantification of API by alkalimetry, it was demonstrated that boric acid is more intensively and completely released from the emulsion base oil / water from the complex emulsifier Emulpharma 1000 (Cetearyl alcohol, Glyceryl stearate, Cebitary).
Discussion. It is determined that the release of salicylic and boric acid from ointments on emulsion bases is more complete in comparison with vaseline base. There is a direct relationship between the degree of release of the active substance and the type of emulsion base. It is shown that a well-soluble in oil active substance will be more fully released when using emulsion bases of the second type, in turn, the water-soluble substance is better released from the bases of the first type.
Acknowledgment
The research was conducted within the framework of a cooperation agreement with the Lithuanian University of Health Sciences. The authors thank prof. Jurga
dialysis fluid, is in the range from 0.6 to 2.83% with the maximum release of API from sample № 2. Vaseline base (sample № 8) very slowly releases boric acid, so at 24 hours of the experiment from this basis released only 0.68% of API.
Bernatoniene, head of the Department of Drug Technology and Social Pharmacy and prof. Valdas Jakstas, Department of Pharmacognosy Lithuanian University of Health Sciences, for the presentation of the base and equipment for research.
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