The Development of Identification and Quantitative Definition Methodic of Active substances of the Ointment “Allergolic”

 

Rukhmakova Olga, Yarnykh Tatyana, Tykhonov Olexander

National University of Pharmacy, Valentynovskaya Str. 4, Kharkov, Ukraine 61168  

*Corresponding Author E-mail: rukhmakovaolga@gmail.com

 

ABSTRACT:

Objectives: to develop identification and quantitative definition methodic of active substances of the ointment “Allergolic”. Materials and Methods: the development of identification and quantitative definition methodic of licorice root dry extract and terbinafine hydrochloride in the experimental series of the ointment “Allergolic” was carried out by HPLC method. To determine the glycyrrhizin acid (GA) used the modification of the procedure described in the State Pharmacopoeia, validation was carried out only for proof of specificity. The method for determining terbinafine hydrochloride has been validated according to the «Specificity» and «Linearity» indices. To confirm the presence of lavender essential oil in the medicine, its identification and quantitative determination using the gas chromatography method was performed in accordance with the requirements of the State Pharmacopoeia. Results and Discussions: based on the conducted research we have developed identification and quantitative definition methodic of active substances of the ointment “Allergolic”, description of which is given in the article. Conclusion: using the HPLC method, an identification was made and the content of GA and terbinafine hydrochloride in the medicine was determined. The content of GA was 13.12 mg, the content of terbinafine hydrochloride - 5.07 mg per gram of the ointment. The methodic of identification and quantitative determination of lavender essential oil in the ointment by the gas chromatography method was proposed. Its content ranged from 4.5 mg to 5.75 mg.

 

KEYWORDS: ointment, development, identification, quantitative definition, methodic, active substances.

 

 


INTRODUCTION:

Currently, the problem of the treatment of allergic skin diseases is of particular importance in connection with the increasing number of patients and the increased prevalence of severe forms of dermatitis, difficult to treat [1-3].

 

Quite often, allergic dermatitis is complicated by secondary fungal infection. The main pathogens of this form of disease are the various types of Trichophyton, Microsporum, as well as several species of Epidermophyton, combined under the general name of dermatophytes. In addition, there are lesions of the skin with fungi of the genus Candida [4, 5].

 

 

Taking into account the existing approaches to the treatment of allergic dermatitis complicated by fungal infection, it should be noted that in the development of medicines for their therapy, they must have anti-allergic, anti-inflammatory and antifungal effects [6].

 

Today, more and more dermatologists recommend using in the treatment of skin diseases medicines based on natural plant components, the benefits of which consist of a fairly wide therapeutic effect and virtually complete absence of side effects [7].

 

Thus, at the National University of Pharmacy, a dermatological ointment under the conventional name “Allergolic” with licorice root dry extract, terbinafine hydrochloride and essential oil of lavender is created [8].

 

The objective of this work is to develop identification and quantitative definition methodic of active substances of the ointment “Allergolic”.

 

MATERIALS AND METHODS:

The development of identification and quantitative definition methodic of glycyrrhizin acid (GA) of licorice root dry extract and terbinafine hydrochloride in the medicine was carried out on the Varian ProStar analytical chromatograph (ProStar 210 pump; ProStar 330 spectrophotometric detector; ProStar 410 autosampler with 20 μl dosing loop volume), and Columns Nucleosil 100-3C18100*4.6 with pre-colon and Microsorb 100*5C8250*4.6.

 

The following solvents and reagents were used: acetonitrile “gradient grade” (Sigma-Aldrich), water for chromatography (Millipore Direct-Q5), acetic acid (chemically pure), ammonia water (chemically pure), trifluoroacetic acid (chemically pure). The standard samples that were used: the Pharmacopoeia Standard Sample of monoammonium glycyrrhizate, the working standard sample of the licorice root dry extract, the working standard sample of terbinafine hydrochloride.

 

The development of identification and quantitative definition methodic was carried out using the experimental series of the ointment “Allergolic” and several placebo (without licorice root dry extract and terbinafine hydrochloride; without licorice root dry extract; without terbinafine hydrochloride) by HPLC method.

 

The first stage in the development of methodic for the determination of GA and terbinafine hydrochloride was to determine the requirements for the selection of active components from the matrix consisting of an emulsion ointment base (soybean oil, emulsifier No. 1, isopropyl myristate, and propylene glycol) and essential oil of lavender. It was necessary to propose a methodic for sample preparation, which would ensure the complete release of active substances and prevent the matrix components from being sampled. As a solvent for the determination of GA was chosen an aqueous ammonia solution, capable of forming an easily soluble monoammonium glycyrrhizate with it, and for the determination of terbinafine hydrochloride - a solution of hydrochloric acid.

 

To prevent the components of the oil emulsion from entering the sample, after dispersing the suspension, the solution was centrifuged for 5 minutes at 5000 min-1 and filtered through a membrane filter with a pore size of no more than 0.45 microns.

 

To determine the GA used the modification of the procedure described in the State Pharmacopoeia, validation was carried out only for proof of specificity.

 

The method for determining terbinafine hydrochloride has been validated according to the «Specificity» and «Linearity» indices.

 

To confirm the presence of lavender essential oil in the medicine, its identification and quantitative determination using the gas chromatography method was performed in accordance with the requirements of the State Pharmacopoeia.

 

Studies were conducted based on the State scientific-research laboratory of National University of Pharmacy for medicinal substances quality control.

 

RESULTS:

After making changes in the pharmacopoeial methodic for sample preparation in determining the GA in the ointment “Allergolic”, the methodic is proposed as follows.

 

Sample mixture. Mix 25 ml of ammonia concentrated solution and 975 ml of water.

 

Mobile phase. Mix 55 ml of acetic acid, 335 ml of acetonitrile and 610 ml of water.

 

Test solution. 1.0 g of the medicine place in a 100 ml volumetric flask, add 50 ml of the sample mixture, sonicate until complete dispersion of the sample, bring to the mark with the same solvent and centrifuge. Filter the supernatant liquid through a nylon membrane filter with a pore size of 0.45 microns, rejecting the first portions of the filtrate.

 

Comparison solution. Approximately 50 mg of Pharmacopoeia Standard Sample of monoammonium glycyrrhizate place in a 50 ml volumetric flask, dissolve in a sample mixture; bring to the mark with the same solvent. 5.0 ml of the resulting solution adjust to a volume of 50 ml and filter through nylon membrane filter with a pore size of 0.45 microns.

 

Chromatography is carried out on a liquid chromatograph with UV-detector under the following conditions: a column 0.10 m×4.6 cm filled with silica gel for chromatography, with a particle size of 3.0 microns, with a pre-column (Nucleosil 100-3C18100*4.6); speed of the mobile phase: 1.5 ml/min; column temperature: 25 °C; detection at wavelength: 254 nm; injection volume: 20 μl.

 

The content of GA (X), in mg per gram of the ointment, is calculated by the formula:

 

 

 

where: S1 – average value of peak area of the test substance, calculated from the chromatogram of the test solution; S0 – average value of peak area of the test substance, calculated from the chromatogram of the comparison solution; m1 – weight of the sample of the medicine, g; m0 – weight of Pharmacopoeia Standard Sample of monoammonium glycyrrhizate, mg; P – content of the active substance in Pharmacopoeia Standard Sample of monoammonium glycyrrhizate, %; 823 – molecular weight of GA; 840 – molecular weight of monoammonium glycyrrhizate (without taking into account crystallization water).

 

The content of GA should be at least 7.5 mg per gram of the ointment.

 

Identification:.

The maintenance time of the main peaks on the chromatograms of the test solution obtained with the quantitative determination of GA coincides with the maintenance periods of peaks on the chromatograms of the solution of Pharmacopoeia Standard Sample of monoammonium glycyrrhizate (Fig. 1).

 

The methodic of determination of terbinafine hydrochloride in the ointment “Allergolic” is proposed in the following form.

 

Sample mixture. Mix 900 ml of a 0.1 M solution of hydrochloric acid and 100 ml of acetonitrile.

 

Mobile phase. Mix 10 ml of trifluoroacetic acid, 720 ml of acetonitrile and 270 ml of water.

 

Test solution. Approximately 0.3 g (precise weight) of the medicine place in a 25 ml volumetric flask, add 15 ml of the sample mixture, sonicate until complete dispersion of the sample, bring to the mark with the same solvent and centrifuge. Filter the supernatant liquid through a nylon membrane filter with a pore size of 0.45 microns, rejecting the first portions of the filtrate.

 

Comparison solution. Approximately 50 mg of the

standard of terbinafine hydrochloride place in a 100 ml volumetric flask, dissolve in the sample mixture; bring to the mark with the same solvent. 5.0 ml of the resulting solution adjust to a volume of 50 ml and filter through a nylon membrane filter with a pore size of 0.45 microns.

 

Chromatography is carried out on a liquid chromatograph with UV-detector under the following conditions: a column 0.25 m×4.6 cm filled with silica gel for chromatography, with a particle size of 5.0 microns, with a pre-column (Microsorb 100x5С8250x4.6); speed of the mobile phase: 1.3 ml/min; column temperature: 30 °C; detection at wavelength: 283 nm; injection volume: 20 μl.

 

The content of terbinafine hydrochloride (X), in mg per gram of the ointment, is calculated by the formula:

 

 

where: S1 – average value of peak area of terbinafine hydrochloride, calculated from the chromatogram of the test solution; S0 – average value of peak area of terbinafine hydrochloride, calculated from the chromatogram of the comparison solution; m1 – weight of the sample of the medicine, g; m0 – weight of standard of terbinafine hydrochloride, mg; P – content of the active substance in standard of terbinafine hydrochloride, %.

 

The content of terbinafine hydrochloride should be from 4.5 mg to 5.5 mg per gram of the ointment.

 

Identification.:

The maintenance time of the main peaks on the chromatograms of the test solution obtained with the quantitative determination of terbinafine hydrochloride coincides with the maintenance periods of peaks on the chromatograms of the solution of standard of terbinafine hydrochloride (Fig. 2).

 

The methodic of determining the essential oil of lavender in the ointment “Allergolic” is proposed in the following form.

 

Test solution. Approximately 30.0 g (precise weight) of the medicine place in a 500 ml round-bottomed flask, add 300 ml of 0.5 % sodium chloride solution and attach to the apparatus for determining the essential oils.

 

Into the receiver place 2.0 ml of the internal standard solution, heat the sample flask to boil and continue distillation for 60 minutes. The resulting detachment is then passed through 0.5 g of anhydrous sodium sulfate, collecting the resulting filtrate in a 5 ml volumetric flask, washing the filter with 2 ml of toluene, combining the wash solution with the filtrate in the flask, adjusting the solution to the toluene, and mixing.

 

Comparison solution. Approximately 150 mg (precise weight) of essential oil of lavender place in a 5 ml volumetric flask, add 2.0 ml of internal standard solution, adjust the volume of the solution with toluene to the mark and mix.

 

Internal standard solution. 0.50 ml of heptanol-1 place in a 25 ml volumetric flask, add 20 ml of toluene, mix, adjust the volume of the solution with toluene to the mark and mix.

 

Comparison internal standard solution. 2.0 ml of the internal standard solution place in a 5 ml volumetric flask, adjust the volume of the solution with toluene to the mark and mix.

For 1 μl of the test solution, comparison solution and internal standard solution is chromatographed on a gas chromatograph with a flame-ionization detector under the following conditions: a quartz capillary column, 50 m x 0.25 mm in diameter with a stationary phase of FFAP; a layer thickness of 0,33 microns; the temperature of the column is programmed: 70 °C is maintained for 5 minutes, then the temperature is raised at a rate of 3 °C/min to a temperature of 220 °С and maintain for 10 minutes; unit temperature - sample injection - 230 °C; temperature of the detector - 240 °С; speed of carrier gas (nitrogen) - 0.9 ml/min; flow separation - 1:50.

 

The content of essential oil of lavender (X), in mg per gram of the ointment, is calculated by the formula:

,

 

where: Bi – the average value of the ratio of the sum of the areas of components of the essential oil of lavender to the peak area of the internal standard, calculated from the chromatograms of the test solution; Bo – the average value of the ratio of the sum of the areas of components of the essential oil of lavender to the peak area of the internal standard, calculated from the chromatograms of the comparison solution of essential oils of lavender; mo – weight of lavender essential oils, in mg; m – weight of the sample of the medicine, in g.

 

The content of essential oil of lavender should range

from 4.5 mg to 5.75 mg.

 

Identification of the essential oil of lavender in the

developed medicine was carried out at the main peaks of linalool and linalool acetate, the retention times of which should coincide with the maintenance times of the peaks of linalool and linalool acetate on the chromatograms of the comparison solution (Fig. 3).

 

DISCUSSION:

Using the developed methodic, the content of GA and terbinafine hydrochloride was controlled in the experimental series of the medicine “Allergolic”. The content of GA was 13.12 mg per gram of the ointment, the content of terbinafine hydrochloride was 5.07 mg per gram of the ointment.

 

Typical chromatograms obtained in the determination of GA are given in Fig. 1.

 

Typical chromatograms obtained in the determination of terbinafine hydrochloride are given in Fig. 2.


 

          

Fig. 1. Chromatogram of the comparison solution (GA)                         Chromatogram of the test solution (GA)

 

                

Fig 2. Chromatogram of the comparison solution (terbinafine hydrochloride) Chromatogram of the test solution (terbinafine hydrochloride)


To demonstrate the specificity of the methodic for determining GA and terbinafine hydrochloride, solutions of placebo were prepared and analyzed. Based on the conducted researches it was established that the determination of these active substances in the ointment according to the proposed methodic does not interfere with the placebo components.

 

For the proposed methodic for determining terbinafine hydrochloride, linearity was checked. When quantified, the application range should be at least 80 % to 120 % of the selected concentration of the test substance.

 

Nine model solutions were prepared, in which the concentration of terbinafine hydrochloride changed monotonically within the range of use. For model solutions using the least squares method, calculates the linear dependency parameters: the free term a (│a│ = 0.14), the residual standard deviation (S0 = 0.17), the correlation coefficient (r > 0.9999). The eligibility criteria are fulfilled (for our conditions, they are │a│≤ 5.1, S0 ≤ 1.69, r > 0.99236).

 

Thus, the proposed methodic in the range of concentrations from 80 % to 120 % of the nominal value for terbinafine hydrochloride is linear.

 

Using the developed methodic for determining the essential oil of lavender in the ointment “Allergolic”, its content in the experimental series of the medicine was monitored. Its content ranged from 4.5 mg to 5.75 mg.

Chromatograms of test solution, comparison solution and comparison internal standard solution are given in Fig. 3.

 

 

Fig. 3. Chromatograms of test solution, comparison solution and comparison internal standard solution

 

CONCLUSIONS:

1.    Methodic of identification and quantitative determination of active substances of dermatological ointment “Allergolic” are developed.

2.    Using the HPLC method, an identification was made and the content of GA and terbinafine hydrochloride in the medicine was determined. The content of GA was 13.12 mg, the content of terbinafine hydrochloride - 5.07 mg per gram of the ointment.

3.    For the methodic of determining the GA, as one of the validation characteristics, specificity is determined. The methodic for determining terbinafine hydrochloride has been validated according to the “Specificity” and “Linearity” indices.

4.    The methodic of identification and quantitative determination of lavender essential oil in the ointment by the gas chromatography method are proposed. Its content ranged from 4.5 mg to 5.75 mg.

 

REFERENCES:

1.     Allergology and Immunology: National leadership; Moscow, GEOTAR-Media, 2009: 649.

2.     Kogan B.G., Verba E.A. New European approaches to the treatment of resistant forms of allergodermatosis. Ukr. J. of Dermatol., Venereol., Cosmetol. 2013; 1(48): 137-143.

3.     Ryznichenko N.Yu. Allergic contact dermatitis: contemporary notions of treatment based on the review of scientific literature. Act. Quest. of Pharm. and Med. Sci. and Pract. 2013; 3(13): 69-72.

4.     Tanei R., Katsuoka К. Clinical analyses of atopic dermatitis in the aged. J. Dermatol. 2008; 35: 562-569.

5.     Sehgal V.N., Srivastava G., Dogra S. Atopic dermatitis: current options and treatment plan. Skinmed. 2010; 8 (6): 335-344.

6.     Shishkina A.V. Analysis of the domestic pharmaceutical market of soft medicinal forms. Pharmacy. 2013; 1: 28-30.

7.     Medicinal products from plants (index): Textbook for students of Pharmacy Faculty; Irkutsk, IGMU, 2011: 74.

8.     Rukhmakova О.А., Yarnykh T.G. Development of laboratory technology of ointment “Allergolic”. Pharm. J. 2014; 5: 41-47.

 

 

 

 

Received on 15.07.2018          Modified on 05.09.2018

Accepted on 30.09.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(12): 5332-5336.

DOI: 10.5958/0974-360X.2018.00970.8