INTRODUCTION:

Pain that occurs during human life activities is one of the major problems of medicine. However, acute pain can reasonably be entertained a symptom of a disease or injury, chronic and recurring pain is a specific health problem, the disease itself^1,2. Worldwide, it has been estimated that one in five adults suffer from pain and that one in ten adults is diagnosed with chronic pain annually. Although pain affects all populations, regardless of age, gender, income, ethnicity, or geographical location, it is unevenly distributed throughout the world^1,3. Those who experience pain can experience acute, chronic or intermittent pain, or a combination of them. The four main causes of pain are cancer, osteo and rheumatoid arthritis, operations and injuries, as well as problems with the spine, which makes the etiology of pain a complex, transdisciplinary matter. The pain is accompanied by many serious consequences, including depression, inability to work, disruption of social relations and suicidal thoughts.

Of those living with chronic pain, the average exposure time is 7 years^3-5. The treatment of pain is a rather difficult task, due to the variety of its causes and subjectivity of sensations. Currently, pharmaceutical companies produce a huge number of painkillers, often differing only in their trade name, while their analgesic effect may practically not differ from each other^6,7.

Drugs of the non-narcotic analgesics and antispasmodic group are often used to alleviate the pain. The non-narcotic analgesics stop the pain and are anesthetizing, anti-inflammatory, antipyretic. The antispasmodics help restore blood circulation. Non-narcotic analgesics in their pharmacological properties differ significantly from narcotic analgesics. These drugs are significantly inferior to narcotic painkillers by the strength of the analgesic effect. Their effect is manifested mainly in inflammatory pains (arthritis, myositis, neuralgia, etc.). The advantage of these drugs is the lack of narcotic effect, which gave reason to widely use them in ambulatory practice^6-8. Effective and promising approach in the treatment of pain is the creation and use of combination preparations comprising fixed combinations of active pharmaceutical ingredients (APhI) from different pharmacological groups like non-narcotic analgesics, spasmolytics and others. Since, the source of pain is different factors, the pain can be caused by spasm or become the result of more serious problems. The combination of active pharmaceutical ingredients of different pharmacological groups expands the mechanisms of analgesic action, reduces the frequency of side effects^5-8.

In this regard, at the Department of Industrial Technology of Medicines of the Tashkent Pharmaceutical Institute, together with “SAMO” LLC (Tashkent), the composition and technology of a multi-component analgesic drug was developed in the form of tablets under the conditional name “Analfenon”. The composition of the tablets as APhI includes metamizole sodium (non-narcotic analgesic), drotoverin hydrochloride (antispasmodic), diphydramine hydrochloride (antihistamine), as well as auxiliary substances approved for medical use: potato starch, calcium stearate.

Given the above, an urgent task is to increase the effectiveness of existing methods of analysis of analgesic drugs. The development of quality control methods for new painkillers is mandatory for implementation. Among modern methods of pharmaceutical analysis, optical control methods occupy an important place, which are widely used both for the purposes of quantitative determination and for the control of purity and identification of drugs^9-14. Spectrophotometric analysis is one of the widespread optical research methods, which today is widely used to assess the quantitative content of medicinal substances¹¹. The aim of this work is to develop methods for the quantitative content of APhIs of the new combined analgesic drug "Analfenon" in the form of tablets.

MATERIAL AND METHODS:

Chemicals and reagents:

Metamizole sodium (Analgin) was purchased from Hebei Jiheng (Group) Pharmaceutical Co., Ltd, No. 368 Jianshe Street, Hengshui City, Hebei Province, 053000 P.R. China. Drotaverine hydrochloride and diphenhydramine hydrochloride were purchased from Xi’an Accenture Biological Technology Co., Ltd, Room703, Unit 1, No.1, Weilanchuncheng Residential Quarter, Taoyuan North Road 355, Lianhu District, Xi'an.

The objects of our research were metamizole sodium, drotoverin hydrochloride, diphydramine hydrochloride (Fig.1). All reagents and chemicals used were of analytical or pharmaceutical grade and all solutions were prepared fresh daily. Distilled water was used throughout the investigation.

Fig. 1: Structural formula of Metamizole sodium (structure I), Drotoverin hydrochloride (structure II), Diphenhydramine hydrochloride (structure III).

Metamizole sodium ([(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl) methylamino] methanesulfonic acid sodium salt (in the form of a monohydrate)) is a pyrosolone derivative. White or almost imperceptible yellowish-white crystalline powder, quickly decomposes under the influence of moisture. Easily soluble in water (1: 1.5), when dissolved in water forms a solution with a pH of 6-7.5. It is soluble in ethanol (1: 60-1: 80), practically insoluble in ether, chloroform, acetone. Metamizole sodium belongs to the following therapeutic groups: non-steroidal anti-inflammatory drugs (NSAIDs), non-narcotic analgesics. ATC code N02BB02^10,11.

Drotverin hydrochloride 1- [3,4-Diethoxyphenyl) methylene] -6,7-diethoxy-1,2,3,4-tetrahydroisoquinoline (as hydrochloride) is a crystalline powder of a light yellow color, odorless. Soluble in water and alcohol. Myotropic antispasmodic. In chemical structure and pharmacological properties it is close to papaverine, but has a stronger and longer lasting effect^9,11.

Diphenhydramine hydrochloride 2- (diphenylmethoxy) -N, N-dimethylethanamine (and in the form of hydrochloride) - a white fine crystalline powder with a bitter taste, causing onemenia in the oral cavity. Hygroscopic. Soluble in water (1g in 1ml), alcohol (1g in 2ml), chloroform (1g in 2ml), acetone (1g in 50ml). Very insoluble in benzene and ether. The pH of a 1% aqueous solution is 5.5¹¹.

Preparation of a solution of a working standard sample (WSS) of metamizole sodium:

About 200mg (accurately weighed) of metamizole sodium (BP CRS, USP RS) is placed in a 100ml volumetric flask, 80ml of water are added, dissolved, the solution volume is adjusted to the mark and stirred. 1.0 ml of the resulting solution was placed in a volumetric flask with a capacity of 100ml, the volume of the solution was adjusted to the mark with water and stirred (0.02mg/ml). The solution is used freshly prepared.

Preparation of a solution of a working standard sample (WSS) drotaverine hydrochloride:

About 160mg (accurately weighed) of drotaverine hydrochloride, in terms of 100%, the substance is placed in a 100ml volumetric flask, 20ml of a 0.1mol/l hydrochloric acid solution is added, and it is heated in a water bath with shaking until dissolved. It is cooled to room temperature, the solution volume is adjusted with a 0.1mol/l hydrochloric acid solution to the mark and stirred.

1ml of the resulting solution was placed in a 100ml volumetric flask, the volume of the solution was adjusted with 0.1mol/l hydrochloric acid solution and the mark and mixed. The shelf life of the solution is 1 day.

Preparation of a solution of mercury oxide acetate:

5g of mercury oxide acetate is placed in a 100ml volumetric flask and dissolved in warm glacial acetic acid. After cooling, the volume of the solution was adjusted to the mark with glacial acetic acid. The mercury content of oxide acetate is not less than 97.0%.

Methods:

The study of the physical and chemical properties of the resulting compounds were carried out according to the methods presented in the European Pharmacopeia [13,14]. Analysis of metamizole sodium and drotaverine hydrochloride in a tablet was carried out on a dual-beam spectrophotometer Ultraviolet (UV)-1800 from Shimadzu (Japan).

Quantitative determination of API of metamizole sodium:

About 500mg (accurately weighed) of the powder of the crushed tablets is placed in a volumetric flask with a capacity of 200ml, shaken with 100ml of purified water for 5 minutes, the volume of the solution is adjusted to the mark with water and mixed, settled for 5 minutes and filtered through a blue paper filter tape".

1ml of the resulting solution is placed in a 100ml volumetric flask, the volume of the solution is adjusted to the mark with water, mixed, and the optical density of the obtained solution is determined on a spectrophotometer at a wavelength of 229nm in a cuvette with a layer thickness of 10mm (test solution). As a comparison solution, water is used.

At the same time, the optical density of the solution of the WSS metamizole sodium is measured. The content of metamizole sodium, (X) in one tablet, in milligrams, is calculated by the formula:

D₁ – is the optical density of the test solution;

D₀ – is the optical density of the solution of the WSS metamizole sodium;

а₀ – is the mass of a portion of the WSS of metamizole sodium, in mg;

Р – is the content of metamizole sodium in WSS, in %;

a₁ – is the mass of the sample, in mg;

b – is the average weight of one tablet, in mg.

The content of С₁₃H₁₆N₃NaO₄S × H₂O (metamizole sodium) should be from 380 to 420mg, based on the average weight of one tablet.

Quantitative determination of API of drotoverin hydrochloride:

About 280mg (accurately weighed) of the powder of the crushed tablets is placed in a 100ml volumetric flask, 50 ml of a 0.1mol/l hydrochloric acid solution is added, and it is heated in a water bath with shaking for 20 minutes. Cool to room temperature, adjust the volume of the 0.1 mol/l hydrochloric acid solution to the mark, and mix. The solution is filtered through a paper filter, discarding the first portions of the filtrate.

1ml of the resulting solution was placed in a volumetric flask with a capacity of 50ml, the solution volume was adjusted with 0.1mol/l hydrochloric acid solution to the mark and stirred. The optical density of the resulting solution is measured at the absorption maximum at a wavelength of 353nm in a cell with a layer thickness of 10mm. At the same time, the optical density of the solution of the WSS of drotaverine hydrochloride is measured. The measurement of optical density is carried out relative to 0.1mol/l of a solution of hydrochloric acid.

The content of drotaverine hydrochloride in one tablet, in milligrams (X), is calculated by the formula:

D₁ – is the optical density of the test solution;

D₀ – is the optical density of the solution of the WSS drotaverin hydrochloride;

а₀ – is the mass of a sample of WSS drotaverine hydrochloride, in mg;

a₁ – is the mass of the sample, in mg;

b – is the average weight of one tablet, in mg.

The content of С₂₄H₃₁NO₄S × HCl (drotaverine hydrochloride) in one tablet should be from 38mg to 42 mg.

Quantitative determination of API diphenhydramine hydrochloride:

About 1000mg (accurately weighed) of the powder of the crushed tablets is placed in a flask with a capacity of 50ml, 10ml of chloroform are added and shaken for 10 minutes, then filtered through a paper filter. The flask and the filter residue are washed 2 times with 2ml of chloroform and attached to the filter. The resulting chloroform extract was evaporated to dryness in a water bath. The resulting residue is dissolved in 10ml of anhydrous acetic acid, 5ml of a solution of mercury oxide acetate are added and triturated from a microburette with a 0.01mol/l solution of perchloric acid until a greenish-blue color, using a crystalline violet indicator.

Parallel conducted a control experiment. 1ml of a 0.01 mol/l perchloric acid solution corresponds to 2.918g of С₁₇H₂₁NO × HCl (diphenhydramine hydrochloride).

The content of С₁₇H₂₁NO × HCl (diphenhydramine hydrochloride) should be from 9 mg to 11 mg, based on the average weight of one tablet.

RESULT AND DISCUSSION:

All working standard samples (WSS) and test solutions of API was chromatographed 5 times. At the beginning of the experiment, a standard and test solution of metamizole sodium in water was prepared and the UV spectrum was studied in the region of 190-279nm, in a cuvette with a layer thickness of 1cm.

The ultraviolet spectrum of metamizole sodium in the RSO and test solution is shown in Fig.2-3.

Fig. 2: Chromatogram of UV spectrum of metamizole sodium WSS solution, 229 nm

Fig. 3: Chromatogram of UV spectrum of the “Analfenon” test solution in the purified water, 229 nm

From the obtained spectrum it was determined that the maximum absorption of metamizole sodium is observed at a wavelength of 229 nm, and at a wavelength of 253 nm - the minimum absorption. These results were used by us in further studies.

For research, the WSS solution and a test solution of drotaverine hydrochloride with a 0.1 mol/l hydrochloric acid solution were prepared. Subsequently, a UV spectrum was obtained on a spectrophotometer in the wavelength range 300-360 nm of the test solution shown in Fig.4-5.

Fig. 4: Chromatogram of UV spectrum of Drotaverine hydrochloride WSS solution, 353 nm

Fig.5: Chromatogram of UV spectrum of the “Analfenon” test solution in the 0.1 mol/l hydrochloric acid solution, 353 nm

The UV spectrum of drotaverine hydrochloride has two absorption maxima, the first at a wavelength of 303nm, and the second at a wavelength of 353nm. The obtained spectra of a solution of drotaverine hydrochloride in the UV region are characteristic and specific for the study drug and can be used as one of the methods for drug identification.

The presented chromatograms (Fig.2-5) of standard samples of metamizole sodium, drotoverin hydrochloride and the analyzed solution of “Analfenon” tablets, which contain metamizole sodium, drotoverin hydrochloride, diphenhydramine hydrochloride as APIs, confirm the identity of the results and confirm the presence of active components in the developed tablets. The components of the tablet do not affect the identification and quantification of the active substance in the drug. Using the developed technique, the content of metamizole sodium, drotoverin hydrochloride, diphenhydramine hydrochloride in 5 samples was determined. The results of quantitative determination and their metrological characteristics are presented in Table 1.

Table 1: Metrological characteristics of the method for determining the active pharmaceutical ingredients of “Analfenon” tablets (n=5)

№	Weight, g	Defined		Metrological characteristics
№	Weight, g	g	%	Metrological characteristics
Metamizole sodium
1.	0,5058	0,415	103,75	P=95,00, X_ср=101,75 f=4, S²=0,84 S=0,93, S_x=0,35 E=0,88
2.	0,5102	0,392	98
3.	0,5075	0,418	104,5
4.	0,4918	0,402	100,5
5.	0,5167	0,408	102
Drotoverin hydrochloride
1.	0,2939	0,0413	103	P=95,00; X_ср=100,5 f=4; S²=0,82 S=0,91, S_x=0,34 E=0,86
2.	0,3098	0,0392	98
3.	0,2910	0,0396	99
4.	0,2779	0,0408	102
5.	0,2786	0,0402	100,5
Diphydramine hydrochloride
1.	0,9823	0,0095	95	P=95,00, X_ср=99 f=4, S²=0,0002 S=0,002; S_x=0,001 E=2,35
2.	1,0550	0,0098	98
3.	0,9560	0,0102	102
4.	1,1543	0,0093	93
5.	1,0733	0,0107	107

The content of С₁₃H₁₆N₃NaO₄S × H₂O (metamizole sodium) should be from 380 to 420mg, and С₂₄H₃₁NO₄S × HCl (drotaverine hydrochloride) should be from 38mg to 42mg, taking into account the norms of permissible deviations (± 10%) and taking into account the average weight of one tablet.

The research results (Tab.1) showed that the content of APIs, namely metamizole sodium, drotaverine hydrochloride and diphenhydramine hydrochloride (calculated on 100% substance) in the combined dosage form of tablets is within the permissible limits. The developed methods for the identification and quantitative content of APIs in “Analfenon” tablets are included in the document “Drug Quality Control Methods” combined analgesic tablets “Analfenon”.

According to the data given in the table, the content of metamizole sodium in tablet formulations is from 0.392 to 0.418g, the content of drotaverine hydrochloride is from 0.0392 to 0.0413g, the content of diphenhydramine hydrochloride is from 0.0093 to 0.0107g and is within normal limits^15,16.

CONCLUSION:

Therefore, based on the data obtained, the following conclusions can be drawn. For the first time, a technique has been developed for the identification and quantification of the active pharmaceutical ingredients (metamizole sodium, drotoverin hydrochloride and diphenhydramine hydrochloride) of combined “Analfenon” tablets by spectrophotometric and titrimetric methods. The proposed methods for the analysis of analgesic combined tablets provide reliable and well reproducible results. The developed methods formed the basis of regulatory documentation for a new analgesic drug - Analfenon tablets.

CONFLICT OF INTEREST:

There is no conflict of interests to be reported.

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Received on 07.04.2020 Modified on 11.05.2020

Research J. Pharm. and Tech. 2020; 13(12):5749-5753.

DOI: 10.5958/0974-360X.2020.01001.X