Quantitative determination of albendazole forced degradation percentages by densitometric thin layer chromatographic method

 

Mohammed Khanji¹, Ghoufran Kawas¹, Mohammad Haroun¹, Mouhammad Abu Rasheed², Amir Alhaj Sakur²

¹Pharmaceutical Chemistry and Quality Control Dept., Faculty of Pharmacy, Al Andalus University,

Tartous, Syria

²Department of Food and Analytical Chemistry, Faculty of Pharmacy, University of Aleppo, Syria.

 

ABSTRACT:

Simple densitometric thin layer chromatographic method has been applied in this study to proceed forced degradation test of albendazole by subjecting drug to acidic, alkaline, oxidative, thermal, and photolytic conditions, and determine the degradation percentages of albendazole in these conditions after 1, 2, 4, and 6 hrs.

Two different mobile phases have been used in this study to separate degradation products. chloroform:diethyl ether:glacial acetic acid (75:12.5:12.5, v/v) as mobile phase 1, and dichloromethane:methanol (90:10, v/v) as mobile phase 2. Silica gel GF-254 TLC-plates were used, and densitometric Area under curve (AUC) and AUC percentages were measured. Densitometric measurements showed that albendazole is unstable in all stress conditions but in different percentages, with the least susceptibility have been reported with acid condition (just 6.13% of drug lost after 6 hrs. of incubation). Acidic medium caused reversible degradation with homogeneity in the structures of degradation products, Unlike Alkaline medium which showed heterogeneity in structures detected through the change in the number and Rf-values of degradation spots against time. Both mobile phases were suitable for detecting albendazole degradation percentages under different conditions, as quantitative measurements were closely similar for the two phases, but mobile phase 1 was better to differentiate base-induced degradation products and mobile phase 2 was better for photolytic-induced degradation products.

 

 

INTRODUCTION:

Albendazole is a broad-spectrum anti-helminthic agent of the benzimidazole type, and used to treat certain tapeworm infections, such as neurocysticercosis and hydatid disease¹.

 

Determination of impurities in active pharmaceutical ingredients is considered an important test to verify the quality of manufacturing processes and storage efficiency.

 

As the presence of impurities in above Pharmacopoeial limits may significantly affect the effectiveness and safety of the final product. According to The International Conference on Harmonization guidelines for impurities in drug substance (ICH Q3A-R2)² and drug product (ICH Q3B-R2)³, impurities defined as any component of the new drug substance which is not the chemical entity defined as the new drug substance, or any component of the drug product which is not the chemical entity defined as the drug substance or an excipient in the drug product. These impurities are classified in three main categories, which are inorganic impurities, organic impurities, and residual solvents. Inorganic impurities may be derived from the manufacturing process and are normally known and identified as inorganic salts, heavy metals, catalysts, filter aids and charcoal. Organic impurities include starting raw materials, by-products, degradation products, and organic reagents or ligands. Residual solvents normally arise from solvents used during the synthesis processes^4-6. These impurities have acceptable limits according to pharmacopoeias, which are estimated in different methods, mostly chromatographic methods, in so-called limit tests^7-8.

 

Although Limit tests in most recent pharmacopoeial monographs are performed by liquid chromatographic methods, thin layer chromatography is still incorporated for many pharmaceutical ingredients limit tests due to its low cost and high sensitivity characteristics^7-8.

 

It is well known that organic impurities are the most important type due to their wide diversity of chemical structures and almost unpredictable toxicity profile. In particular, degradation products resulting from storage or formulation to different dosage forms or aging are a common source of organic impurities in the medicines⁵. The definition of degradation product in the ICH guidelines is a molecule resulting from a chemical change in the substance brought about by overtime or due to the action of light, temperature, pH or water or by reaction with excipient and/or the intermediate container closure system^2-3. This kind of impurities is verified by stress testing. According to Q1A-R2 ICH guideline⁹, stress testing defined as studies undertaken to elucidate the intrinsic stability of the drug substance. Such testing is part of the development strategy and is normally carried out under more severe conditions than those used for accelerated testing. Methods for stress stability studies can be developed which measure the amount of drug remaining, the amount of drug lost (or the appearance of degradation products), or both. These tests are essential to determine the optimal conditions appropriate for the manufacturing, packaging, storage, and pharmaceutical formulation of the drug substance to ensure the full effectiveness and safety of the final product¹⁰.

 

Albendazole has been determined by TLC-densitometric and chemometric methods in the presence of its alkaline degradation products¹¹. Ljubas D et al¹² have studied degradation possibilities of albendazole by UV-Based Advanced Oxidation Processes to determine the conditions that result in the highest rate of degradation for environmental reasons. In addition, Patel AK et al¹³ estimated the degradation percentages of albendazole in forced conditions by RP-HPLC method, and this study showed that albendazole degraded in acid, alkali, oxidation and dry heat while it was found stable in photolytic condition. While Ragno G et al. studied only photo- and thermal- degradation of albendazole and other benzimidazole anti-helminthics in solid and ethanolic solutions by monitoring their degradation products by HPLC, and identified two major degradation products formed by carbamic group hydrolysis by GC-MS¹⁴. Another research group has developed HPLC method to determine and separate process related impurities of albendazole in bulk drugs¹⁵.

 

To the best of our knowledge, there is no stress stability testing for albendazole using TLC-densitometric method until now. According to US. Pharmacopoeia, routine limit test for the related organic substances of albendazole is performed by simple thin layer chromatographic method.

 

The aim of this work is to incorporate the US. pharmacopoeial limit test procedure of albendazole⁸ and the method procedure developed by Attia KAS et al¹¹ to evaluate albendazole stability in different stress conditions, and determination of the degradation percentages quantitatively using UV-densitometric TLC method.

 

MATERIALS AND METHODS:

Apparatus:

Shimadzu “dual wavelength flying spot scanning” densitometer CS-9301 PC (Tokyo, Japan, 2000) (program version 2.00) was used for TLC plates scanning. Digital Water Bath of Heidolph Laborota 4001-Rotary Evaporator (Schwa Bach, Germany) was used to incubate solutions. UV-254 nm chamber was used for UV-forced degradation experiments. Pre-coated TLC plates, silica gel 60 GF-254 (20 × 20 cm) (Merck, Germany). Hamilton 25-μL micro-syringe (Germany) was used to apply samples on TLC plates. Glass TLC developing chamber (20 × 20 × 10 cm).

 

Materials and reagents:

Pharmaceutical grade Albendazole (99%) (supplied by Lyphar, Shaanxi, China). Methanol, isocratic HPLC grade (Scharlab S.L., Spain). Glacial acetic acid (Surechem products L.T.D., England) Sodium hydroxide, analytical grade (Medex export Co., UK). Hydrochloric acid, analytical grade (May & Bayer L.T.D., England). Hydrogen peroxide 35%, analytical grade (Merck, Germany).

 

Standard stock solution of albendazole:

Standard Stock solution of albendazole (5 mg/mL) was prepared by dissolving 250 mg of albendazole in least amount of glacial acetic acid and complete to 50 mL with methanol. Two 1 μL-spots from stock solution were applied to two separate TLC plates described below (see procedure for chromatographic separation).

 

Procedure for degraded samples preparation:

1-    Acid-induced degradation:

In four 5 mL-volumetric flask, 1 mL of the standard stock solution of albendazole was added to 1 mL of HCl (0.1N). The prepared mixtures were incubated in a water bath at 60°C for 1, 2, 4, 6 hrs. Then 1 mL of NaOH (0.1N) was added separately to each flask and the volumes were completed to the mark with methanol. The concentration of the end solution is 1 mg/mL. Two 5 μL-spots from each solution were applied to two separate TLC plates described below (see procedure for chromatographic separation).

2-    Base-induced degradation:

Standard solution for the alkaline degradation of albendazole was prepared by dissolving 31.25 mg of albendazole in 10 mL NaOH (0.1N), then the volume was completed to 25 mL in a volumetric flask.

4 mL of the above prepared solution were transferred separately to four 5 mL-volumetric flask and incubated in a water bath at 60°C for 1, 2, 4, 6 hrs. Then the volumes were completed to the mark with glacial acetic acid. The concentration of the end solution is 1 mg/mL. Two 5 μL-spots from each solution were applied to two separate TLC plates described below (see procedure for chromatographic separation).

3-    Hydrogen peroxide-induced degradation:

In four 5 mL-volumetric flask, 1 mL of the standard stock solution of albendazole was added to 1 mL of H₂O₂ (3%). The prepared mixtures were incubated in a water bath at 60°C for 1, 2, 4, 6 hrs. Then the volumes were completed to the mark with methanol. The concentration of the end solution is 1 mg/mL. Two 5 μL-spots from each solution were applied to two separate TLC plates described below (see procedure for chromatographic separation).

4-    Thermal degradation:

In four 5 mL-volumetric flask, 1 mL of the standard stock solution of albendazole were transferred separately and the flasks were incubated in a water bath at 80°C for 1, 2, 4, 6 hrs., Then the volumes were completed to the mark with methanol. The concentration of the end solution is 1 mg/mL. Two 5 μL-spots from each solution were applied to two separate TLC plates described below (see procedure for chromatographic separation).

5-    Photolytic UV-C degradation:

1 mL of the standard stock solution of albendazole were transferred to a quartz cuvette and exposed to ultra-violet light (254 n.m.) for 24 hrs. Then the volume was completed to 5 mL with methanol. The concentration of the end solution is 1 mg/mL. Two 5 μL-spots from solution were applied to two separate TLC plates described below (see procedure for chromatographic separation).

Procedure for chromatographic separation:

Analysis was performed on pre-coated 20 × 20 cm TLC aluminum silica gel 60 GF254 plates. Hamilton micro-syringe (25-μL) was used to apply samples as about 2 mm width spots. Plates were spotted 1.5 cm apart from each other and 1 cm apart from the bottom edge. The chromatographic tank was pre-saturated with the mobile phase for 20 min, then developed by ascending chromatography using two different mobile phases separately, chloroform:diethyl ether:glacial acetic acid (75:12.5:12.5, v/v) (MP-1) which is the mobile phase used in US. Pharmacopoeial limit test of albendazole, and dichloromethane: methanol (90:10, v/v) (MP-2) as obtained by the reference method¹¹. When the solvent front reached 10 cm, the plates were removed and air dried, detected in UV- chamber (254 nm) (figure-1) and scanned by CS-9301 Shimadzu densitometer. The parameters selected for all densitometric measurements were reported in table-1.

 

 

Figure-1: An example of TLC plates display under UV-chamber at 254 nm showed the spots resulted from degradation of albendazole in different stress conditions (O: oxidation ; H: heat ; A: Acid ; B: base) degraded spots after 4, and 6 hrs, respectively – control: spot of non-degraded albendazole solution.

 

RESULTS:

Two TLC-plates were used for each mobile phase used in this study. All spots were scanned by densitometer at 254 nm. The densitograms and results from densitometric measurements for control spots (figure-2, table-2), Acid degradation spots (figure-3, table-3), base degradation spots (figure-4, table-4), hydrogen peroxide degradation spots (figure-5, table-5), thermal degradation spots (figure-6, table-6), and photolytic UV-C degradation spots (figure-7, table-7) are all shown respectively.

 

Table-1: data parameters for all densitometric measurements proceeded

 

Table-2: densitometric measurements of spots from control stock solution

 

 

Figure-2: Densitograms of control stock solution by using (a)-mobile phase-1, and (b)-and mobile phase-2

 

 

Figure-3: Densitograms of Acid-forced degraded solutions after 1, 2, 4, 6 hrs. by using (a)-mobile phase-1, and (b)-mobile phase-2

 

 

Figure-4: Densitograms of base-forced degraded solutions after 1, 2, 4, 6 hrs. by using (a)-mobile phase-1, and (b)-mobile phase-2

 

 

Figure-5: Densitograms of hydrogen peroxide-forced degraded solutions after 1, 2, 4, 6 hrs. by using (a)-mobile phase-1, and (b)-mobile phase-2

 

Table-3: densitometric measurements of spots from acid-forced degradation solutions

Table-4: densitometric measurements of spots from base-forced degradation solutions

 

Table-5: densitometric measurements of spots from hydrogen peroxide-forced degradation solutions

 

Figure-6: Densitograms of thermal-forced degraded solutions after 1, 2, 4, 6 hrs. by using (a)-mobile phase-1, and (b)-mobile phase-2

 

Table-6: densitometric measurements of spots from thermal-forced degradation solutions

 

 

Figure-7: Densitograms of UV-forced degraded solutions after 24 hrs. by using (a)-mobile phase-1, and (b)-mobile phase-2

Table-7: densitometric measurements of spots from UV-forced degradation solutions

 

 

DISCUSSION:

According to US. Pharmacopoeia⁸, albendazole is considered stable in low acidic conditions, where its test solutions are prepared in glacial acetic acid, and that is confirmed by densitometric measurements of control stock solution (table-2). However, densitometric measurements of acid-induced degradation products spots showed that albendazole is unstable in severe acidic conditions (HCl 0.1N). The mean percentage of drug lost was 15.74% during the first two hours, followed by a decrease to a mean of 7.80% after 4 hrs. and to a lower value (6.13%) after 6 hrs. This reversible degradation may be explained by hydrolysis of carbamic bond in albendazole (figure-8) as demonstrated in previous study¹⁴. Results showed homogeneity in the structure of the degradation products within the acidic medium, due to existence of single impurities spot with retardation factor (R_f) of 0.012 and 0.013 using the mobile phases 1 and 2, respectively, in all experiments at different incubation intervals.

 

Figure-8: chemical structure of albendazole

 

Densitometric measurements of base-induced degradation products spots showed that albendazole is susceptible to alkaline conditions (NaOH 0.1N). The mean percentage of drug lost was 23.22% during the first hour of incubation, and then decreased to a mean of 7.20% after 2 hours, followed by gradual increase to 23.12% and 31.66% after 4 and 6 hours of incubation, respectively.

 

Results showed heterogeneity in the structure of the degradation products of albendazole within the alkaline medium. Albendazole gave two spots of impurities in alkaline medium after one hour of incubation (R_f = 0.012 – 0.558) (using mobile phase 1). This is followed by disappearance of the second impurities spot and appearance of another spot (R_f = 0.184) after 2, 4, and 6 hrs. When mobile phase 2 has been used, one spot of impurities was produced after an hour of incubation (R_f=0.013), and a second spot (R_f = 0.035) emerged after 2, 4, and 6 hrs.

 

On mobile phase 1, reversible degradation mechanisms may be the reason for disappearance of the second spot of impurities (R_f=0.558) and increasing of albendazole remaining percentage after 2 hrs. compared with the percentage after 1 hr. This value then decreased because of elevation of the second spot percentages (R_f = 0.184) after 4 and 6 hrs. of incubation.

Densitometric measurements of H₂O₂-induced degradation products spots showed that albendazole is susceptible to oxidation. The mean percentages of drug remained were decreased gradually against incubation time (79.57% - 75.18% - 73.23% - 71.98%) after 1, 2, 4, and 6 hrs. respectively.

 

Results showed homogeneity in the structure of the oxidation-induced degradation products, due to existence of two impurities spots (R_f=0.013 – 0.570) when mobile phase 1 was used, and (R_f=0.013 – 0.520) when mobile phase 2 was used in all experiments at different incubation intervals, With irregular fluctuation in the percentage of each spot depending on the incubation time.

 

Measurements of thermal-induced degradation products spots showed that albendazole is unstable in high temperature. The mean percentages of drug remained were decreased to 69.35% - 67.71% - 66.78% - 66.24% after 1, 2, 4, and 6 hrs. respectively.

 

Results showed homogeneity in the structure of the thermal-induced degradation products, due to existence of two impurities spots with (R_f=0.013 – 0.553) when mobile phase 1 was used, and (R_f=0.013 – 0.515) when mobile phase 2 was used in all experiments at different incubation intervals, with general tendency of values to decrease for the first impurities spot and increase for the second spot against incubation time.

 

Densitometric measurements of photolytic-induced degradation products spots showed that albendazole is susceptible to UV-C (254 nm). The mean percentage of drug remained decreased to 33.48% after incubation for 24 hrs. There was two degradation spots by using mobile phase 1 (R_f=0.015 – 0.556) and three spots on mobile phase 2 (R_f=0.013 – 0.059 – 0.489).

 

As a conclusion, forced-degradation testing of albendazole showed that this drug is unstable in all stress conditions but in different percentages. Albendazole is less susceptible to acidic condition in contrast with alkaline, oxidation, thermal, and photolytic conditions. Acidic medium caused reversible degradation with homogeneity in the structures of degradation products, Unlike Alkaline medium which showed heterogeneity in structures detected through the change in the number and R_f-values of degradation spots against time.

 

Several base-induced degradation spots were observed, but the main degradation spot was that with retardation factor of 0.035 and 0.184 when using mobile phase 1 and 2, respectively. On the other hand, only one degradation spot was observed in acidic conditions with retardation factor of 0.012 and 0.013 on mobile phase 1 and 2, respectively.

 

Both mobile phases were suitable for detecting albendazole degradation percentages under different conditions, as quantitative measurements were close for the two phases. However, mobile phase 1 was able to give better differentiation between degradation products in alkaline condition, while mobile phase 2 was better for photolytic-induced degradation products differentiation.

 

Retardation factor values and the number of degradation products spots were similar for thermal- and oxidation-induced forced degradation on both mobile phases. This may be result of the chemical similarity in the degradation products in both conditions. Although the results of photolytic-induced degradation experiments were similar to that of thermal- and oxidation-induced degradation on mobile phase 1, results by using mobile phase 2 showed a difference in products of photolytic-induced degradation due to appearance of additional degradation spot (R_f=0.059).

 

More studies should be performed to detect the accurate chemical structures of degradation products in different stress conditions and studying degradation kinetics and mechanisms.

 

REFERENCES:

1-     Jaime ND, and William AR. Textbook of organic medicinal and pharmaceutical chemistry. 10th Edition. Philadelphia, PA: Lippincott Raven Publishers, 1988.

2-     ICH Harmonized Tripartite Guideline. Impurities in New Drug Substances Q3A (R2), October 2006.

3-     ICH Harmonized Tripartite Guideline. Impurities in New Drug Products Q3B (R2), June 2006.

4-     Roy J. Pharmaceutical impurities-a mini-review. AAPS Pharm. Sci. Tech., 2002; 3(2):1-8.

5-     Grekas N. organic impurities in chemical drug substances. Pharmaceutical Technology Europe, 2005; 17(10):24-32.

6-     Rousseau G. international conference on harmonization impurity guidelines- the industry perspective. Drug Information Journal, 2000; 34:903-907.

7-     British Pharmacopoeia. 7^th edition. TSO Information and Publishing Solutions, 2013.

8-     United States Pharmacopoeia and National Formulary USP 38 – NF 33, 2015.

9-     ICH Topic Q1A (R2) Stability Testing of new Drug Substances and Products. August 2003.

10-   Baertschi SW. The Role of Stress Testing in Pharmaceutical Product Development. The American Association of Pharmaceutical Scientists. Midwest Regional Meeting. Chicago, 1996.

11-   Attia KAS, Mohamad AAH, Emara MS. Determination of Albendazole in the Presence of its Alkaline Degradation Product Using TLC-Densitometric and Chemometric Methods: A Comparative Study. Eurasian Journal of Analytical Chemistry, 2017; 12(4):365-383.

12-   Ljubas D, Čizmić M, Vrbat K, Stipaničev D, Repec S, Ćurković L, Babić S. Albendazole Degradation Possibilities by UV-Based Advanced Oxidation Processes. International Journal of Photoenergy, 2018; 6181747.

13-   Patel AK, Joshi HV, Patel JK. Development and validation of stability indicating RP-HPLC method for estimation of ivermectin and albendazole in pharmaceutical dosage form. Indian Journal of Drugs, 2015; 3(3), 57-70.

14-   Ragno G, Risoli A, Ioele G, De Luca M. Photo- and Thermal-Stability Studies on Benzimidazole Anthelmintics by HPLC and GC-MS. Chem. Pharm. Bull., 2006; 54(6) 802-806.

15-   Gomes AR, Nagaraju V. High-performance liquid chromatographic separation and determination of the process related impurities of mebendazole, fenbendazole and albendazole in bulk drugs. Journal of Pharmaceutical and Biomedical Analysis, 2001; 26: 919-927.

 

Accepted on 28.08.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(5): 2207-2213.