Introduction

Albendazole (ALB) Figure 1, a benzimidazole, is used in the treatment of broad-spectrum intestinal helminthic infections (NTD, 2006; WHO, 2010; WHO, 2017). The drug is most effective for the treatment of intestinal helminthic infections caused by nematode (Necator americanus, Ancylostoma duodenale, Ascaris lumbericoides, Trichuris trichiura, Trichinella spiralis, Loa loa, Onchocerca volvulus) and cestode (Taenia saginata, Taenia solium) (Lemke, Williams, Roche, & Zito, 2013). The drug is effective in mixed infections also. The main mechanism of action of albendazole is its inhibitory action on tubulin polymerization, which results in the loss of cytoplasmic microtubules. Albendazole is also the preferred drug for mass drug administration in school going children (WHO, 2017).

Anything made by humans is subject to decay with time, and pharmaceuticals are no exception. The drugs decompose to degradation products (DPs) on storage, and the presence of the latter in pharmaceutical products beyond certain limits is not only quality but also a safety issue for the regulatory agencies. This is because DPs may be more potent and/or more toxic than the drug.

Keeping in mind the above, the project was undertaken to study the behavior of the drug (ALB) under different conditions when in the solution state. No report exists in literature providing such a comprehensive study of degradation behaviour in the solution state.

Materials and Methods

Reagents and chemicals

Pure albendazole (ALB), was obtained from Mahalaxmi Chemicals (Hyderabad, India). Analytical reagent (AR) grade boric acid (H₃BO₃) was purchased from s.d. Fine-chem Ltd. (Boisar, India), while sodium carbonate (Na₂CO₃) was sourced from Rankem (Avantor Performance Materials, Maharashtra, India). Buffer salts and all other chemicals of AR grade were bought from s.d. Fine-chem Ltd. (Boisar, India). HPLC grade ACN and methanol were procured from Fischer Scientific (Mumbai, India). Water for HPLC studies was obtained from the ELGA water purification unit (Bucks, England).

Apparatus and equipment

Separation behavior of the drugs and degradation products was studied using a Prominence liquid chromatography system that was equipped with a photodiode detector (SPD-M20A), and the same was controlled by CBM 20 A software, version 3 (all from Shimadzu, Kyoto, Japan). The column used was Inertsil ODS-3 C-18 (250 nm x 4.6 nm, i.d., particle size 5 µ).

A pH/Ion analyzer (Seven Easy, Mettler Toledo, Schwerzenbach, Switzerland) was used to check and adjust the pH of buffer solutions. Other small equipments were sonicator (YJ 5200 DT, Citizen Scale India Pvt. Ltd., India), precision analytical balance (XP 205 and AG204, Mettler Toledo, Schwerzenbach, Switzerland) and autopipettes (Eppendorf, Hamburg, Germany). LC-MS/TOF studies were carried out on a system in which AQUITY UPLC (I Class, Waters Corporations, USA) was hyphenated to XEVO G2XS QTOF spectrometer (Waters Corporations, USA). It was controlled using Masslynx (version 4.0). The column used was XBridge C18-5µ-4.6*250mm.

Solution State Stress Degradation Studies

The stressors, choice of their concentration, and preparation of samples were based on the protocol described in the publication (Alsante, Martin, & Baertschi, 2003; Baertschi, Alsante, & Reed, 2011; Klick et al., 2005; Singh & Bakshi, 2000). As the drug, namely albendazole (Europe, Council of Europe, 2016), was slightly soluble in water but more so under acidic conditions, so the method of preparation of drug samples for ALB was varied according to the conditions as given in Table 1. The stock solution was not prepared, but the drug was added to a mixture of diluent and stressor. The concentration of ALB was 2 mg/ml.

The drug was kept at 80 °C for 72 hrs. For hydrolytic, along with study, the effect of thermal stress also. In the oxidative study, the study was conducted with 15% H₂O₂ for 7 d at room temperature. Parallel blanks were kept for all the stress conditions. The optimized stress conditions for the drugs are enlisted in Table 2. The stress samples were withdrawn at suitable time intervals and diluted with the suitable anti stressor (50:50 v/v) before analysis by HPLC. The final concentration of the drug in all the samples was 1mg/ml for injection in HPLC.

Method Optimization

To achieve satisfactory separation of the drugs from the degradation products (DPs) in the stressed samples, the composition of the mobile phase including the ratio of polar to the non-polar component, buffer concentration, pH, flow rate, column temperature, and injection volume were suitably optimized. Various possible permutations and combinations were used (Singh & Bakshi, 2000; Singh et al., 2013; Snyder, Kirkland, & Glajch, 1997). A desirable separation and resolution were obtained using 10 mM potassium dihydrogen orthophosphate buffer (pH 3.0), which was run in a gradient mode (Table 3) at a flow rate of 1 ml/min. The column temperature was set at 25°C. The analysis was performed using the PDA detector and the wavelength finally selected was 254 nm and 295 nm.

MS Studies on the Drugs

The elucidation of the mass fragmentation pathway of the drug was achieved with the help of MS/TOF. The studies were performed in ESI positive mode (Kurmi, Golla, Kumar, Sahu, & Singh, 2015; Singh et al., 2013). The instrument parameters were first optimized to get the molecular ion peak of the drugs. The same was subsequently modified to get the complete fragmentation profile of each drug. The instrument parameters, at which mass studies were done for all the drugs, are listed in Table 4.

LC-MS Studies on the Degradation Products

The stressed samples were subjected to LC-MS/TOF analyses (Kurmi et al., 2015) using the optimized MS/TOF parameters listed in Table 4. The optimized method was used with the replacement of buffer with ammonia buffer (Snyder et al., 1997). For internal calibration, Leucine Enkephalin was injected through a diverter in a specific segment near the peak of interest.

Results and Discussion

HPLC-PDA method

The HPLC chromatograms for the degradation of ALB under different stress conditions are shown in Figure 2. They showed that the drug eluted at a retention time of 30 min when the samples were analyzed at 254 nm using the optimized method. The drug was reduced to 15 degradation products (ALB1-ALB15) under different solution-state stress conditions, as shown in Table 5. The products were formed upon degradation by both the hydrolytic as well as the oxidative conditions. The peak at around 19.283 min (ALB8) was present in almost every sample, as supported by LC-HRMS data. The peak at around 17.5 min (ALB5, ALB6) was present in almost every sample, but after analysis of LC-HRMS the sample, it was found that peak in the oxidative condition has different patterns, thus given a separate number. The peak at RT around 15.593 (ALB4) and another peak at 22.800 min (ALB12) were observed under all the hydrolytic conditions. The peak at 14.686 (ALB3), ALB2 (observed in LC-HRMS), ALB15 (observed in LC-HRMS) are exclusively present under degradation in oxidative conditions. The peak at RT 23.577 (ALB13) is present only under acidic conditions. Finally, the drug in oxidative condition was completely degraded while under hydrolytic conditions, it was present in a sufficient amount.

LC-HRMS method

The comparative charts of RRT (Relative retention time) of HPLC-PDA and LC-HRMS data are compiled in Table 6. The data of HRMS supports the HPLC-PDA data, so in all sixteen degradation products are separated. Other peaks are also seen in the chromatogram, but they can be due to H₂O_2, blank of the other components (solvent, salts) used to carry out the study. The comparative data includes only acidic and oxidative conditions as all the peaks are covered under the two. Some of the peaks in LC-HRMS are there, which are not in HPLC-PDA as the LC used in HRMS is UPLC, thus causing more refined separation.

The compiled Table 7 shows the comparison of all the conditions with respect to the degradation products formed. This comparative data shows the interrelation of the formation of various degradation products in more than one condition (Raijada, Prasad, Paudel, Shah, & Singh, 2010; Singh et al., 2013; Sonawane & Gide, 2011).

The mass spectrum of the drug and the degradation products separated by LC is shown in Figure 7; Figure 6; Figure 5; Figure 4; Figure 3 and the detailed fragmentation is highlighted in Figure 12; Figure 11; Figure 10; Figure 9; Figure 8.

Conclusion

The systematic solution state degradation studies of drug albendazole were studied. Degradation products in all the stressed samples were separated by an optimized HPLC- PDA system at 254 nm. Overall degradation products were formed under every condition-acidic, neutral, basic, and oxidative. The oxidative products were formed readily in 2 h as well as the basic hydrolysis was also achieved in 6 h at 0.1 N NaOH. The hydrolytic condition for neutral as well as acidic was taking a long time of 72 h. The hydrolysis at -NH end, oxidation at -S (mono- and di- are possible). The alkylation of aliphatic -N is also seen in some of the products formed as supported by ALB4, ALB5, and ALB15 peaks in HRMS data. The study emphasized the care to be taken while manufacturing and storing the dosage form so as to prevent the formation of degradation products that are toxic or otherwise unwanted. The study has been further carried out using the drug in combination with other drugs of the above category.