An Overview of Several Innovative Analytical Techniques Presented on Triclabendazole Estimation

Abstract

Analytical technique development and validation are part of the ongoing and interrelated processes associated with research and development, quality assurance, and control. These processes are essential for risk management and equivalence evaluations because they support the creation of standards for product-specific acceptance and yield reliable results. Analytical methods' suitability for their intended uses is ascertained through validation procedures. A thorough literature review states that analytical techniques like UV spectroscopy, RP-HPLC, and HPTLC can be used to analyze Triclabendazole either by itself or in conjunction with other drugs. The metrics, including accuracy, precision, robustness, and other aspects of analytical validation, were thoroughly examined in compliance with ICH guidelines. Because the techniques are straightforward, sensitive, and reproducible, they can be used to both bulk and tablet dose versions of Triclabendazole. The study also highlights the suitability and limitations of several established analytical techniques for Triclabendazole analysis. This comprehensive report will be extremely useful to researchers working on Triclabendazole trials.

Keywords

Introduction

Triclabendazole is a benzimidazole derivative primarily used as an anthelmintic drug, especially effective against Fasciola hepatica (liver flukes). It works by disrupting the microtubule function in parasites. Due to its clinical importance, especially in veterinary and human parasitic infections, reliable analytical methods for its quantification and quality control are crucial. Triclabendazole (TCBZ, 6-chloro-5(2-3 dichlorophenoxy) 2-methyl thiobenzimidazole), an halogenated benzimidazole (BZD) thiol derivative, shows high efficacy against both the immature and mature stages of Fasciola hepatica in sheep and cattle, which is a differential feature compared to other available trematodicidal drugs ^[1]. One of the primary factors causing economic losses in the sheep breed business is intestinal nematode infections. Veterinarians are now employing a variety of anthelmintic medication in combination to promptly treat animals as the incidence of parasite resistance continues to rise ^[3].

Analytical Methods Reported on Triclabendazole

1. Shrivatsava, et al., (2016) have developed a new, less time consuming and cost-effective UV spectrophotometric method for the quantification of Triclabendazole in bulk. Triclabendazole (TCBZ, 6-chloro-5(2-3 dichlorophenoxy) 2-methyl thio-benzimidazole), a halogenated benzimidazole (BZD) thiol derivative, shows high efficacy against both the immature and mature stages of Fasciola hepatica in sheep and cattle, which is a differential feature compared to other available trematodicidal drugs. Triclabendazole is effective in humans presenting with infection by the liver fluke Fasciola hepatica or with the lung fluke Paragonimus sp. Preliminary pharmacokinetics in European patients indicate that the absorption of TCBZ might be higher in postprandial than in fasting administration conditions. The pharmacokinetics of triclabendazole in sheep and goat have been studied. Like other sulfide benzimidazoles, triclabendazole is oxidized to sulfoxide and sulfone metabolites in sheep, goat, and cattle. The purpose of the present study was to develop a rapid and simple UV spectroscopic method for assaying triclabendazole in bulk and pharmaceutical dosage forms.
2. R. MRUTHUNJAYA RAO et al., (2008) have developed a new, less time consuming and cost effective visible spectrophotometric method for the quantification of Triclabendazole in bulk. Triclabendazole is an antifungal drug. It is chemically known as 1H benzimidazole, 5-chloro-6-(2,3-dichlorophenoxy), -2-(methyl thio-benzimidazole) and it is only available in Egypt as oral tablets fasinex (250 mg). A number of methods such as HPLC1-11 and UV1,2 were reported for estimation of triclabendazole. The present paper describes three simple and sensitive spectrophotometric methods (A, B and C) for the determination of triclabendazole based on its tendency to form chloroform extractable ion-association complexes with acidic dyes belonging to different chemical classes, namely, wool fast blue BL (WFB BL, method A), tropaeolin OOO (TPOOO, method B). Method C is based on the oxidative coupling of drug with brucine and sodium metaperiodate under acidic conditions forming coloured bruciquinone derivatives.
3. Khalid A. M. Attia et al., (2023) have developed a HPLC procedure, it is optimized to finalize a different approach for the estimation of Triclabendazole in tablet dosage form. One of the primary factors causing economic losses in the sheep breed business is intestinal nematode infections. Veterinarians are now employing a variety of anthelmintic medication in combination to promptly treat animals as the incidence of parasite resistance continues to rise. Martibendazene is an oral suspension formula that consists of two active ingredients with distinct pharmacological actions on sheep GIT worms. The anthelmintic drugs used are levamisole hydrochloride (LEVA) and triclabendazole (TCBZ), their chemical structures are illustrated. The concurrent administration of LEVA with TCBZ has been observed to result in superior therapeutic outcomes and accelerated amelioration of hepatic pathologies in naturally infected sheep afflicted with Fasciola species.
4. Fathalla F et al., (2020) have developed A simple, rapid and highly sensitive spectrofluorimetric method for the determination of triclabendazole (TCB). Triclabendazole, (6-chloro-5-(2,3-dichlorophenoxy)- 2- methyl thio-benzimidazole) is a halogenated benzimidazole thiol derivative.1It has a high efficacy against both the mature and immature stages of Fasciola hepatica in sheep and cattle which is a feature that differentiates it from other available trematodicidal drugs. TCB is effective in humans presenting with infection by the liver fluke Fasciola hepatica or with the lung fluke Paragonimus species. Benzimidazoles such as TCB are generally accepted to bind to beta tubulin and prevent the polymerization of the micro tubules of which they are part.8 A literature survey revealed many methods for the analysis of TCB, these methods include; spectrophotometric methods, HPLC methods with UV detection and with mass spectrometric detection. This paper presents a simple, sensitive and accurate method for the determination of pure TCB and for TCB in laboratory prepared tablets, in addition to the application of stability studies.
5. Kandukuru Sunil Kumar et al., (2023) have developed a FT-IR method was developed for the quantitative estimation of triclabendazole in bulk and pharmaceutical dosage form. Targeted delivery of a drug molecule to specific organ sites is one of the most challenging research areas in pharmaceutical sciences. By developing colloidal delivery systems such as liposomes, micelles, and nanoparticles, new frontiers have opened for improving drug delivery. Nanoparticles, with their unique characteristics of small particle size, large surface area, and the capability of changing their surface properties, have numerous advantages compared with other delivery systems. Nanoparticles are solid colloidal particles ranging from 10 to 1000 nm (1.0 μm), in which the active principles (drug or biologically active material) are dissolved, entrapped, and to which the functional code is adsorbed or attached. In recent years, significant effort has been devoted to developing nanotechnology for drug delivery since it offers a suitable means of delivering small molecular weight drugs, as well as macromolecules such as proteins, peptides, or genes to cells and tissues and prevents them against enzymatic degradation. The advantages of nanoparticles as drug delivery systems are that they are biodegradable, non-toxic, and can be stored for extended periods as they are more stable.
6. Noha Salem Rashed et al., (2020) have developed a rapid, simple and sensitive micellar liquid chromatographic method was developed for the quantification of triclabendazole. Helminths are worm-like parasites that survive by feeding on a living host to gain nourishment and protection, sometimes resulting in illness in host. Their effects inside their host vary, causing a wide spectrum of diseases, from mild to potentially deadly. Helminths include three types; cestodes (tapeworms), nematodes (roundworms) and trematodes (liver flukes). Anthelmintic drugs have been widely used on pets to treat or prevent parasitic diseases. In some cases, combinations of these drugs are required to enlarge their spectrum of action. They include benzene sulphonamide like clorsulon (CLO), benzimidazoles like albendazole (ABZ) and triclabendazole (TCB) and macro cyclic lactones like ivermectin (IVM). CLO is 4-amino-6-(1,2,2 trichloroethenyl)-benzene-1,3-disulfonamide has good efficacy against mature rather than immature flukes. CLO is recommended for treat ment and control of adult liver (Fasciola hepatica, Fasciola gigantica) in cattle as suspension or injectable formulation.
7. MAHER SHURBAJI et al., (2010) have developed A rapid, simple, and sensitive RP HPLC analytical method was developed for the determination of triclabendazole. Triclabendazole [5-chloro-6-(2.3-dichlorophenoxy)-2 (methylthio)-1H benzimidazole; Figure 1] is a member of the benzimidazole family of anthelmintics. Ivermectin [5-O demethyl-22,23-dihydroavermectin Ala] is an antiparasite medication that is effective against most common intestinal worms (except tapeworms), most mites, and some lice. A combination of triclabendazole and ivermectin is a new and important veterinary drug treatment used against liver flukes and gastrointestinal nematodes in cattle and sheep. Several methods for determination of triclabendazole and ivermectin in biological matrixes have been developed, including HPLC. Ivermectin was determined by HPLC, capillary electrophoresis, use of an optical immunobiosensor, and spectrophotometry. To date, there is no reported method for the determination of triclabendazole and ivermectin in combination in a pharmaceutical preparation. Therefore, it was the purpose of this research to develop a simple, sensitive, reliable, and validated analytical method for the measurement of both drugs, which will be the first for their simultaneous analysis in a formulation or biological matrixes.
8. Rosella Ferretti A et al., (2013) have developed a simple, precise and accurate HPLC method has been developed and validated for assay of Triclabendazole. Halogenated sulfanyl-benzimidazole derivative triclabendazole (TCBZ) [5-Chloro-6-(2,3 dichlorophenoxy)-2-(methylthio) 1H-benzimidazole], is the WHO-recommended essential medicine for the treatment of fascioliasis, a disease affecting millions of people worldwide. Fascioliasis is acquired through the ingestion of food contaminated with the larval stages of trematode parasites that mainly affect the liver. Recently, TCBZ has been efficaciously tested on the larval stage of the cestode parasite Echinococcus multilocularis and has also been evaluated for the treatment of paragonimiasis in humans, a parasitic infection that mainly causes subacute and chronic inflammatory disease of the lungs. Control trials have shown that the efficacy, safety and tolerability of TCBZ were comparable to those of praziquantel which is the drug of choice for both pulmonary and extra pulmonary paragonimiasis.
9. A. Negro et al., (1998) have developed An ion-pair high-performance liquid chromatography method for measuring the concentration of triclabendazole metabolites. Triclabendazole [4-chl.oro-5-(2,3-dichlorophenoxy)-2-methylthiobenzimidazole] benzimidazole anthehnintic shown to exhibit high activity against immature and mature stages of Fasciola hepatica and Fasciola gigantica in sheep and cattle. After oral administration to these species it undergoes rapid and exensive metabolism, first to its sulphoxide and sulphone, with the parent compound remaining undetectable in the plasma of treated animals. The purpose of this study was to develop a reversed phase ion-pair chromatographic high-performance liquid chromatograpy (HPLC) method for determining triclabendazole metabolites in serum and urine samples. To this end we studied the influence on their retention time of pH, mobile phase composition and the nature of sodium alkyl sulphonate added to the mobile phase.
10. Kazue Takeba et al., (2000) have developed a sensitive and validated HPLC method for simultaneous determination of triclabendazole and its metabolites (sulphoxide and sulphone) in bovine milk. Triclabendazole is a benzimidazole anthelmintic widely used in the control of the liver fluke, Fasciola hepatica, in sheep and cattle. After oral administration, it has been found that triclabendazole is oxidized to sulphoxide and sulphone metabolites in animals, similarly to other benzimidazole sulphide compounds. FAO/WHO had reported the recommended maximum residue limits (MRLs) for triclabendazole in animal foods. The MRLs of triclabendazole are scheduled to be adopted after June of 2000 in Japan. In order to monitor the residual triclabendazole and its metabolites in commercial milk samples, a simple, rapid and sensitive method is needed. Methods have been published for the determination of triclabendazole and its metabolites in biological samples using HPLC with UV or fluorescence detection in pharmacokinetic studies. A method has been published for the determination of eight benzimidazole anthelmintics including triclabendazole in meat samples. But, the metabolites of triclabendazole could not be simultaneously studied by this method. At present no method is available for the simultaneous determination of triclabendazole and its metabolites in milk. The purpose of this study was to develop a simple method for the simultaneous determination of triclabendazole and its metabolites in milk.
11. Khalid A.M. Attia et al., (2016) have developed a e simple, accurate, and precise for the determination of levamisole and triclabendazole. Intestinal nematode infections are a major source of financial loss in the sheep breeding industry..As the prevalence of parasite resistance keeps increasing to treat animals promptly, veterinarians are combining many anthelmintic drugs together. Oral suspension martibendazene medication has two active ingredients that have various effects on sheep GIT worms with distinct pharmacological actions. The anthelmintic medications employed in this study include levamisole HCl (LEV) and triclabendazole (TCB). The administration of LEV and TCB together has been found to yield enhanced therapeutic effects and expedited improvement of hepatic diseases in sheep infected by species of Fasciola. LEV (C11H12N2S) has been successful in significantly reducing the prevalence of ascariasis, a parasitic infection, among the people in the world. Levamisole was given FDA approval in 1990 to be used as an adjuvant therapy for colon cancer. Levamisole was previously used as a treatment for rheumatoid arthritis. The results show the immunomodulatory properties of levamisole, which make it useful for enhancing immune response even in immunocompromised folks. The results of the trial also revealed the therapeutic efficacy of LEV in the treatment of individuals afflicted with mild cases of coronavirus infections (COVID-19). TCB (C14H9Cl3N2OS), is an anthelmintic drug belonging to the benzimidazole class, and it has been approved for use in the treatment of worms in sheep and other bovine animals. The efficacy of this treatment in eradicating early immature and mature Fasciola species has been demonstrated in ovine and bovine animals. Since the 1980’s, research has demonstrated that triclabendazole can be used to effectively treat Fasciola infections in livestock.
12. Nischal K et al, (2011) have developed a simple method for reversed-phase High Performance Liquid Chromatography (HPLC) for r the estimation of triclabendazole and ivermectin in a pharmaceutical suspension dosage form present in 50:1 ratio respectively. Triclabendazole (TCBZ) has been the drug of choice to treat liver fluke infections in livestock for more than 20 years, due to its high activity against both adult and juvenile flukes. TCBZ is a benzimidazole derivative and, by analogy with what is known about other benzimidazole drugs, it would be anticipated that TCBZ might bind to the β-tubulin molecule and so disrupt microtubule-based processes. On the other hand ivermectin (IVR) has potent activity at GABA receptors in both invertebrates and mammals, and GABA is known to be the primary inhibitory neurotransmitter in the nematode somatic neuromuscular system. However, subsequent work by Merck scientists identified glutamate-gated Cl¯ channels as the more likely physiological targets of ivermectin and related drugs.

RESULTS AND DISCUSSION

A variety of analytical methods have been developed for the estimation of Triclabendazole (TCBZ). Early UV and visible spectrophotometric methods provided simple, rapid, and low-cost analysis suitable for routine quality control of bulk drug and dosage forms. Later studies introduced more sensitive approaches such as spectrofluorimetric and FT-IR, improving detectability and reducing sample preparation. HPLC became the preferred technique in many investigations due to superior accuracy, precision, and ability to analyze TCBZ in complex mixtures, combination formulations, and biological fluids. Several methods enabled simultaneous determination of TCBZ with other anthelmintic drugs (e.g., levamisole, ivermectin) and also facilitated monitoring of metabolites in milk, serum, and urine. Overall, spectrophotometric methods are practical for rapid routine analysis, while chromatographic techniques offer higher sensitivity and selectivity for pharmacokinetic studies, residue monitoring, and multicomponent dosage forms.

CONCLUSION

The review shows that multiple analytical methods exist for the determination of Triclabendazole, ranging from basic spectrophotometric techniques to advanced chromatographic and spectrofluorimetric methods. Simple UV and visible spectrophotometric methods are reliable, economical, and suitable for routine quality control. However, HPLC and related techniques provide higher accuracy, sensitivity, and suitability for complex dosage forms, combination therapies, and biological samples. Overall, method selection depends on analytical needs, with spectrophotometry preferred for rapid routine analysis and chromatographic methods recommended for high-precision regulatory and pharmacokinetic applications.

REFERENCES

1. Shrivastava A, Kumar S, Jain A. Spectrophotometric method for quantitative determination of triclabendazole in bulk and pharmaceutical. Chronicles of Young Scientists. 2011 Apr 1;2(2):90.
2. Rao RM. Determination of triclabendazole by visible spectrophotometry. Asian Journal of Chemistry. 2008 Aug 20;20(6):4536.
3. Attia KA, El-Desouky EA, Abdelfatah AM, Abdelshafi NA. Simultaneous analysis of the of levamisole with triclabendazole in pharmaceuticals through developing TLC and HPLC–PDA chromatographic techniques and their greenness assessment using GAPI and AGREE methods. BMC chemistry. 2023 Nov 24;17(1):163.
4. Belal FF, El-Din MK, El Enany NM, Saad S. Stability-indicating spectroflurometric method for determination of triclabendazole in pure form and tablets. Analytical Methods. 2014;6(2):615-22.
5. Kumar KS, Bhargavi MN. Formulation and evaluation of triclabendazole nanoparticles. World Journal of Advanced Research and Reviews. 2023;19(2):505-18.
6. Shurbaji M, Al Rub MH, Saket MM, Qaisi AM, Salim ML, Abu-Nameh ES. Development and validation of a new HPLC-UV method for the simultaneous determination of triclabendazole and ivermectin B1a in a pharmaceutical formulation. Journal of AOAC International. 2010 Nov 1;93(6):1868-73.
7. Ferretti R, Carradori S, Guglielmi P, Pierini M, Casulli A, Cirilli R. Enantiomers of triclabendazole sulfoxide: Analytical and semipreparative HPLC separation, absolute configuration assignment, and transformation into sodium salt. Journal of Pharmaceutical and Biomedical Analysis. 2017 Jun 5; 140:38-44.
8. Negro A, Alvarez-Bujidos ML, Ortiz AI, Cubria JC, Mendez R, Ordonez D. Reversed phase ion-pair high-performance liquid chromatographic determination of triclabendazole metabolites in serum and urine. Journal of Chromatography B: Biomedical Sciences and Applications. 1992 Apr 15;576(1):135-41.
9. Takeba K, Fujinuma K, Sakamoto M, Miyazaki T, Oka H, Itoh Y, Nakazawa H. Simultaneous determination of triclabendazole and its sulphoxide and sulphone metabolites in bovine milk by high-performance liquid chromatography. Journal of Chromatography A. 2000 Jun 16;882(1-2):99-107.
10. Attia KA, Abolmagd E, Abdelfatah AM, Abdelshafi N. Simultaneous Determination of Levamisole and Triclabendazole by Multivariate Calibration Models using Second Derivative Spectrophotometry in Veterinary Pharmaceutical Formulation. Egyptian Journal of Chemistry. 2024 Jul 1;67(7):331-40.
11. Ramadan NK, Mohamed AO, Shawky SE, Salem MY. Spectrophotometric determination of triclabendazole by acid-dye complexation method in bulk and pharmaceutical formulation. Journal of Applied Pharmaceutical Science. 2012 Jan 30(Issue):128-33.
12. Rahman TU, Zaman A, Bahadur A, Zeb MA, Liaqat W, Santali EY, Alharthi S, Omar RM, Alharthy SA, Ali A. Development of RP?HPLC Method for Simultaneous Determination of Triclabendazole and Ivermectin in Pharmaceutical Suspension Dosage Form. Journal of Analytical Methods in Chemistry. 2025;2025(1):5522915.
13. Cai C, Zhang L, Xue F, Qiu M, Zheng W. Simultaneous determination of triclabendazole and its metabolites in bovine and goat tissues by liquid chromatography–tandem mass spectrometry. Journal of Chromatography B. 2010 Nov 15;878(30):3106-12.
14. Zhang P, Tian J, Rustum A. Development and Validation of a Fast Stability-Indicating Ion Paired Reversed-Phase HPLC Method for the Assay of Thiabendazole and Estimation of Its Related Compounds. Journal of AOAC International. 2017 Jan 1;100(1):74-81.
15. Belal F, Sharaf El?Din MK, Elenany N, Saad S. Application of liquid chromatographic method with fluorescence detection for the determination of triclabendazole in tablets and biological fluids. Luminescence. 2014 Sep;29(6):559-65.
16. Canas-Mueller A, Vargas Del Campo M, Richter P. Determination of triclabendazole in cattle plasma as its sulphoxide and sulphone metabolites by rotating disk sorptive extraction combined with high-performance liquid chromatography and its application to pharmacokinetic studies. Journal of the Chilean Chemical Society. 2016 Dec;61(4):3195 200.