Identification of major degradation products of Clorsulon drug substance including its degradation pathways by high resolution mass spectrometry and NMR.

Clorsulon is an effective anthelmintic drug substance extensively used in the treatment of parasitic infestations in both cattle and sheep. An in-depth investigation of Clorsulon's degradation products (DPs) was carried out through forced degradation study to comprehend its degradation path. A total of eight degradation products were separated under various stress degradation conditions. Structural elucidation of these DPs was conducted using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS), and their fragmentation patterns were compared to that of the parent compound. Adequate amount of DP-4 was isolated and purified by semi-preparative high-performance liquid chromatography (HPLC) methods. Subsequently, it was examined in detail using both 1D and 2D NMR (nuclear magnetic resonance spectroscopy). Most probable mechanistic pathways for the formation of degradation products under various stress degradation conditions were proposed to better understand the degradation profile. Based on the results of the stress study, Clorsulon drug substance was found to be unstable under photolytic and oxidative conditions. Understanding Clorsulon's degradation pathway is essential for determining shelf-life prediction of the finished product, safety and efficacy assurance, and guiding the development of stable, high-quality formulations.

Simultaneous Determination of Fipronil, Permethrin, and Their Key Related Substances in a Topical Drug Product by a Single Stability-Indicating High-Performance Liquid Chromatography Method.

BACKGROUND: The topical veterinary drug product containing fipronil and permethrin provides an effective repellent protection and high insecticidal efficacy for dogs. OBJECTIVE: The objective of this study was to develop a stability-indicating high-performance liquid chromatography (HPLC) method for simultaneous detection and quantification of fipronil, permethrin, their key degradation products, and butylated hydroxytoluene (BHT) in a topical drug product. METHOD: The two active ingredients, their degradation products, and the antioxidant (BHT) were separated by a gradient elution on a Phenomenex Kinetex C18 column (150 × 3 mm, 2.6 µm particle size) maintained at 37°C with H2O acetonitrile isopropyl alcohol 85% H3PO4 (65.5 + 32.5 + 4/0.0053, v/v/v/v) as mobile phase A and acetonitrile (100%) as mobile phase B. The flow rate was 0.9 mL/min, and analytes were detected and quantified at 235 nm. RESULTS: The specificity of the method was demonstrated by adequate separation of fipronil, permethrin, their degradation products, and BHT in the forced degraded finished product. The linearity of the method was demonstrated in the range of 0.2% to 150% of target analytical concentration of both active ingredients and 50% to 150% for BHT. Excellent recoveries of fipronil, permethrin, and BHT in placebo spiked active ingredient solutions in the linearity range showed sufficient accuracy of the method. The LOQ and LOD of the method were determined to be 0.2% and 0.07% of the analytical concentration. A robustness study did not identify any critical parameter that adversely affected the separation and quantification. CONCLUSIONS: Here, we report the development and validation of a robust, stability-indicating HPLC method for identification and assay of fipronil, permethrin, and BHT, including estimation of fipronil's and permethrin's degradation products in a topical drug product for dogs. HIGHLIGHTS: The new HPLC method permits the acquisition of data for all analytes of interest for a topical finished drug product containing fipronil, permethrin, and BHT.

Development of a Reversed-Phase UPLC Method for Assay of Fipronil Including Determination of Its Related Substances in Bulk Batches of Fipronil Drug Substance.

BACKGROUND: Fipronil is a commonly used pesticide in the agricultural and animal health industries for the protection of crops and control of fleas, ticks, and chewing lice. It is difficult to obtain reproducible retention time and relative retention time (RRT) for a common hydrolytic degradation product of fipronil with the current European Pharmacopeia (EP) monograph for assay and estimation of related substances of fipronil. This situation causes misidentification, mislabeling, and/or false out-of-specification results for this hydrolytic degradation product of fipronil in bulk commercial batches during batch release and/or in the stability samples during the shelf life of a released batch. OBJECTIVE: This study aimed to develop a reversed-phase ultra performance liquid chromatography (UPLC) method for assay and identification of fipronil including identification and estimation of its related substances in bulk drug substance batches of fipronil and provide consistent retention time of the hydrolytic degradation product. METHODS: Fipronil and its related substances were separated by gradient elution on a Halo C18 column (50 mm × 2.1 mm id, 2.0 µm particle size) maintained at 40°C with 0.1% H3PO4 in H2O as mobile phase-A and acetonitrile-methanol (50 + 50, v/v) as mobile phase-B. Fipronil and its related substances were detected and quantified at 280 nm with a quantitation limit of 0.05% of the target (analytical) concentration. RESULTS: The UPLC method was able to separate all analytes of interest by gradient elution with a total run time of 7 min (approximately 40% faster than EP). CONCLUSION: In this paper, we report the development and validation of a fast, stability-indicating reversed-phase UPLC method for assay and estimation of related substances of fipronil in stability samples and bulk batches of fipronil. HIGHLIGHTS: The new UPLC method is approximately 40% faster than the current Ph. Eur. monograph for fipronil assay and the new method provides reproducible retention of a common hydrolytic degradation product of fipronil.

Determination of Clorsulon and its related substances in commercial bulk drug substance batches by a rapid ion-pair UPLC method.

Clorsulon is a potent anthelmintic agent and is widely used for the treatment and control of parasites including adult liver flukes in cattle and sheep. A rapid ion-paired reversed phase ultraperformance liquid chromatography (IP-UPLC) method has been developed and validated for determination of Clorsulon and its related substances in bulk drug substance batches of Clorsulon with a short octadecyl column. Analytes were eluted by a gradient elution on a Acquity UPLC® BEH C18 column (50 mm × 2.1 mm i.d., 1.7 µm particle size). Column temperature was maintained at 55 °C and all analytes were monitored by UV detection at 268 nm. Mobile phase-A constitutes 3 mM tetrabutylammonium hydroxide in H2O and mobile phase-B constitutes acetonitrile/methanol (50/50, v/v), respectively. All analytes of interest were adequately separated within 5 min. The analytes were quantitated against an external reference standard of Clorsulon. The chemical structures of degradation products of Clorsulon were proposed based on two-dimensional UPLC-MS data. The IP-UPLC method has been successfully validated and demonstrated to be accurate, sensitive, robust, and specific.

Development of Relatively Simple Sample Pretreatment Strategies to Selectively Remove Chromatographic Interfering Peaks of Polysorbate 80 from Liquid Oral Finished Drug Product.

Polysorbate 80 (PS 80) is a nonionic surfactant, used in myriad of pharmaceutical, food and cosmetic formulations. PS 80 components have strong UV absorbance and retain under reversed-phase chromatographic conditions, significantly masking sections of the chromatogram. PS 80-related peaks interferences in a sample are common and can be difficult to separate from the analyte peaks. A liquid oral finished product (LOFP) containing PS 80 and Ivermectin as the active pharmaceutical ingredient (API) was selected for this study. Herein, we report two sample pretreatment strategies focusing on the selective removal of PS 80 from the LOFP. Both methods significantly reduce and/or practically eliminate excipients and PS 80-related peaks interferences from the LOFP without a negative impact on the API and its key-related substances recovery. The solid-phase extraction (SPE) strategy uses a C18 SPE followed by a silica gel SPE, whereas the liquid-liquid extraction strategy uses in situ-generated sodium caprylate for the removal of formulation excipients and PS 80. These methods can significantly increase the reliability of high-performance liquid chromatography methods and decrease false positive out-of-specifications events because of coelution of PS 80-related peaks with peaks of interest.

Improved Stability-Indicating RP-UPLC Method for the Levamisole Hydrochloride Assay and Estimation of Its Related Compounds.

BACKGROUND: Levamisole hydrochloride (LVM) is an anthelmintic drug substance with immunomodulatory and anticancer activities. LVM has also found usage as a cutting agent in street cocaine. OBJECTIVE: This study was aimed to develop and validate an alternative and improved stability-indicating reversed-phase ultraperformance liquid chromatography (RP-UPLC) method for the determination of LVM and the estimation of its related compounds. METHOD: The UPLC method for the assay was optimized in terms of organic solvents consumed, pH, column temperature, and flow rate. Determination of LVM and its related compounds was performed using a gradient elution on a Waters ACQUITY UPLC® BEH C18 (50 mm × 2.1 mm i.d., 130 Å). The column temperature was maintained at 35°C. Mobile phase A was composed of aqueous 5 mM ammonium hydroxide, and mobile phase B was composed of acetonitrile. All the analytes were monitored by UV detection at 215 nm with a flow rate of 0.7 mL/min. The total runtime of the method with column equilibration is 4.0 min. RESULTS: The developed method met all the acceptance criteria of the current International Council for Harmonization [ICH Q2 (R1)] guidelines. The method was tested in terms of specificity, linearity (R2 > 0.999), limit of detection (LOD; 0.06 µg/mL), limit of quantitation (LOQ; 0.2 µg/mL), accuracy, precision, and robustness. With a short analysis time (<2.5 min) and reduced consumption of organic solvents, the proposed method is considered a greener alternative to conventional chromatographic methods. CONCLUSIONS: An alternative and improved UPLC method was successfully developed and validated in accordance with the ICH guidelines for the determination of LVM and the estimation of its related compounds. HIGHLIGHTS: Due to its high degree of selectivity, speed, and accuracy, the developed method can significantly benefit the end-users with laboratory efficiency and throughput during routine analysis of production batches and stability monitoring of LVM-related drug products.

Development of a Simple Extraction Procedure and an HPLC Method for the Determination of Delmopinol in a Challenging Rubbery Dental Chew.

BACKGROUND: Delmopinol hydrochloride (delmopinol) is widely used in oral hygiene products such as dental chews for preventing dental plaque buildup and gingivitis. OBJECTIVE: This study aimed to develop a simple and inexpensive extraction method, followed by a stability-indicating reversed-phase HPLC (RP-HPLC) method for the determination of delmopinol from rubbery dental chews. METHODS: The extraction method was optimized in terms of pH, temperature, solvents, repeatability, and reproducibility. Delmopinol was extracted from the rubbery dental chews using 6 N NaOH-MeOH (1 + 1, v/v) at 65°C. The delmopinol peak was eluted in about 7 min by gradient elution on a pH-stable (alkaline) Phenomenex Gemini-NX-C18 column (50 × 4.6 mm id, 110 Å, 3 µm particle size) maintained at 50°C. Mobile phase A was composed of aqueous 10 mM ammonium hydroxide and mobile phase B was acetonitrile. The analyte was monitored by UV detection at 220 nm with a flow rate of 2.0 mL/min. RESULTS: The method was successfully validated as per the current International Conference on Harmonization guidelines for delmopinol with respect to specificity, linearity (R2 >0.999), accuracy, precision, LOQ (2 µg/mL), LOD (0.6 µg/mL), and robustness. The reliability of the method was also demonstrated by batch analysis of 132 dental chews. CONCLUSION: A simple extraction method and RP-HPLC method were successfully developed and validated for the determination of delmopinol from rubbery dental chews. HIGHLIGHTS: The overall method was demonstrated to be accurate, robust, specific, and stability-indicating. Therefore, the developed method is suitable and highly desirable for routine analysis of delmopinol in its finished drug products.

Development and Validation of a Stability-Indicating Reversed-Phase HPLC Method for Assay and Estimation of Related Substances of Ivermectin in an Oral Paste.

Ivermectin is a potent semi-synthetic antiparasitic drug used in veterinary medicine. A reversed-phase high performance liquid chromatography (HPLC) method has been developed and validated for the identification and assay of Ivermectin, including the identification and estimation of its related impurities in an oral paste. Analytes were separated using a gradient elution at a flow rate of 1.5 mL/min on a Zorbax Extend-C18 column (150 mm × 4.6 mm i.d., 3.5-µm particle size) maintained at 30 °C. The mobile phase was composed of water as mobile-phase A and acetonitrile/methanol (85/15, v/v) as mobile-phase B. Ultraviolet detection at 245 nm was employed to monitor the analytes. Limit of quantitation (LOQ) and limit of detection (LOD) of the method are 0.6 and 0.2 µg/mL, respectively. The validation results demonstrated excellent linearity of the method in the range of 0.1-150% of the analytical concentration (0.6 mg/mL) of the method. The stability-indicating capability of the method has been demonstrated by adequately separating the degradation products from the stress degraded samples of the oral paste as per method validation requirements prescribed in the current International Council for Harmonisation guidelines.

Comprehensive study on degradation profile of firocoxib and structural elucidation of its key degradation products.

Firocoxib is widely used in veterinary medicine as a non-steroidal analgesic and anti-inflammatory drug substance. Herein, a comprehensive study on the degradation profile of firocoxib was performed through force degradation studies to understand its degradation profile and characterize its major degradation products (DPs). Firocoxib drug substance was subjected to acidic, alkaline, oxidation (H2O2, KMnO4, and K2Cr2O7), thermal (solid and solution state), and photolytic (solid and solution state) stress degradation, as recommended in the ICH guidelines. Firocoxib and its major DPs were adequately separated by investigational HPLC method, which utilized a HALO C18 (100 × 2.1 mm, 2.0 µm) column. Mobile phase-A for the HPLC method is composed of 0.1% formic acid in water and mobile phase-B acetonitrile. A total of six major DPs were observed for firocoxib drug substance under these stress degradation conditions. Structural elucidation of the DPs performed using liquid chromatography-high resolution mass spectrometry and comparison of their fragmentation profile with that of the parent compound. For further structural confirmation of two major DPs, DP-2 and DP-6 were isolated and purified from the stressed samples using a preparative HPLC and analyzed by comprehensive nuclear magnetic resonance (NMR) spectroscopy studies. Most probable mechanistic pathways for the formation of DPs of firocoxib under various stress degradation conditions were postulated to understand its degradation profile. Based on the results from forced degradation, firocoxib was found to be quite stable under basic and thermal conditions, and somewhat unstable under acidic, oxidative, and photolytic conditions. The results of this study should facilitate quality monitoring and establish a stability profile of firocoxib drug substance and drug products. These results may also assist in the design and development of new formulations made with firocoxib drug substance with desired shelf life.

Simultaneous Determination of Esafoxolaner, Eprinomectin, Praziquantel, BHT and Their Related Substances in a Topical Finished Product by a Single Reversed-Phase High-Performance Liquid Chromatography Method.

The topical product with three active pharmaceutical ingredients (APIs), namely, esafoxolaner, eprinomectin and praziquantel has demonstrated its efficacy in the treatment of cats with mixed infections with ectoparasites and nematodes and cestodes. A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for assay and determination of related substances peaks of three APIs including the assay of antioxidant butylated hydroxytoluene (BHT) in the finished product. Analytes were separated on a short Zorbax SB-C18 column (50 × 4.6 mm I.D., 5 µm particle size, pore size: 80 Å) with gradient elution at 40 °C column temperature. Analytes were detected at 245 nm for praziquantel, esafoxolaner, eprinomectin and their degradation products. BHT and eprinomectin degradation product 8a-oxo-B1a were detected at 280 nm. All analytes of interest were adequately separated within 40 min. The assay for praziquantel, esafoxolaner, eprinomectin and BHT was conducted against their corresponding external reference standards. The related substances peaks of each API were determined by peak area and relative response factor against total peak area of their corresponding API peak in sample solution. This method has been demonstrated to be accurate, robust, specific and stability indicating.

Determination of Firocoxib and Its Related Substances in Bulk Drug Substance Batches of Firocoxib by a High-Speed Reversed-Phase HPLC Method With a Short Fused-Core Biphenyl Column.

Firocoxib is a nonsteroidal anti-inflammatory drug. It provides control of postoperative pain and inflammation associated with soft tissue and orthopedic surgery in dogs, and control of pain and inflammation associated with osteoarthritis in horses. A high-speed stability-indicating reversed-phase high-performance liquid chromatography method was developed to determine firocoxib and its related substances in bulk batches of firocoxib drug substance. Firocoxib was dissolved in neat acetonitrile (ACN) and analyzed on a short HALO (fused-core) biphenyl column (30 × 4.6 mm i.d., 2.7-µm particle size) at flow rate of 2.5 mL/min. Column temperature was maintained at 50°C. Mobile phase A is composed of 0.1% of H3PO4 in water and mobile phase B is composed of ACN. Analytes were detected with UV detection at 240 nm and quantitated against an external reference standard. Firocoxib and its related compounds were adequately separated within 4 min by a gradient elution. The method was validated for specificity, linearity, accuracy, precision and robustness according to method validation guidelines described in The International Conference on Harmonization. The validation data demonstrated that this method is sensitive, accurate, robust, specific and stability-indicating.

Structural elucidation of major degradation products of milbemycin oxime drug substance using LC-MS and NMR.

Milbemycin oxime (MO) drug substance is a 16-membered macrocyclic lactone that exhibits a broad spectrum of biological activity and high potency towards parasites. In this study, a comprehensive forced degradation study was carried out on MO drug substance to identify and characterize its major degradation products (DPs). MO drug substance was subjected to acid, base, oxidation (H2O2), heat (solid and solution state), and photolytic (solid and solution state) stress degradation as per the ICH guidelines. Chromatographic separation of the drug substance (MO A3 and MO A4) and its DPs was achieved using a gradient elution on a HALO C18 column (100 × 4.6 mm, 2.7 µm). Mobile phase A consisted of water/acetonitrile (60/40, v/v) and mobile phase B consisted of ethanol/isopropanol (50/50, v/v). A total of twelve major DPs were observed for MO drug substance under various stress conditions. These DPs were further identified and characterized using liquid chromatography-high resolution mass spectrometry and comparison of their fragmentation profile with MO A4 and MO A3 using tandem mass spectrometry. Of these, H2O2 induced oxidative degradation product (3,4-dihydroperoxide MO A4) was isolated using semi-preparative HPLC and characterized by comparison of its nuclear magnetic resonance spectroscopy data with MO A4. The proposed structures of the DPs have been rationalized by appropriate degradation pathways for MO A4 and MO A3.

Identification and characterization of major degradation products of Eprinomectin drug substance including degradation pathways using LC-HRMS and NMR.

Eprinomectin (EPM) is a semi-synthetic potent antiparasitic drug widely used in veterinary medicine. In this study, a comprehensive forced degradation study was carried out on EPM drug substance as per ICH guidelines. Generation of adequate quantities of major degradation products of EPM via forced degradation studies was necessary for identification, structure elucidation, and understanding its degradation mechanism and degradation pathways. EPM drug substance was subjected to acid, base, oxidation (H2O2 and K2Cr2O7), thermal (solid and solution state), and photolytic (solid and solution state) stress degradation. The degradation products (DPs) formed in the stressed degraded samples were successfully separated using a gradient elution on a HALO C18 column (100 × 4.6 mm, 2.7 µm). Mobile phase A consisted of water and mobile phase B consisted of ethanol/isopropanol (98/2, v/v). A total of six major DPs of EPM drug substance formed under various stress conditions. The chemical structures of DPs were determined using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and characterized through comparison of their fragmentation profile with EPM B1a using tandem mass spectrometry (MS/MS). Additionally, two solvates (methanol adduct B1a #1 and methanol adduct B1a #2) were observed during the acid-stressed degradation study of EPM in presence of methanol. To confirm the chemical structure, these products were isolated with semi-preparative HPLC and characterized by using a combination of LC-MS/MS and nuclear magnetic resonance spectroscopy. The elucidated chemical structure of the degradation products of EPM was also justified through mechanistic explanations. Identification and characterization of the DPs including degradation mechanism(s) of EPM should facilitate the understanding of the stability behavior of EPM drug substances as well as aid in the design of new formulations made with EPM.

A comprehensive study to identify and characterize major degradation products of Ivermectin drug substance including its degradation pathways using LC-HRMS and NMR.

Ivermectin (IVM) drug substance is a semi-synthetic macrocyclic lactone that exhibits a broad spectrum of activity and high potency towards endo- and ectoparasites. In this study, a comprehensive forced degradation study was carried out on IVM drug substance (under the conditions recommended in the ICH guidelines) to identify and characterize its major degradation products (DPs). IVM drug substance was subjected to acidic, alkaline, oxidation (H2O2 and K2Cr2O7), thermal (solid and solution state), and photolytic (solid and solution state) stress degradations. Chromatographic separation of the drug substance and its DPs was achieved using a gradient elution on a HALO C18 column (150 × 4.6 mm, 2.7 µm). A total of five major DPs were observed for IVM drug substance under various stressed conditions. Additionally, ivermectin API lots exhibited instability when stored under room temperature and at 45% relative humidity for two years. These DPs were identified and characterized using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and a comparison of their fragmentation profile with IVM H2B1a using tandem mass spectrometry. Of these, H2O2 induced oxidative degradation product (3,4-epoxide H2B1a) was isolated using semi-preparative HPLC and its structure was elucidated comprehensively using LC-HRMS and nuclear magnetic resonance spectroscopy. The proposed structures of the DPs have been rationalized by appropriate degradation pathways of IVM H2B1a. Comprehensive degradation profile of IVM drug substance should facilitate the understanding of the stability profile of IVM drug substance, setting the specification of DPs in finished products as well as aid in the design of generic formulation made with IVM.

Simultaneous determination of ivermectin, clorsulon and their related substances in an injectable finished product by a stability-indicating RP-HPLC method.

A reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for the determination of ivermectin and clorsulon, including the identification and estimation of their related impurities in an ivermectin and clorsulon injectable finished product. Chromatographic separation was achieved using a gradient elution on a Supelco Ascentis® Express C18 column (150 mm × 4.6 mm i.d., 5 µm particle size) maintained at 55 °C. Mobile phase-A is composed of water and mobile phase-B is composed of acetonitrile/methanol (65/35, v/v). Analytes were monitored by UV detection at 245 nm. The stability-indicating capability of this method has been demonstrated by the adequate separation of all the process related impurities and degradation products of ivermectin and clorsulon from the stress degraded samples of the injectable product. This method was also successfully validated as per the current ICH guidelines with respect to specificity, linearity (R2> 0.999), limit of detection (0.3 µg/mL), limit of quantitation (1.0 µg/mL), accuracy, precision, and robustness for both APIs. This proposed method can significantly benefit the end-users in QC laboratories with laboratory efficiency and throughput during routine analysis and stability monitoring of the injectable product.

Development and Validation of a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Identification, Assay and Estimation of Related Substances of Ivermectin in Bulk Drug Batches of Ivermectin Drug Substance.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for the identification and assay of Ivermectin, including the identification and estimation of its process-related impurities and degradation products in bulk drug substance of Ivermectin. Analytes were separated on a HALO C18 column (100 mm × 4.6 mm I.D., 2.7 µm particle size) maintained at 40 °C (column temperature) with gradient elution. All analytes of interests were adequately separated within 25 min. All degradation products, process-related impurities and assay were monitored by ultraviolet detection at 254 nm. The new HPLC method described here successfully separated an isomer peak of the active pharmaceutical ingredient (API) from the major API peak. This newly separated isomer peak is around 1.2 to 1.5% (peak area) in typical API samples, and coelutes with the major API peak by all current HPLC methods. Quantitation limit of the HPLC method is 0.1% of target analytical concentration (~1.0 µg/mL). This method has been demonstrated to be accurate, robust, significantly higher degree of selectivity compared to the HPLC methods of Ivermectin drug substance reported in the literature and in the compendial HPLC methods prescribed in the current USA and European Pharmacopeia.

Development and Validation of a Stability-Indicating Reversed-Phase Ultra High Pressure Liquid Chromatography Method for Assay of Delmopinol Hydrochloride and Estimation of Its Related Substances in Commercial Bulk Batches.

Delmopinol hydrochloride is widely used as an active ingredient (AI) in many human oral hygiene products. To the best of our knowledge (via literature search), there is no stability-indicating liquid chromatography method available in the public domain for assay and estimation of related substances of Delmopinol hydrochloride. A fast stability-indicating Reversed-Phase Ultra High-Performance Liquid Chromatography (RP-UHPLC) method has been developed and validated for identification, assay and estimation of related substances in commercial bulk batches of this AI. The major peak of the AI and its related compounds are adequately separated in 6 min by a gradient elution on a hybrid silica-based C18 column (Waters Acquity UPLC® BEH C18, 50mm × 2.1 mm I.D., 1.7 µm particle size) maintained at 50°C. Mobile phase-A is composed of aqueous 10 mM NH4OH and mobile phase-B is ACN. The AI and its related compounds are detected with UV detection at 220 nm and quantitated by an external Delmopinol hydrochloride reference standard. The quantitation limit of the method is 0.1% of the target analytical concentration. This UHPLC method is fast and green and has been demonstrated to be specific, accurate, linear, precise, sensitive and robust as per ICH guidelines.

Assay of afoxolaner and determination of its related substances in commercial bulk batches of afoxolaner by reversed-phase HPLC method based on a short octadecyl column.

Afoxolaner is a new insecticidal and acaricidal active pharmaceutical ingredient (API) belonging to the isoxazoline family, widely prescribed for the control of fleas and ticks in dogs. A stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for the assay of afoxolaner and determination of its related compounds in bulk API lots of afoxolaner. The chromatographic separation of afoxolaner and its related compounds was achieved by gradient elution on a Zorbax-SB C18 column (50 mm × 4.6 mm i.d., 5 µm particle size) maintained at 40 °C. Mobile phase-A is composed of water and mobile phase-B is composed of acetonitrile/methanol (50/50, v/v). Analytes were monitored by UV detection at 225 nm with a flow rate of 2.0 mL/min. The stability-indicating capability of the method has been demonstrated by adequate separation of all the process related impurities and degradation products of afoxolaner generated by stress degradation of afoxolaner bulk drug substance under various stress conditions. This method was also successfully validated as per the current ICH guidelines for afoxolaner and Q6S07 (afoxolaner related substance) with respect to specificity, linearity (R2 > 0.999), detection limit (∼0.21 µg/mL), quantitation limit (∼0.70 µg/mL), accuracy, precision, and robustness. Due to its speed, high degree of selectivity, and accuracy, the proposed method is suitable and highly desirable in quality control laboratories for routine analysis of afoxolaner.

Development and Validation of a Stability-Indicating Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Identification, Assay, and Estimation of Related Substances of Ivermectin Drug Substance.

BACKGROUND: Ivermectin is a potent semi-synthetic antiparasitic drug used in veterinary medicine. It is widely used for the treatment of parasites. OBJECTIVE: This study aimed to develop a stability-indicating reversed-phase HPLC (RP-HPLC) method for assay and identification of ivermectin including identification and estimation of its related substances in bulk drug substance batches of ivermectin. METHOD: Ivermectin and its related substances were separated on an Ascentis Express C18 column (100 mm × 4.6 mm id, 2.7 µm particle size) maintained at 45°C (column temperature) on an HPLC system with gradient elution. The mobile phase was composed of water - acetonitrile (ACN; 50 + 50, v/v) as mobile phase A, and isopropanol - ACN (15 + 85, v/v) as mobile phase B. Analytes were detected with a detection wavelength of 252 nm and quantitated against an external reference standard of ivermectin with a quantitation limit of 0.1% of the target (analytical) concentration. RESULTS: The HPLC method was able to separate all analytes of interest by gradient elution within 25 min. The method was validated according to the guidelines described in the International Conference on Harmonization guideline Q2(R1). CONCLUSIONS: The HPLC method for assay of ivermectin and estimation of its related substances was successfully developed, validated, and demonstrated to be accurate, robust, specific, and stability indicating. HIGHLIGHTS: The performance of the HPLC method is significantly faster and possesses a higher degree of selectivity. Implementation of this method for routine analysis in QC laboratories would save significant time, resources and solvents.

Alternative and Improved Stability-Indicating HPLC Method for the Assay of Eprinomectin and Determination of Its Related Compounds in Bulk Batches of Eprinomectin Drug Substance.

BACKGROUND: Eprinomectin is used as an active pharmaceutical ingredient (API) in various drug products by the animal health industry. Several major related impurities of eprinomectin API are not separated and coelute by the current United States Pharmacopeia (USP) method for eprinomectin. OBJECTIVE: The aim was to develop and validate a true and reliable stability-indicating reversed-phase (RP) HPLC method for assay and determination of related substances of eprinomectin in bulk batches of eprinomectin API. METHOD: HPLC analysis is carried out using a Kinetex C8 column (100 mm × 4.6 mm i.d. , 2.6 µm particle size) maintained at 30°C with water-acetonitrile-isopropanol (48 + 42 + 10, v/v/v) as mobile phase A and 100% acetonitrile as mobile phase B. Analytes are separated by gradient elution at a flow rate of 0.7 mL/min and detected by UV at 252 nm. The total run time of the method is 30 min. Eprinomectin assay and estimation of all eprinomectin-related substances are obtained in a single HPLC run. RESULTS: The HPLC method was able to separate all analytes of interest by gradient elution. The new method was successfully validated according to current The Internal Council for Hamonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and International Cooperation on Hamonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guidelines and was found to be specific, linear, accurate, precise, robust, and sensitive. CONCLUSIONS: An HPLC method for assay of eprinomectin and estimation of its related substances was successfully developed, validated, and demonstrated to be accurate, robust, specific, and stability-indicating. HIGHLIGHTS: The HPLC method presented in this paper is more desirable as compared to USP and suitable for routine analysis of bulk batches of eprinomectin API and stability samples in QC laboratories.